COMPOSITIONS FOR RESTORING GENE EXPRESSION IN NEUROPSYCHIATRIC OR NEURODEGENERATIVE DISORDERS

Abstract

Methods for increasing reelin (RELN) levels in the brain of a subject in need thereof, as well as compositions for use in such methods, are provided. In addition, methods and compositions are described herein which may be used in the treatment of neuropsychiatric or neurodegenerative disorders, such as schizophrenia and Alzheimer's disease (AD). Compositions described herein may comprise whey protein isolate and/or whey protein concentrate, a source of the glutathione precursor cysteine. The provided methods and compositions are not limited to increasing RELN levels, and may be used to correct a number of other neurological disregulations or abnormalities occurring in a subject in need thereof.

Description

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/398,892, filed Sep. 23, 2016, which is herein incorporated by reference in its entirety.

FIELD OF INVENTION

[0002] The present invention relates generally to methods and compositions for treating neurological or neurodegenerative diseases or conditions. More specifically, the present invention relates to compositions comprising whey protein isolate and/or concentrate, and their use in increasing reelin (RELN) levels and/or in restoring other imbalances or abnormalities of gene expression linked with a neuropsychiatric or neurodegenerative disorder in a subject in need thereof.

BACKGROUND

[0003] Neuropsychiatric and neurodegenerative disorders represent a significant and ongoing concern for public health and wellbeing. Millions of individuals have suffered, or will suffer from, some form of mental disorder during their lifetime. These diseases and conditions are highly complex, and as a result successful treatment can be quite challenging. Schizophrenia (SCZ), for example, is a complex neuropsychiatric condition including disordered thought and behavior that afflicts approximately 1% of the population.sup.1. Alzheimer's disease (AD), as another example, affects millions of people worldwide and has only limited treatment options available.

[0004] While the majority of neuropsychiatric and neurodegenerative disorders are believed to stem from diseases of the nervous system, their root causes and mechanisms of disease progression have proven complex and difficult to treat. Even still, a number neuropsychiatric and neurodegenerative disorders have been linked or attributed at least in part to imbalances or genetic abnormalities arising in connection with environmental factors, gene mutations, and/or dysregulation of expression of particular genes.

[0005] Indeed, a host of prenatal and early postnatal stressors have been implicated as risk factors or triggers for human neurodevelopmental disorders.sup.2,3 such as SCZ.sup.4,5, autism.sup.6,7 and attention deficit-hyperactivity disorder (ADHD).sup.8. Candidate perinatal stressors include exposure to noxious chemicals during pregnancy, maternal infection (bacterial, viral, or parasitic) and attendant immune activation, and maternal psychotrauma and associated activation of the hypothalamic-pituitary-adrenal axis. While the etiopathogenesis of SCZ, as with many other neuropsychiatric and neurodegenerative disorders, remains incompletely understood, there is evidence suggesting that many genetic and perinatal risk factors converge on limited neurodevelopmental pathways to elicit the disease.

[0006] Alzheimer's disease is another particularly relevant example of a complex neurodegenerative disease which affects millions worldwide, and for which treatment has proven difficult. Hallmarks of AD include amyloid plaques (insoluble deposits of amyloid beta peptide) and neurofibrillary tangles containing hyper-phosphorylated tau protein. Treatment options are limited, and are generally not curative.

[0007] Substantial research efforts have been directed toward the identification of genes which are linked with neuropsychiatric and neurodegenerative disorders, and toward the identification of therapeutic agents capable of restoring such genes to healthy expression and/or activity levels. Although certain genes believed to be involved in the onset and/or progression of particular neuropsychiatric and neurodegenerative disorders have been identified, treatment options successfully taking advantage of these gene targets are still lacking.

[0008] Reelin (RELN; human NCBI Gene ID: 5649; human mRNA and amino acid sequences shown in FIG. 11 as SEQ ID NO:1 and SEQ ID NO: 2, respectively; mouse NCBI Gene ID: 19699; mouse genome ID: gi:372099105; mouse mRNA and amino acid sequences shown in FIG. 11 as SEQ ID NO: 23 and SEQ ID NO: 24, respectively) is an example of a gene which has been linked to a variety of different neuropsychiatric and neurodegenerative disorders. According to the NCBI, RELN may be involved in schizophrenia, autism, bipolar disorder, and major depression. RELN expression levels are significantly reduced in both schizophrenia and Alzheimer's disease, for example. Reelin overexpression may protect against, or rescue, certain aspects of Alzheimer's disease (see Pujadas, L., et al., 2013, Reelin delays amyloid-beta fibril formation and rescues cognitive deficits in a model of Alzheimer's disease, Nature Communications, DOI: 10.1038/ncomms4443), for example.

[0009] Reelin is secreted by specific cells within the central nervous system, and plays a key role in patterning and layering of the cerebral cortex and other regions of the brain during development. In adults, Reelin is thought to be a central regulator of synapse formation and critical neuronal processes required for learning and memory. Alzheimer's disease is the most prevalent cognitive disorder in adults and is characterized by substantial deficits in learning and memory. The entorhinal cortex layer II neurons are one of the first populations to die in AD, resulting in a severe loss of synaptic contacts to the dentate gyrus. Many of the entorhinal cortex layer II neurons express Reelin, and these Reelin-expressing cells are significantly reduced in the brains of human amyloid precursor protein (hAPP) transgenic mice expressing the Swedish and Indiana mutant form of the hAPP gene (J20 strain). Similar loss of Reelin-expressing entorhinal cortex layer II neurons is also observed in the brains of patients with AD (Chin, J., et al., 2007, J Neurosci, 27(11): 2727-2733; Herring, A., et al., 2012, J Alz Dis, 30(4): 963-979). In a transgenic rat model of AD (McGill-R-Thy1-APP strain). Reelin-expressing neurons of the entorhinal cortex layer II were found to selectively express increased levels of soluble intracellular A.beta. early in disease, prior to the deposition of amyloid plaques (Kobro-Flatmoen, A., et al., 2016, Neurobiol Dis 93: 172-183). Collectively, these studies suggest that Reelin-expressing neurons of entorhinal cortex layer II play a central role in the early pathogenic changes in AD, and that loss of these Reelin-expressing cells and their synaptic projections to the hippocampus are early markers of disease (Krstic, D., et al., 2013, Neuroscience 246: 108-116).

[0010] Alternative, additional, and/or improved methods and compositions for restoring healthy levels of gene products linked to neuropsychiatric and neurodegenerative disorders, such as RELN, are highly desirable.

SUMMARY OF INVENTION

[0011] Methods and compositions for treating neurological, neuropsychiatric, or neurodegenerative diseases or conditions in a subject in need thereof are provided. It has been found that, as described in detail herein, compositions comprising whey protein isolate and/or whey protein concentrate allow for restoration of reelin (RELN) levels in the brain of a subject in need thereof. The provided methods and compositions are not limited to increasing or restoring RELN levels, and may also, or alternatively, be used to correct a number of other neurological conditions, imbalances, dysregulations, or abnormalities occurring in a subject. Subjects suffering from neuropsychiatric or neurodegenerative diseases may particularly benefit from treatment with such compositions, however other subjects may also benefit from such treatment with whey protein isolate and/or concentrate compositions as described herein.

[0012] In an embodiment, there is provided herein a method for increasing reelin (RELN) levels in the brain a subject in need thereof, said method comprising:

[0013] administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

[0014] In another embodiment of the above method, the method may further comprise a step of:

[0015] measuring an initial RELN level of the subject and comparing the measured initial RELN level to a healthy RELN level,

[0016] wherein the subject is identified as having a reduced RELN level in need of increase when the measured initial RELN level is less than the healthy RELN level.

[0017] In still another embodiment of the above method or methods, administration of the composition comprising whey protein isolate and/or whey protein concentrate may increase expression levels of GAD67, a gene acting downstream of RELN.

[0018] In still another embodiment of the above method or methods, the subject in need thereof may be a subject suffering from a neuropsychiatric or neurodegenerative disorder.

[0019] In yet another embodiment of the above method or methods, the subject in need thereof may be a subject suffering from schizophrenia.

[0020] In another embodiment of the above method or methods, the subject in need thereof may be a subject sufferning from Alzheimer's disease (AD).

[0021] In still another embodiment of the above method or methods, the subject in need thereof may be a subject suffering from a neurological disorder characterized by enhanced expression of heme oxygenase-1 and/or increased oxidative stress in brain such as Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis, other motor neuron disorders, Down's syndrome, Creutzfeldt-Jakob disease, other prion diseases, multiple sclerosis, cerebral ischemia, cerebral hemorrhage, traumatic brain injury, spinal cord injury, cerebral malaria, schizophrenia, bipolar disease with psychosis, or autism.

[0022] In yet another embodiment of the above method or methods, the reelin (RELN) levels may be neuronal reelin (RELN) levels.

[0023] In another embodiment, there is provided herein a method for treating, preventing, or ameliorating symptoms of a neuropsychiatric disease or a neurodegenerative disorder in a subject in need thereof, said method comprising:

[0024] administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

[0025] In another embodiment of the above method, the composition comprising whey protein isolate and/or whey protein concentrate may be administered to increase reelin (RELN) levels in the brain of the subject.

[0026] In still another embodiment of the above method, the method may further comprise a step of:

[0027] measuring an initial RELN level of the subject and comparing the measured initial RELN level to a healthy RELN level,

[0028] wherein the subject is identified as being particularly susceptible to treatment when the measured initial RELN level is less than the healthy RELN level.

[0029] In yet another embodiment of the above method or methods, the neuropsychiatric disease or neurodegenerative disorder may be schizophrenia or Alzheimer's disease (AD).

[0030] In still another embodiment of the above method or methods, the neuropsychiatric disease or neurodegenerative disorder may be a neurological disorder characterized by enhanced expression of heme oxygenase-1 and/or increased oxidative stress in brain such as Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis, other motor neuron disorders, Down's syndrome, Creutzfeldt-Jakob disease, other prion diseases, multiple sclerosis, cerebral ischemia, cerebral hemorrhage, traumatic brain injury, spinal cord injury, cerebral malaria, schizophrenia, bipolar disease with psychosis, or autism.

[0031] In yet another embodiment of the above method or methods, the reelin (RELN) levels may be neuronal reelin (RELN) levels.

[0032] In another embodiment, there is provided herein a use of a composition comprising whey protein isolate and/or whey protein concentrate for increasing reelin (RELN) levels, such as but not limited to neuronal reelin levels, in a subject in need thereof.

[0033] In yet another embodiment, there is provided herein a use of a composition comprising whey protein isolate and/or whey protein concentrate in the manufacture of a medicament for increasing reelin (RELN) levels, such as but not limited to neuronal reelin levels, in a subject in need thereof.

[0034] In another embodiment, there is provided herein a use of a composition comprising whey protein isolate and/or whey protein concentrate for treating, preventing, or ameliorating symptoms of a neuropsychiatric disease or a neurodegenerative disorder in a subject in need thereof.

[0035] In still another embodiment, there is provided herein a use of a composition comprising whey protein isolate and/or whey protein concentrate in the manufacture of a medicament for treating, preventing, or ameliorating symptoms of a neuropsychiatric disease or a neurodegenerative disorder in a subject in need thereof.

[0036] In another embodiment, there is provided herein a method for restoring serotonin levels; normalizing MnSOD mRNA levels; augmenting glutathione (GSH) levels in brain cells; augmenting whole brain GSH/glutathione disulfide (GSSG) ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; improving HMOX1-related changes in brain dopamine, dopamine metabolites, serotonin, norephinephrine, and epinephrine; restoring deficient DOPAC/DA ratios; augmenting norepinephrine and dopamine concentrations in the prefrontal cortex; and/or correcting prefrontal cortex hypodopaminergia in a subject in need thereof, said method comprising:

[0037] administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

[0038] In yet another embodiment, there is provided herein a method for augmenting GSH reserves in the brain; normalizing brain MnSOD mRNA levels; restoration of redox homeostasis; normalization of brain dopamine, serotonin, norepinephrine, and/or epinephrine levels; normalization of brain DOPAC/DA ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; and/or ameliorating hyperlocomotion and/or stereotypic behaviour; or any combination thereof; in a subject in need thereof, said method comprising:

[0039] administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

[0040] In another embodiment of the above method or methods, the subject may be a subject suffering from, or may be at risk of developing, a neuropsychiatric or neurodegenerative disorder.

[0041] In yet another embodiment of the above method, the neuropsychiatric or neurodegenerative disorder may be schizophrenia or Alzheimer's disease (AD).

[0042] In still another embodiment of the above method or methods, the neuropsychiatric or neurodegenerative disorder may be a neurological disorder characterized by enhanced expression of heme oxygenase-1 and/or increased oxidative stress in brain such as Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis, other motor neuron disorders, Down's syndrome, Creutzfeldt-Jakob disease, other prion diseases, multiple sclerosis, cerebral ischemia, cerebral hemorrhage, traumatic brain injury, spinal cord injury, cerebral malaria, schizophrenia, bipolar disease with psychosis, or autism.

[0043] In yet another embodiment of the above method or methods, the composition comprising whey protein isolate and/or whey protein concentrate may be administered for restoring serotonin levels; normalizing MnSOD mRNA levels; augmenting glutathione (GSH) levels in brain cells; augmenting whole brain GSH/glutathione disulfide (GSSG) ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; improving HMOX1-related changes in brain dopamine, dopamine metabolites, serotonin, norephinephrine, and epinephrine; restoring deficient DOPAC/DA ratios; augmenting norepinephrine and dopamine concentrations in the prefrontal cortex; and/or correcting prefrontal cortex hypodopaminergia in the subject.

[0044] In still another embodiment of the above method or methods, the composition comprising whey protein isolate and/or whey protein concentrate may be administered for augmenting GSH reserves in the brain; normalizing brain MnSOD mRNA levels; restoration of redox homeostasis; normalization of brain dopamine, serotonin, norepinephrine, and/or epinephrine levels; normalization of brain DOPAC/DA ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; and/or ameliorating hyperlocomotion and/or stereotypic behaviour; or any combination thereof; in the subject.

[0045] In an embodiment, there is provided herein a use of a composition comprising whey protein isolate and/or whey protein concentrate for restoring serotonin levels; normalizing MnSOD mRNA levels; augmenting glutathione (GSH) levels in brain cells; augmenting whole brain GSH/glutathione disulfide (GSSG) ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression;

[0046] improving HMOX1-related changes in brain dopamine, dopamine metabolites, serotonin, norephinephrine, and epinephrine; restoring deficient DOPAC/DA ratios; augmenting norepinephrine and dopamine concentrations in the prefrontal cortex; and/or correcting prefrontal cortex hypodopaminergia in a subject in need thereof.

[0047] In still another embodiment, there is provided herein a use of a composition comprising whey protein isolate and/or whey protein concentrate for augmenting GSH reserves in the brain; normalizing brain MnSOD mRNA levels; restoration of redox homeostasis; normalization of brain dopamine, serotonin, norepinephrine, and/or epinephrine levels; normalization of brain DOPAC/DA ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; and/or ameliorating hyperlocomotion and/or stereotypic behaviour; or any combination thereof; in a subject in need thereof.

[0048] In yet another embodiment, there is provided herein a use of a composition comprising whey protein isolate and/or whey protein concentrate in the manufacture of a medicament for restoring serotonin levels; normalizing MnSOD mRNA levels; augmenting glutathione (GSH) levels in brain cells; augmenting whole brain GSH/glutathione disulfide (GSSG) ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; improving HMOX1-related changes in brain dopamine, dopamine metabolites, serotonin, norephinephrine, and epinephrine; restoring deficient DOPAC/DA ratios; augmenting norepinephrine and dopamine concentrations in the prefrontal cortex; and/or correcting prefrontal cortex hypodopaminergia in a subject in need thereof.

[0049] In another embodiment, there is provided herein a use of a composition comprising whey protein isolate and/or whey protein concentrate in the manufacture of a medicament for augmenting GSH reserves in the brain; normalizing brain MnSOD mRNA levels; restoration of redox homeostasis; normalization of brain dopamine, serotonin, norepinephrine, and/or epinephrine levels; normalization of brain DOPAC/DA ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; and/or ameliorating hyperlocomotion and/or stereotypic behaviour; or any combination thereof; in a subject in need thereof.

[0050] In another embodiment, there is provided herein a method for increasing GAD67 levels, such as but not limited to neuronal GAD67 levels, in a subject in need thereof, said method comprising:

[0051] administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

[0052] In still another embodiment, there is provided herein a use of a composition comprising whey protein isolate and/or whey protein concentrate for increasing GAD67 levels, such as but not limited to neuronal GAD67 levels, in a subject in need thereof.

[0053] In yet another embodiment, there is provided herein a use of a composition comprising whey protein isolate and/or whey protein concentrate in the manufacture of a medicament for increasing GAD67 levels, such as but not limited to neuronal GAD67 levels, in a subject in need thereof.

[0054] In another embodiment of the above methods or uses, the RELN levels may be levels in the prefrontal cortex of the brain.

[0055] In still another embodiment of the above methods or uses, the subject may be a mammal, such as a human.

[0056] In another embodiment, there of provided herein a method for inducing or restoring RELN expression and/or signalling in the entorhinal cortex and/or hippocampus of a subject in need thereof, said method comprising:

[0057] administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

[0058] In yet another embodiment of the above method, the subject may have or may be at risk of developing schizophrenia or Alzheimer's Disease.

[0059] In still another embodiment, there is provided herein a use of a composition comprising whey protein isolate and/or whey protein concentrate for inducing or restoring RELN expression and/or signalling in the entorhinal cortex and/or hippocampus of a subject in need thereof.

[0060] In yet another embodiment, there is provided herein a use of a composition comprising whey protein isolate and/or whey protein concentrate in the manufacture of a medicament for inducing or restoring RELN expression and/or signalling in the entorhinal cortex and/or hippocampus of a subject in need thereof.

[0061] In another embodiment of the above uses, the subject may have or may be at risk of developing schizophrenia or Alzheimer's Disease.

[0062] In another embodiment, there is provided herein a method for treating, preventing, or ameliorating symptoms of Alzheimer's Disease in a subject in need thereof, said method comprising:

[0063] administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

[0064] In another embodiment, there is provided herein a use of a composition comprising whey protein isolate and/or whey protein concentrate for treating, preventing, or ameliorating symptoms of Alzheimer's Disease in a subject in need thereof.

[0065] In still another embodiment, there is provided herein a use of a composition comprising whey protein isolate and/or whey protein concentrate in the manufacture of a medicament for treating, preventing, or ameliorating symptoms of Alzheimer's Disease in a subject in need thereof.

[0066] In another embodiment of the above methods or uses, the composition maybe for preserving or restoring cognitive function in the subject.

[0067] In yet another embodiment of the above methods or uses, the composition may be administered in an amount sufficient to provide at least one of:

[0068] an increase in RELN levels in the entorhinal cortex;

[0069] an increase in RELN levels in the dentate gyrus;

[0070] an increase in RELN levels in the CA1 region;

[0071] an increase in RELN levels in the CA3 region;

[0072] a correction in a deficit in cortical GSH levels;

[0073] a restoration of GAD67 expression in the hippocampal-entorhinal cortex sub-region;

[0074] a restoration of p-CREB levels in the hippocampal-entorhinal cortex sub-region;

[0075] an increase in RELN levels in layer II of the entorhinal cortex;

[0076] a prevention of loss of RELN positive neurons in the entorhinal cortex; or

[0077] a restoration of GAD67 expression in the dentate gyrus and/or CA3 region of the hippocampus;

[0078] or any combination thereof.

[0079] In still another embodiment of the above methods or uses, the subject may be a mammal, such as a human.

BRIEF DESCRIPTION OF DRAWINGS

[0080] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0081] FIG. 1 shows results of prepulse inhibition studies. (A.) Casein-treated (control) GFAP.HMOX1 transgenic (TG) and wild-type (WT) male (top panel) and female (bottom panel) mice. (B.) Immunocal-treated TG and WT male (top panel) and female (bottom panel) mice. (C.) Comparison of Immunocal- and casein-treated WT mice (top panel, males; bottom panel, females). (D.) Comparison of Immunocal- and casein-treated TG mice (top panel, male; bottom panel, female).

[0082] FIG. 2 shows results of locomotor activity studies. (A.) Male; (B.) Female. Total distance (top panels); STRCNT-stereotypy count, STRNO-stereotypy number, and MOVNO-movement number (middle panels); RESTIME-rest time, STRTIME-stereotypy time, and MOVTIME-movement time (bottom panels). *P<0.05, **P<0.01, ***P<0.001, relative to WT controls.

[0083] FIG. 3 shows results of whole brain glutathione concentration studies. (A.) Casein treatment (control); (B.) Immunocal treatment; (C.) Comparison of Immunocal- and casein-treated WT mice; (D.) Comparison of Immunocal- and casein-treated TG mice. Top panels, male and female mice; middle panels, males; bottom panels, females. *P<0.05, **P<0.01, ***P<0.001, relative to WT preparations.

[0084] FIG. 4 shows results of sub-regional brain glutathione concentration studies. (A.) PFC, HC, and STM of casein-treated (control) group. (B.) PFC, HC, and STM of Immunocal-treated group. (C.) Comparison of Immunocal- and casein-treated WT mice. (D.) Comparison of Immunocal- and casein-treated TG mice. *P<0.05, **P<0.01, ***P<0.001, relative to WT preparations.

[0085] FIG. 5 shows results of studies of glutathione levels in remaining brain tissue after removal of the PFC, HC, and STM. (A.) Casein-treated (control) group; (B.) Immunocal-treated group; (C.) Comparison of Immunocal- and casein-treated WT mice. (D.) Comparison of Immunocal- and casein-treated GFAP.HMOX1 mice.*P<0.05, **P<0.01, ***P<0.001, relative to WT preparations.

[0086] FIG. 6 shows results of studies of content of brain DA and metabolites. (A.) DA and metabolites. (B.) Ratio of DA to DOPAC or HVA. Number of animals per group are indicated in the bars. *P<0.05, **P<0.01, relative to WT preparations.

[0087] FIG. 7 shows results of studies of content of brain serotonin (5-HT), serotonin metabolite (5-HIAA), norepinephrine (NE) and epinephrine (E). (A.) Serotonin and metabolites; (B.) Norepinephrine and epinephrine. Number of animals per group are indicated in the bars. *P<0.05, relative to WT preparations.

[0088] FIG. 8 shows results of studies of brain histomorphology. (A.) Six-micron thick coronal sections (bregma--4.52 mm) stained with H & E. Note dilatation of lateral ventricles in the GFAP.HMOX1 preparations. (B.) Morphometrics of hippocampus and dentate gyrus. *P<0.05, relative to WT preparations.

[0089] FIG. 9 shows results of studies of brain mRNA profiles. (A.) MnSOD. (B.) Neuronal reelin (RELN). (C.) GAD67. (D.) Nrxn 1. E. Nlgn 2. *P<0.05, **P<0.01, ***P<0.001, relative to WT preparations.

[0090] FIG. 10 shows results of studies of brain miRNA profiles. (A.) mmu-miR-137-5p. (B.) mmu-miR-137-3p. (C.) mmu-miR-181a. (D.) mmu-miR-138. (E.) mmu-miR-128-1-5p. (F.) mmu-miR-128-3p. (G.) mmu-miR-200c. *P<0.05, **P<0.01, ***P<0.001, relative to WT preparations.

[0091] FIG. 11 provides the sequences of certain nucleic acids and amino acids referred to herein.

[0092] FIG. 12 shows distinct pathways of APP proteolytic processing. APP processing has two different pathways once it has been inserted into a lipid membrane. The non-amyloidogenic pathway utilizes .alpha.-secretase to produce soluble amyloid-beta precursor protein-alpha (sAPP.sub..alpha.) and C-terminal fragment 83 (CTF83). .gamma.-secretase then cleaves the CTF83 into a soluble p3 fragment and the amyloid precursor protein intracellular domain (AICD). The amyoidogenic pathway utilizes processing by .beta.-secretase, which produces the soluble amyloid-beta precursor protein-beta (sAPP.sub..beta.) and C-terminal fragment 99 or 89 (CTF99/89), depending on tissue type. .gamma.-secretase then cleaves CTF99/89 into A.beta..sub.40 or A.beta..sub.42, which become extracellular, and the AICD. Figure adapted from Muller et al. (2008).

[0093] FIG. 13 shows the canonical Reelin signaling pathway. Reelin binds to one of two cell surface receptors, VLDLR or Apoer2, and induces tyrosine phosphorylation of the adapter protein, Dab 1, via Src family kinase (SFK) activity. Phosphorylated Dab 1 acts as a docking site to initiate multiple downstream signaling cascades. Figure adapted from Wasser and Herz (2017).

[0094] FIG. 14 shows results of studies in which Immunocal.RTM. treatment increased Reelin expression in vitro in hippocampal-entorhinal cortex slices. A) Brain slices were incubated for 24 h in either control medium alone (Con) or containing recombinant Reelin (recRln). Following incubation, slices were lysed and DAB1 was immunoprecipitated (IP). The immune complexes were resolved by SDS-PAGE and immunoblotted (IB) for phosphotyrosine (PY) followed by stripping and reprobing for DAB1. B) Brain slices were incubated for 24 h in either Con medium or containing Immunocal.RTM. (ICAL). Protein lysates were IB for Reelin.

[0095] FIG. 15 shows results of studies in which Immunocal.RTM. treatment increased Reelin mRNA expression in vitro in hippocampal-entorhinal cortex slices. Brain slices were incubated for 24 h in either control medium (Con), medium containing Immunocal.RTM. (ICAL), recombinant Reelin protein (Rln), or a combination of the two. Total RNA was extracted from the slices, cDNA was prepared and subjected to qPCR using primers and probes for rat Reelin and GAPDH (as a control housekeeping gene). Data are expressed as the .DELTA..DELTA.Ct levels of the Reelin transcript normalized to GAPDH. **p<0.01 compared to Con; #p<0.05 versus Rln alone (n=5 independent rat brain slice preparations).

[0096] FIG. 16 shows results of studies in which Immunocal.RTM. treatment increased Reelin expression in vitro in hippocampal-entorhinal cortex slices. Brain slices were incubated for 24 h in either control medium alone (Con) or containing Immunocal.RTM. (ICAL). Following treatment, free floating slices were stained for Reelin (shown in green) and NeuN (shown in red). Images shown represent specific brain regions including entorhinal cortex (A), dentate gyrus (B), and CA1 region of hippocampus (C). z-stacked images were captured on a laser scanning confocal microscope using identical laser intensities and exposure times for each tissue slice.

[0097] FIG. 17 depicts Barnes maze apparatus employed for cognitive testing. During the acquisition phase of testing, the mouse is placed in one of the four quadrants as indicated, and allowed 90 seconds to explore the maze and find the escape pod, as indicated by the line and filled arrows. During the probe phase, the mouse is placed in the center of the maze and the time it takes for the mouse to find the escape pod or the hole to either side of the escape pod is recorded.

[0098] FIG. 18 shows cyto-architecture of the hippocampal-entorhinal cortex region of mouse brain. A control (non-carrier) mouse brain was co-stained for Reelin (shown in green), NeuN (shown in red), and Draq (a nuclear marker, shown in blue). The cartoon at top left shows the cyto-architecture of this region schematically.

[0099] FIG. 19 shows results of studies in which Immunocal.RTM. treatment preserved brain GSH in J20 AD model mice. Brain (cortical) tissue was harvested from 5 month-old hemizygous J20 AD mice (either untreated (Unt) or treated with Immunocal.RTM. (ICAL) for 2 months) and non-carrier control mice. Tissue was extracted and reduced GSH was measured by HPLC with electrochemical detection. Values are normalized to protein content and represent the mean.+-.SEM for n=3 mice per group. Statistical differences were calculated by one-way ANOVA with a post hoc Tukey's test. *significantly different than non-carrier at p<0.05. ##significantly different than Hemi (Unt) at p<0.01.

[0100] FIG. 20 shows results of studies in which Immunocal.RTM. treatment rescued Reelin expression and increased GAD67 expression in J20 AD model mice. Brain tissue was harvested from 5 month-old hemizygous J20 AD mice (either untreated (Unt) or treated with Immunocal.RTM. (ICAL) for 2 months) and non-carrier control mice. Tissue was micro-dissected to enrich for the hippocampal-entorhinal cortex sub-region. Brain lysates were resolved by SDS-PAGE and immunoblotted for (A) Reelin (Rln) and (B) GAD67. The Reelin blot was stripped and reprobed for OPAL The graph shows the densitometric analysis of the 180 kDa Reelin bands in arbitrary units (n=3 mice per group). Statistical differences were calculated by one-way ANOVA with a post hoc Tukey's test.

[0101] FIG. 21 shows results of studies in which Immunocal.RTM. treatment preserved brain p-CREB in J20 AD model mice. Brain tissue was harvested from 5 month-old hemizygous J20 AD mice (either untreated (Unt) or treated with Immunocal.RTM. (ICAL) for 2 months) and non-carrier control mice. Tissue was micro-dissected to enrich for the hippocampal-entorhinal cortex sub-region. Brain lysates were resolved by SDS-PAGE and immunoblotted for CREB phosphorylated on Ser133 (p-CREB). Brain lysates from two mice per treatment group are shown.

[0102] FIG. 22 shows results of studies in which Immunocal.RTM. treatment rescued Reelin expression in entorhinal cortex layer II of J20 AD model mice. Brain tissue was harvested from 5 month-old hemizygous (Hemi) J20 AD mice (either untreated (Unt) or treated with Immunocal.RTM. (ICAL) for 2 months) and non-carrier control mice. Formalin-fixed sections were co-stained with antibodies to Reelin (Rln, shown in green) and NeuN (shown in red), along with Draq (nuclear stain, shown in blue). The demarcated areas in the middle panels encompass an approximation of layer II of entorhinal cortex and are represented in the lower panels. The mean fluorescence intensity (per pixel area) of Reelin staining in this region was calculated using Adobe Photoshop. The untreated hemizygous J20 AD mouse showed an approximately 30% reduction in Reelin staining in layer II of entorhinal cortex compared to the non-carrier control. This deficit was prevented by treatment with Immunocal.RTM.. Similar results were observed in four independent sets of mice consisting of a non-carrier control, hemizygous untreated and hemizygous Immunocal.RTM.-treated.

[0103] FIG. 23 shows results of studies in which Immunocal.RTM. treatment increased Reelin expression in dentate gyrus of J20 AD model mice. Brain tissue was harvested from 5 month-old hemizygous (Hemi) J20 AD mice (either untreated (Unt) or treated with Immunocal.RTM. (ICAL) for 2 months) and non-carrier control mice. Formalin-fixed sections were co-stained with antibodies to Reelin (Rln, shown in green) and NeuN (shown in red), along with Draq (nuclear stain, shown in blue).

[0104] FIG. 24 shows results of studies in which Immunocal.RTM. treatment rescued Reelin expression in hippocampus CA1 of J20 AD model mice. Brain tissue was harvested from 5 month-old hemizygous (Hemi) J20 AD mice (either untreated (Unt) or treated with Immunocal.RTM. (ICAL) for 2 months) and non-carrier control mice. Formalin-fixed sections were co-stained with antibodies to Reelin (Rln, shown in green) and NeuN (shown in red), along with Draq (nuclear stain, shown in blue).

[0105] FIG. 25 shows results of studies in which Immunocal.RTM. treatment preserved Reelin expression in hippocampus CA3 of J20 AD model mice. Brain tissue was harvested from 5 month-old hemizygous (Hemi) J20 AD mice (either untreated (Unt) or treated with Immunocal.RTM. (ICAL) for 2 months) and non-carrier control mice. Formalin-fixed sections were co-stained with antibodies to Reelin (Rln, shown in green) and NeuN (shown in red), along with Draq (nuclear stain, shown in blue).

[0106] FIG. 26 shows results of studies in which Immunocal.RTM. treatment increased GAD67 expression in dentate gyrus of J20 AD model mice. Brain tissue was harvested from 5 month-old hemizygous (Hemi) J20 AD mice (either untreated (Unt) or treated with Immunocal.RTM. (ICAL) for 2 months) and non-carrier control mice. Formalin-fixed sections were co-stained with antibodies to GAD67 (shown in green) and NeuN (shown in red), along with Draq (nuclear stain, shown in blue).

[0107] FIG. 27 shows results of studies in which Immunocal.RTM. treatment preserved GAD67 expression in hippocampus CA3 of J20 AD model mice. Brain tissue was harvested from 5 month-old hemizygous (Hemi) J20 AD mice (either untreated (Unt) or treated with Immunocal.RTM. (ICAL) for 2 months) and non-carrier control mice. Formalin-fixed sections were co-stained with antibodies to GAD67 (shown in green) and NeuN (shown in red), along with Draq (nuclear stain, shown in blue).

[0108] FIG. 28 shows results of studies in which Immunocal.RTM. treatment enhanced co-staining of Reelin and phospho-DAB1 in hippocampus CA1 of J20 AD model mice. Brain tissue was harvested from 5 month-old hemizygous (Hemi) J20 AD mice (either untreated (Unt) or treated with Immunocal.RTM. (ICAL) for 2 months) and non-carrier control mice. Formalin-fixed sections were co-stained with antibodies to Reelin (shown in green) and phospho-DAB1 (pTyr232; shown in red), along with Draq (nuclear stain, shown in blue).

[0109] FIG. 29 shows results of studies in which Immunocal.RTM. treatment improved cognitive performance in the Barnes maze in J20 AD model mice. Following two months of Immunocal.RTM. treatment from 3 months-old to 5 months-old, hemizygous, female J20 AD mice were subjected to the Barnes maze to test spatial learning and memory. A) The delay in seconds for mice to find the escape pod during days 5 and 6 of the acquisition phase (combined) is shown. ##significantly different than hemizygous untreated (Hemi Unt) at p<0.01. B) The delay in seconds for mice to find the escape pod during the probe phase (day 7) is shown. *significantly different than non-carrier control at p<0.05. #significantly different than Hemi Unt at p<0.05. ICAL=Immunocal.RTM. treated, NC=non-carrier. All statistical comparisons were made by one-way ANOVA with a post-hoc Tukey's test (n=4 mice per group).

DETAILED DESCRIPTION

[0110] Described herein are methods for increasing reelin (RELN) levels and/or in restoring other imbalances or abnormalities of gene expression in a subject in need thereof, as well as compositions for use in such methods. Methods and compositions provided herein may be used in the treatment of neuropsychiatric or neurodegenerative disorders, such as schizophrenia and Alzheimer's disease, for example. Compositions described herein may comprise whey protein isolate and/or whey protein concentrate, which is a source of the glutathione precursor cysteine. The provided methods and compositions are not limited to increasing or restoring RELN levels, and may alternatively or additionally be used to correct a number of other neurological dysregulations or abnormalities occurring in a subject in need thereof as described in detail herein.

[0111] It will be appreciated that embodiments and examples are provided herein for illustrative purposes intended for those skilled in the art, and are not meant to be limiting in any way. One or more illustrative embodiments have been described by way of example. It will be understood to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.

[0112] In an embodiment, there is provided herein a method for increasing or restoring reelin (RELN) levels in the brain of a subject in need thereof, said method comprising:

[0113] administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

[0114] As will be understood, in certain embodiments, a subject in need thereof may include any subject for which increasing or restoring reelin (RELN) levels (and/or other gene expression imbalances or abnormalities as described in further detail hereinbelow) in the brain may be beneficial or desirable. By way of example, in certain embodiments, the subject may include a subject exhibiting at least one symptom or characteristic of a disease or disorder associated with low RELN levels. In certain embodiments, the subject may be a subject exhibiting at least one symptom or characteristic of a neuropsychiatric or neurodegenerative disorder such as schizophrenia or Alzheimer's disease, for example. By way of example, in certain embodiments, a subject in need of treatment may be determined as simply a subject exhibiting one or more symptoms of a disease or disorder for which the compositions described herein may prevent, ameliorate, reduce, or treat as further described hereinbelow.

[0115] As will be understood, an increase in RELN levels may refer to any increase in RELN gene expression in brain cells of the subject in need thereof. In certain embodiments, the RELN levels may be, for example, neuronal RELN levels, although RELN levels in other brain cells is also contemplated. In certain embodiments, the increase in RELN levels may be sufficient to restore RELN levels to, near, or above those of a healthy control subject or group. Gene expression may refer to the production of a polypeptide from the nucleic acid sequence of a gene. As well, gene expression may include both transcription and translation processes, and so an increase in RELN levels may refer to any increase in the production of a nucleic acid sequence such as a RELN mRNA (i.e. transcription), production of a RELN protein (i.e. translation), or both. In certain embodiments, RELN expression may include expression of full-length RELN protein (388 kDa), RELN cleavage product(s) (including, for example, 310 kDa and/or 180 kDa cleavage products), or any combination thereof. Increases in gene expression may be determined with reference to wild-type, healthy, baseline, or untreated levels, or levels measured at a previous time point, for example, as would be understood by a person of skill in the art.

[0116] Without wishing to be bound by theory, it will be understood that effective gene expression levels of a particular gene may also be considered increased if the rate of protein turnover/degradation of the expression product of the gene can be slowed or prevented.

[0117] Regardless of the underlying mechanism, references herein to increasing levels of a particular gene may include any suitable increase in mRNA levels and/or protein levels and/or activity levels of the gene as compared to wild-type, healthy, baseline, or untreated levels, or levels measured at a previous time point, or with reference to another suitable comparator level selected by the skilled person to suit a particular application.

[0118] As will also be understood, in additional embodiments, increases in RELN levels may refer to increases in signalling pathway activation, either as a result of increased RELN gene expression, or as a result of other factors resulting in increased pathway activation. In certain embodiments, a RELN level increase may be accompanied by, signalled by, or phenotypically represented by increased mRNA expression levels of GAD67, a gene acting downstream of RELN.

[0119] It will be understood that increases in neuronal RELN levels may occur generally throughout the brain of the subject, or may be limited to certain regions of the brain. In certain embodiments, RELN levels may be generally increased throughout the brain, or may be increased in particular brain regions such as the prefrontal cortex (PFC), the striatum (STM), the substantia nigra (SN), or the entorhinal cortex layer II, for example. In certain embodiments, a RELN level increase in a brain region may be accompanied by, signalled by, or phenotypically linked with increased mRNA expression levels of GAD67, a gene acting downstream of RELN. For example, GAD67 expression levels may be increased in the STM and/or the SN following treatment. In certain embodiments, neuronal RELN levels may be increased.

[0120] Compositions comprising whey protein isolate and/or whey protein concentrate may comprise any suitable composition comprising whey protein isolate and/or whey protein concentrate which may serve as a glutathione precursor by providing an enriched source of bioavailable cysteine after administration. As will be understood, whey proteins may generally be considered as a group a milk proteins which remain soluble in "milk serum" or whey after precipitation of caseins at pH 4.6 and 20.degree. C. Major whey proteins in cow's milk, for example, may include beta-lactoglobulin (.beta.L), alpha-lactalbumin (.alpha.L), immunoglobulin, and serum albumin (SA). The product of industrial separation of this protein mixture from whey is typically referred to as whey protein isolate (WPI; also known as whey protein concentrate, WPC).

[0121] Compositions may, optionally, additionally comprise one or more pharmaceutically acceptable excipients, diluents, and/or carriers, one or more vitamins, essential amino acids, or minerals, one or more antioxidants, one or more additional glutathione precursors, and/or one or more nutritional diet supplement components, for example.

[0122] Compositions may also include, and/or be used in simultaneous or sequential combination with, one or more other drugs, pharmaceutical compositions, or therapies used in the treatment or management of neuropsychiatric diseases or neurodegenerative disorders known to the person of skill in the art, as will be selectable by the skilled person to suit the particular subject and/or application. By way of example, drugs used in the treatment of schizophrenia may include antipsychotics such as amisulpride, olanzapine, risperidone, and clozapine, and neuroleptics for controlling psychosis in schizophrenia. Drugs used in the treatment of Alzheimer's disease may include acetylcholinesterase inhibitors such as tacrine, rivastigmine, galantamine, and donepezil, and/or NMDA receptor antagonists such as memantine. Drugs used in the treatment of Parkinson's disease may include 1-dopa replacement therapy, for example.

[0123] A pharmaceutically acceptable carrier, diluent, or excipient may include any suitable carrier, diluent, or excipient known to the person of skill in the art. Examples of pharmaceutically acceptable excipients may include, but are not limited to, cellulose derivatives, sucrose, and starch. The person of skill in the art will recognize that pharmaceutically acceptable excipients may include suitable fillers, binders, lubricants, buffers, glidants, and disentegrants known in the art (see, for example, Remington: The Science and Practice of Pharmacy (2006); herein incorporated by reference in its entirety). Examples of pharmaceutically acceptable carriers, diluents, and excipients may be found in, for example, Remington's Pharmaceutical Sciences (2000--20th edition) and in the United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

[0124] In certain embodiments, a whey protein isolate or a whey protein concentrate as described herein may include any suitable extract, isolate, concentrate, or other product which is obtainable from whey protein. As will be understood, whey protein comprises a mixture of milk proteins that remain soluble in milk serum or whey after precipitation of caseins, for example. Whey is often encountered as a by-product of cheese or casein manufacture. Major whey protein components may include, for example but without wishing to be limiting, beta-lactoglobulin, alpha-lactalbumin, immunoglobulin, and serum albumin. Although bovine milk is commonly used for obtaining whey protein, it will be understood that other sources of milk are also contemplated. Whey protein isolate (WPI) is generally considered in the field as having >90% protein, while whey protein concentrate (WPC) may have protein concentrations below 90%; however, for the present purposes, WPI and WPC may be considered as generally interchangeable unless otherwise explicitly specified.

[0125] In particular embodiments, a whey protein isolate or whey protein concentrate as described herein is preferably an undenatured whey protein isolate or whey protein concentrate. Undenatured isolates and concentrates are those in which one or more of the protein component(s) obtainable from whey protein remain substantially undenatured (i.e. tertiary protein structure is substantially maintained and/or disulfide bonds between cysteine residues remain substantially intact) in the whey protein isolate or whey protein concentrate.

[0126] Whey proteins contain sulfur-containing amino acids such as cysteine (Cys). These Cys amino acid residues may occur as free residues (i.e. --SH; reduced), or two Cys residues may form intramolecular disulfide bonds (S--S; oxidized) so as to produce cystine dimers. Such disulfide bonds play a role in protein folding. In certain embodiments, undenatured whey protein isolates or whey protein concentrates as described herein may include those having at least about 2 wt % cystine dimer. Examples of undenatured whey protein isolates and whey protein concentrates may include those having about 2 wt % cystine dimer, or more than about 2 wt % cystine dimer. For example, the wt % of cystine dimer may be about 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 wt %, or the wt % cystine dimer may fall within a range spanning between any two such values, or a range bounded at the lower end by any such value.

[0127] Whey protein isolates and whey protein concentrates may be obtained using any suitable technique(s) as will be known to the person of skill in the art having regard to the teachings herein. Such techniques may include ultrafiltration using membranes, ion exchange methods, and membrane methods, for example. Discussions of suitable techniques may be found in, for example, Advanced Dairy Chemistry, McSweeney and Mahony (Ed.), Volume 1B: Proteins: Applied Aspects, 4th Edition, Springer, ISBN: 978-1-4939-2799-9 (herein incorporated by reference in its entirety).

[0128] Examples of suitable compositions comprising whey protein isolate and/or whey protein concentrate are described in Canadian patent nos. 1,333,471, 1,338,682, 2,142,277, and 2,090,186, each of which is herein incorporated by reference in its entirety. CA 2,142,277, for example, provides detailed preparation processes and analytical characterization of particularly preferred compositions comprising whey protein isolate, including the composition known as Immunocal.RTM.. This exemplary whey protein isolate composition as described in CA 2,142,277 may be characterized by having a solubility index of about 99.5% at pH 4.6; about 58% .beta.L (beta-lactoglobulin) protein composition, about 11% .alpha.L (alpha-lactalbumin) protein composition, about 10% serum albumin (i.e. BSA) protein composition, and about 22% immunoglobulin (i.e. Ig) protein composition. A process for preparing such a composition is also described in detail in CA 2,142,277. Immunocal.RTM. (Natural Product Number (NPN) 80004370 issued with Health Canada) is now a commercially available whey protein isolate composition available from Immunotec.RTM..

[0129] Further description of whey protein isolates and concentrates may be found in Example 2 below.

[0130] In an embodiment, compositions as described herein may be administered orally. For example, compositions as described herein may be reconstituted in, or may comprise, a liquid carrier (for example, water or juice), allowing for straightforward oral administration. The person of skill in the art having regard to the teachings herein will be able to select a suitable administration to suit a particular subject and/or particular therapeutic application.

[0131] In certain non-limiting embodiments, it is contemplated that compositions as described herein may be administered orally in an amount suitable for achieving a desired effect. In certain non-limiting embodiments, compositions as described herein may be administered orally in a dosage of about 20-40 grams per day, for example, and may be administered once or more than once daily, for example.

[0132] Compositions as described herein may, in certain embodiments, be used in combination with a nucleic acid or expression vector which can cause overexpression of RELN or a functional RELN mimic, or may be used in combination with RELN protein or a functional RELN protein mimic, in order to increase effective RELN levels in the subject. The nucleic acid and amino acid sequences of human RELN are provided in FIG. 11 as SEQ ID NOs. 1 and 2, respectively. Murine RELN sequences are also shown in FIG. 11, and homologs in other species are available from the national center for biotechnology information (NCBI).

[0133] In a further embodiment of the methods described above, the methods may further comprise an additional step of:

[0134] measuring an initial RELN level of the subject and comparing the measured initial RELN level to a healthy RELN level, wherein the subject is identified as having a reduced RELN level in need of increase when the measured RELN level is less than the healthy RELN level.

[0135] As will be understood, a healthy RELN level may be a RELN level as determined in a wild type, control, or healthy subject or a group of healthy subjects, or as determined from the subject at a previous time point where the subject was healthy, for example. The healthy level may be a specific or approximate threshold level, or may be a range spanning between upper and lower thresholds.

[0136] It will be understood that such a step of measuring may be performed either before or after administration of the composition comprising the whey protein isolate and/or whey protein concentrate, or both. Where the step of measuring is performed before the administration, the measuring step may be considered as a screening step, allowing for the identification of subjects in need of the treatment, of subjects who may particularly benefit from the treatment, and/or of subjects belonging to a patient subpopulation which may be particularly susceptible to the treatment. Where the step of measuring is performed after the administration, the measuring step may be considered as a step of determining treatment efficacy, allowing for the identification of subjects in need of a subsequent, repeated, or adjusted treatment, of subjects who may particularly benefit from a repeated treatment, and/or of subjects belonging to a patient subpopulation which may be particularly susceptible to the treatment.

[0137] As will be recognized, the RELN level of the subject may be measured using any suitable method capable of identifying a reduced neuronal RELN level. RELN levels generally representative of whole-brain RELN levels may be determined, or RELN levels in particular brain regions may be determined. In certain embodiments, neuronal RELN levels may be determined. Methods for measuring RELN levels may include those quantitating RELN mRNA levels, RELN protein levels, or both, in the brain or in relevant brain region(s). Methods may involve PCR, ELISA, mass spectrometry, and/or neuroimaging methods, among others. While direct monitoring of Reelin levels in brain parenchyma of living humans may be challenging, indirect monitoring may be possible by analyzing cerebrospinal fluid (CSF), which may be used to correlate with brain tissue concentrations by, for example, immunoassay and/or mass spectrometry.

[0138] In certain embodiments, neuronal RELN levels of the subject may be determined through analysis of a sample obtained from the subject, such as a biopsy sample, or a fluid sample such as a blood sample or cerebrospinal fluid sample. In certain embodiments, it is contemplated herein that neuronal RELN levels of the subject may be measured by determining RELN levels in neuronal exosomes circulating in bodily fluids such as blood. As will be understood, neuronal exosomes carry particular cell surface markers allowing their isolation, at which point RELN levels may be quantitated using techniques such as ELISA or others.

[0139] In certain embodiments, it is contemplated that reelin expression levels may be ascertained or estimated by determining reelin expression levels in neural-derived exosomes isolated from human plasma, for example. Such plasma exosomes may be enriched in neural sources by, for example, anti-human L1 CAM antibody immunoabsorption. By way of example, reelin and other extracted exosomal proteins may be quantified by ELISA and normalized with the exosomal marker, CD81 (Goetzl et al., Neurology, 85:40-47, 2015; herein incorporated by reference in its entirety).

[0140] In certain embodiments of the above methods, the subject in need of treatment may be any subject having a reduced RELN level which is below that of a healthy subject or healthy control group. The reduced RELN level may occur generally throughout the brain tissue of the subject, or may be localized to particular regions such as the prefrontal cortex, the striatum, the substantia nigra, and/or the entorhinal cortex layer II, for example. The RELN level may be, for example, a neuronal RELN level. In certain further embodiments, the subject in need of treatment may be a subject suffering from, or at risk of developing, a neuropsychiatric or neurodegenerative disorder. The neuropsychiatric or neurodegenerative disorder may be, for example, schizophrenia, Alzheimer's disease, bipolar disease with psychosis, Parkinson's disease (PD), or another such disease or condition.

[0141] In certain embodiments, a neuropsychiatic or neurodegenerative disorder may include neurological disorders characterized by enhanced expression of heme oxygenase-1 and/or increased oxidative stress in brain. Such diseases may include, but are not limited to, Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis, other motor neuron disorders, Down's syndrome, Creutzfeldt-Jakob disease, other prion diseases, multiple sclerosis, cerebral ischemia, cerebral hemorrhage, traumatic brain injury, spinal cord injury, cerebral malaria, schizophrenia, bipolar disease with psychosis, and/or autism.

[0142] In still another embodiment, there is provided herein a method for treating, preventing, or ameliorating/reducing the symptoms of, a neuropsychiatric disease or a neurodegenerative disorder in a subject in need thereof, said method comprising:

[0143] administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

[0144] As will be understood, the composition comprising whey protein isolate and/or whey protein concentrate, which has already been described in detail hereinabove, may be administered to increase reelin (RELN) levels, for example neuronal RELN levels, in the subject, or to restore RELN levels in the subject to or above a healthy level, or to within a healthy range as determined by measuring RELN levels in a healthy subject, or in a group of healthy subjects, for example.

[0145] In a further embodiment, the method may further comprise a step of:

[0146] measuring an initial RELN level of the subject and comparing the measured RELN level to a healthy RELN level, wherein the subject is identified as having a reduced RELN level in need of increase when the measured initial RELN level is less than the healthy RELN level.

[0147] It will be understood that such a step of measuring may be performed either before or after administration of the composition comprising the whey protein isolate and/or whey protein concentrate, or both. Where the step of measuring is performed before the administration, the measuring step may be considered as a screening step, allowing for the identification of subjects in need of the treatment, of subjects who may particularly benefit from the treatment, and/or of subjects belonging to a patient subpopulation which may be particularly susceptible to the treatment. Where the step of measuring is performed after the administration, the measuring step may be considered as a step of determining treatment efficacy, allowing for the identification of subjects in need of a subsequent, repeated, or adjusted treatment, of subjects who may particularly benefit from a repeated treatment, and/or of subjects belonging to a patient subpopulation which may be particularly susceptible to the treatment. Examples of methods for measuring neuronal RELN levels have already been described hereinabove.

[0148] As will be understood, neuropsychiatric and neurodegenerative disorders may be characterized by multiple imbalances or abnormalities in gene expression. Without wishing to be bound by theory or considered limiting in any manner, the results presented herein suggest that treatment with a composition comprising whey protein isolate may not only increase or restore RELN levels, but may also, or alternatively, be used to at least partially alleviate or otherwise restore several imbalances or abnormalities in gene expression which are believed to be involved in certain neuropsychiatric and neurodegenerative disorders. As such, in certain alternative, or additional embodiments, the compositions comprising whey protein isolate as described herein may be administered for restoring serotonin levels; normalizing MnSOD mRNA levels; restoring GAD67 levels; augmenting GSH reserves in the brain; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related disregulation of miR-128 expression; improving HMOX1-related changes in brain dopamine, dopamine metabolites, serotonin, norephinephrine, and epinephrine; restoring deficient DOPAC/DA ratios; augmenting norepinephrine and dopamine concentrations in the prefrontal cortex; and/or correcting prefrontal cortex hypodopaminergia in the subject. As well, in certain further alternative or additional embodiments, the compositions comprising whey protein isolate as described herein may be administered for augmenting GSH reserves in the brain; normalizing brain MnSOD mRNA levels; restoration of redox homeostasis; normalization of brain dopamine, serotonin, norepinephrine, and/or epinephrine levels; normalization of brain DOPAC/DA ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; and/or ameliorating hyperlocomotion and/or stereotypic behaviour; or any combination thereof; in the subject.

[0149] As will be understood, the neuropsychiatric disease or neurodegenerative disorder may be, for example, schizophrenia or Alzheimer's disease, wherein one or more of these imbalances or abnormalities in gene expression may be occurring. In certain embodiments, it is contemplated that a neuropsychiatic or neurodegenerative disorder may include neurological disorders characterized by enhanced expression of heme oxygenase-1 and/or increased oxidative stress in brain. Such diseases may include, but are not limited to, Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis, other motor neuron disorders, Down's syndrome, Creutzfeldt-Jakob disease, other prion diseases, multiple sclerosis, cerebral ischemia, cerebral hemorrhage, traumatic brain injury, spinal cord injury, cerebral malaria, schizophrenia, bipolar disease with psychosis, and/or autism.

[0150] In certain embodiments, compositions comprising whey protein isolate and/or whey protein concentrate as described herein may be used for increasing reelin (RELN) levels (such as, for example, neuronal RELN levels) in a subject in need thereof, or may be used in the manufacture of a medicament for increasing reelin (RELN) levels in a subject in need thereof, for example. Alternatively, or in addition, such compositions may be used for treating, preventing, or ameliorating the symptoms of, a neuropsychiatric disease or a neurodegenerative disorder, or may be used in the manufacture of a medicament for treating, preventing, or ameliorating the symptoms of a neuropsychiatric disease or a neurodegenerative disorder, in a subject in need thereof, for example.

[0151] In still other embodiments, compositions comprising whey protein isolate and/or whey protein concentrate as described herein may be for use in restoring serotonin levels; augmenting GSH reserves in the brain; normalizing MnSOD mRNA levels; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; improving HMOX1-related changes in brain dopamine, dopamine metabolites, serotonin, norephinephrine, and epinephrine; restoring deficient DOPAC/DA ratios; augmenting norepinephrine and dopamine concentrations in the prefrontal cortex; and/or correcting prefrontal cortex hypodopaminergia in a subject in need thereof, or in the manufacture of a medicament for achieving such a result.

[0152] In still other embodiments, compositions comprising whey protein isolate and/or whey protein concentrate as described herein may be for use in augmenting GSH reserves in the brain; normalizing brain MnSOD mRNA levels; restoration of redox homeostasis; normalization of brain dopamine, serotonin, norepinephrine, and/or epinephrine levels; normalization of brain DOPAC/DA ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; and/or ameliorating hyperlocomotion and/or stereotypic behaviour; or any combination thereof; in a subject in need thereof, or in the manufacture of a medicament for achieving such a result.

[0153] It will be understood that compositions as described herein may be administered as part of a treatment regimen including other drugs, pharmaceutical compositions, or therapies used in the treatment of neuropsychiatric diseases or neurodegenerative disorders. Compositions as described herein may be for administration simultaneously, sequentially, in combination with, or separately from such other drugs, pharmaceutical compositions, or therapies.

[0154] Compositions as described herein may be for use in treating a neuropsychiatric or neurodegenerative disease or disorder, or may be for use as part of a preventative strategy for preventing or delaying onset of a neuropsychiatric or neurodegenerative disease or disorder such as schizophrenia or Alzheimer's disease, or both.

[0155] In certain embodiments, compositions as described herein may be for use in treating neurological disorders characterized by enhanced expression of heme oxygenase-1 and/or increased oxidative stress in brain. Such diseases may include, but are not limited to, Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis, other motor neuron disorders, Down's syndrome, Creutzfeldt-Jakob disease, other prion diseases, multiple sclerosis, cerebral ischemia, cerebral hemorrhage, traumatic brain injury, spinal cord injury, cerebral malaria, schizophrenia, bipolar disease with psychosis, and/or autism.

[0156] In still another embodiment, there is provided herein a method for restoring serotonin levels; normalizing MnSOD mRNA levels; augmenting GSH reserves in the brain; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; improving HMOX1-related changes in brain dopamine, dopamine metabolites, serotonin, norephinephrine, and epinephrine; restoring deficient DOPAC/DA ratios; augmenting norepinephrine and dopamine concentrations in the prefrontal cortex; and/or correcting prefrontal cortex hypodopaminergia in a subject in need thereof, said method comprising:

[0157] administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

[0158] In certain embodiments of the above method, the subject may be a subject suffering from, or at risk of developing, a neuropsychiatric or neurodegenerative disorder such as, for example, schizophrenia or Alzheimer's disease.

[0159] In certain embodiments of the above method, the subject may be suffering from, or at risk of developing, a neurological disorder characterized by enhanced expression of heme oxygenase-1 and/or increased oxidative stress in brain. Such diseases may include, but are not limited to, Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis, other motor neuron disorders, Down's syndrome, Creutzfeldt-Jakob disease, other prion diseases, multiple sclerosis, cerebral ischemia, cerebral hemorrhage, traumatic brain injury, spinal cord injury, cerebral malaria, schizophrenia, bipolar disease with psychosis, and/or autism.

[0160] In yet another embodiment, there is provided herein a method for augmenting GSH reserves in the brain; normalizing brain MnSOD mRNA levels; restoration of redox homeostasis; normalization of brain dopamine, serotonin, norepinephrine, and/or epinephrine levels; normalization of brain DOPAC/DA ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; and/or ameliorating hyperlocomotion and/or stereotypic behaviour; or any combination thereof; in a subject in need thereof, said method comprising:

[0161] administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

[0162] In certain embodiments of the above method, the subject may be a subject suffering from, or at risk of developing, a neuropsychiatric or neurodegenerative disorder such as, for example, schizophrenia or Alzheimer's disease.

[0163] In certain embodiments of the above method, the subject may be suffering from, or at risk of developing, a neurological disorder characterized by enhanced expression of heme oxygenase-1 and/or increased oxidative stress in brain. Such diseases may include, but are not limited to, Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis, other motor neuron disorders, Down's syndrome, Creutzfeldt-Jakob disease, other prion diseases, multiple sclerosis, cerebral ischemia, cerebral hemorrhage, traumatic brain injury, spinal cord injury, cerebral malaria, schizophrenia, bipolar disease with psychosis, and/or autism.

[0164] As will be understood, compositions comprising whey protein isolate and/or whey protein concentrate as described herein may serve as a glutathione precursor by providing an enriched source of bioavailable cysteine following administration. Several neuropsychiatric and neurodegenerative disorders have been linked to oxidative stress and/or glutathione (GSH) deficits in brain tissue. As discussed in detail herein, treatment with a composition comprising whey protein isolate has been observed to restore GSH homeostasis in the CNS of a mouse disease model, and to augment GSH reserves in the brains of wild-type animals. These results demonstrate that compositions as described herein may be used to augment GSH stores and antioxidant defenses in the healthy and diseased brain.

[0165] As such, in certain embodiments, there is provided herein a method for augmenting glutathione (GSH) levels in brain cells, or augmenting whole brain GSH/glutathione disulfide (GSSG) ratios, of a subject in need thereof, said method comprising:

[0166] administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

[0167] As will be understood, in certain embodiments, such augmentation of glutathione (GSH) levels, or GSH/GSSG ratios, in the subject may accompany an increase or restoration of RELN levels or other imbalances or abnormalities in gene expression achieved using any of the other methods as described hereinabove. In additional embodiments, compositions as described herein may be used simultaneously, or sequentially, with other glutathione precursor compounds or compositions.

[0168] In still another embodiment, there is provided herein a kit comprising a composition comprising whey protein isolate and/or whey protein concentrate as described hereinabove, and one or more of a pharmaceutically acceptable excipient, diluent, carrier, vitamin, essential amino acid, mineral, antioxidant, glutathione precursor, nutritional diet supplement component, or drug, pharmaceutical composition, or therapy used in the treatment of a neuropsychiatric disease or neurodegenerative disorder.

[0169] Such kits may additionally, or alternatively, comprise instructions for use of the kit in the treatment of a neuropsychiatric disease or neurodegenerative disorder, or for use of the kit in increasing or restoring RELN levels (for example, but not limited to, neuronal RELN levels); augmenting GSH reserves in the brain; restoring serotonin levels; normalizing MnSOD mRNA levels; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; improving HMOX1-related changes in brain dopamine, dopamine metabolites, serotonin, norephinephrine, and epinephrine; restoring deficient DOPAC/DA ratios; augmenting noepinrephrine and dopamine concentrations in the prefrontal cortex; and/or correcting prefrontal cortex hypodopaminergia in a subject in need thereof.

[0170] In further embodiments, such kits may additionally, or alternatively, comprise instructions for use of the kit in the treatment of a neuropsychiatric disease or neurodegenerative disorder, or for use of the kit in augmenting GSH reserves in the brain; normalizing brain MnSOD mRNA levels; restoration of redox homeostasis; normalization of brain dopamine, serotonin, norepinephrine, and/or epinephrine levels; normalization of brain DOPAC/DA ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; and/or ameliorating hyperlocomotion and/or stereotypic behaviour; or any combination thereof; in a subject in need thereof.

[0171] In yet another embodiment, there is provided herein a method for measuring reelin (RELN) levels in a subject, said method comprising:

[0172] isolating or enriching a neuronal exosome sample from a bodily fluid sample obtained from the subject using one or more neuronal exosome-specific cell surface markers; and

[0173] measuring a RELN level of the neuronal exosome sample.

[0174] In an embodiment of the method above, the bodily fluid sample may be a cerebrospinal fluid (CSF) sample, whole blood sample, plasma sample, or another processed or unprocessed blood sample obtained from the subject. In still another embodiment, the step of isolating or enriching may involve a pull-down type assay targeting the one more neuronal exosome-specific cell surface markers, or another suitable isolating method. In still another embodiment, the RELN level may be measured by quantitating RELN mRNA levels, protein levels, or both, and may be determined using a suitable PCR assay, immunoassay such as ELISA, mass spectrometry, or another suitable assay for quantitating RELN.

[0175] As will be understood, neuronal exosomes carry particular cell surface markers allowing their isolation. Neuronal exosomes may be isolated/enriched by, for example, anti-human L1CAM antibody immunoabsorption techniques. By way of example, reelin and other extracted exosomal proteins may be quantified by ELISA and normalized with the exosomal marker, CD81 (Goetzl et al., Neurology, 85:40-47, 2015; herein incorporated by reference in its entirety).

Example 1--Amelioration of Deficits in GFAP.HMOX1 Mouse Model of Schizophrenia

[0176] The following example describes experimental results obtained from treating wild type mice, and a mouse model of schizophrenia, with Immunocal.RTM., a composition comprising whey protein isolate. Under the experimental conditions used, a number of beneficial effects were observed in the animals, indicating the potential such compositions may possess in the treatment of neuropsychiatric or neurodegenerative disorders and/or in restoring neuronal imbalances or gene expression abnormalities.

[0177] Schizophrenia is a neuropsychiatric disorder that features neural oxidative stress and glutathione (GSH) deficits. Oxidative stress is augmented in brain tissue of GFAP.HMOX1 transgenic mice which exhibit schizophrenia-relevant characteristics. The whey protein isolate, Immunocal.RTM., serves as a GSH precursor upon oral administration. In this Example, GFAP.HMOX1 transgenic mice were treated daily with Immunocal between the ages of 5 and 6.5 months. Immunocal attenuated many of the behavioral, neurochemical and redox abnormalities observed in GFAP.HMOX1 mice. In addition to restoring GSH homeostasis in the CNS of the transgenic mice, the whey protein augmented GSH reserves in the brains of wild-type animals. These results demonstrate that whey protein consumption augments GSH stores and antioxidant defenses in the healthy and diseased mammalian brain. Furthermore, administration of Immunocal was found to increase reelin (RELN) levels in the animal models tested, a particularly interesting experimental finding. Results obtained indicate that whey protein supplementation may constitute a safe and effective modality for the management of schizophrenia and other neuropsychiatric and neurodegenerative disorders.

[0178] The HMOX1 gene coding for the 32 kDa stress protein, heme oxygenase-1 (HO-1) is exquisitely sensitive to induction by stressors implicated in the development of SCZ.sup.9. We previously demonstrated that the accumulation of heme-derived ferrous iron and carbon monoxide accruing from transfection of HMOX1 in cultured rat astrocytes promotes mitochondrial damage and predisposes co-cultured neuronal elements to oxidative injury. Our laboratory recently engineered a conditional GFAP.HMOX1 transgenic mouse that selectively over-expresses human HO-1 in the astrocytic compartment under temporal control by the Tet-Off system. After 48 weeks of continuous HMOX1 induction, these mice exhibit a set of robust behavioural (some sex-specific), neurochemical, neuropathological and developmental features reminiscent of human SCZ and animal models of the disease. Specific abnormalities documented in the GFAP.HMOX1 mice germane to SCZ include hyperlocomotion, behavioral stereotypy and impaired prepulse inhibition to acoustic startle; increased basal ganglia dopamine (DA) and serotonin concentrations; suppressed neuronal reelin immunoreactivity; dysgenesis of the hippocampal dentate gyrus.sup.9; and ventriculomegaly (unpublished results). As in the HMOX1-transfected glial cultures (vide supra), pathological deposition of redox-active iron, oxidative mitochondrial damage and mitophagy are clearly demonstrable in the brains of GFAP.HMOX1 mice.sup.9,10. Depletion of the intracellular antioxidant tripeptide, glutathione (GSH) and oxidative stress have been documented in SCZ-affected human neural tissues.sup.11-13. Moreover, administration of glutathione precursors has been shown to improve symptomatology in animal models of SCZ and in patients with the disease.sup.14.

[0179] The delivery of the amino acids, cysteine and cystine is believed to be the rate-limiting factor for the synthesis of intracellular glutathione in brain and other tissues. Immunocal.RTM. is an un-denatured bovine whey protein isolate which serves as a glutathione precursor by providing an enriched source of bioavailable cysteine after oral administration. Immunocal has been tested in human clinical trials for its role in the management of diverse conditions including HIV/AIDS, cancer and cystic fibrosis, and the optimization of sports performance in healthy subjects.sup.15-18.

[0180] As part of the studies described herein, experiments were performed in order to ascertain, among other things, whether (i) GSH concentrations and GSH:GSSG ratios are deficient in salient brain regions of GFAP.HMOX1 mice by 6.5 months of age; (ii) whether Immunocal treatment augments brain GSH stores and alleviates SCZ-like abnormalities in these animals; and (iii) whether Immunocal treatment is able to restore neuronal levels of RELN and/or other imbalnances or abnormalities of gene expression in these animal models of neuropsychiatric or neurodegenerative disorders.

[0181] Materials and Methods

[0182] The GFAP.HMOX1 Mouse:

[0183] Transgenic (TG) mice (FVB strain) were generated expressing GFAP.tTA.TRE.Flag.hHO-1 final constructs, as previously described.sup.9. Incorporation of the Glial Fibrillary Acidic Protein (GFAP) promoter selectively targets human HMOX1 gene expression to the astrocytic compartment. To permit conditional expression of the transgene during select periods of neuroembryogenesis, perinatal and mature life, a tetracycline-suppressible (`tet-off`) promoter element was included in the experimental design.sup.10.

[0184] Whey Protein (Immunocal) Supplementation:

[0185] Immunocal.RTM. is a dietary natural health product with an NPN 80004370 issued by Natural Health Product Directorate (NHPD) Health Canada. It is a natural source of the glutathione precursor, cysteine. Immunocal is fat-free, contains less than 1% lactose and has a high protein biological value (>110 BV) providing all essential amino acids. It has been tested in experimental animals.sup.19-21 and human clinical trials.sup.22-24 and is marketed worldwide for enhancement of the immune system.

[0186] Experimental protocols have been approved by the Animal Care Committee of McGill University in accordance with the guidelines of the Canadian Council on Animal Care. Mice were kept at a room temperature of 21.+-.1.degree. C. with a 12 h light/dark schedule. All the mice were bred and cared for in the Animal Care Facilities at the Lady Davis Institute for Medical Research. Male and female heterozygous GFAP.HMOX1 (continuously expressing the HMOX1 transgene) and wild-type (WT) mice at 5 months of age were treated daily with Immunocal at 33 mg/ml drinking water vs. drinking water containing 33 mg/ml casein (control). Daily drinking volume per mouse was recorded. After 4-6 weeks of treatment, all animals were assessed for the behavioral, neurochemical and neuropathological endpoints described below. Fur texture, body weight and survival rates were monitored as indices of general health.

[0187] Behavioral Tests:

[0188] GFAP.HMOX1 mice and their WT littermates were transferred to the Neurophenotyping Centre of the Douglas Mental Health University Institute (Montreal) for behavioral analyses. The animals were tested for locomotor activity 25 and startle response [prepulse inhibition (PPI)].sup.26.

[0189] Surgical Procedures:

[0190] (1) Mouse brains were fixed by transcardial perfusion as previously described.sup.27 with minor modifications.sup.9. Briefly, the animals were deeply anesthetized with rodent mixture containing ketamine, xylazine, acepromazine and saline and perfused with 200 ml of ice-cold saline followed by 250 ml of cold 4% paraformaldehyde in 0.1M PBS, pH 7.4, for light-microscopic analysis, or cold 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.5, containing 0.1% CaCl2 for transmission electron microscopy (TEM). The brains were removed and immersed in the same fixatives for 24 h at 4.degree. C. For histomorphology, brains were embedded in paraffin. For RNA and protein expression assays, mouse brains were frozen in dry ice immediately after transcardial perfusion with 200 ml of ice-cold PBS and stored at -80.degree. C. (2) For HPLC and glutathione assays, animals were decapitated and brains were removed and frozen in 2-methylbutane at -40.degree. C. and stored at -80.degree. C. until use.sup.28.

[0191] Glutathione Assay:

[0192] An HPLC method was utilized to measure reduced (GSH) and oxidized (GSSG) glutathione concentrations in brain hemispheres.sup.29. GSH and GSSG levels were determined in four sub-regions: prefrontal cortex (PFC), hippocampus (HC), striatum (STM) and the remainder of the hemisphere (excluding cerebellum; REM).

[0193] Neurotransmitter Measurement:

[0194] Brains were cut in 400-500 micron serial sections using a cryostat and selected regions (PFC, HC, STM, and substantia nigra (SN)) were dissected using 0.5-2.0 mm micropunches.sup.30, 31 Tissues were homogenized in 0.25 M perchloric acid and centrifuged at 4.degree. C. (10,000 rpm, 15 min), and supernatants were collected. The concentrations of monoamines [i.e., dopamine (DA), norepinephrine (NE), epinephrine (E), and 5-hydroxytryptamine (5-HT)] and monoamine metabolites [i.e., 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA)] were determined using HPLC with electrochemical detection (HPLC-EC), in the laboratory of Dr. A. Gratton (Douglas Hospital, Montreal), as previously described.sup.9,32.

[0195] mRNA and miRNA Expression:

[0196] Total RNA extraction, polyadenylation, and cDNA synthesis--Total RNA from each dissected brain region was extracted in Trizol according to the manufacturer's instructions (Invitrogen). Two and half micrograms of total RNA were subjected to RT-qPCR using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher) and anchored-oligo-dT18 primer. miRNA polyadenylation was performed followed by cDNA synthesis using 2 .mu.g of polyadenylated total RNA with miScript II RT Kit (Qiagen).

[0197] mRNA and miRNA RT-qPCR--The Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems by Life Technologies) was used to quantify mRNA and miRNA with EvaGreen RT-qPCR Mastermix-Low ROX reagent (Diamed) according to manufacturer's instructions. Twenty nanograms (ng) and 2.5 ng of cDNA were quantified for mRNA and miRNA, respectively, using the above reagent (Diamed) via RT-qPCR. The forward (F) and reverse (R) primer sequences used to detect mouse mRNA were a) provided by OriGene Technologies (Rockville, Md.), which were designed to span an exon-intron boundary and the possible contaminating genomic DNA was not amplified because the primer cannot anneal to the template.sup.33, b) designed with Primer Express Software Version 3.0 (Applied Biosystems by Life Technologies) and validated by published study, c) validated by published study. Additional checks of melting curve for each reaction was always carried out to assess contamination of genomic DNA or poor primer design (primer dimer formation) (Applied Biosystems by Life Technologies). Primer sequences were as follows:

TABLE-US-00001 (1) manganese superoxide dismutase (MnSOD).sup.b: (SEQ ID NO: 3) 5'-GCTGCACCACAGCAAGCA-3' (F) and (SEQ ID NO: 4) 5'-TCGGTGGCGTTGAGATTGT-3' (R); (2) Reelin (Reln).sup.b: (SEQ ID NO: 5) 5'-GCCACGCCACAATGGAA-3' (F) and (SEQ ID NO: 6) 5'-CGACCTCCACATGGTCCAA-3' (R); (3) Glutamate Decarboxylase 1 (Brain, 67 kDa; gAD-1/67).sup.a: (SEQ ID NO: 7) 5'-CGCTTGGCTTTGGAACCGACAA-3' (F) and (SEQ ID NO: 8) GAATGCTCCGTAAACAGTCGTGC-3' (R); (4) Neurexin 1 (Nrxn1).sup.a: (SEQ ID NO: 9) 5'-ACCGTGCCTTAGCAATCCTTGC-3' (F) and (SEQ ID NO: 10) 5'-GTCGTAGCTCAAAACCGTTGCC-3' (R); (5) Neuroligin 2 (Nlgn 2).sup.a: (SEQ ID NO: 11) 5'-CGATGTCATGCTCAGCGCAGTA-3' (F) and (SEQ ID NO: 12) 5'-CCACACTACCTCTTCAAAGCGG-3' (R); (6) As an internal reference, .beta.-Actin.sup.c mRNA was used and probed using a pair of primers [5'-CAGCAGATGTGGATCAGCAAG-3' (F) (SEQ ID NO: 13) and (SEQ ID NO: 14) 5'-GCATTTGCGGTGGACAGAT-3' (R)].sup.34.

[0198] Mature DNA sense sequences (obtained from miRBase; http://microrna.sanger.ac.uk/) were used as forward primers to detect miRNA. The miRNA primer sequences used were mmu-miR-137-5p (5'-acgggtattcttgggtggataat-3') (SEQ ID NO: 15), mmu-miR-137-3p (5'-ttattgcttaagaatacgcgtag-3') (SEQ ID NO: 16), mmu-miR-181a (5'-aacattcaacgctgtcggtgagt-3') (SEQ ID NO: 17), mmu-miR-128-1-5p (5'-cggggccgtagcactgtctga-3') (SEQ ID NO: 18), mmu-miR-128-3p (5'-tcacagtgaaccggtctcttt-3') (SEQ ID NO: 19), mmu-miR-138 (5'-agctggtgttgtgaatcaggccg-3') (SEQ ID NO: 20), and mmu-miR-200c (5'-taatactgccgggtaatgatgga-3') (SEQ ID NO: 21).

[0199] As a reference sequence, mouse small nucleolar RNA 202 (snoRNA-202) was probed using an internal forward primer (5'-agtacttttgaaccatttcca-3').sup.35. (SEQ ID NO: 22) mRNA and miRNA expression fold changes between groups were calculated using the .DELTA..DELTA.Ct method relative to controls following normalization with levels of snoRNA-202.sup.36.

[0200] Data mining for candidate targets of lead miRNAs--microRNA target predictions were adduced from the literature or the following databases: miRBase website (http://microrna.sanger.ac.uk) 37; TargetScan (http://www.targetscan.org), RNA22 (http://cbcsrv.watson.ibm.com/rna22 targets.html) and PicTar (http://pictar.mdcberlin.de).

[0201] Neuromorphological Analysis:

[0202] Coronal brain sections (4 .mu.m) were deparaffinized in toluene and rehydrated in a series of graded alcohol solutions followed by H.sub.2O. Sections were stained with hematoxylin and eosin (H&E). The preparations were examined using a Leica DM LB2 microscope. Bregma coordinates were identified using the mouse brain atlas of Paxinos and Franklin.sup.38. The lateral ventricles of left and right hemispheres were examined at +0.50 mm from bregma. The width and height of the hippocampus and the length of the dentate gyrus granule cell layer were measured at -1.55 mm to -1.99 mm from bregma with the aid of an ocular grid by a single investigator unaware of the tissue source.

[0203] Statistical Analyses:

[0204] Data are expressed as means.+-.SEM. For locomotor activity, analyses were performed in cases with more than two groups using a genotype (TG and WT) by treatment (Immunocal and casein) ANOVA followed by Newman-Keuls post hoc comparisons to assess significant main effects within groups. For PPI assessment of WT and TG mice (with Immunocal or casein), two-way ANOVA was used to analyze serial intensity tests considering two factors (genotype and intensity). For GSH assay and quantitative hippocampal pathology, the comparison was made between two genotypes for each item using Student's t test (one or two-tailed with 95% confidence intervals). Fold changes in TG mice versus WT mice for qPCR assays were analyzed with paired Student's t test (two-tailed). Unless stated otherwise in the figure legends, statistical significance was set at P<0.05.

[0205] Results

[0206] Toxicity:

[0207] Immunocal exposure resulted in no overt toxicity as evidenced by normal body weights, fur texture and survival rates relative to age-matched, casein-treated (current study) and untreated 9 FVB control mice (data not shown).

[0208] Behavior:

[0209] Prepulse inhibition (FIG. 1)--PPI occurred in male and female WT mice treated with casein (controls), although the effect was less robust in the females (FIG. 1A) as previously reported in rodents.sup.9 and humans.sup.39. Relative to casein-treated WT animals, PPI was significantly attenuated in male TG mice (FIG. 1A). We observed a trend towards impairment of PPI in casein-treated female TG mice (P=0.06, relative to WT subjects, FIG. 1A), particularly following exposure to high pre-pulse levels, as noted in an earlier report.sup.40. Impairment of PPI was significantly ameliorated in female TG mice receiving Immunocal treatment (P<0.0001 relative to casein-treated TG group, FIG. 1D). In male mice, no significant differences in PPI rescue could be evinced between the Immunocal and casein-treated TG mice because the baseline PPI level of WT mice exposed to Immunocal was lower (albeit not statistically significantly) than that of the WT-casein group (FIG. 1C), whereas the PPI levels of the TG-Immunocal and TG-casein groups were comparable (FIG. 1D).

[0210] Locomotor Activity--

[0211] Casein-treated male TG mice displayed a robust hyperkinetic profile as reflected in all locomotor measurements, whereas female TG mice exhibited partial hyperlocomotor activity (FIG. 2). Immunocal treatment significantly attenuated the hyperlocomotor activity in male TG mice, as evidenced by changes in total distance, stereotypy count and time, movement time, and rest time (FIG. 2A). Certain measures of locomotor activity were enhanced in Immunocal-treated female WT mice relative to those exposed to casein, thereby masking potential differences in locomotor activity between the Immunocal-exposed TG and WT females (FIG. 2B).

[0212] Brain GSH Concentrations:

[0213] Whole brain measurements revealed no significant differences in GSH concentrations, GSSG concentrations or GSH/GSSG ratios between casein-treated WT and TG mice, and glutathione levels were similar between the males and females (p>0.05 for all comparisons) (FIG. 3A). A sub-regional analysis of the brain samples showed a significant reduction of GSH content in the HC of casein-treated TG mice relative to WT values (p<0.05; FIG. 4A), and a trend towards lower GSH/GSSG ratios in the TG HC and STM compared with WT preparations (FIG. 4A). Glutathione values in the WT and TGPFC were similar (p>0.05, FIG. 4A, B).

[0214] Immunocal supplementation significantly augmented whole brain GSH/GSSG ratios in both WT and TG mice compared with casein-treated animals (p<0.01-0.001; FIG. 3C, D). The latter was achieved mainly through marked elevations of GSH concentrations (634.2-676 vs. 188.5-201.1 nmol/mg, P<0.001) accruing from exposure to the whey protein. A significant reduction of brain GSSG content following Immunocal supplementation was observed in WT but not TG brains (WT: 4.9.+-.0.5 vs. 7.7.+-.0.5 nmol/mg, p<0.05; TG: 5.7.+-.0.4 vs. 6.1.+-.0.5 nmol/mg, p>0.05.). There were similar alterations in GSH content and GSH/GSSG ratios between male and female mice (p>0.05), and the diminished GSSG concentrations resulting from Immunocal treatment were observed in both WT males and females, but only in TG males (p<0.05 for each comparison; FIG. 3C, D).

[0215] Immunocal treatment diminished the differences in hippocampal GSH content and GSH/GSSG ratio between the WT and TG groups (FIG. 4B). Immunocal treatment significantly elevated striatal GSH/GSSG ratios in TG mice relative to WT preparations (p<0.01, FIG. 4B). Immunocal treatment significantly increased GSH/GSSG ratios in all three brain regions surveyed of both WT and TG mice compared with casein-treated preparations (83.7-117.2 vs. 0.6-4.1, P<0.01-0.001; FIG. 4C, D).

[0216] The GSH/GSSG ratio in the remainder of the hemisphere (REM) was greater in the TG mice relative to the WT animals (P<0.01), was not significantly affected by Immunocal exposure (FIG. 5), and likely accounts for the absence of differences in whole brain glutathione concentrations between these groups (FIG. 3A).

[0217] Hippocampal and Ventricular Pathology:

[0218] H&E staining of coronal brain sections revealed markedly enlarged lateral ventricles (ventriculomegaly) (FIG. 8A) and altered hippocampal cytoarchitectonics (dentate gyrus dysgenesis) (FIG. 8B) in both male and female TG mice, features characteristic of human SCZ neuropathology.sup.41-43. A morphometric analysis of the HC showed that the granule cell layer of the dentate gyrus in TG mice was significantly diminished in size compared to WT mice (p<0.05, FIG. 8B). The ventriculomegaly and dentate gyrus dysgenesis observed in both male and female TG mice were not improved by Immunocal supplementation (FIG. 8). In contradistinction to 12 month old GFAP.HMOX1 TG mice.sup.9, Gallyas-positive (degenerate) neurites were rarely encountered in the Immunocal- or casein-treated 6.5 month-old TG animals.

[0219] Neurotransmitters:

[0220] A) DA and Metabolites--

[0221] Among the casein-treated groups, DOPAC and HVA were significantly increased in the TG HC compared to WT preparations (p<0.05-0.01, FIG. 6A). The ratios of hippocampal DOPAC/DA and HVA/DA were also significantly augmented in the TG mice (p<0.05, FIG. 6B). No significant changes in the levels of DA or DA metabolites were found in PFC, STM and SN of TG brains relative to WT mice (FIG. 6A). The DOPAC/DA ratio was reduced in the TG PFC (p<0.05) without significant alterations in the concentrations of DA or DA metabolites per se (FIG. 6B). All HMOX1-related changes in brain DA and DA metabolites were attenuated by Immunocal supplementation (FIG. 6A, B). Of note, the DA content of the TG PFC was significantly increased after Immunocal treatment (p<0.05, FIG. 1A).

[0222] B) Serotonin and Metabolites--

[0223] Among the casein-treated animals, serotonin levels were significantly greater in the TG PFC compared to its WT counterpart (p<0.05, FIG. 7A). Immunocal treatment restored serotonin concentrations in the TG PFC to WT values (FIG. 7A).

[0224] C) NE and E--

[0225] Among the casein-treated mice, levels of NE and E were significantly higher in the TG STM compared to WT STM (p<0.05, FIG. 7B). Concentrations of NE and E in the TG STM were normalized following Immunocal exposure (FIG. 7B).

[0226] mRNA and miRNA Expression Levels:

[0227] A) Neuronal Reelin (RELN), GAD67 and MnSOD--

[0228] MnSOD mRNA, a marker of oxidative stress, was significantly up-regulated in casein-treated TG PFC, STM, and SN compared to WT preparations (p<0.05-0.01, FIG. 9A). The mRNA expression levels of reelin (RELN; a protein involved in the regulation of neuronal migration and positioning in the developing brain.sup.44) and GAD67 (a GABA-synthesizing enzyme that is co-regulated with reelin.sup.45) were significantly reduced in casein-treated TG PFC and STM compared to WT PFC and STM (p<0.05-0.001, FIG. 9B, C). In the GFAP.HMOX1 mice, Immunocal treatment normalized the MnSOD mRNA levels in the PFC and STM (but not SN); restored reelin and GAD67 expression in the PFC (p>0.05 relative to casein-treated WT mice; FIG. 9A-C); and augmented GAD67 mRNA levels in the STM and SN (p<0.01-0.001 relative to casein-treated WT mice; FIG. 9C). The Immunocal-treated TG mice also exhibited a trend towards recovery of reelin expression in the STM, although reelin mRNA concentrations in this brain region remained significantly (p<0.05) below WT values (FIG. 9B).

[0229] B) miR-137, Nrxn1 and Nlgn2--

[0230] Mutations in Nrxn1 and Nlgn2 genes have previously been linked to SCZ, autism and intellectual disability.sup.46-52. GFAP.HMOX1 mice exhibited significant down-regulation of Nrxn1 in PFC and STM, and Nlgn2 in PFC, relative to WT values (p<0.05-0.01, FIG. 9D, E). Administration of Immunocal reversed the reduction of Nrxn1 and Nlgn2 in the TG brains (FIG. 9D, E). The TG mice exhibited up-regulation of miR-137, a putative SCZ susceptibility gene.sup.53 and predicted suppressor of Nrxn1 (www.targetscan.org), in STM and suppression of miR-137 in the PFC and SN (p<0.01-0.001, FIG. 10A). The differences in neural miR-137 expression between the GFAP.HMOX1 and WT animals were obviated by Immunocal treatment (FIG. 10A).

[0231] C) Mir-181A & Mir-138--

[0232] miR-181a may play important roles in SCZ because it regulates synaptic plasticity, is induced by dopamine signaling in hippocampal neurons.sup.54 and is predicted to suppress reelin and sirt1 gene expression. In the casein-treated groups, the expression of mmu-miR-181a was significantly up-regulated in the TG PFC and STM relative to their WT counterparts (p<0.05-0.01, FIG. 10C) and correlated inversely with reelin gene expression which it may target (www.targetscan.org). Similarly, mmu-miR-138, a miRNA implicated in several human neuropsychiatric disorders.sup.55,56 and impacted by HMOX1 transfection in cultured astroglia.sup.57, was up-modulated in the TG STM vs. WT values (p<0.01, FIG. 10D).

[0233] D) Mir-128 & Mir-200c--

[0234] miR-128 is highly expressed during neuronal differentiation.sup.58 and de-regulated in patients with SCZ.sup.59. miR-200c is up-regulated by oxidative stress.sup.60 and suppresses reelin.sup.61, a protein implicated in the pathogenesis of SCZ. miR-128 was up-regulated in the GFAP.HMOX1 PFC (miR-128-1-5p and miR-128-3p) and STM (miR-128-1-5p), and down-regulated in SN (miR-128-1-5p and miR-128-3p) compared to WT controls (p<0.05-0.001, FIG. 10E, F). Immunocal treatment prevented HMOX1-related dysregulation of miR-128 expression in the TG brains, as evidenced by unaltered levels of miR-128-1-5p between the WT and TG animals and enhancement of miR-128-3p expression in TG SN (FIG. 10E, F). Expression of miR-200c, an miRNA that targets reelin.sup.61, remained unchanged in all three TG brain regions relative to WT controls (FIG. 10G) and was not affected by Immunocal exposure (FIG. 10G).

DISCUSSION

[0235] In these experiments, the 6.5 month-old GFAP.HMOX1 transgenic mice exhibited schizophrenia-relevant behavioral, neuropathological and neurochemical features akin to those previously reported by our laboratory in these mice at 12 months of age.sup.9. Documentation of the full neuroendophenotype at this earlier, 6.5-month time point is significant because it is equivalent to approximately 30 human years.sup.62 when first psychotic presentation (diagnosis) of schizophrenia is often manifest.sup.63. Behavioral abnormalities in the 6.5 month-old GFAP.HMOX1 TG mice included hyperkinesia, stereotypy and impaired PPI of the acoustic startle response. As previously noted in the 12 month-old animals.sup.9 and in human SCZ.sup.39, the behavioral deficits were often more prominent in the males (see below). In contrast, there were no sex predilections for the striking neuromorphological anomalies which consisted of dysgenesis of the hippocampal dentate gyrus and enlargement of the lateral ventricles. Dysregulation of neurotransmitter systems in 6.5 month-old GFAP.HMOX1 TG mice included elevated serotonin content in the PFC, augmented norepinephrine and epinephrine concentrations in the STM and a trend towards increased dopamine levels in the HC. Dopamine turnover was accelerated in the HC, as evidenced by significant increases in the DA metabolites, DOPAC and HVA as well as in DOPAC/DA and HVA/DA ratios. These changes in DOPAC and HVA are indicative of enhanced dopaminergic activity in this brain region.sup.64-67. By contrast, the DOPAC/DA ratio in the PFC of TG mice was significantly lower than that of WT mice, similar to what has been observed in the isolation-reared rat model of schizophrenia.sup.68. This reduced DA turnover may point to PFC hypodopaminergia which is a characteristic feature ("hypofrontality") of the human schizophrenic brain.sup.69. Altered PFC physiology may contribute to the behavioral sensitization and hyperkinesia observed in psychotic states.sup.70. Furthermore, the cognitive deficits characteristic of SCZ may be due, at least partly, to aberrant catecholaminergic transmission within the lateral PFC and its interactions with related brain regions.sup.71. Conceivably, the elevated serotonin content in the GFAP.HMOX1 PFC, possibly recapitulating the 5-HT1A receptor increases in PFC of schizophrenic patients.sup.72,73, may have exerted an inhibitory effect on dopamine turnover in this region.sup.74, thereby contributing to the hypodopaminergia.

[0236] The 6.5 month-old GFAP.HMOX1 TG mice exhibited altered gene expression profiles of key neurodevelopmental proteins implicated in the etiopathogenesis of SCZ including reelin (RELN), GAD67, Nrxn1 and Nlgn2. As previously hypothesized.sup.9, abnormal expression of these genes may contribute to the neuroanatomical anomalies and aberrant neurotransmission characteristic of the GFAP.HMOX1 mice. In addition, neural concentrations of miR-137, miR-181a, miR-138, and miR-128, miRNAs known or predicted to regulate the expression of these neurodevelopmental genes, and reportedly altered in the brains of persons with SCZ and related disorders.sup.46,49,52,59,61,75-77, deviated substantially from WT values. Annotation of the targeted genes relevant to the etiopathogenesis of human neurodevelopmental disorders is provided in Table 1. Of note, 30 predicted targets have been implicated in SCZ whereas 1-15 gene targets may contribute to autism and other developmental CNS conditions. Among the former, 50% are purportedly regulated by miR-137, a SCZ susceptibility gene.sup.53 heavily impacted in the GFAP.HMOX1 basal ganglia and PFC. A smaller proportion (13.3-33.3%) of the putative gene targets are regulated by the other miRNAs surveyed here (i.e. miR-138, -128, -181a, and -200c).

TABLE-US-00002 TABLE 1 Data mining for predicted targets of miRNAs implicated in neurodevelopmental and neuropsychiatric disorders. A) and B): neurodevelopmental and neuropsychiatric disorders listed alphabetically; C) major neuropathology; D) Footnotes for Table 1; E) References cited in Table 1; F) Gene names for Table 1. (A) miR-137 miR-181a miR-138 miR-128 miR-200c AD (10) GRIN2A (Hu).sup.90 Kcnn3 (Ms).sup.91* PTP4A1 (Hu, Rt, Ms).sup.67* Kcnn3 (Ms).sup.91* DRD2 (Hu).sup.48* PHF3 (Hu, Rt, Ms).sup.67* SLC6A11 (Hu, Rt, Ms).sup.67* PXN (Hu).sup.56* KIAA0040 (Hu).sup.67* PXN (Hu).sup.56* STK40 (Hu, Rt, Ms).sup.67* TPH2 (Hu).sup.57* STK40 (Hu, Rt, Ms) .sup.67* ADHD (8) NF1 (Hu).sup.83* KCNJ5 (Hu).sup.65* SNAP25 (Hu).sup.85* KCNJ5 (Hu).sup.65* NCAM1 (Hu).sup.76* MAP1B (Hu).sup.62* NCAM1 (Hu).sup.76* SNAP25 (Hu).sup.85* NF1 (Hu).sup.83* STX1A (Hu).sup.85* SNAP25 (Hu).sup.85* TPH1 (Hu).sup.58* STX1A (Hu).sup.85* YWHAQ (Hu).sup.58* ASD (7) CSMD1 (Hu).sup.4* EN2 (Hu, Ms).sup.50 EN2 (Hu, Ms).sup.50 EN2 (Hu, Ms).sup.50 EN2 (Hu, Ms).sup.50 EN2 (Hu, Ms).sup.50 RORA (Hu).sup.52 RORA (Hu).sup.52 F0XG1 (Hu).sup.51 RORA (Hu).sup.52 NLGN4X (Hu).sup.39* STXBP5 (Hu).sup.4* NRG1 (Hu).sup.43* BD (12) GSK3B (Hu).sup.87* BRD1 (Hu).sup.14* RELN (Hu).sup.89* GSK3B (Hu).sup.87* NDUFS1 (Hu).sup.38 IMPA2 (Hu).sup.87* CDH13 (Hu).sup.77* HLA-DRA (Hu).sup.80* SYNJ1 (Hu).sup.40* NRXN1 (Hu).sup.36 CNTNAP2 (Hu).sup.88* YWHAG (Hu).sup.58* HLA-C (Hu).sup.80* NRXN1 (Hu).sup.36 YWHAG (Hu).sup.58* IDD (ID) (6) CSMD1 (Hu).sup.4* ARID2 (Hu).sup.75* KDM5C (Hu).sup.63* ARID2 (Hu).sup.75* DDX3X (Hu).sup.86* DDX3X (Hu).sup.86* DDX3X (Hu).sup.86* NRXN1 (Hu).sup.64* IQSEC2 (Hu).sup.63* NRXN1 (Hu).sup.64* (B) miR-137 miR-181a miR-138 miR-128 miR-200c MDD (13) CACNA1C (Hu).sup.2* CNTNAP2 (Hu).sup.88* PTK2 (Hu).sup.78 GRM5 (Hu).sup.82 YWHAQ (Hu).sup.58* GRIA2 (Hu).sup.82 ESR1 (Hu).sup.81* GSK3B (Hu).sup.10* GRIN2A (Hu).sup.82 GRM1 (Hu).sup.82 KCNK2 (Hu).sup.66* GRM5 (Hu).sup.82 GRM5 (Hu).sup.82 GSK3B (Hu).sup.10* GRM7 (Hu).sup.82 SLIT3 (Hu).sup.4* OCD (15) CHGA (Hu).sup.93* MYCBP2 (Hu).sup.93* CLN5 (Hu).sup.93* ATP9A (Hu).sup.93* GPC6 (Hu).sup.79* DLGAP1 (Hu).sup.71, 72* PBX1-LMX1A (Hu).sup.74* .RTM. LSAMP (Hu).sup.93* LSAMP (Hu).sup.93* PVRL1 (Hu).sup.93* RYR3 (Hu).sup.71, 73* MEIS2 (Hu).sup.74* TNF (Hu).sup.70* NFATC2 (Hu).sup.93* NGFR (Hu).sup.92* WDR7 (Hu).sup.93* PTHS (1) TCF4 (Hu).sup.1 ? TCF4 (Hu).sup.49 TCF4 (Hu).sup.49 TCF4 (Hu).sup.49 SCZ (30) CACNA1C (Hu).sup.2* BRD1 (Hu).sup.14* Erbb4 (Rt).sup.5 C10orf26 (Hu).sup.7* CACNA1C (Hu).sup.2* CSMD1 (Hu).sup.4* CNTNAP2 (Hu).sup.88* NEUROD1 (Hu).sup.16 Grm5 (Ms, Rt).sup.9 C10orf26 (Hu).sup.7* C10orf26 (Hu).sup.7* GABRA1 (Hu).sup.6 RELN (Hu).sup.16, 89* GSK3B (Hu).sup.10* Erbb4 (Rt).sup.5 Erbb4 (Rt).sup.5 Grm5 (Ms, Rt).sup.9 TCF4 (Hu).sup.1 HLA-DRA (Hu).sup.80* MAP2 (Hu).sup.42 GABRA1 (Hu).sup.6 HLA-C (Hu).sup.80* RELN (Hu).sup.16 NRG1 (Hu).sup.44* GRIN2B (Hu).sup.8* NRXN1 (Hu).sup.36 TCF4 (Hu).sup.1 RELN (Hu).sup.16 Grm5 (Ms, Rt).sup.9 TNF-.alpha. (Hu).sup.15 YWHAB (Hu).sup.58* SLC6A1 (Hu).sup.41 GSK3B (Hu).sup.10* WDR60 (Hu).sup.4* TCF4 (Hu).sup.1 HTR2C (Hu).sup.11 YWHAB (Hu).sup.58* TPH1 (Hu).sup.58* NRG2 (Hu).sup.12* YWHAZ (Hu).sup.58* YWHAB (Hu).sup.58* NRG3 (Hu).sup.34* NRXN1 (Hu).sup.36 NRXN3 (Hu).sup.28* TCF4 (Hu).sup.1 ZNF804A (Hu).sup.3 TS (10) CHGA (Hu).sup.93* DPP6 (Hu).sup.86* CLN5 (Hu).sup.93* ATP9A (Hu).sup.93* DRD2 (Hu).sup.47* PVRL (Hu).sup.93* KCNJ5 (Hu).sup.65* KCNJ5 (Hu).sup.65* LSAMP (Hu).sup.93* MYCBP2 (Hu).sup.93* LSAMP (Hu).sup.93* PVRL (Hu).sup.93* TNF (Hu).sup.70* (C) miR-137 miR-181a miR-138 miR-128 miR-200c OS (18) GPD2 (Hu).sup.33 Gpx1 (Rt).sup.54 Gnai2 (Rt).sup.22 PARK7 (Hu).sup.13 HYOU1 (Hu).sup.45 GPX7 (Hu).sup.31 GRM1 (Hu).sup.17 Psen1 (Ms).sup.23 RARA (Hu).sup.24 Maf (Ms).sup.46 .COPYRGT. Mtfr1 (Ms).sup.29 IL1A (Hu).sup.18 RARA (Hu).sup.24 Sirt1 (Ms).sup.26 Sin3a (Dm).sup.25 Rgs6 (Ms).sup.32 MMP14 (Hu).sup.19 Sin3a (Dm).sup.25 Sirt1 (Ms).sup.26 Serp1 (Yst).sup.30.sup.@ Sirt1 (Ms).sup.26 Sirt1 (Ms).sup.26 Tnf-.alpha. (Rt).sup.20 Mitochondrial NOTCH1 (Hu).sup.68 Bcl-2 (Ms).sup.37 PPARD (Hu).sup.27 Bnip3 (Ms).sup.59 Bnip3 (Ms).sup.59 dysfunction (8) Mcl-1 (Ms).sup.37 PARK7 (Hu).sup.13 Tnf-.alpha. (Rt).sup.20 Macroautophagy (9) ATG14 (Hu).sup.35 Atg5 (Ms).sup.21, 84 Atg7 (Ms).sup.69 Bnip3 (Ms).sup.59 Arntl (Ms).sup.60 FUNDC1 (Hu).sup.53 Bnip3 (Ms).sup.59 NIX (Hu).sup.53 Ctse (Ms).sup.61 UBQLIN1 (Hu).sup.54 (D) Table 1 Footnotes Genes referenced without underlining were down-regulated in literature reports. Genes referenced with underlining were reportedly up-regulated. Genes referenced with character shading were either up- or down-modulated contingent on clinical/experimental context. Genes referenced with stars are considered susceptibility genes with associated risk variants including single nucleotide polymorphism (SNP) and copy number variants (CNVs). Interrogation marks imply absence of published reports or uncertain relationships to neuropathological processes. Number of predicted targets for each disorder are listed in parentheses below disorder names. Prediction algorithms for miR-181a and -200c are derived from their broadly conserved miRNA families: miR-181abcd/4262 and miR-200bc/429/548a. (Reference index and full names for the genes listed in the table are provided in Table 1 E) and F) below). Species associated with studied targets (genes) are also listed. AD, Alcohol dependence; ASD, Autism spectrum disorder; BP, bipolar disorder; Dm, Drosophila melanogaster; Hu, human; ID, Intellectual disability; IDD, Intellectual development disorder; MDD, Major depression disorder; Ms, mouse; OCD, Obsessive compulsive disorder; OS, oxidative stress; PTHS, Pitt-Hopkins syndrome; Rt, rat; SCZ, schizophrenia; TS, Tourette syndrome. Yst, yeast. Sirt1 (underlined) exhibits dual effects, i.e. moderate overexpression of Sirt1 reduced oxidative stress and high levels of Sirt1 increased oxidative stress. @, Serp1 SERP1 represents an oxidative stress- associated endoplasmic reticulum (ER) protein with chaperone-like functions thought to play a cytoprotective role against ER stress. .COPYRGT., Small Maf proteins variably impact gene expression: complexes with Bach1 repress MARE-dependent gene expression, whereas heterodimers with NF-E2 p45 or related factors (Nrf1, Nrf2, and Nrf3) activate MARE-driven genes.sup.85(Igarashi, K., Sun, J., Antioxid. 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and glutamatergic pathways in lithium response: association with IMPA2, INPP1, and GSK3B genes. J Clin Psychopharmacol. (2015) Aug 11. [Epub ahead of print] 88. Chen, X., et al. A novel relationship for schizophrenia, bipolar and major depressive disorder Part 7: A hint from chromosome 7 high density association screen. Behav Brain Res. (2015) 293: 241- 251. 89. Ovadia, G., Shifman, S. The genetic variation of RELN expression in schizophrenia and bipolar disorder. PLoS One. (2011) 6(5): e19955. 90. Xiang Y., et al. Ethanol Upregulates NMDA Receptor Subunit Gene Expression in Human Embryonic Stem Cell-Derived Cortical Neurons. PLoS One. (2015) 10(8): e0134907 91. Padula A E., et al. KCNN Genes that Encode Small-Conductance Ca2+-Activated K+ Channels Influence Alcohol and Drug Addiction. Neuropsychopharmacology. (2015) 40(8): 1928-39. 92. Gasso, P., et al. Association between genetic variants related to glutamatergic, dopaminergic and neurodevelopment pathways and white matter microstructure in child and adolescent patients with obsessive-compulsive disorder. J Affect Disord. (2015) 186: 284-292. 93. Yu D., et al. Cross-disorder genome-wide analyses suggest a complex genetic relationship between Tourette's syndrome and OCD. Am J Psychiatry. (2015) 172(1): 82-93. (F) Table 1 Gene Names AP4M1, Adaptor-Related Protein Complex 4, Mu 1 Subunit ARIN2, AT Rich Interactive Domain 2 (ARID, RFX-Like) ARNTL (BMAL1), Aryl Hydrocarbon Receptor Nuclear Translocator-Like ATG5, Autophagy Related 5 ATG14, Autophagy Related 14 ATP9A, ATPase, Class II, Type 9A BCL2, B-Cell CLL/Lymphoma 2 BNIP3 (NIP3), BCL2/Adenovirus E1B 19 kDa Interacting Protein 3 BNIP3L (NIX), BCL2/Adenovirus E1B 19 kDa Interacting Protein 3-Like BRD1, Bromodomain Containing 1 CACNA1C, Calcium Channel, Voltage-Dependent, L Type, Alpha 1C Subunit CDH13, Cadherin 13 CHGA, Chromogranin A CLN5, Ceroid-Lipofuscinosis, Neuronal 5 CNTNAP2, Contactin Associated Protein-Like 2 CSMD1, CUB and Sushi Multiple Domains 1 CTSE, Cathepsin E DDX3X, DEAD (Asp-Glu-Ala-Asp) Box Helicase 3, X-Linked DLGAP1, Discs, Large (Drosophila) Homolog-Associated Protein 1 DPP6, Dipeptidyl-Peptidase 6 DRD2, Dopamine Receptor D2 EN2, Engrailed-2 Erbb4, Erb-B2 Receptor Tyrosine Kinase 4 ESR1, Estrogen Receptor 1 FOXG1, Forkhead Box G1 FUNDC1, FUN14 Domain Containing 1 GABRA1, Gamma-Aminobutyric Acid (GABA) A Receptor, Alpha 1 GNAI2, Guanine Nucleotide Binding Protein (G Protein), Alpha Inhibiting Activity Polypeptide 2 GPC6, Glypican 6 GPX1, Glutathione Peroxidase 1 GPX7, Glutathione peroxidase 7 GRIA2, Glutamate Receptor, Ionotropic, AMPA 2 GRIN2A, Glutamate Receptor, Ionotropic, N-Methyl D-Aspartate 2A GRIN2B, Glutamate Receptor, Ionotropic, N-Methyl D-Aspartate 2B GRM1, Glutamate Receptor, Metabotropic 1 GRM5, Glutamate Receptor, Metabotropic 5 GRM7, Glutamate Receptor, Metabotropic 7 GSK3B, Glycogen Synthase Kinase 3 Beta HLA-C, Major Histocompatibility Complex, Class I, C HLA-DRA, Major Histocompatibility Complex, Class II, DR Alpha HTR2C, 5-Hydroxytryptamine (Serotonin) Receptor 2C, G Protein-Coupled HYOU1, Hypoxia Up-Regulated 1 IMPA2, Inositol(Myo)-1(Or 4)-Monophosphatase 2 KCNJ5, Potassium Channel, Inwardly Rectifying Subfamily J, Member 5 KCNK2, Potassium Channel, Two Pore Domain Subfamily K, Member 2 KCNN3, Potassium Channel, Calcium Activated Intermediate/Small Conductance Subfamily N Alpha, Member 3 KDM5C, Lysine (K)-Specific Demethylase 5C KLAA0040, KIAA0040 IL1A, Interleukin 1, Alpha IQSEC2, IQ Motif and Sec7 Domain 2 LMX1A, LIM Homeobox Transcription Factor 1, Alpha LSAMP, Limbic System-Associated Membrane Protein MAF, V-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homolog MAP1B, Microtubule-Associated Protein 1B MAP2, Microtubule-Associated Protein 2 MCL-1, Myeloid Cell Leukemia 1 MEIS2, Meis Homeobox 2 MMP14, Matrix Metallopeptidase 14 (Membrane-Inserted) MTFR1, Mitochondrial Fission Regulator 1 MYCBP2, MYC Binding Protein 2, E3 Ubiquitin Protein Ligase NCAM1, Neural Cell Adhesion Molecule 1 NDUFS1, NADH Dehydrogenase (Ubiquinone) Fe--S Protein 1, 75 kDa (NADH-Coenzyme Q Reductase) NEUROD1, Neuronal Differentiation 1 NFATC2, Nuclear Factor of Activated T-Cells, Cytoplasmic, Calcineurin-Dependent 2 NF1, Neurofibromin 1 NGFR, Nerve Growth Factor Receptor NLGN4X, Neuroligin 4, X-linked NOTCH1, Notch1 NRG1, Neuregulin 1 NRG2, Neuregulin 2 NRG3, Neuregulin 3 NRXN1, Neurexin 1 NRXN3, Neurexin 3 OFCC1, Orofacial Cleft 1 Candidate 1 PARK7, Parkinson Protein 7 PBX1, Pre-B-Cell Leukemia Homeobox 1 PHF3, PHD Finger Protein 3 PPARD, Peroxisome Proliferator-Activated Receptor Delta PSEN1, Presenilin 1 PTK2, Protein Tyrosine Kinase 2 PTP4A1, Protein Tyrosine Phosphatase Type IVA, Member 1 PVRL1, Poliovirus Receptor-Related 1 (Herpesvirus Entry Mediator C) PXN, Paxillin RARA, Retinoic Acid Receptor, Alpha RELN, Reelin RGS6, Regulator of G-Protein Signaling 6 RORA, RAR-Related Orphan Receptor A RYR3, Ryanodine Receptor 3 SERP1, Stress-associated endoplasmic reticulum protein 1 SIN3, SIN3 Transcription Regulator Family Member A SIRT1, Sirtuin 1 SLC6A1, Solute Carrier Family 6 (Neurotransmitter Transporter), Member 1 SLC6A11, Solute Carrier Family 6 (Neurotransmitter Transporter), Member 11 SLIT3, Slit Homolog 3 (Drosophila) SNAP25, Synaptosomal-Associated Protein 25 kDa STK40, Serine/Threonine Kinase 40 STX1A, Syntaxin 1A STXBP5, Syntaxin Binding Protein 5 (Tomosyn) SYNJ1, Synaptojanin 1 TCF4, Transcription Factor 4 TNFA, Tumor Necrosis Factor-Alpha TPH2, Tryptophan Hydroxylase 2 UBQLIN1, Ubiquilin 1 WDR7, WD Repeat Domain 7 WDR60, WD Repeat Domain 60 YWHAB, Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein, Beta YWHAG, Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein, Gamma YWHAQ, Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein, Theta YWHAZ, Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein, Zeta ZNF804A, Zinc Finger Protein 804A

[0237] MnSOD mRNA expression levels in the PFC, STM and SN of the GFAP.HMOX1 mice were significantly augmented in comparison to the WT counterparts. Furthermore, glutathione reserves were depleted in the HC and STM of TG brains, as evidenced by diminished GSH concentrations and/or low GSH:GSSG ratios relative to WT preparations. These finding further corroborate the contention that oxidative stress downstream from sustained or repeated over-expression of HO-1 in astrocytes may be a pivotal player leading to neuronal dysfunction in a host of developmental and degenerative brain disorders.sup.78.

[0238] A major finding of these experiments was that Immunocal treatment attenuated many of the behavioural, neurochemical and redox abnormalities characteristic of the GFAP.HMOX1 mouse. These observations are consistent with previous reports of symptom amelioration in human SCZ patients and animal models of the disease following the administration of glutathione precursors.sup.14. In GFAP.HMOX1 mice of both sexes, Immunocal supplementation restored redox homeostasis by significantly increasing GSH concentrations and GSH:GSSG ratios in whole brain and in discrete regions (PFC, HC, STM) implicated in the etiopathogenesis of SCZ. The restoration of brain GSH homeostasis in the GFAP.HMOX1 mice by exposure to Immunocal was sufficient to alleviate cellular oxidative stress as evidenced by normalization of MnSOD mRNA expression in the transgenic PFC and STM. Immunocal also bolstered GSH reserves in the brains of WT animals relative to the casein-treated controls. Thus, by serving as a source of bioavailable cysteine, believed to be the rate-limiting amino acid required for GSH biosynthesis, results herein indicate that whey protein consumption augments GSH stores and antioxidant defenses in the healthy and diseased mammalian CNS.

[0239] Immunocal treatment significantly ameliorated the hyperlocomotion and stereotypy in male GFAP.HMOX1 mice and improved PPI in the transgenic females. Without wishing to be bound by theory, these benefits may be attributed to the positive impact of Immunocal exposure on aberrant monoaminergic neurotransmission observed in the TG mice. All HMOX1-related changes in brain dopamine, dopamine metabolites, serotonin, norepinephrine and epinephrine were improved by Immunocal supplementation. An interesting neurotransmitter profile was observed in the PFC of Immunocal-treated TG mice where Immunocal not only restored excessive serotonin levels and deficient DOPAC/DA ratios to WT values, but also concurrently augmented norepinephrine and dopamine concentrations in this brain region. Without wishing to be bound by theory, it is thought that by normalizing serotonin content in the TG PFC, Immunocal may have released the local dopamine system from serotonergic inhibition (as evidenced by the increased dopamine turnover and enhanced dopamine content).sup.74, thereby correcting the PFC hypodopaminergia. Additionally, the Immunocal-mediated increase in NE may have exerted an additive effect on DA release in the TG PFC, in keeping with the previously reported positive regulation of DA by NE in rat PFC.sup.79.

[0240] Data sets indicate further that these beneficial effects of whey protein supplementation on dopaminergic tone are mediated, at least in part, by correction of deviant mRNA and miRNA expression profiles known to regulate the growth, function and maintenance of catecholaminergic circuitry in the developing mammalian CNS. Noteworthy in this regard is the full or partial normalization of mRNA expression profiles for neural reelin (RELN), GAD67, Nrxn1, Nlgn2 and several of their targeting miRNAs in salient regions of the GFAP.HMOX1 brain. PFC hypodopaminergia is thought to underlie the `negative` symptoms of schizophrenia (apathy, social withdrawal, etc.).sup.74.

[0241] As previously reported.sup.9, the wild-type and GFAP.HMOX1 mice exhibited several sex-specific differences on behavioural testing. Thus, PPI was more robust in males than females (as seen in human schizophrenia.sup.39) as was hyperlocomotor activity. Interestingly, Immunocal treatment significantly suppressed hyperlocomotion in male GFAP.HMOX1 mice, whereas in female WT mice certain indices of locomotor activity were enhanced by whey protein exposure. These results suggest that the development of nutrition-based strategies may allow for the management of neuropsychiatric disorders in a more nuanced, gender-specific fashion.

[0242] Immunocal supplementation in these mouse model studies exerted no appreciable effects on the dysgenesis of the hippocampal dentate gyrus and ventriculomegaly which characterize the GFAP.HMOX1 mouse.sup.9 and human SCZ brain.sup.81,82. This outcome is not surprising inasmuch as these midline brain defects are established during neurodevelopment prior to the Immunocal treatment window and are likely to be irreversible. It is hypothesized that these structural anomalies may be obviated by Immunocal treatment of pregnant GFAP.HMOX1 dams and prepubertal transgenic pups. Such findings would suggest whey protein supplementation as a potential safe and effective intervention for diminishing the likelihood of SCZ in persons at-risk for this disease.sup.83,84.

[0243] One study analyzing reelin levels in different brain regions of schizophrenia subjects found a significant decrease in reelin in 5 key brain regions, namely the prefrontal cortex (PFC), the striatum (STM), hippocampus (HC), temporal cortex, and cerebellum, compared to control subjects (Impagnatiello et al., 1998, A decrease of reelin expression as a putative vulnerability factor in schizophrenia, Proc. Natl. Acad. Sci., USA, vol. 95: pp. 15718-15'723; herein incorporated by reference in its entirety). In GFAP.HMOX1 mouse model of schizophrenia, a significant decline in reelin protein immunoreactivity in the PFC, STM (caudate-putamen), and HC has been observed (Song et al. J Neurosci 32:10841-10853, 2012).

[0244] As described hereinabove, reelin (RELN) mRNA was analyzed in 3 brain regions relevant to schizophrenia in younger GFAP.HMOX1 mice: the PFC, STM and the substantia nigra (SN). Immunocal treatment induced a statistically significant increase in RELN, as observed in the PFC. Moreover, mRNA expression levels of GAD67, a gene acting downstream of RELN, were significantly increased in the STM and SN after Immunocal treatment. Without wishing to be bound by theory, these data suggest that Immunocal treatment may have salutary effects in schizophrenia and related neurodevelopmental disorders (e.g. autism) by enhancing brain reelin/GAD67 expression. The STM and SN are also key loci of pathology in Parkinson's disease and other extrapyramidal disorders; therefore it is contemplated that Immunocal-mediated upregulation of GAD67 in these regions may ameliorate such conditions as well. Again, without wishing to be bound by theory, upregulation of reelin/GAD67 in the PFC and HC may also benefit Alzheimer's dementia since degeneration in these brain regions is believed to be important, respectively, for the executive dysfunction and memory deficits characteristic of this condition. The role of reelin/GAD67 and Immunocal supplementation in Alzheimer disease could be further investigated in, for example, APPswe/PS1.DELTA.E9 double-transgenic mice (e.g. J Neurochem 131: 778-790, 2014; herein incorporated by reference in its entirety) and other established rodent models of Alzheimer disease.

[0245] Without wishing to be bound by theory, Immunocal treatment may confer neuroprotection in human neurodegenerative disorders at least in part because (i) the glial HO-1 response is an important transducer of environmental and endogenous stressors into patterns of neural damage (pathological iron deposition, mitochondrial injury, mitophagy, etc.) characteristic of Alzheimer's disease and Parkinson's disease (Schipper H M, Song W. A Heme Oxygenase-1 Transducer Model of Degenerative and Developmental Brain Disorders. International journal of molecular sciences 2015; 16:5400-5419), (ii) central oxidative stress and glutathione deficiency figure prominently in the pathophysiology of the latter conditions (Gu M, Owen A D, Toffa S E, et al. Mitochondrial function, GSH and iron in neurodegeneration and Lewy body diseases. Journal of the neurological sciences 1998; 158:24-29; Jomova K, Vondrakova D, Lawson M, Valko M. Metals, oxidative stress and neurodegenerative disorders. Mol Cell Biochem 2010; 345:91-104; Mandal P K, Saharan S, Tripathi M, Murari G. Brain Glutathione Levels--A Novel Biomarker for Mild Cognitive Impairment and Alzheimer's Disease. Biological psychiatry 2015; and Schulz J B, Lindenau J, Seyfried J, Dichgans J. Glutathione, oxidative stress and neurodegeneration. Eur J Biochem 2000; 267:4904-4911) and, (iii) as documented herein, Immunocal administered orally markedly improved GSH homeostasis in the brains of GFAP.HMOX1 mice.

[0246] The results obtained in these experiments indicate that compositions comprising whey protein isolate and/or whey protein concentrate, such as Immunocal.RTM., may be used for treating, ameliorating, or preventing neurological or neurodegenerative diseases or conditions in a subject. It has been found that, as described in detail herein, compositions comprising whey protein isolate and/or whey protein concentrate may be used for restoration of neuronal reelin (RELN) levels. The effects of treatment with such compositions were not limited to increasing or restoring RELN levels, indicating that such compositions may also, or alternatively, be used to correct a number of other neurological imbalances, dysregulations, or abnormalities occurring in a subject as described hereinabove. Subjects suffering from neuropsychiatric or neurodegenerative diseases may particularly benefit from treatment with such compositions, however other subjects may also benefit from such treatment with whey protein isolate.

Example 2--Whey Characteristics and Whey Protein Isolate Production

[0247] An example of whey protein isolate production is provided below for illustrative purposes intended for the person of skill in the art.

[0248] As will be understood, whey may be considered as a by-product of cheese or of casein manufacture. Whey typically contains soluble proteins of milk, so-called whey proteins. Cheese whey, for example, typically contains 5-8 g/l of proteins (N.times.6.38), among which .beta.-lactoglobulin (.beta.-lg) and .alpha.-lactalbumin (.alpha.-la) are the most abundant (accounting for 50-55% and 15-20% of total whey proteins, respectively) and bovine serum albumin (BSA), lactoferrin (LF) and immunoglobulins (IgG) are considered as minor whey proteins (accounting each for 3-5%). Whey may also comprise protein fragments or polypeptides such as so-called proteose-peptones (PP-4, PP-5, PP-8f) resulting from proteolysis of milk proteins by lactic starters in cheesemaking or by psychrotrophic bacteria during cold storage of raw milk. These proteinaceous compounds are not completely characterized, and their concentration in whey is highly variable. Finally, non-protein nitrogen (NPN) group may comprise a large number of molecules in whey, among which urea may account for 50-60%.

[0249] For illustrative purposes, Table 2 below provides some characteristics of some of the major proteins and polypeptides found in an exemplary whey sample (in this case, bovine sweet whey).

TABLE-US-00003 TABLE 2 Some Characteristics of Major Proteins and Polypeptides in an Exemplary Whey Sample Weight Protein or contribution Molecular polypeptide (g/l) (approx.) weight .beta.-lactoglobulin 3.0 18 400 .alpha.-lactalbumin 1.2 14 200 BSA 0.3 69 000 Lactoferrin 0.2 77 000 IgG 0.2 160 000 PP-3 0.6 22 000 PP-5 14 300 PP-8f 4 100 NPN 1.6

[0250] In this example, whey protein isolate may be obtained from whey, such as the whey exemplified above in Table 2. As will be understood, process steps involved in the manufacture of whey protein isolate (WPI) may lead to compositional differences in terms of protein profile between whey protein isolates. Thus, the specific components and their abundance are not meant to be considered limiting in any manner. Factors influencing whey protein isolate characteristics may include, for example:

[0251] [1] Source of the whey proteins: For example, sweet- or acid-whey may be used as starting material for the manufacture of WPI;

[0252] [2] Pasteurization: For example, the proteins in cheese whey-derived ingredients may be submitted to two (2) pasteurization (i.e. 72-75.degree. C.--12-16 sec.) treatments at a cheese plant where milk is pasteurized (Canada and US regulation) before cheesemaking, or at the ingredient manufacturing plant, or before transportation of drained whey to this plant, in order to reduce bacterial count before membrane processing or ion exchange chromatography; and

[0253] [3] Defatting: For example, centrifugal clarification is typically used to reduce the fat content of whey to 0.8-1.2%. However, an additional defatting step is often performed to further decrease the fat content to 0.3-0.5% in order to increase membrane separation performance or to prevent an irreversible fouling or clugging of ion-exchange resins with polar lipids. Defatting typically involves holding whey at 50-55.degree. C. for 30 to 90 min. in order to promote aggregation of fat particles (optionally in the presence of added CaCl.sub.2)). The product will thereafter be submitted to centrifugal separation or MF in order to remove the agglomerated material.

[0254] In this example, high-protein concentration (>90% dry basis) whey protein isolate may typically be prepared from whey such as that exemplified in Table 2 by either of two methods: membrane processing or ion-exchange chromatography. In membrane processing, microfiltration (MF) and/or ultrafiltration (UF) membranes may be used for concentrating whey. In ion-exchange chromatography, cationic- and/or anionic-exchange chromatography may be used to purify whey proteins.

[0255] In this example, obtained samples may be submitted to spray drying conditions. Where a substantially undenatured isolate is to be prepared, the obtained concentrated liquid may be, for example, sprayed in a hot air current (inlet T.degree.: 180-200.degree. C., outlet T.degree.: 80-100.degree. C.) circulating in a spray drying tower. A combination of dehydration and gravity may allow the collection of dry particles (4-8% humidity) at the bottom of the spray dryer. Estimates obtained from mathematical modeling of such drying processes suggest that the droplet temperature does not exceed about 80-85.degree. C. during the few seconds used for dehydration, providing for an example of low impact spray drying which may not substantially denature whey protein.

[0256] As will be understood, ingredients having high-protein contents may generally be more difficult to rehydrate (possibly because of their low lactose and minerals content). For certain applications where rapid rehydration of the powder obtained from spray drying is desired, the powder may be submitted to agglomeration. Such steps may involve a final drying of the powder (from 12-15% to 4% humidity) on a fluid bed, generating agglomerated particles having better sinkability in water. In products containing fat (which is generally not the case for high protein ingredients), lecithin may be injected during fluid bed drying. Lecithin may cover fat droplets and improve their wettability. Instantization step(s) may also be used, although such steps are generally uncommon in the manufacture of high-protein ingredients.

[0257] As a result of the above steps, an example of a whey protein isolate may be prepared from the whey protein starting material exemplified in Table 2 above. It will be understood that this example is provided for illustrative and non-limiting purposes, and that many alternative, substituted, or modified whey protein sources and/or processing steps known to the person of skill in the art having regard to the teachings herein are also contemplated.

Example 3--Effects of Whey Protein Isolate in Cell and Mouse Models of Alzheimer's Disease (AD)

[0258] As discussed above, deficits in Reelin expression and/or Reelin signaling play a pathogenic role in several nervous system disorders including schizophrenia and Alzheimer's disease (AD).

[0259] As described hereinabove, the cysteine-rich whey protein supplement, Immunocal.RTM., rescues Reelin expression, particularly in the prefrontal cortex, of a mouse model of schizophrenia. Given that Reelin expressing neurons of the entorhinal cortex layer II are a highly vulnerable population of cells that are lost very early in AD, the presently described experiments sought to evaluate the effects of whey protein isolate on Reelin expression and signaling in vitro in hippocampal-entorhinal cortex rat brain slices and in vivo in the hAPPSweInd mutant (J20) mouse model of AD.

[0260] As discussed below, incubation of hippocampal-entorhinal cortex slices with Immunocal.RTM. increased Reelin expression at the mRNA and protein levels. In addition, immunostaining of slices revealed a striking increase in the intensity and number of neurons staining positively for Reelin within the entorhinal cortex, dentate gyrus and CA1 region of the hippocampus following Immunocal.RTM. treatment in vitro. These studies next evaluated the effects of whey protein isolate in vivo by treating hemizygous J20 AD mice from 3 months-old to 5 months-old with Immunocal.RTM.. Reelin expression and signaling was then assessed by western blotting and immunofluorescence microscopy and cognitive function using the Barnes maze to test spatial learning and memory. Immunocal.RTM. treatment corrected a deficit in cortical GSH levels observed in the brains of untreated hemizygous J20 mice. Western blotting of brain sections micro-dissected to enrich for the hippocampal-entorhinal cortex sub-region revealed a decrease in Reelin and GAD67 expression in untreated hemizygous J20 AD mice compared to non-carrier control mice and this effect was prevented by treatment with Immunocal.RTM.. In addition, untreated hemizygous J20 AD mice displayed a marked reduction in p-CREB immunoreactivity in the hippocampal-entorhinal cortex sub-region of the brain and this deficit was essentially rescued by treatment with Immunocal.RTM.. In a similar manner, using immunofluorescence microscopy, Reelin expression was diminished in the entorhinal cortex, dentate gyrus and CA1/CA3 regions of the hippocampus in untreated hemizygous J20 AD mice compared to non-carrier control mice. In contrast, Immunocal.RTM. treatment largely rescued these deficits in Reelin expression. In particular, Immunocal.RTM. treated J20 mice displayed robust Reelin staining in layer II of entorhinal cortex, apparently rescuing the loss of Reelin positive neurons observed in this brain region in untreated J20 AD mice. In parallel with the observed rescue in Reelin expression in J20 mice, Immunocal.RTM. also preserved GAD67 expression in the dentate gyrus and CA3 region of the hippocampus and markedly enhanced the co-staining of Reelin and phospho-DAB1 in the CA1 of these mice. Finally, Immunocal.RTM. treatment had a statistically significant positive effect on Barnes maze performance, both during the late stages of the acquisition phase and during the probe phase, in female, hemizygous J20 AD mice. Collectively, these findings indicate that Immunocal.RTM. induces Reelin expression in vitro in hippocampal-entorhinal cortex brain slices and rescues Reelin expression and signaling in vivo within the entorhinal cortex and hippocampus in the J20 mouse model of AD.

[0261] Alzheimer's Disease: Phenotypic and Pathological Characteristics. Alzheimer's disease (AD) is the leading cause of dementia and cognitive decline with over 5 million patients currently diagnosed in the United States and approximately 500,000 new cases each year. Aging is the most significant risk factor for developing sporadic AD. According to the Alzheimer's Association, approximately 13% of people age 65 and older have AD and this fraction increases to approximately 45% of people over the age of 85. Given the demographic shift to advancing age in our population, there is predicted to be a very significant increase in the number of people diagnosed with AD in the next several decades. By 2025 the number of people age 65 and older suffering with AD is predicted to be approximately 7.7 million and this number is expected to perhaps double by 2050. The average life expectancy once a patient is diagnosed with AD is approximately 8 years and AD has risen to the 6th leading cause of death in the United States. Alzheimer's disease was first described in 1906 by Dr. Alois Alzheimer and is characterized phenotypically by progressive memory loss and cognitive decline. Pathological hallmarks of the disease include amyloid plaques consisting of insoluble deposits of amyloid beta (A.beta.) peptide and neurofibrillary tangles containing hyper-phosphorylated tau protein. These plaques and tangles are found throughout the brain parenchyma and are believed to play a significant role in the neuronal loss and atrophy that are characteristic of the AD brain. Of the diverse neuronal cell types that die in AD, hippocampal pyramidal cells, cortical pyramidal cells, and basal forebrain cholinergic neurons are among the most severely affected. The death of these populations of neurons leads to profound synaptic loss and significant neurotransmitter deficits, particularly in cholinergic pathways. Although 3 genes (amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2)) have been shown to be mutated in early onset, autosomal dominant, familial AD, these inherited forms of the disease make up less than 5% of AD cases, with the remaining 95% of cases being sporadic in nature. Current FDA approved drugs for AD are limited to acetylcholinesterase inhibitors and the NMDA antagonist memantine. These drugs are minimally effective and treat the symptoms of AD while having no effect on the underlying etiology of the disease. As such, current therapies do not significantly delay or halt the progression of AD.

[0262] The Amyloid Cascade Hypothesis. The amyloid cascade hypothesis has dominated the field of AD research for the past two decades and is founded on the premise that deposition of A.beta. peptide is the initiating event in disease pathogenesis, ultimately leading to neurofibrillary tangle formation, synaptic loss, and neuronal cell demise (Hardy and Higgins, 1992). Throughout the years, the molecular form of A.beta. thought to initiate the toxic cascade responsible for AD pathology has shifted from insoluble plaques to protofibrils to soluble oligomers (Klein et al., 2001; Naylor et al., 2008; Ferreira and Klein, 2011; Larson and Lesne, 2012). Regardless of which particular form(s) of A.beta. leads to disease pathology, for more than a decade, the amyloidogenic processing of APP to form A.beta. has been recognized as a viable molecular target for AD therapeutic development (Vassar, 2001; Evin et al., 2006). The amyloidogenic pathway generates beta amyloid peptide (of which the A.beta..sub.1-42 form (here referred to simply as A.beta.) is commonly regarded as the most likely toxic species) through the sequential cleavage of APP by the beta-site APP cleaving enzyme (BACE; also known as .beta.-secretase) and .gamma.-secretase (FIG. 12). Alternatively, the sequential cleavage of APP by .alpha.-secretase and .gamma.-secretase is considered non-amyloidogenic and primarily produces a soluble non-aggregating form of APP (sAPP.alpha.). Based largely on the amyloid cascade hypothesis and more recent studies showing that soluble A.beta. oligomers are intrinsically neurotoxic, selective inhibitors of BACE and .gamma.-secretase have been developed as drugs to decrease the tissue load of A.beta. in AD brain (Vassar et al., 2009; D'Onofrio et al., 2012). The development of selective BACE inhibitors that are orally bioavailable and penetrate the blood brain barrier has been slow; however, this class of compounds has recently begun clinical testing in AD (Ghosh et al., 2012). The Merck compound, MK-8931 (verubecestat), initially completed Phase I testing and reportedly was capable of reducing cerebral spinal fluid amyloid levels by up to 90% in rats, monkeys, healthy human volunteers, and AD patients (Menting and Claassen, 2014; Kennedy et al., 2016). A Phase II/III clinical trial of this drug (EPOCH) completed enrollment in early 2016 in a population of patients with mild-to-moderate AD. Unfortunately, in February of 2017, Merck announced that it was terminating the trial after an interim analysis suggested little chance of discerning any positive therapeutic benefit. This was a significant setback for the AD field; however, Merck has continued another trial of verubecestat in patients with prodromal AD (APECS). In a similar manner to the recent failure of BACE inhibitors, .gamma.-secretase inhibitors have been largely disappointing in the clinic. Most notably, two large Phase III clinical trials of the Eli Lilly compound, semagacestat, in mild-to-moderate AD patients were terminated early due to a statistically significant worsening of clinical measures of cognition and ability to perform activities associated with daily living (D'Onofrio et al., 2012). It is presently unclear why this compound failed clinically but it may be an off target effect related to modulation of Notch signaling as Notch is also a substrate for .gamma.-secretase-mediated cleavage. Thus, Notch-sparing .gamma.-secretase inhibitors are currently under development in hopes of circumventing possible toxic effects of this class of drugs (Augelli-Szafran et al., 2010). As a result of these recent clinical disappointments, and due to the observation that brain amyloid load does not necessarily correlate with the severity of cognitive deficits in AD (as well as other findings contrary to an amyloid-centric view), some have begun to question whether the amyloid cascade hypothesis is sufficient to explain the underlying pathogenesis of late onset, sporadic AD (Giannakopoulos et al., 2003; Pimplikar et al., 2010; Castellani and Smith, 2011; Karran et al., 2011; Mullane and Williams, 2013; Karran and De Strooper, 2016; Swerdlow et al., 2017; Tse and Herrup, 2017).

[0263] The Complex Role Of Reelin In Alzheimer's Disease. Reelin is a large glycoprotein secreted by specific cells within the central nervous system that plays a key role in patterning and layering of the cerebral cortex and other regions of the brain during development. In adults, Reelin plays a central role in processes that influence synapse formation and neuronal plasticity required for learning and memory, such as the regulation of dendritic spine architecture and the maintenance of long term potentiation (Niu et al., 2004; Beffertt al., 2006; Kim et al., 2015; Bosch et al., 2016). Alzheimer's disease is the most prevalent cognitive disorder in adults and is characterized by substantial deficits in learning and memory. The pathological basis of AD is complex and is characterized by the formation of senile plaques made up of misfolded A.beta. protein and neurofibrillary tangles consisting of hyper-phosphorylated tau protein. Specific populations of neurons in the brain die in AD including most prominently, forebrain cholinergic neurons, hippocampal neurons, and cortical pyramidal neurons. In particular, neurons of the entorhinal cortex project to the hippocampus and are involved in declarative memory formation and consolidation. Entorhinal cortex layer II neurons provide the principal excitatory glutamatergic input to the dentate gyrus of the hippocampus. The entorhinal cortex layer II neurons are one of the first neuronal populations to die in AD, resulting in a severe loss of synaptic contacts to the dentate gyrus. Many of the entorhinal cortex layer II neurons express Reelin and these Reelin-expressing cells are significantly reduced in the brains of human amyloid precursor protein (hAPP) transgenic mice expressing the Swedish and Indiana mutant form of the hAPP gene (J20 strain). In accordance with the loss of these Reelin-expressing entorhinal cortex layer II neurons, Reelin levels in the hippocampus of J20 mice are also significantly reduced, compared to nontransgenic controls (Chin et al., 2007). Similar loss of Reelin-expressing entorhinal cortex layer II neurons is also observed in the brains of patients with AD (Chin et al., 2007; Herring et al., 2012). Finally, in a transgenic rat model of AD (McGill-R-Thy1-APP strain), Reelin-expressing neurons of the entorhinal cortex layer II were found to selectively express increased levels of soluble intracellular A.beta. early in disease, prior to the deposition of amyloid plaques (Kobro-Flatmoen et al., 2016). Collectively, these studies suggest that Reelin-expressing neurons of entorhinal cortex layer II play a central role in the early pathogenic changes in AD and loss of these Reelin-expressing cells and their synaptic projections to the hippocampus are early markers of disease (Krstic et al., 2013). Thus deficits in Reelin signaling to the hippocampus likely underlie some of the cognitive deficits observed in patients with AD (Cuchillo-Ibanez et al., 2016; Yu et al., 2016).

[0264] A Brief Overview Of Reelin Signaling. Reelin is secreted into the extracellular space where it interacts with one of two cell surface receptors on target cells, either the very low density lipoprotein receptor (VLDLR) or the apolipoprotein E receptor-2 (ApoER2). Upon binding its receptor, Reelin induces tyrosine phosphorylation of the adapter protein, Disabled-1 (DAB1), via the nonreceptor tyrosine kinases, Src or Fyn. Phosphorylated DAB1 acts as a docking site to initiate multiple downstream signal transduction cascades such as those involved in cell survival (PI3K/AKT) and regulation of actin assembly (Cdc42/PAK/cofilin) (FIG. 13) (Wasser and Herz, 2017).

[0265] The pathway shown in FIG. 13 represents the canonical Reelin signaling pathway and is the one most commonly attributed to Reelin's actions in the central nervous system. However, other (non-canonical) Reelin signaling pathways do exist, as well as additional Reelin receptors (Bock and May, 2016; Lee and D'Arcangelo, 2016).

[0266] Modulation of Reelin Expression and Signaling as a Means of Altering the Course of Alzheimer's Disease.

[0267] As noted above, Reelin-expressing cells of the entorhinal cortex layer II are significantly reduced in the brains of hAPP transgenic mice expressing the Swedish and Indiana mutant form of the hAPP gene (J20 strain). In accordance with the loss of these Reelin-expressing entorhinal cortex layer II neurons, Reelin levels in the hippocampus of J20 mice are also significantly reduced, compared to nontransgenic controls. Moreover, further reducing Reelin in these mice by crossing J20 AD model mice with heterozygous reeler mice accelerates amyloid plaque formation and tau pathology (Kocherhans et al., 2010). On the other hand, Reelin overexpression in J20 AD model mice significantly delays A.beta. fibril formation and rescues cognitive deficits in these mice (Pujadas et al., 2014). Thus, J20 mice are an established model of familial AD and the disease course of these mice is significantly impacted by alterations in Reelin expression, making this an excellent model system to investigate the effects of Immunocal.RTM. on these processes. The whey protein supplement, Immunocal.RTM., is a rich source of the glutathione precursor, cysteine, and is known to boost antioxidant levels in vivo. In addition, as described in detail hereinabove, in a schizophrenia mouse model characterized by low Reelin levels, Immunocal.RTM. elevated Reelin in the brain.

[0268] Loss of Reelin-expressing entorhinal cortex layer II neurons has also been observed in the brains of patients with AD, and therefor studies investigating whether Immunocal.RTM. elevates Reelin expression and signaling in the entorhinal cortex-hippocampus of J20 mice and mitigates cognitive dysfunction in these animals, was performed as described herein.

[0269] The present studies have been performed determine if supplementation with a whey protein isolate such as Immunocal.RTM. increases Reelin expression in vitro in a hippocampal-entorhinal cortex slice model and in vivo within the brain of an Alzheimer's disease mouse model (J20 strain). In addition, Immunocal.RTM. administration to J20 AD model mice studies were performed to determine whether enhanced cognitive function and diminished amyloid load would be observed when compared to untreated mice.

Experimental Methods and Results

[0270] I. In Vitro Studies

[0271] Experimental Model:

[0272] The effects of Immunocal.RTM. on Reelin expression in vitro were investigated. Organotypic hippocampal-entorhinal cortex slices prepared from postnatal day 25 rats were utilized. Brain slices were prepared using a vibrating microtome essentially as described by Leutgeb et al. (2003).

[0273] Treatment and Analysis of Brain Slices:

[0274] Hippocampal-entorhinal cortex slices (.about.400 micron thickness) were cultured in a humidified incubator in tissue culture medium with 5% CO2/95% air at 37.degree. C. After equilibration overnight, slices were subsequently treated with either culture medium alone or containing Immunocal.RTM. (3.3% w/v) for 24 h, followed by measurement of Reelin expression assessed at the mRNA level using quantitative real-time polymerase chain reaction (qPCR) and at the protein level using western blotting. In addition, the expression of Reelin within entorhinal cortex layer II neurons was evaluated specifically using immunofluorescence microscopy after co-staining slices for Reelin and NeuN. As a control to assess viability of the slices and functionality of the Reelin signaling pathway, DAB1 was immunoprecipitated and tyrosine phosphorylation of this adapter protein was measured by western blotting following incubation with recombinant Reelin.

[0275] Results:

[0276] Incubation of hippocampal-entorhinal cortex slices with recombinant Reelin for 24 h induced a marked increase in the tyrosine phosphorylation of the adapter protein DAB1 (FIG. 14A). This result demonstrates two points; first, the brain slices were viable under the incubation conditions used and second, the Reelin signaling pathway is intact in the slice culture as addition of recombinant Reelin stimulated (Src/Fyn-dependent) tyrosine phosphorylation of DAB1 via binding to its cell surface receptors. Next, hippocampal-entorhinal cortex slices were incubated for 24 h in either tissue culture medium alone or containing Immunocal.RTM.. Immunocal.RTM. treatment induced an increase in the full length Reelin protein (388 kDa) and in two prominent Reelin cleavage products (310 kDa and 180 kDa) (FIG. 14B). In addition, the Reelin transcript was measured by qPCR after incubation of brain slices in control medium, medium containing Immunocal.RTM., recombinant Reelin, or a combination of the two. Incubation with Immunocal.RTM. induced a statistically significant, approximately two-fold increase in Reelin mRNA transcript levels in either the absence or presence of recombinant Reelin protein (FIG. 15). Incubation with recombinant Reelin protein alone had no significant effect on the amount of Reelin transcript detected in the slices, although a trend towards decreased Reelin mRNA levels was observed under these conditions.

[0277] Next, the effects of Immunocal.RTM. treatment on Reelin expression were assessed in hippocampal-entorhinal cortex slices by co-staining for Reelin and NeuN using specific antibodies and immunofluorescence microscopy. Incubation of brain slices with Immunocal.RTM. induced a striking increase in Reelin immunoreactivity in the entorhinal cortex, dentate gyrus, and CA1 region of the hippocampus (FIG. 16). Some, but not all, of the Reelin positive cells also co-stained for NeuN.

[0278] II. In Vivo Studies

[0279] Experimental Model:

[0280] To investigate the effects of Immunocal.RTM. on Reelin expression in vivo, the hAPP(Swe/Ind) mutant transgenic mouse model of AD (J20 strain) was utilized. This mouse model is commercially available from Jackson Laboratories (B6.Cg-Tg(PDGFB-APPSwInd) 20Lms/2Mmjax) and displays significant brain pathology, amyloid plaques, and cognitive deficits that recapitulates multiple aspects of AD in humans (Mucke et al., 2000; Karl et al., 2012; Diaz-Hernandez et al., 2012). According to previous reports, these transgenic mice typically show significant cognitive deficits, diminished numbers of Reelin-positive, entorhinal cortex layer II neurons, and decreased Reelin expression in the hippocampus by approximately 4-5 months of age. Amyloid plaques are visible in the brains of these mice by around 12 months of age. All animal studies were conducted in accordance with a protocol approved by the University of Denver Institutional Animal Care and Use Committee.

[0281] Studies were performed to determine whether Immunocal.RTM. treatment would rescue the Reelin-expressing neurons of the entorhinal cortex layer II and as a result, enhance Reelin expression and signaling in the hippocampus of J20 AD model mice. As well, studies were performed to investigate whether Immunocal.RTM. treatment corrects or delays the cognitive deficits observed in J20 AD model mice.

[0282] Immunocal.RTM. Treatment:

[0283] To perform these studies, three groups of mice were used: hAPP(Swe/Ind) mutant hemizygous mice (J20 strain) treated with Immunocal.RTM. (3.3% w/v in drinking water ad libitum, as previously described by Ross et al., 2014), untreated hemizygous J20 mice, and untreated nontransgenic (non-carrier) control mice. Immunocal.RTM. treatment was initiated at 3 months-old and continued until the mice were 5 months-old.

[0284] Behavioral Cognitive Testing:

[0285] During the final week of treatment, mice were evaluated for spatial learning and memory performance using the Barnes maze (FIG. 17). The mice were subjected to a six-day acquisition phase followed by a single day of probe testing, as previously described by Mouzon et al. (2012).

[0286] Immunostaining and Western Blotting for Reelin and its Signaling Pathway Components:

[0287] Following behavioral testing, mice were euthanized and biochemical and immunohistochemical analysis was conducted on brain tissue to evaluate Reelin-expressing neurons in the entorhinal cortex layer II and Reelin expression and signaling in the dentate gyrus, CA1, and CA3 of the hippocampus. For western blotting, brains were micro-dissected to obtain tissue samples enriched for the hippocampal-entorhinal cortex architecture. For immunofluorescence staining of brain tissue, a composite of several sections from a single non-carrier control mouse stained for Reelin, NeuN, and Draq (a nuclear stain) is shown in FIG. 18 to demonstrate the type of staining that was obtained and to orient the reader to the architecture of the hippocampal-entorhinal cortex region.

[0288] Analysis of Brain GSH by HPLC with Electrochemical Detection (HPLC-ECD):

[0289] Tissue Processing

[0290] Cortical tissue was obtained from mice and immediately frozen in liquid nitrogen. For HPLC-ECD analysis, 2.5M perchloric acid was added and the brains were roughly chopped using pointed surgical scissors. Samples were then sonicated 3 times for 15 s intervals. Samples were then centrifuged for 5 min at 13,000 rpm and the supernatant was removed. A 20 .mu.L aliquot of the supernatant was used for a BCA protein assay. The remainder of each solution was neutralized with 50 .mu.L of 4M KOH and vortexed thoroughly. Samples were then centrifuged for 15 min at 13,000 rpm, and stored at -80.degree. C. until separation and analysis by HPLC-ECD.

[0291] HPLC-ECD

[0292] GSH in samples and known standards were separated by reversed-phase HPLC on a C18 bonded silica column at 35.degree. C. (5 .mu.m, 4.6.times.250 mm) from Dionex, Inc. (Sunnyvale, Calif.). Analytes were detected using a CoulArray.RTM. detector (model 5600, ESA) on three coulometric array cells in series; electrochemical detectors were set between 0 and 900 mV at increments of 75 mV. Concentrations were determined with a standard curve of each identified analyte. Mobile phase consisted of 50 mM lithium acetate and 1% acetonitrile in water, pH 3.8. The flow rate was set to 0.4 mL/min for all samples. CoulArray.RTM. software was used for baseline correction and peak analysis.

[0293] Results:

[0294] First, the brain levels of reduced GSH in cortical tissue from the mice were measured using HPLC-ECD. Untreated hemizygous J20 mice displayed a statistically significant decrease in cortical GSH at 5 months-old when compared to non-carrier control mice (FIG. 19). However, hemizygous J20 mice treated with Immunocal.RTM. from 3 months-old to 5 months-old showed a complete preservation of cortical GSH which was statistically significantly greater than the levels observed in untreated hemizygous mice (FIG. 19). Thus, Immunocal.RTM. treatment corrected a deficit in brain GSH observed in AD model mice.

[0295] The expression of Reelin and the glutamic acid decarboxylase protein (GAD67) was next evaluated by western blotting of mouse brain lysates. GAD67 is expressed in GABAergic neurons and its expression is regulated in a manner parallel with Reelin. For example, both Reelin and GAD67 expression are significantly decreased in the prefrontal cortex of patients with schizophrenia and both gene promoters are similarly regulated by drugs that influence epigenetic modifications such as DNA methyltransferase inhibitors and histone deacetylase inhibitors (Guidotti et al., 2000; Kundakovic et al., 2009). Brain lysates enriched for the hippocampal-entorhinal cortex sub-region were immunoblotted for Reelin and GAD67. Reelin expression was significantly reduced in untreated hemizygous J20 AD mice compared to non-carrier controls and Immunocal.RTM. treatment largely corrected this deficiency (FIG. 20A). Only the 180 kDa cleavage fragment of Reelin was detected in these mouse brain lysates. Quantitative densitometric analysis of the 180 kDa Reelin band revealed an approximate 50% reduction in Reelin protein expression in hemizygous untreated AD mice compared to non-carrier controls, and a complete preservation of Reelin expression in Immunocal.RTM.-treated mice (FIG. 20A, graph). The Reelin blots were stripped and reprobed for the inner mitochondrial membrane protein, OPA1, initially as a loading control. Interestingly, OPA1 levels were substantially reduced in 5-month-old hemizygous J20 mice when compared to non-carrier control mice. We have previously shown OPA1 to be susceptible to cleavage and degradation in the brains of old mice and rats (Gray et al., 2013). Nonetheless, Immunocal.RTM. treatment did not appear to rescue OPA1 levels in the brains of hemizygous J20 mice as evidenced by both untreated and Immunocal.RTM.-treated hemizygous mice displaying equivalent amounts of this mitochondrial protein in brain lysates (FIG. 20A). Thus, the changes observed in Reelin expression in these same brain lysates were not due to discrepancies in protein loading. The expression of GAD67 was somewhat variable between untreated hemizygous AD mice and non-carrier control mice; however, Immunocal.RTM. treatment of hemizygous mice markedly increased GAD67 protein levels (FIG. 20B).

[0296] One of the principal transcription factors known to regulate Reelin expression is the cAMP response element-binding protein (CREB) (Grayson et al., 2006). Therefore, the phosphorylation of CREB on Ser133 (p-CREB) which is necessary for its transcriptional activity was evaluated. Brain lysates enriched for the hippocampal-entorhinal cortex sub-region were immunoblotted for p-CREB. Phosphorylation of CREB on Ser133 was relatively high in non-carrier control mice but was dramatically reduced in hemizygous untreated J20 AD mice (FIG. 21). Treatment of hemizygous J20 mice with Immunocal.RTM. from 3 months-old to 5 months-old resulted in a nearly complete preservation of p-CREB expression in the hippocampal-entorhinal cortex sub-region (FIG. 21). Thus, Immunocal.RTM. treatment corrected a major deficiency in brain p-CREB observed in AD model mice.

[0297] Next, immunofluorescence microscopy was used to evaluate Reelin expression in the entorhinal cortex, dentate gyrus, and CA1/CA3 regions of the hippocampus in J20 AD model mice. The entorhinal cortex of non-carrier control mice showed significant Reelin immunoreactivity, particularly in layer II, the area demarcated in the middle panels of FIG. 22 and reproduced in the lower panels. In comparison to the non-carrier control, untreated hemizygous J20 AD mice displayed a marked (.about.30%) reduction in Reelin immunoreactivity in layer II of entorhinal cortex. The measured reduction in Reelin immunoreactivity is comparable to previously reported reductions in Reelin positive layer II neurons in 6-7 month-old J20 mice (Chin et al., 2007). Intriguingly, treatment with Immunocal.RTM. from 3 months-old to 5 months-old essentially rescued this deficit in Reelin expression within layer II of the entorhinal cortex of J20 AD mice (FIG. 22).

[0298] Next, Reelin staining in the dentate gyrus and CA1/CA3 regions of the hippocampus in J20 AD model mice was assessed. In the dentate gyrus, Reelin expression was diffuse and moderate in intensity in non-carrier control mice (FIG. 23). Untreated hemizygous J20 AD mice showed a slight overall reduction in Reelin staining in the dentate gyrus, whereas Immunocal.RTM.-treated hemizygous J20 mice displayed a striking increase in the intensity of Reelin staining in this brain region (FIG. 23).

[0299] In the CA1 region of the hippocampus, Reelin expression was very high in non-carrier control mice and was markedly reduced in untreated hemizygous J20 AD mice (FIG. 24). Notably, this deficit in Reelin staining was essentially prevented by treatment of hemizygous J20 mice with Immunocal.RTM. for 2 months (FIG. 24). A very similar effect on Reelin expression was observed in the CA3 region of the hippocampus. In particular, Reelin expressing neurons in the middle section of this sub-region were particularly evident in non-carrier control mice, essentially absent in untreated hemizygous J20 AD mice, and largely preserved in Immunocal.RTM.-treated J20 mice (FIG. 25).

[0300] Next, GAD67 staining and Reelin co-staining with phospho-DAB1 in the dentate gyrus and CA1/CA3 regions of the hippocampus in J20 AD model mice was evaluated. GAD67 staining in the dentate gyrus of non-carrier control mice was relatively diffuse and the overall intensity was somewhat diminished in untreated hemizygous J20 AD mice (FIG. 26). In contrast, GAD67 staining was markedly increased in the dentate gyrus of hemizyous J20 mice which had been treated with Immunocal.RTM. (FIG. 26). A similar pattern of GAD67 staining in the CA3 region of the hippocampus was observed. In particular, there were several prominent GAD67 expressing neurons clearly visible in the middle section of the CA3 region of non-carrier control mice (FIG. 27). These GAD67-positive cells were essentially absent from the CA3 of untreated hemizygous J20 AD mice, while Immunocal treatment largely rescued GAD67 expression in this region (FIG. 27). Finally, co-staining of Reelin and phospho-DAB1 (pTyr232; a Src/Fyn phosphorylation site) in the CA1 region of the hippocampus in J20 AD model mice was evaluated. Compared to non-carrier control mice, untreated hemizygous J20 AD mice displayed a subtle decrease in the intensity of Reelin and phospho-DAB1 co-staining in the CA1 (FIG. 28). In contrast, Immunocal.RTM. treatment for two months induced a marked increase in Reelin and phospho-DAB1 co-staining in this region of the hippocampus of hemizygous J20 mice (FIG. 28).

[0301] Finally, the effects of Immunocal.RTM. treatment were evaluated for two months on cognitive function in J20 AD model mice at approximately 5 months of age. In particular, the Barnes maze was utilized to test for spatial learning and memory performance. During the six-day acquisition phase, mice in all groups progressively learned to find the escape pod on the maze. No significant differences were observed between groups in the time delay to find the escape pod on days 1-4 of the acquisition phase. However, on days 5 and 6 of the acquisition phase (combined), the Immunocal.RTM.-treated, hemizygous J20 female mice were significantly quicker at finding the escape pod than the untreated hemizygous female group (FIG. 29A). Moreover, during the probe phase, the untreated hemizygous female mice were significantly slower than the non-carrier female group at finding the escape pod and this deficit was corrected in the Immunocal.RTM. treated hemizygous female group (FIG. 29B).

[0302] Previous work has shown that deficits in Reelin expression and/or Reelin signaling play a pathogenic role in several nervous system disorders including schizophrenia and AD. Thus, strategies aimed at correcting these deficits are desirable in this field of study. The cysteine-rich, whey protein supplement, Immunocal.RTM., has been shown to elevate glutathione in the brain and spinal cord, and is neuroprotective and therapeutically efficacious in mouse models of schizophrenia and amyotrophic lateral sclerosis (Ross et al., 2014; examples above). As described hereinabove, Immunocal.RTM. treatment is shown to rescue Reelin expression at the mRNA and protein level in the prefrontal cortex of a mouse model of schizophrenia. Given that Reelin expressing neurons of the entorhinal cortex layer II are a highly vulnerable population of cells that are lost very early in AD, the effects of Immunocal.RTM. on Reelin expression and signaling in vitro in hippocampal-entorhinal cortex rat brain slices and in vivo in the hAPPSweInd mutant (J20) mouse model of AD was investigated in the experiments provided herein.

[0303] Incubation of hippocampal-entorhinal cortex slices with Immunocal.RTM. increased Reelin expression at the mRNA and protein levels, as assessed by qPCR and western blotting. In addition, immunostaining of slices revealed a striking increase in the intensity and number of neurons staining positively for Reelin within the entorhinal cortex, dentate gyrus and CA1 region of the hippocampus following Immunocal.RTM. treatment in vitro.

[0304] The effects of Immunocal.RTM. treatment in vivo was next evaluated by treating hemizygous J20 AD mice from 3 months-old to 5 months-old. Reelin expression and signaling was assessed by western blotting and immunofluorescence microscopy and cognitive function using the Barnes maze was also measured to test spatial learning and memory. In the studies described herein, Immunocal.RTM. treatment corrected a deficit in cortical GSH levels observed in the brains of untreated hemizygous J20 mice. Western blotting of brain sections micro-dissected to enrich for the hippocampal-entorhinal cortex sub-region revealed a decrease in Reelin and GAD67 expression in untreated hemizygous J20 AD mice compared to non-carrier control mice and these effects were prevented by treatment with Immunocal.RTM.. In addition, untreated hemizygous J20 AD mice displayed a marked reduction in p-CREB immunoreactivity in the hippocampal-entorhinal cortex sub-region of the brain and this deficit was essentially rescued by treatment with Immunocal.RTM.. In a similar manner, using immunofluorescence microscopy, Reelin expression was diminished in the entorhinal cortex, dentate gyrus and CA1/CA3 regions of the hippocampus in untreated hemizygous J20 AD mice compared to non-carrier control mice. In contrast, Immunocal.RTM. treatment largely rescued these deficits in Reelin expression and in some cases, markedly increased the expression of Reelin even above what was observed in the non-carrier control mice. In particular, Immunocal.RTM. treated J20 mice displayed robust Reelin staining in layer II of entorhinal cortex, apparently rescuing the loss of Reelin positive neurons observed in this brain region in untreated J20 AD mice. In parallel with the observed rescue of Reelin expression in the hippocampus-entorhinal cortex of J20 mice, Immunocal.RTM. also preserved GAD67 expression in the dentate gyrus and CA3 region of the hippocampus and markedly enhanced the co-staining of Reelin and phospho-DAB1 in the CA1 of these mice.

[0305] Further, Immunocal.RTM. treatment had a statistically significant positive effect on Barnes maze performance, both during the late stages of the acquisition phase and during the probe phase, in female, hemizygous J20 AD mice. Collectively, these findings indicate that treatment with Immunocal.RTM. induces Reelin expression in vitro in hippocampal-entorhinal cortex brain slices and rescues Reelin expression and signaling in vivo within the entorhinal cortex and hippocampus of the J20 mouse model of AD in these studies.

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[0306] References cited in Table 3 abvoe, and those cited elsewhere herein, are herein incorporated by reference in their entireties.

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[0382] 76. Wright C, Turner J A, Calhoun V D, Perrone-Bizzozero N. Potential Impact of miR-137 and Its Targets in Schizophrenia. Front Genet 2013; 4:58.

[0383] 77. Abu-Elneel K, Liu T, Gazzaniga F S, et al. Heterogeneous dysregulation of microRNAs across the autism spectrum. Neurogenetics 2008; 9:153-161.

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[0385] 79. Gresch P J, Sved A F, Zigmond M J, Finlay J M. Local Influence of Endogenous Norepinephrine on Extracellular Dopamine in Rat Medial Prefrontal Cortex. Journal of neurochemistry 1995; 65:111-116.



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[0387] 81. Tamminga C A, Stan A D, Wagner A D. The hippocampal formation in schizophrenia. Am J Psychiatry 2010; 167:1178-1193.

[0388] 82. Walton N M, Zhou Y, Kogan J H, et al. Detection of an immature dentate gyrus feature in human schizophrenia/bipolar patients. Translational psychiatry 2012; 2:e135.

[0389] 83. Brown A S. The environment and susceptibility to schizophrenia. Prog Neurobiol 2011; 93:23-58.

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[0393] All references cited herein, cited in Tables 1 and 3, and cited elsewhere in the specification are herein incorporated by reference in their entirety.

Sequence CWU 1

1

24111580DNAhomo sapien 1cacgcgtggg ctcggcgggg gcccgctccc aggcccgctc ccgagcccgt tccgctcccg 60tccgccttct tctcgccttc tctccgcgtg gctcctccgt cccggcgtct ccaaaactga 120atgagcgagc ggcgcgtagg gcgscggcgg cggcggcggc ggcggcggcg gcggcatgga 180gcgcagtggc tgggcccggc agactttcct cctagcgctg ttgctggggg cgacgctgag 240ggcgcgcgcg gcggctggct attacccccg cttttcgccc ttctttttcc tgtgcaccca 300ccacggggag ctggaagggg atggggagca gggcgaggtg ctcatttccc tgcatattgc 360gggcaacccc acctactacg ttccgggaca agaataccat gtgacaattt caacaagcac 420cttttttgac ggcttgctgg tgacaggact atacacatct acaagtgttc aggcatcaca 480gagcattgga ggttccagtg ctttcggatt tgggatcatg tctgaccacc agtttggtaa 540ccagtttatg tgcagtgtgg tagcctctca cgtgagtcac ctgcccacaa ccaacctcag 600tttcatctgg attgctccac ctgcgggcac aggctgtgtg aatttcatgg ctacagcaac 660acaccggggc caggttattt tcaaagatgc tttagcccag cagttgtgtg aacaaggagc 720tccaacagat gtcactgtgc acccacatct agctgaaata catagtgaca gcattatcct 780gagagatgac tttgactcct accaccaact gcaattaaat ccaaatatat gggttgaatg 840taacaactgt gagactggag aacagtgtgg cgcgattatg catggcaatg ccgtcacctt 900ctgtgaacca tatggcccac gagaactgat taccacaggc cttaatacaa caacagcttc 960tgtcctccaa ttttccattg ggtcaggttc atgtcgcttt agttattcag accccagcat 1020catcgtgtta tatgccaaga ataactctgc ggactggatt cagctagaga aaattagagc 1080cccttccaat gtcagcacaa tcatccatat cctctacctt cctgaggacg ccaaagggga 1140gaatgtccaa tttcagtgga agcaggaaaa tcttcgtgta ggtgaagtgt atgaagcctg 1200ctgggcctta gataacatct tgatcatcaa ttcagctcac agacaagtcg ttttagaaga 1260tagtctcgac ccagtggaca caggcaactg gcttttcttc ccaggagcta cagttaagca 1320tagctgtcag tcagatggga actccattta tttccatgga aatgaaggca gcgagttcaa 1380ttttgccacc accagggatg tagatctttc cacagaagat attcaagagc aatggtcaga 1440agaatttgag agccagccta caggatggga tgtcttggga gctgtcattg gtacagaatg 1500tggaacgata gaatcaggct tatcaatggt cttcctcaaa gatggagaga ggaaattatg 1560cactccatcc atggacacta ccggttatgg gaacctgagg ttttactttg tgatgggagg 1620aatttgtgac cctggaaatt ctcatgaaaa tgacataatc ctgtatgcaa aaattgaagg 1680aagaaaagag catataacac tggataccct ttcctattcc tcatataagg ttccgtcttt 1740ggtttctgtg gtcatcaatc ctgaacttca gactcctgct accaaatttt gtctcaggca 1800aaagaaccat caaggacata ataggaatgt ctgggctgta gactttttcc atgtcttgcc 1860tgttctccct tctacaatgt ctcacatgat acagttttcc atcaatctgg gatgtggaac 1920gcatcagcct ggtaacagtg tcagcttgga attttctacc aaccatgggc gctcctggtc 1980cctccttcac actgaatgct tacctgagat ctgtgctgga ccccacctcc cccacagcac 2040tgtctactcc tctgaaaact acagtgggtg gaaccgaata acaattcccc ttcctaacgc 2100agcactaacc cggaacacca ggattcgctg gagacaaaca ggaccaatcc ttggaaacat 2160gtgggcaatt gataatgttt atattggccc gtcatgtctc aaattctgtt ctggcagagg 2220acagtgcact agacatggtt gcaagtgtga ccctggattt tctggcccag cttgtgagat 2280ggcatcccag acattcccaa tgtttatttc tgaaagcttt ggcagttcca ggctctcctc 2340ttaccataac ttttactcta tccgtggtgc tgaagtcagc tttggttgtg gtgtcttggc 2400cagtggtaag gccctggttt tcaacaaaga agggcggcgt cagctaatta catctttcct 2460tgacagctca caatccaggt ttctccagtt cacactgaga ctggggagca aatctgttct 2520gagcacgtgc agagcccctg atcagcctgg tgaaggagtt ttgctgcatt attcttatga 2580taatgggata acttggaaac tcctggagca ttattcatat ctcagctatc atgagcccag 2640aataatctcc gtagaactac caggtgatgc aaagcagttt ggaattcagt tcagatggtg 2700gcaaccgtat cattcttccc agagagaaga tgtatgggct attgatgaga ttatcatgac 2760atctgtgctt ttcaacagca ttagtcttga ctttaccaat cttgtggagg tcactcagtc 2820tctgggattc taccttggaa atgttcagcc atactgtggc cacgactgga ccctttgttt 2880tacaggagat tctaaacttg cctcaagtat gcgctatgtg gaaacacaat caatgcagat 2940aggagcatcc tatatgattc agttcagttt ggtgatggga tgtggccaga aatacacccc 3000acacatggac aaccaggtga agctggagta ctcaaccaac cacggcctta cctggcacct 3060cgtccaagaa gaatgccttc caagtatgcc aagttgtcag gaatttacat cagcaagtat 3120ttaccatgcc agtgagttta cacagtggag gagagtcata gtgcttcttc cccagaaaac 3180ttggtccagt gctacccgtt tccgctggag ccagagctat tacacagctc aagacgagtg 3240ggctttggac agcatttaca ttgggcagca gtgccccaac atgtgcagtg ggcatggctc 3300atgcgatcat ggcatatgca ggtgtgacca ggggtaccaa ggcactgaat gccacccaga 3360agctgccctt ccgtccacaa ttatgtcaga ttttgagaac cagaatggct gggagtctga 3420ctggcaagaa gttattgggg gagaaattgt aaaaccagaa caagggtgtg gtgtcatctc 3480ttctggatca tctctgtact tcagcaaggc tgggaaaaga cagctggtga gttgggacct 3540ggatacttct tgggtggact ttgtccagtt ctacatccag ataggcggag agagtgcttc 3600atgcaacaag cctgacagca gagaggaggg cgtcctcctt cagtacagca acaatggggg 3660catccagtgg cacctgctag cagagatgta cttttcagac ttcagcaaac ccagatttgt 3720ctatctggag cttccagctg ctgccaagac cccttgcacc aggttccgct ggtggcagcc 3780cgtgttctca ggggaggact atgaccagtg ggcagtcgat gacatcatca ttctgtccga 3840gaagcagaag cagatcatcc cagttatcaa tccaacttta cctcagaact tttatgagaa 3900gccagctttt gattacccta tgaatcagat gagtgtgtgg ttgatgttgg ctaatgaagg 3960aatggttaaa aatgaaacct tctgtgctgc cacaccatca gcaatgatat ttggaaaatc 4020agatggagat cgatttgcag taactcgaga tttgaccctg aaacctggat atgtgctaca 4080gttcaagcta aacataggtt gtgccaatca attcagcagt actgctccag ttcttcttca 4140gtactctcat gatgctggta tgtcctggtt tctggtgaaa gaaggctgtt acccggcttc 4200tgcaggcaaa ggatgcgaag gaaactccag agaactaagt gagcccacca tgtatcacac 4260aggggacttt gaagaatgga caagaatcac cattgttatt ccaaggtctc ttgcatccag 4320caagaccaga ttccgatgga tccaggagag cagctcacag aaaaacgtgc ctccatttgg 4380tttagatgga gtgtacatat ccgagccttg tcccagttac tgcagtggcc atggggactg 4440catttcagga gtgtgtttct gtgacctggg atatactgct gcacaaggaa cctgtgtgtc 4500aaatgtcccc aatcacaatg agatgttcga taggtttgag gggaagctca gccctctgtg 4560gtacaagata acaggtgccc aggttggaac tggctgtgga acacttaacg atggcaaatc 4620tctctacttc aatggccctg ggaaaaggga agcccggacg gtccctctgg acaccaggaa 4680tatcagactt gttcaatttt atatacaaat tggaagcaaa acttcaggca ttacctgcat 4740caaaccaaga actagaaatg aagggcttat tgttcagtat tcaaatgaca atgggatact 4800ctggcatttg cttcgagagt tggacttcat gtccttcctg gaaccacaga tcatttccat 4860tgacctgcca caggacgcga agacacctgc aacggcattt cgatggtggc aaccgcaaca 4920tgggaagcat tcagcccagt gggctttgga tgatgttctt ataggaatga atgacagctc 4980tcaaactgga tttcaagaca aatttgatgg ctctatagat ttgcaagcca actggtatcg 5040aatccaagga ggtcaagttg atattgactg tctctctatg gatactgctc tgatattcac 5100tgaaaacata ggaaaacctc gttatgctga gacctgggat tttcatgtgt cagcatctac 5160ctttttgcag tttgaaatga gcatgggctg tagcaagccc ttcagcaact cccacagtgt 5220acagctccag tattctctga acaatggcaa ggactggcat cttgtcaccg aagagtgtgt 5280tcctccaacc attggctgtc tgcattacac ggaaagttca atttacacct cggaaagatt 5340ccagaattgg aagcggatca ctgtctacct tccactctcc accatttctc ccaggacccg 5400gttcagatgg attcaggcca actacactgt gggggctgat tcctgggcga ttgataatgt 5460tgtactggcc tcagggtgcc cttggatgtg ctcaggacga gggatttgtg atgctggacg 5520ctgtgtgtgt gaccggggct ttggtggacc ctattgtgtt cctgttgttc ctctgccctc 5580gattcttaaa gacgatttca atgggaattt acatcctgac ctttggcctg aagtgtatgg 5640tgcagagagg gggaatctga atggtgaaac catcaaatct ggaacatctc taatttttaa 5700aggggaagga ctaaggatgc ttatttcaag agatctagat tgtacaaata caatgtatgt 5760ccagttttca cttagattta tagcaaaaag taccccagag agatctcact ctattctgtt 5820acaattctcc atcagtggag gaatcacttg gcacctgatg gatgaatttt actttcctca 5880aacaacgaat atacttttca tcaatgttcc cttgccatac actgcccaaa ccaatgctac 5940aagattcaga ctctggcaac cttataataa cggtaagaaa gaagaaatct ggattgttga 6000tgacttcatt atcgatggaa ataatgtaaa caaccctgtg atgctcttgg atacatttga 6060ttttgggccc agagaagaca attggttttt ctatcctggt ggtaacatcg gtctttattg 6120tccatattct tcaaaggggg cacctgaaga agattcagct atggtgtttg tttcaaatga 6180agttggtgag cattccatta ccacccgtga cctaaatgtg aatgagaaca ccatcataca 6240atttgagatc aacgttggct gttcgactga tagctcatcc gcggatccag tgagactgga 6300attttcaagg gacttcgggg cgacctggca ccttctgctg cccctctgct accacagcag 6360cagccacgtc agctctttat gctccaccga gcaccacccc agcagcacct actacgcagg 6420aaccatgcag ggctggagga gggaggtcgt gcactttggg aagctgcacc tttgtggatc 6480tgtccgtttc agatggtacc agggatttta ccctgccggc tctcagccag tgacatgggc 6540cattgataat gtctacatcg gtccccagtg tgaggagatg tgtaatggac aggggagctg 6600tatcaatgga accaaatgta tatgtgaccc tggctactca ggtccaacct gtaaaataag 6660caccaaaaat cctgattttc tcaaagatga tttcgaaggt cagctagaat ctgatagatt 6720cttattaatg agtggtggga aaccatctcg aaagtgtgga atcctttcta gtggaaacaa 6780cctctttttc aatgaagatg gcttgcgcat gttgatgaca cgagacctgg atttatcaca 6840tgctagattt gtgcagttct tcatgagact gggatgtggt aaaggcgttc ctgaccccag 6900gagtcaaccc gtgctcctac agtattctct caacggtggc ctctcgtgga gtcttcttca 6960ggagttcctt ttcagcaatt ccagcaatgt gggcaggtac attgccctgg agataccctt 7020gaaagcccgt tctggttcta ctcgccttcg ctggtggcaa ccgtctgaga atgggcactt 7080ctacagcccc tgggttatcg atcagattct tattggagga aatatttctg gtaatacggt 7140cttggaagat gatttcacaa cccttgatag taggaaatgg ctgcttcacc caggaggcac 7200caagatgccc gtgtgtggct ctactggtga tgccctggtc ttcattgaaa aggccagcac 7260ccgttacgtg gtcagcacag acgttgccgt gaatgaggat tccttcctac agatagactt 7320cgctgcctcc tgctcagtca cagactcttg ttatgcgatt gaattggaat actcagtaga 7380tcttggattg tcatggcacc cattggtaag ggactgtctg cctaccaatg tggaatgcag 7440tcgctatcat ctgcaacgga tcctggtgtc agacactttc aacaagtgga ctagaatcac 7500tctgcctctc cctccttata ccaggtccca agccactcgt ttccgttggc atcaaccagc 7560tccttttgac aagcagcaga catgggcaat agataatgtc tatatcgggg atggctgcat 7620agacatgtgc agtggccatg ggagatgcat ccagggaaac tgcgtctgtg atgaacagtg 7680gggtggcctg tactgtgatg accccgagac ctctcttcca acccaactca aagacaactt 7740caatcgagct ccatccagtc agaactggct gactgtgaac ggagggaaat tgagtacagt 7800gtgtggagcc gtggcgtcgg gaatggctct ccatttcagt gggggttgta gtcgattatt 7860agtcactgtg gatctaaacc tcactaatgc tgagttcatc caattttact tcatgtatgg 7920gtgcctgatt acaccaaaca accgtaacca aggtgttctc ttggaatatt ctgtcaatgg 7980aggcattacc tggaacctgc tcatggagat tttctatgac cagtacagta agcccggatt 8040tgtgaatatc cttctccctc ctgatgctaa agagattgcc actcgcttcc gctggtggca 8100gccaagacat gacggcctgg atcagaacga ctgggccatt gacaatgtcc tcatctcagg 8160ctctgctgac caaaggaccg ttatgctgga caccttcagc agcgccccag taccccagca 8220cgagcgctcc cctgcagatg ccggccctgt cgggaggatc gcctttgaca tgtttatgga 8280agacaaaact tcagtgaatg agcactggct attccatgat gattgtacag tagaaagatt 8340ctgtgactcc cctgatggtg tgatgctctg tggcagtcat gatggacggg aggtgtatgc 8400agtgacccat gacctgactc ccactgaagg ctggattatg caattcaaga tctcagttgg 8460atgtaaggtg tctgaaaaaa ttgcccagaa tcaaattcat gtgcagtatt ctactgactt 8520cggtgtgagt tggaattatc tggtccctca gtgcttgcct gctgacccaa aatgctctgg 8580aagtgtttct cagccatctg tattctttcc aactaaaggg tggaaaagga tcacctaccc 8640acttcctgaa agcttagtgg gaaatccggt aaggtttagg ttctatcaga agtactcaga 8700catgcagtgg gcaatcgata atttctacct gggccctgga tgcttggaca actgcagggg 8760ccatggagat tgcttaaggg aacagtgcat ctgtgatccg ggatactcag ggccaaactg 8820ctacttgacc cacactctga agactttcct gaaggaacgc tttgacagtg aagaaatcaa 8880acctgactta tggatgtcct tagaaggtgg aagtacttgc actgagtgtg gaattcttgc 8940cgaggacact gcactctatt ttgggggatc cactgtgaga caagcggtta cacaagattt 9000ggatcttcga ggtgcaaagt tcctgcaata ctgggggcgc atcggtagtg agaacaacat 9060gacctcttgc catcgtccca tctgccggaa ggaaggcgtg ctgttggact actctaccga 9120tggaggaatt acctggactt tgctccatga gatggattac cagaaataca tttctgttag 9180acacgactac atacttcttc ctgaagatgc cctcaccaac acaactcgac ttcgctggtg 9240gcagcctttt gtgatcagca atggaattgt ggtctctggg gtggagcgtg ctcagtgggc 9300actggacaac attttgattg gtggagcaga aatcaatccc agccaattgg tggacacttt 9360tgatgatgaa ggcacttccc atgaagaaaa ctggagtttt taccctaatg ctgtaaggac 9420agcaggattt tgtggcaatc catcctttca cctctattgg ccaaataaaa agaaggacaa 9480gactcacaat gctctctcct cccgagaact cattatacag ccaggataca tgatgcagtt 9540taaaattgtg gtgggttgtg aagccacttc ttgtggtgac cttcattccg taatgctgga 9600atacactaag gatgcaagat cggattcctg gcagctcgta cagacccagt gccttccttc 9660ctcttctaac agcattggct gctccccttt ccagttccat gaagccacca tctacaactc 9720tgtcaacagc tcaagctgga aaagaatcac catccagctg cctgaccatg tctcctctag 9780tgcaacacag ttccgctgga tccagaaggg agaagaaact gagaagcaaa gctgggcaat 9840tgaccacgtg tacattggag aggcttgccc caagctctgc agcgggcacg gatactgcac 9900gaccggtgcc atctgcatct gcgacgagag cttccaaggt gatgactgct ctgttttcag 9960tcacgacctt cccagttata ttaaagataa ttttgagtcc gcaagagtca ccgaggcaaa 10020ctgggagacc attcaaggtg gagtcatagg aagtggctgt gggcagctgg ccccctacgc 10080ccatggagac tcactgtact ttaatggctg tcagatcagg caagcagcta ccaagcctct 10140ggatctcact cgagcaagca aaatcatgtt tgttttgcaa attgggagca tgtcgcagac 10200ggacagctgc aacagtgacc tgagtggccc ccacgctgtg gacaaggcgg tgctgctgca 10260atacagcgtc aacaacggga tcacctggca tgtcatcgcc cagcaccagc caaaggactt 10320cacacaagct cagagagtgt cttacaatgt ccccctggag gcacggatga aaggagtctt 10380actgcgctgg tggcaaccac gccacaatgg aacaggtcat gatcaatggg ctttggacca 10440tgtggaggtc gtcctagtaa gcactcgcaa acaaaattac atgatgaatt tttcacgaca 10500acatgggctc agacatttct acaacagaag acgaaggtca cttaggcgat acccatgaag 10560aatcaaaaag tttatttttt ttcttccaac atgtgatgtg ttgctctcca ttcttttaaa 10620tctcgcacta catctgatat caggaaatat ctgtgaagga cttggtgatt acctgaaagc 10680ccttctcaag accgagtgta caccactttc ccacactgtg aactaatgac aagtgactta 10740tttgctcata agtaaatgtc ttcatgttga tgtgtccgtg aaagttgtga tctgttgtaa 10800tatcagttac agtggcagta ttgacaataa gaaacagttt aacagaaaaa tgaaatttaa 10860gcacaaaaaa tttaagagat tttatgttta aaatggcatt tagcacagta tttaacattc 10920ttggtcacaa agctatttaa gtggactgta tttcagctat gtctcatgtt ttatatgatt 10980aaattatcat tgtttgtcct ttatgtattc tcttctacaa tacaacacat tgaaactgta 11040tttacttgtt atgttgtaat attttgctgc tgaatttggg gctacttata ttctgcagaa 11100aattaattga aatacctatt caagaagata gttgtaaaga tattgtatct cctttaatat 11160actccttaaa aatgtatgtt ggtttagcgt tgttttgtgg ataagaaaaa tgcttgaccc 11220tgaaatattt tctactttaa attgtggatg aagaccctat ctcccacaaa taagttccca 11280tttccttgtc taaagatctt tttttaagtg ttctgtggct gatttactaa cagtaactgc 11340cattttttgt ctgtgataac agagtgattt gtaaaacagt ggttgttttt tcattgtgtt 11400ttcttcgtgg attgtttttt ctgcgggtca tattcatacc ttctgatgaa gttgtacaac 11460accagcaaca ttataatggc cctgtagctc tgaatgctat ttgtgtaact gaaaggttgc 11520actctagggt gaaccaagct ataaaagccc atgcttaaat aaaaattatg tccaaaagcc 1158023460PRThomo sapien 2Met Glu Arg Ser Gly Trp Ala Arg Gln Thr Phe Leu Leu Ala Leu Leu1 5 10 15Leu Gly Ala Thr Leu Arg Ala Arg Ala Ala Ala Gly Tyr Tyr Pro Arg 20 25 30Phe Ser Pro Phe Phe Phe Leu Cys Thr His His Gly Glu Leu Glu Gly 35 40 45Asp Gly Glu Gln Gly Glu Val Leu Ile Ser Leu His Ile Ala Gly Asn 50 55 60Pro Thr Tyr Tyr Val Pro Gly Gln Glu Tyr His Val Thr Ile Ser Thr65 70 75 80Ser Thr Phe Phe Asp Gly Leu Leu Val Thr Gly Leu Tyr Thr Ser Thr 85 90 95Ser Val Gln Ala Ser Gln Ser Ile Gly Gly Ser Ser Ala Phe Gly Phe 100 105 110Gly Ile Met Ser Asp His Gln Phe Gly Asn Gln Phe Met Cys Ser Val 115 120 125Val Ala Ser His Val Ser His Leu Pro Thr Thr Asn Leu Ser Phe Ile 130 135 140Trp Ile Ala Pro Pro Ala Gly Thr Gly Cys Val Asn Phe Met Ala Thr145 150 155 160Ala Thr His Arg Gly Gln Val Ile Phe Lys Asp Ala Leu Ala Gln Gln 165 170 175Leu Cys Glu Gln Gly Ala Pro Thr Asp Val Thr Val His Pro His Leu 180 185 190Ala Glu Ile His Ser Asp Ser Ile Ile Leu Arg Asp Asp Phe Asp Ser 195 200 205Tyr His Gln Leu Gln Leu Asn Pro Asn Ile Trp Val Glu Cys Asn Asn 210 215 220Cys Glu Thr Gly Glu Gln Cys Gly Ala Ile Met His Gly Asn Ala Val225 230 235 240Thr Phe Cys Glu Pro Tyr Gly Pro Arg Glu Leu Ile Thr Thr Gly Leu 245 250 255Asn Thr Thr Thr Ala Ser Val Leu Gln Phe Ser Ile Gly Ser Gly Ser 260 265 270Cys Arg Phe Ser Tyr Ser Asp Pro Ser Ile Ile Val Leu Tyr Ala Lys 275 280 285Asn Asn Ser Ala Asp Trp Ile Gln Leu Glu Lys Ile Arg Ala Pro Ser 290 295 300Asn Val Ser Thr Ile Ile His Ile Leu Tyr Leu Pro Glu Asp Ala Lys305 310 315 320Gly Glu Asn Val Gln Phe Gln Trp Lys Gln Glu Asn Leu Arg Val Gly 325 330 335Glu Val Tyr Glu Ala Cys Trp Ala Leu Asp Asn Ile Leu Ile Ile Asn 340 345 350Ser Ala His Arg Gln Val Val Leu Glu Asp Ser Leu Asp Pro Val Asp 355 360 365Thr Gly Asn Trp Leu Phe Phe Pro Gly Ala Thr Val Lys His Ser Cys 370 375 380Gln Ser Asp Gly Asn Ser Ile Tyr Phe His Gly Asn Glu Gly Ser Glu385 390 395 400Phe Asn Phe Ala Thr Thr Arg Asp Val Asp Leu Ser Thr Glu Asp Ile 405 410 415Gln Glu Gln Trp Ser Glu Glu Phe Glu Ser Gln Pro Thr Gly Trp Asp 420 425 430Val Leu Gly Ala Val Ile Gly Thr Glu Cys Gly Thr Ile Glu Ser Gly 435 440 445Leu Ser Met Val Phe Leu Lys Asp Gly Glu Arg Lys Leu Cys Thr Pro 450 455 460Ser Met Asp Thr Thr Gly Tyr Gly Asn Leu Arg Phe Tyr Phe Val Met465 470 475 480Gly Gly Ile Cys Asp Pro Gly Asn Ser His Glu Asn Asp Ile Ile Leu 485 490 495Tyr Ala Lys Ile Glu Gly Arg Lys Glu His Ile Thr Leu Asp Thr Leu 500 505 510Ser Tyr Ser Ser Tyr Lys Val Pro Ser Leu Val Ser Val Val Ile Asn 515 520 525Pro Glu Leu Gln Thr Pro Ala Thr Lys Phe Cys Leu Arg Gln Lys Asn 530 535 540His Gln Gly His Asn Arg Asn Val Trp Ala Val Asp Phe Phe His Val545 550 555 560Leu Pro Val Leu Pro Ser Thr Met Ser His

Met Ile Gln Phe Ser Ile 565 570 575Asn Leu Gly Cys Gly Thr His Gln Pro Gly Asn Ser Val Ser Leu Glu 580 585 590Phe Ser Thr Asn His Gly Arg Ser Trp Ser Leu Leu His Thr Glu Cys 595 600 605Leu Pro Glu Ile Cys Ala Gly Pro His Leu Pro His Ser Thr Val Tyr 610 615 620Ser Ser Glu Asn Tyr Ser Gly Trp Asn Arg Ile Thr Ile Pro Leu Pro625 630 635 640Asn Ala Ala Leu Thr Arg Asn Thr Arg Ile Arg Trp Arg Gln Thr Gly 645 650 655Pro Ile Leu Gly Asn Met Trp Ala Ile Asp Asn Val Tyr Ile Gly Pro 660 665 670Ser Cys Leu Lys Phe Cys Ser Gly Arg Gly Gln Cys Thr Arg His Gly 675 680 685Cys Lys Cys Asp Pro Gly Phe Ser Gly Pro Ala Cys Glu Met Ala Ser 690 695 700Gln Thr Phe Pro Met Phe Ile Ser Glu Ser Phe Gly Ser Ser Arg Leu705 710 715 720Ser Ser Tyr His Asn Phe Tyr Ser Ile Arg Gly Ala Glu Val Ser Phe 725 730 735Gly Cys Gly Val Leu Ala Ser Gly Lys Ala Leu Val Phe Asn Lys Glu 740 745 750Gly Arg Arg Gln Leu Ile Thr Ser Phe Leu Asp Ser Ser Gln Ser Arg 755 760 765Phe Leu Gln Phe Thr Leu Arg Leu Gly Ser Lys Ser Val Leu Ser Thr 770 775 780Cys Arg Ala Pro Asp Gln Pro Gly Glu Gly Val Leu Leu His Tyr Ser785 790 795 800Tyr Asp Asn Gly Ile Thr Trp Lys Leu Leu Glu His Tyr Ser Tyr Leu 805 810 815Ser Tyr His Glu Pro Arg Ile Ile Ser Val Glu Leu Pro Gly Asp Ala 820 825 830Lys Gln Phe Gly Ile Gln Phe Arg Trp Trp Gln Pro Tyr His Ser Ser 835 840 845Gln Arg Glu Asp Val Trp Ala Ile Asp Glu Ile Ile Met Thr Ser Val 850 855 860Leu Phe Asn Ser Ile Ser Leu Asp Phe Thr Asn Leu Val Glu Val Thr865 870 875 880Gln Ser Leu Gly Phe Tyr Leu Gly Asn Val Gln Pro Tyr Cys Gly His 885 890 895Asp Trp Thr Leu Cys Phe Thr Gly Asp Ser Lys Leu Ala Ser Ser Met 900 905 910Arg Tyr Val Glu Thr Gln Ser Met Gln Ile Gly Ala Ser Tyr Met Ile 915 920 925Gln Phe Ser Leu Val Met Gly Cys Gly Gln Lys Tyr Thr Pro His Met 930 935 940Asp Asn Gln Val Lys Leu Glu Tyr Ser Thr Asn His Gly Leu Thr Trp945 950 955 960His Leu Val Gln Glu Glu Cys Leu Pro Ser Met Pro Ser Cys Gln Glu 965 970 975Phe Thr Ser Ala Ser Ile Tyr His Ala Ser Glu Phe Thr Gln Trp Arg 980 985 990Arg Val Ile Val Leu Leu Pro Gln Lys Thr Trp Ser Ser Ala Thr Arg 995 1000 1005Phe Arg Trp Ser Gln Ser Tyr Tyr Thr Ala Gln Asp Glu Trp Ala 1010 1015 1020Leu Asp Ser Ile Tyr Ile Gly Gln Gln Cys Pro Asn Met Cys Ser 1025 1030 1035Gly His Gly Ser Cys Asp His Gly Ile Cys Arg Cys Asp Gln Gly 1040 1045 1050Tyr Gln Gly Thr Glu Cys His Pro Glu Ala Ala Leu Pro Ser Thr 1055 1060 1065Ile Met Ser Asp Phe Glu Asn Gln Asn Gly Trp Glu Ser Asp Trp 1070 1075 1080Gln Glu Val Ile Gly Gly Glu Ile Val Lys Pro Glu Gln Gly Cys 1085 1090 1095Gly Val Ile Ser Ser Gly Ser Ser Leu Tyr Phe Ser Lys Ala Gly 1100 1105 1110Lys Arg Gln Leu Val Ser Trp Asp Leu Asp Thr Ser Trp Val Asp 1115 1120 1125Phe Val Gln Phe Tyr Ile Gln Ile Gly Gly Glu Ser Ala Ser Cys 1130 1135 1140Asn Lys Pro Asp Ser Arg Glu Glu Gly Val Leu Leu Gln Tyr Ser 1145 1150 1155Asn Asn Gly Gly Ile Gln Trp His Leu Leu Ala Glu Met Tyr Phe 1160 1165 1170Ser Asp Phe Ser Lys Pro Arg Phe Val Tyr Leu Glu Leu Pro Ala 1175 1180 1185Ala Ala Lys Thr Pro Cys Thr Arg Phe Arg Trp Trp Gln Pro Val 1190 1195 1200Phe Ser Gly Glu Asp Tyr Asp Gln Trp Ala Val Asp Asp Ile Ile 1205 1210 1215Ile Leu Ser Glu Lys Gln Lys Gln Ile Ile Pro Val Ile Asn Pro 1220 1225 1230Thr Leu Pro Gln Asn Phe Tyr Glu Lys Pro Ala Phe Asp Tyr Pro 1235 1240 1245Met Asn Gln Met Ser Val Trp Leu Met Leu Ala Asn Glu Gly Met 1250 1255 1260Val Lys Asn Glu Thr Phe Cys Ala Ala Thr Pro Ser Ala Met Ile 1265 1270 1275Phe Gly Lys Ser Asp Gly Asp Arg Phe Ala Val Thr Arg Asp Leu 1280 1285 1290Thr Leu Lys Pro Gly Tyr Val Leu Gln Phe Lys Leu Asn Ile Gly 1295 1300 1305Cys Ala Asn Gln Phe Ser Ser Thr Ala Pro Val Leu Leu Gln Tyr 1310 1315 1320Ser His Asp Ala Gly Met Ser Trp Phe Leu Val Lys Glu Gly Cys 1325 1330 1335Tyr Pro Ala Ser Ala Gly Lys Gly Cys Glu Gly Asn Ser Arg Glu 1340 1345 1350Leu Ser Glu Pro Thr Met Tyr His Thr Gly Asp Phe Glu Glu Trp 1355 1360 1365Thr Arg Ile Thr Ile Val Ile Pro Arg Ser Leu Ala Ser Ser Lys 1370 1375 1380Thr Arg Phe Arg Trp Ile Gln Glu Ser Ser Ser Gln Lys Asn Val 1385 1390 1395Pro Pro Phe Gly Leu Asp Gly Val Tyr Ile Ser Glu Pro Cys Pro 1400 1405 1410Ser Tyr Cys Ser Gly His Gly Asp Cys Ile Ser Gly Val Cys Phe 1415 1420 1425Cys Asp Leu Gly Tyr Thr Ala Ala Gln Gly Thr Cys Val Ser Asn 1430 1435 1440Val Pro Asn His Asn Glu Met Phe Asp Arg Phe Glu Gly Lys Leu 1445 1450 1455Ser Pro Leu Trp Tyr Lys Ile Thr Gly Ala Gln Val Gly Thr Gly 1460 1465 1470Cys Gly Thr Leu Asn Asp Gly Lys Ser Leu Tyr Phe Asn Gly Pro 1475 1480 1485Gly Lys Arg Glu Ala Arg Thr Val Pro Leu Asp Thr Arg Asn Ile 1490 1495 1500Arg Leu Val Gln Phe Tyr Ile Gln Ile Gly Ser Lys Thr Ser Gly 1505 1510 1515Ile Thr Cys Ile Lys Pro Arg Thr Arg Asn Glu Gly Leu Ile Val 1520 1525 1530Gln Tyr Ser Asn Asp Asn Gly Ile Leu Trp His Leu Leu Arg Glu 1535 1540 1545Leu Asp Phe Met Ser Phe Leu Glu Pro Gln Ile Ile Ser Ile Asp 1550 1555 1560Leu Pro Gln Asp Ala Lys Thr Pro Ala Thr Ala Phe Arg Trp Trp 1565 1570 1575Gln Pro Gln His Gly Lys His Ser Ala Gln Trp Ala Leu Asp Asp 1580 1585 1590Val Leu Ile Gly Met Asn Asp Ser Ser Gln Thr Gly Phe Gln Asp 1595 1600 1605Lys Phe Asp Gly Ser Ile Asp Leu Gln Ala Asn Trp Tyr Arg Ile 1610 1615 1620Gln Gly Gly Gln Val Asp Ile Asp Cys Leu Ser Met Asp Thr Ala 1625 1630 1635Leu Ile Phe Thr Glu Asn Ile Gly Lys Pro Arg Tyr Ala Glu Thr 1640 1645 1650Trp Asp Phe His Val Ser Ala Ser Thr Phe Leu Gln Phe Glu Met 1655 1660 1665Ser Met Gly Cys Ser Lys Pro Phe Ser Asn Ser His Ser Val Gln 1670 1675 1680Leu Gln Tyr Ser Leu Asn Asn Gly Lys Asp Trp His Leu Val Thr 1685 1690 1695Glu Glu Cys Val Pro Pro Thr Ile Gly Cys Leu His Tyr Thr Glu 1700 1705 1710Ser Ser Ile Tyr Thr Ser Glu Arg Phe Gln Asn Trp Lys Arg Ile 1715 1720 1725Thr Val Tyr Leu Pro Leu Ser Thr Ile Ser Pro Arg Thr Arg Phe 1730 1735 1740Arg Trp Ile Gln Ala Asn Tyr Thr Val Gly Ala Asp Ser Trp Ala 1745 1750 1755Ile Asp Asn Val Val Leu Ala Ser Gly Cys Pro Trp Met Cys Ser 1760 1765 1770Gly Arg Gly Ile Cys Asp Ala Gly Arg Cys Val Cys Asp Arg Gly 1775 1780 1785Phe Gly Gly Pro Tyr Cys Val Pro Val Val Pro Leu Pro Ser Ile 1790 1795 1800Leu Lys Asp Asp Phe Asn Gly Asn Leu His Pro Asp Leu Trp Pro 1805 1810 1815Glu Val Tyr Gly Ala Glu Arg Gly Asn Leu Asn Gly Glu Thr Ile 1820 1825 1830Lys Ser Gly Thr Ser Leu Ile Phe Lys Gly Glu Gly Leu Arg Met 1835 1840 1845Leu Ile Ser Arg Asp Leu Asp Cys Thr Asn Thr Met Tyr Val Gln 1850 1855 1860Phe Ser Leu Arg Phe Ile Ala Lys Ser Thr Pro Glu Arg Ser His 1865 1870 1875Ser Ile Leu Leu Gln Phe Ser Ile Ser Gly Gly Ile Thr Trp His 1880 1885 1890Leu Met Asp Glu Phe Tyr Phe Pro Gln Thr Thr Asn Ile Leu Phe 1895 1900 1905Ile Asn Val Pro Leu Pro Tyr Thr Ala Gln Thr Asn Ala Thr Arg 1910 1915 1920Phe Arg Leu Trp Gln Pro Tyr Asn Asn Gly Lys Lys Glu Glu Ile 1925 1930 1935Trp Ile Val Asp Asp Phe Ile Ile Asp Gly Asn Asn Val Asn Asn 1940 1945 1950Pro Val Met Leu Leu Asp Thr Phe Asp Phe Gly Pro Arg Glu Asp 1955 1960 1965Asn Trp Phe Phe Tyr Pro Gly Gly Asn Ile Gly Leu Tyr Cys Pro 1970 1975 1980Tyr Ser Ser Lys Gly Ala Pro Glu Glu Asp Ser Ala Met Val Phe 1985 1990 1995Val Ser Asn Glu Val Gly Glu His Ser Ile Thr Thr Arg Asp Leu 2000 2005 2010Asn Val Asn Glu Asn Thr Ile Ile Gln Phe Glu Ile Asn Val Gly 2015 2020 2025Cys Ser Thr Asp Ser Ser Ser Ala Asp Pro Val Arg Leu Glu Phe 2030 2035 2040Ser Arg Asp Phe Gly Ala Thr Trp His Leu Leu Leu Pro Leu Cys 2045 2050 2055Tyr His Ser Ser Ser His Val Ser Ser Leu Cys Ser Thr Glu His 2060 2065 2070His Pro Ser Ser Thr Tyr Tyr Ala Gly Thr Met Gln Gly Trp Arg 2075 2080 2085Arg Glu Val Val His Phe Gly Lys Leu His Leu Cys Gly Ser Val 2090 2095 2100Arg Phe Arg Trp Tyr Gln Gly Phe Tyr Pro Ala Gly Ser Gln Pro 2105 2110 2115Val Thr Trp Ala Ile Asp Asn Val Tyr Ile Gly Pro Gln Cys Glu 2120 2125 2130Glu Met Cys Asn Gly Gln Gly Ser Cys Ile Asn Gly Thr Lys Cys 2135 2140 2145Ile Cys Asp Pro Gly Tyr Ser Gly Pro Thr Cys Lys Ile Ser Thr 2150 2155 2160Lys Asn Pro Asp Phe Leu Lys Asp Asp Phe Glu Gly Gln Leu Glu 2165 2170 2175Ser Asp Arg Phe Leu Leu Met Ser Gly Gly Lys Pro Ser Arg Lys 2180 2185 2190Cys Gly Ile Leu Ser Ser Gly Asn Asn Leu Phe Phe Asn Glu Asp 2195 2200 2205Gly Leu Arg Met Leu Met Thr Arg Asp Leu Asp Leu Ser His Ala 2210 2215 2220Arg Phe Val Gln Phe Phe Met Arg Leu Gly Cys Gly Lys Gly Val 2225 2230 2235Pro Asp Pro Arg Ser Gln Pro Val Leu Leu Gln Tyr Ser Leu Asn 2240 2245 2250Gly Gly Leu Ser Trp Ser Leu Leu Gln Glu Phe Leu Phe Ser Asn 2255 2260 2265Ser Ser Asn Val Gly Arg Tyr Ile Ala Leu Glu Ile Pro Leu Lys 2270 2275 2280Ala Arg Ser Gly Ser Thr Arg Leu Arg Trp Trp Gln Pro Ser Glu 2285 2290 2295Asn Gly His Phe Tyr Ser Pro Trp Val Ile Asp Gln Ile Leu Ile 2300 2305 2310Gly Gly Asn Ile Ser Gly Asn Thr Val Leu Glu Asp Asp Phe Thr 2315 2320 2325Thr Leu Asp Ser Arg Lys Trp Leu Leu His Pro Gly Gly Thr Lys 2330 2335 2340Met Pro Val Cys Gly Ser Thr Gly Asp Ala Leu Val Phe Ile Glu 2345 2350 2355Lys Ala Ser Thr Arg Tyr Val Val Ser Thr Asp Val Ala Val Asn 2360 2365 2370Glu Asp Ser Phe Leu Gln Ile Asp Phe Ala Ala Ser Cys Ser Val 2375 2380 2385Thr Asp Ser Cys Tyr Ala Ile Glu Leu Glu Tyr Ser Val Asp Leu 2390 2395 2400Gly Leu Ser Trp His Pro Leu Val Arg Asp Cys Leu Pro Thr Asn 2405 2410 2415Val Glu Cys Ser Arg Tyr His Leu Gln Arg Ile Leu Val Ser Asp 2420 2425 2430Thr Phe Asn Lys Trp Thr Arg Ile Thr Leu Pro Leu Pro Pro Tyr 2435 2440 2445Thr Arg Ser Gln Ala Thr Arg Phe Arg Trp His Gln Pro Ala Pro 2450 2455 2460Phe Asp Lys Gln Gln Thr Trp Ala Ile Asp Asn Val Tyr Ile Gly 2465 2470 2475Asp Gly Cys Ile Asp Met Cys Ser Gly His Gly Arg Cys Ile Gln 2480 2485 2490Gly Asn Cys Val Cys Asp Glu Gln Trp Gly Gly Leu Tyr Cys Asp 2495 2500 2505Asp Pro Glu Thr Ser Leu Pro Thr Gln Leu Lys Asp Asn Phe Asn 2510 2515 2520Arg Ala Pro Ser Ser Gln Asn Trp Leu Thr Val Asn Gly Gly Lys 2525 2530 2535Leu Ser Thr Val Cys Gly Ala Val Ala Ser Gly Met Ala Leu His 2540 2545 2550Phe Ser Gly Gly Cys Ser Arg Leu Leu Val Thr Val Asp Leu Asn 2555 2560 2565Leu Thr Asn Ala Glu Phe Ile Gln Phe Tyr Phe Met Tyr Gly Cys 2570 2575 2580Leu Ile Thr Pro Asn Asn Arg Asn Gln Gly Val Leu Leu Glu Tyr 2585 2590 2595Ser Val Asn Gly Gly Ile Thr Trp Asn Leu Leu Met Glu Ile Phe 2600 2605 2610Tyr Asp Gln Tyr Ser Lys Pro Gly Phe Val Asn Ile Leu Leu Pro 2615 2620 2625Pro Asp Ala Lys Glu Ile Ala Thr Arg Phe Arg Trp Trp Gln Pro 2630 2635 2640Arg His Asp Gly Leu Asp Gln Asn Asp Trp Ala Ile Asp Asn Val 2645 2650 2655Leu Ile Ser Gly Ser Ala Asp Gln Arg Thr Val Met Leu Asp Thr 2660 2665 2670Phe Ser Ser Ala Pro Val Pro Gln His Glu Arg Ser Pro Ala Asp 2675 2680 2685Ala Gly Pro Val Gly Arg Ile Ala Phe Asp Met Phe Met Glu Asp 2690 2695 2700Lys Thr Ser Val Asn Glu His Trp Leu Phe His Asp Asp Cys Thr 2705 2710 2715Val Glu Arg Phe Cys Asp Ser Pro Asp Gly Val Met Leu Cys Gly 2720 2725 2730Ser His Asp Gly Arg Glu Val Tyr Ala Val Thr His Asp Leu Thr 2735 2740 2745Pro Thr Glu Gly Trp Ile Met Gln Phe Lys Ile Ser Val Gly Cys 2750 2755 2760Lys Val Ser Glu Lys Ile Ala Gln Asn Gln Ile His Val Gln Tyr 2765 2770 2775Ser Thr Asp Phe Gly Val Ser Trp Asn Tyr Leu Val Pro Gln Cys 2780 2785 2790Leu Pro Ala Asp Pro Lys Cys Ser Gly Ser Val Ser Gln Pro Ser 2795 2800 2805Val Phe Phe Pro Thr Lys Gly Trp Lys Arg Ile Thr Tyr Pro Leu 2810 2815 2820Pro Glu Ser Leu Val Gly Asn Pro Val Arg Phe Arg Phe Tyr Gln 2825 2830 2835Lys Tyr Ser Asp Met Gln Trp Ala Ile Asp Asn Phe Tyr Leu Gly 2840 2845 2850Pro Gly Cys Leu Asp Asn Cys Arg Gly His Gly Asp Cys Leu Arg 2855 2860 2865Glu Gln Cys Ile Cys Asp Pro Gly Tyr Ser Gly Pro Asn Cys Tyr 2870 2875 2880Leu Thr His Thr Leu Lys Thr Phe Leu Lys Glu Arg Phe Asp Ser 2885 2890 2895Glu Glu Ile Lys Pro Asp Leu Trp Met Ser Leu Glu Gly Gly Ser 2900 2905 2910Thr Cys Thr Glu Cys Gly Ile Leu Ala Glu Asp Thr Ala Leu Tyr 2915 2920 2925Phe Gly Gly Ser Thr Val Arg Gln Ala Val Thr Gln Asp Leu Asp 2930 2935 2940Leu Arg Gly Ala Lys Phe Leu Gln Tyr Trp Gly Arg Ile Gly Ser 2945 2950 2955Glu Asn Asn Met Thr Ser Cys His Arg Pro Ile Cys Arg Lys Glu 2960 2965 2970Gly Val Leu Leu Asp Tyr Ser Thr Asp Gly Gly Ile Thr Trp Thr 2975 2980 2985Leu Leu His Glu Met Asp Tyr Gln Lys Tyr Ile Ser Val Arg His 2990 2995 3000Asp Tyr Ile Leu Leu Pro

Glu Asp Ala Leu Thr Asn Thr Thr Arg 3005 3010 3015Leu Arg Trp Trp Gln Pro Phe Val Ile Ser Asn Gly Ile Val Val 3020 3025 3030Ser Gly Val Glu Arg Ala Gln Trp Ala Leu Asp Asn Ile Leu Ile 3035 3040 3045Gly Gly Ala Glu Ile Asn Pro Ser Gln Leu Val Asp Thr Phe Asp 3050 3055 3060Asp Glu Gly Thr Ser His Glu Glu Asn Trp Ser Phe Tyr Pro Asn 3065 3070 3075Ala Val Arg Thr Ala Gly Phe Cys Gly Asn Pro Ser Phe His Leu 3080 3085 3090Tyr Trp Pro Asn Lys Lys Lys Asp Lys Thr His Asn Ala Leu Ser 3095 3100 3105Ser Arg Glu Leu Ile Ile Gln Pro Gly Tyr Met Met Gln Phe Lys 3110 3115 3120Ile Val Val Gly Cys Glu Ala Thr Ser Cys Gly Asp Leu His Ser 3125 3130 3135Val Met Leu Glu Tyr Thr Lys Asp Ala Arg Ser Asp Ser Trp Gln 3140 3145 3150Leu Val Gln Thr Gln Cys Leu Pro Ser Ser Ser Asn Ser Ile Gly 3155 3160 3165Cys Ser Pro Phe Gln Phe His Glu Ala Thr Ile Tyr Asn Ser Val 3170 3175 3180Asn Ser Ser Ser Trp Lys Arg Ile Thr Ile Gln Leu Pro Asp His 3185 3190 3195Val Ser Ser Ser Ala Thr Gln Phe Arg Trp Ile Gln Lys Gly Glu 3200 3205 3210Glu Thr Glu Lys Gln Ser Trp Ala Ile Asp His Val Tyr Ile Gly 3215 3220 3225Glu Ala Cys Pro Lys Leu Cys Ser Gly His Gly Tyr Cys Thr Thr 3230 3235 3240Gly Ala Ile Cys Ile Cys Asp Glu Ser Phe Gln Gly Asp Asp Cys 3245 3250 3255Ser Val Phe Ser His Asp Leu Pro Ser Tyr Ile Lys Asp Asn Phe 3260 3265 3270Glu Ser Ala Arg Val Thr Glu Ala Asn Trp Glu Thr Ile Gln Gly 3275 3280 3285Gly Val Ile Gly Ser Gly Cys Gly Gln Leu Ala Pro Tyr Ala His 3290 3295 3300Gly Asp Ser Leu Tyr Phe Asn Gly Cys Gln Ile Arg Gln Ala Ala 3305 3310 3315Thr Lys Pro Leu Asp Leu Thr Arg Ala Ser Lys Ile Met Phe Val 3320 3325 3330Leu Gln Ile Gly Ser Met Ser Gln Thr Asp Ser Cys Asn Ser Asp 3335 3340 3345Leu Ser Gly Pro His Ala Val Asp Lys Ala Val Leu Leu Gln Tyr 3350 3355 3360Ser Val Asn Asn Gly Ile Thr Trp His Val Ile Ala Gln His Gln 3365 3370 3375Pro Lys Asp Phe Thr Gln Ala Gln Arg Val Ser Tyr Asn Val Pro 3380 3385 3390Leu Glu Ala Arg Met Lys Gly Val Leu Leu Arg Trp Trp Gln Pro 3395 3400 3405Arg His Asn Gly Thr Gly His Asp Gln Trp Ala Leu Asp His Val 3410 3415 3420Glu Val Val Leu Val Ser Thr Arg Lys Gln Asn Tyr Met Met Asn 3425 3430 3435Phe Ser Arg Gln His Gly Leu Arg His Phe Tyr Asn Arg Arg Arg 3440 3445 3450Arg Ser Leu Arg Arg Tyr Pro 3455 3460318DNAArtificial SequenceManganese superoxide dismutase (MnSOD) forward primer 3gctgcaccac agcaagca 18419DNAArtificial SequenceManganese superoxide dismutase (MnSOD) reverse primer 4tcggtggcgt tgagattgt 19517DNAArtificial SequenceReelin forward primer 5gccacgccac aatggaa 17619DNAArtificial SequenceReelin reverse primer 6cgacctccac atggtccaa 19722DNAArtificial SequenceGlutamate decarboxylase 1 (Brain, 67 kDa; Gad-1/67) forward primer 7cgcttggctt tggaaccgac aa 22823DNAArtificial SequenceGlutamate decarboxylase 1 (Brain, 67 kDa; Gad-1/67) reverse primer 8gaatgctccg taaacagtcg tgc 23922DNAArtificial SequenceNeurexin 1 (Nrxn1) forward primer 9accgtgcctt agcaatcctt gc 221022DNAArtificial SequenceNeurexin 1 (Nrxn1) reverse primer 10gtcgtagctc aaaaccgttg cc 221122DNAArtificial SequenceNeuroligin 2 (Nlgn 2) forward primer 11cgatgtcatg ctcagcgcag ta 221222DNAArtificial SequenceNeuroligin 2 (Nlgn 2) reverse primer 12ccacactacc tcttcaaagc gg 221321DNAArtificial SequenceBeta-Actin forward primer 13cagcagatgt ggatcagcaa g 211418DNAArtificial SequenceBeta-Actin reverse primer 14gcatttgcgg tggacgat 181523DNAArtificial Sequencemmu-miR-137-5p primer sequence 15acgggtattc ttgggtggat aat 231623DNAArtificial Sequencemmu-miR-137-3p primer sequence 16ttattgctta agaatacgcg tag 231723DNAArtificial Sequencemmu-miR-181a primer sequence 17aacattcaac gctgtcggtg agt 231821DNAArtificial Sequencemmu-miR-128-1-5p primer sequence 18cggggccgta gcactgtctg a 211921DNAArtificial Sequencemmu-miR-128-3p primer sequence 19tcacagtgaa ccggtctctt t 212023DNAArtificial Sequencemmu-miR-138 primer sequence 20agctggtgtt gtgaatcagg ccg 232123DNAArtificial Sequencemmu-miR-200c primer sequence 21taatactgcc gggtaatgat gga 232222DNAArtificial Sequencesmall nucleolar RNA 202 (snoRNA-202) internal forward primer 22agtacttttg aacccttttc ca 222311673DNAMus musculus 23ggggcgtcgc gtgcacaccg gcggcggcgg cgctcggagg cggacgacgc gctctcggcg 60cccgcggccc cggttccccc cgcgctctcg ctccggcggc ccaaagtaac ttcgggagcc 120tcggtctccc gctaacttcc ccccgcgggc tcggttgccc ggacccgctc ggctcgagcc 180cgccgccggc tcgccttccc cgcacgcggc tcctccgtgc cggtgcctcc gaaagtggat 240gagagagcgc gcggggcgcg cggcggcacg gagcgcggcg gcatggagcg cggctgctgg 300gcgccgcggg ctctcgtcct ggccgtgctg ctgctgctgg cgacgctgag ggcgcgcgcg 360gccaccggct actacccgcg cttctcgcct ttctttttcc tgtgcaccca ccacggggag 420ctggaagggg atggggagca gggcgaggtg ctcatttccc tgcacattgc gggcaacccc 480acctactacg taccgggaca ggaataccat gttacaattt caacaagcac cttctttgat 540ggcttgctgg tgacgggact ctatacctcg acaagcatcc agtcttctca gagcattgga 600ggctccagcg cctttggatt cgggatcatg tccgaccacc agtttggtaa ccagtttatg 660tgcagtgtgg tggcctctca tgtgagtcac ctgcctacaa ccaacctcag ctttgtctgg 720attgccccac cagctggcac aggctgtgtg aatttcatgg ctactgcaac acataggggc 780caggtgattt tcaaagacgc actggcccag cagctgtgtg aacaaggagc tcccacagag 840gccactgctt actcgcacct tgctgaaata cacagtgaca gtgtgatcct acgagatgac 900tttgactcct accagcaact ggaattgaac cccaacatat gggttgaatg cagcaactgt 960gagatgggag agcagtgtgg caccatcatg catggcaatg ctgtcacctt ctgtgagccg 1020tacggccctc gagagctgac caccacatgc ctgaacacaa caacagcatc tgtcctccag 1080ttttccattg ggtcaggatc atgtcgattt agttactctg accccagcat cactgtgtca 1140tacgccaaga acaataccgc tgattggatt cagctggaga aaattagagc cccttccaat 1200gtgagcacag tcatccacat cctgtacctc cccgaggaag ccaaagggga gagcgtgcag 1260ttccagtgga aacaggacag cctgcgagtg ggtgaggtgt atgaggcctg ctgggccctg 1320gataacatcc tggtcatcaa ttcagcccac agagaagtcg ttctggagga caacctcgac 1380ccggtcgaca cgggcaactg gctcttcttc cctggagcaa cggtcaagca tagctgtcag 1440tcagatggga actccattta tttccatgga aatgaaggca gcgagttcaa ttttgccacc 1500acccgggatg tagatctttc tacagaggat attcaagagc agtggtcaga agaatttgag 1560agccagccca caggatggga tatcttggga gcagtagttg gtgcagactg tggaaccgta 1620gaatcaggac tatcactggt gttcctcaaa gatggagaga ggaagctttg caccccctac 1680atggatacaa ctggttatgg caacctgagg ttctacttcg ttatgggagg aatctgtgac 1740cctggagtct ctcatgaaaa cgatatcatc ttatatgcaa agattgaagg aagaaaagaa 1800cacattgcac tggacactct tacctattct tcctataagg ttccgtcttt ggtttctgtg 1860gtcatcaacc ctgaacttca gacacctgcc accaaatttt gtctcaggca aaagagccac 1920caagggtata atcggaatgt ctgggctgtg gacttcttcc atgtgctgcc cgttctccct 1980tcaacaatgt ctcacatgat ccagttttct attaatttgg gatgcggcac acaccagcct 2040gggaacagcg tcagcttgga gttttctact aaccatggac ggtcctggtc cctactccac 2100actgagtgct tgccggagat ctgtgcaggc ccccacctcc cccacagcac tgtctactcc 2160tcagaaaact acagcgggtg gaaccgaatc acgattcctc tccctaatgc agcactcacc 2220cgagacacca ggattcgctg gagacaaaca ggcccaatcc tgggaaatat gtgggcaatt 2280gataatgttt atataggtcc ttcgtgtctc aaattctgtt ctggcagagg acaatgcact 2340cggcatggct gcaagtgtga cccaggattt tctggcccag cttgtgagat ggcatctcag 2400acattcccaa tgtttatttc ggaaagcttt ggcagtgcca gactttcctc ttaccataac 2460ttttactcta tccgtggtgc tgaagtcagc tttggttgtg gtgtcttagc cagtggtaag 2520gctctggttt tcaacaaaga tgggaggcgg cagctaatca cgtcctttct ggacagctcg 2580cagtccaggt ttcttcagtt tacactgagg ctggggagca agtctgtgct gagcacgtgc 2640agagcccctg accagccggg ggagggagtc ctgctgcact attcatatga caacgggata 2700acatggaaac tcctggagca ctattcctac gtcaactacc acgagcccag aataatctct 2760gtagagctac cggatgatgc aagacagttt ggaatccagt tcagatggtg gcagccttac 2820cattcttccc aaggagaaga cgtgtgggcc attgatgaga ttgtcatgac ctcagtcctg 2880ttcaacagca tcagtctcga ctttaccaat cttgtggaag tcactcaatc cctgggattc 2940taccttggca atgttcaacc atactgtggc catgactgga cgctttgttt tacgggagat 3000tctaaacttg cctcaagcat gcgctatgtg gaaacacagt ccatgcagat cggagcatcc 3060tatatgattc agttcagcct agtgatggga tgtggccaga aatacactcc tcacatggac 3120aaccaggtga agctggagta ctcagccaac cacggcctta catggcacct tgtacaagaa 3180gaatgccttc ccagtatgcc aagctgccag gaatttacat ctgccagcat ttaccatgcc 3240agcgagttca cacagtggag aagagtcact gttgttcttc cccagaaaac atggtccggt 3300gccacccgct tccgttggag tcagagctat tacacagccc aggatgagtg ggctttagac 3360aacatttaca ttgggcagca gtgccccaac atgtgcagtg ggcatggctc atgtgaccat 3420ggcgtgtgca ggtgtgacca gggataccag ggcactgaat gccacccaga agctgcactt 3480ccttccacga ttatgtcaga ttttgagaac ccgagcagtt gggaatcaga ctggcaggaa 3540gttattgggg gagaagttgt aaagcctgag caaggctgtg gagtcgtgtc ttctggatct 3600tctctgtact tcagcaaggc tgggaagagg cagctggtga gctgggacct ggacacatcc 3660tgggtggact ttgtccagtt ctacatccag ataggaggag agagtgctgc atgcaacaag 3720cctgacagca gagaggaggg cattctgctc cagtatagca acaacggggg catccagtgg 3780cacctgctgg cagagatgta cttctcagac ttcagcaaac ccagatttgt ctacctggag 3840ctcccagctg ctgggaagac cccttgtacc aggttccgct ggtggaagcc tgtgttctcg 3900ggggaggact atgaccagtg ggccgttgat gatatcatca ttctgtcaga gaagcagaag 3960caggttatcc cagttgtcaa cccaactttg ccccagaact tctatgagaa gccagctttc 4020gattacccta tgaaccaaat gagtgtgtgg ctaatgttgg ccaatgaagg catggctaaa 4080aacgacagct tctgtgcgac cacgccgtca gccatggtgt ttggaaagtc agatggagac 4140cggtttgcag taactcgaga tctgaccctg aaacctggat atgtgctgca gttcaagcta 4200aacataggct gcaccagcca gttcagcagc actgccccgg ttctcctgca gtattcacat 4260gatgccggca tgtcgtggtt tctgttgaag gaaggatgct tcccagcgtc agcagccaaa 4320ggatgtgaag ggaactccag ggaattgagt gagcctactg tctattatac tggggacttc 4380gaagaatgga ctagaatcac cattgccatt ccaaggtccc ttgcatccag caagaccaga 4440ttccgatgga tccaagagag cagctctcag aagaatgtgc ccccgtttgg cttagatggg 4500gtgtacatat ctgagccttg tcccagttac tgcagtggcc atggagactg catctcgggg 4560gtgtgttttt gtgacctggg gtacacagct gcacaaggaa cctgtgtgtc aaacacccct 4620aaccacagtg agatgttcga caggtttgag gggaagctaa gcccactgtg gtacaaaatc 4680accgggggtc aggttggcac gggctgtggc accctcaatg acggcaggtc cctctacttt 4740aatggccttg ggaaaaggga agccaggaca gtcccactgg acaccaggaa tatcagtctt 4800gttcagtttt atatacaaat tggaagtaaa acatcaggga ttacgtacat caccccacgg 4860gctagatatg aggggcttgt tgttcagtat tccaatgata atgggatact ttggcatttg 4920ctgagagagt tggatttcat gtcattcctg gagccacaga tcatttccat tgacctgccc 4980cgggaagcaa agacacctgc cacagctttc cggtggtggc agccgcagca tgggaagcat 5040tcggcccagt gggctttggg tgatgtcctt ataggagtga atgacagctc tcaaactgga 5100tttcaagata aattggatgg ctccatagac ttgcaagcca actggtatcg aatccaggga 5160ggccaagttg atatcgactg cctctctatg gacactgccc ttatattcac tgaaaacata 5220ggaaaccctc gctatgctga gacctgggac ttccatgtgt cagagtcaag cttcttacag 5280tgggaaatga acatgggctg cagcaagcct ttcagtggtg cccacggcat acagctccag 5340tactctctga acaacggcaa ggactggcag cttgtcaccg aagagtgtgt ccctccaacc 5400attgggtgcg tgcactacac agagagttca acttacacat cagaaagatt ccagaactgg 5460aggcgggtca cggtctacct gccactcgcc accaattctc ccaggactcg gttcagatgg 5520attcagacca actatactgt tggagcagat tcctgggcta ttgataatgt catcctggcc 5580tcgggctgtc cttggatgtg ctcaggacga gggatctgtg attcggggcg ctgtgtgtgt 5640gaccggggct tcggtggacc cttctgtgtt cctgttgttc ctcttccctc cattctaaaa 5700gatgatttca atgggaactt acatcctgac ctttggcctg aagtgtacgg ggcagagagg 5760ggcaatctga atggcgaaac catcaaatcc ggaacatgtc tgatctttaa aggggaggga 5820ctaagaatgc ttatttccag agatctagat tgtaccaata ctatgtatgt ccagttctct 5880ctccgattta tagcgaaagg taccccagag aggtctcact ccatccttct acagttctct 5940gtcagtggag gagtcacctg gcacctgatg gatgaattct acttccctca aacgaccagc 6000atacttttca tcaatgttcc cttaccatac ggtgcccaaa ccaacgctac aagattcaga 6060ctctggcaac cgtacaataa tggtaagaaa gaagaaatct ggatcattga tgactttatt 6120attgatggaa acaatttgaa caaccccgtg ctgctgctgg acacgttcga ctttgggccc 6180agggaagaca attggttttt ctatccgggt ggtaatatcg gactttactg cccgtattct 6240tcaaagggag ctcctgagga ggattcggcc atggtgtttg tttcaaacga agttggagaa 6300cactccatta ccacacgaga cctaagtgtg aacgagaaca ccatcattca atttgagatc 6360aatgttggct gctccactga tagttcttct gctgatccgg tcagactgga attctcaagg 6420gactttggag ccacctggca cctgctgctg cctctctgct accacagcag cagcctcgtc 6480agctccttat gctccactga gcatcacccg agcagcacct actacgcggg gaccacccag 6540ggctggcggc gggaggtcgt gcacttcgga aagctgcacc tttgtggatc tgtgcgtttc 6600cgttggtacc agggatttta tcctgctggc tctcagccgg tcacatgggc cattgacaat 6660gtctacattg gtccccagtg tgaagagatg tgctatgggc acgggagctg catcaatgga 6720accaagtgta tatgtgaccc gggctactct gggccaacct gtaaaataag caccaaaaat 6780cctgattttc tcaaagacga ctttgaaggt caactggaat ccgatcgatt cttactgatg 6840agcggtggga agccgtctcg taagtgtggc atcctttcca gtgggaacaa cctcttcttc 6900aatgaggacg gcttgcgcat gctagtaaca cgggacctgg atttatcaca tgcaaggttt 6960gtgcagttct tcatgagact gggatgtggt aaaggtgttc cagaccccag gagccagccc 7020gtgcttctgc agtactccct caatggcggc ctctcctgga gtcttcttca agagttcctc 7080ttcagcaact ccagcaatgt gggcaggtac attgccctgg aaatgcccct gaaagcccgt 7140tctggttcga cacgcctccg ctggtggcag ccatctgaaa atgggcactt ctatagcccc 7200tgggtgatcg accagattct tattggagga aatatctctg gtaatacagt cttagaagat 7260gatttctcaa ctctggacag cagaaagtgg ctgcttcacc caggaggcac caagatgcct 7320gtgtgtggct ccacaggcga tgccctggtc tttattgaaa aggccagcac ccgttacgtg 7380gtcacgacag acatcgctgt gaatgaggac tcattcctac agatagactt tgctgcctcc 7440tgctcagtca cagactcctg ctatgctatt gaactggagt actcggtgga tctcggtctg 7500tcgtggcacc cgctggtgag ggactgcctg cctaccaatg ttgagtgtag tcgttaccac 7560ctgcagcgga tcctggtgtc agatactttc aacaagtgga ccagaatcac tctgcccctg 7620ccttcctaca ccaggtctca agccactcgt ttccgctggc atcagccagc gccttttgac 7680aagcagcaga cctgggcaat agataatgtc tatattgggg atggttgcct agacatgtgc 7740agtggccacg ggagatgcgt ccagggaagc tgtgtctgtg atgaacagtg gggaggcctg 7800tactgtgatg agcctgagac ctcccttccc acccagctca aagacaactt caaccgagcc 7860ccctccaacc agaactggct gactgtgagc ggtgggaagc tgagtacagt gtgtggggct 7920gtggcttccg gcctggctct ccatttcagt gggggctgca gccgattgtt agtcactgtg 7980gatctgaacc tcaccaatgc tgagtttatc cagttttact ttatgtatgg atgcctcatt 8040acgccgagca accgtaacca gggagtcctg ctggagtact ctgtcaatgg aggcatcacc 8100tggaacttgc tgatggagat tttctatgac cagtacagca aacctggatt tgtgaatatc 8160cttctccctc ctgatgctaa agagattgcc actcgcttcc gatggtggca gccacgacat 8220gatggccttg accagaatga ctgggccatt gacaatgtcc tcatctcggg ctctgcggac 8280cagaggacag tcatgctgga cacctttagc agcgccccag taccacagca tgagcgctcc 8340cccgcagacg ctggccctgt tggaagaatt gcttttgaaa tgttcttaga agacaaaact 8400tcagtgaatg agaattggct cttccatgat gactgtacag tggaaagatt ctgtgactcg 8460ccagatggtg tcatgctctg tggcagccat gatggacgag aggtgtatgc agtgactcat 8520gacctgacgc ccactgagaa ctggatcatg cagttcaaga tctctgttgg atgcaaagtg 8580cctgaaaaaa ttgcccagaa tcaaattcac gtgcagtttt ctactgactt tggcgtgagc 8640tggagttatt tagtccctca gtgcttaccc gccgacccaa agtgttctgg aagcgtttct 8700caaccgtctg tgttcttccc aactgaaggg tggaaaagga tcacctaccc gcttcctgaa 8760agcttaacgg ggaatcctgt aagatttagg ttctaccaaa agtactcaga tgtgcagtgg 8820gcaattgaca atttctacct tggccctgga tgtttggaca actgtggagg ccacggagac 8880tgcctaaagg aacagtgtat ctgtgaccca ggctactcag ggccaaactg ctacttaact 8940cacagcctga agactttcct gaaggagcgc tttgacagtg aggagatcaa gcctgactta 9000tggatgtcct tggaaggcgg aagcacttgt acagagtgcg gggtcctcgc cgagaacact 9060gcactctatt ttgggggatc cactgtgaga caagctatta ctcaagactt agatctcaga 9120ggtgcaaaat tcctgcagta ctggggacgt atcggcagtg agaacaacat gacatcttgc 9180catcggcctg tctgccggaa ggaaggcgtg ctgctggact tctctacgga tggaggaatc 9240acttggacct tgcttcacga gatggatttc cagaaataca tttctgtgag gcacgactac 9300atcctcctgc ctgagggggc cctcaccaac acaactcgac ttcgctggtg gcagcctttt 9360gtcatcagca atgggctcgt ggtttccggg gtggagcgtg cgcagtgggc actggacaac 9420attctgattg gtggagcaga aatcaatcca agccaactgg tggacacttt cgatgacgaa 9480ggctcctccc atgaagaaaa ctggagtttt taccctaatg cagtaaggac agcaggattc 9540tgtggcaacc catccttcca cctctactgg ccaaataaaa agaaggacaa gacccacaat 9600gcactctcct cccgagagct cattatacag ccaggataca tgatgcaatt taaaattgtg 9660gtgggttgtg aagccacttc atgtggtgac cttcattccg tgatgctgga gtacaccaag 9720gatgcaaggt ccgattcctg gcagctcgtg cagacccagt gcctaccttc ctcttccaat 9780agcattggct gctccccgtt

ccagttccat gaagccacca tttataatgc tgtcaacagc 9840tcaagctgga agaggatcac catccagctc ccagaccacg tctcgtcaag tgccacacag 9900ttccgctgga tccagaaggg agaagaaacc gagaagcaaa gctgggccat cgaccacgtg 9960tacatcggag aggcttgtcc caagctctgc agcgggcatg gctactgcac cacaggggcc 10020gtctgcatct gcgatgaaag cttccaaggt gacgactgct ctgtcttcag tcacgagctt 10080cctagttaca ttaaagataa ttttgaatca gcaagagtca ctgaagccaa ctgggaaacc 10140atccagggtg gagtgatcgg aagtggctgt gggcagctgg cgccctatgc ccatggagat 10200tcgctctact ttaatggttg tcagataagg caagctgcca ccaagccact ggacctcact 10260cgagcaagca aaattatgtt tgtcttgcaa attgggagcc cagcccagac agacagttgc 10320aacagcgacc tcagcggccc ccacaccgtg gacaaagcag tactgctgca gtacagtgtc 10380aacaatggca tcacctggca cgtcatcgct cagcaccagc cgaaggactt cacacaagct 10440cagcgggtgt cttacaacgt ccccctggaa gctcggatga aaggagttct actgcgctgg 10500tggcagccac gccacaatgg aacaggtcat gatcaatggg ctttggacca tgtggaggtc 10560gtcctagtaa gcactcgcaa acaaaattac atgatgaatt tttcacggca acatgggctc 10620aggcacttct acaacagaag acgaaggtcg cttaggcgat acccatgaag aatccaagtt 10680tatttccctt tccagcgtac aatgtgtccc ttcctggttt tttgaaacac ctctcactgc 10740atctgatatc aggaaacaaa gatgaaggac ttggcgaaca gaaagccctt cgagatcttg 10800tgtaccccac cttcccacac tgtgagctaa tgatgtgtgg tttctctgca cataagtaaa 10860tgtcttcacg tcagtgcgtc cgtggaaatt gtgatctgtt gtaatatcag ttacagtggc 10920agtattgaga ataagaaata gtttaacagg aaaaaacgtt taagcacaaa catttttaag 10980atcttatgtt ttaagtggca ttttagcaca gtatttaaca ttgttggtca ccgagctatt 11040taagtagact gtatttcagc tctgtctctt gtttaatatg aataagttct cgtcgtttgt 11100cctttatgta ttcttctcta ccgtataaca cactgaaact gtatctactt gctgtgttgc 11160aatattttgc tgctggactt tgacctactt gtattatgca gaaagttaat gcagatacct 11220attcaagatg ataactgtaa agacactgct gtctccttaa tatgctcctt aacacgtatg 11280ttgatgtagc atcattttgt ggataggaaa aaaaatgttt gaccttcaga tattttctac 11340ttaaaaaatt gtggatgaac gccctatctc cctcccacag tgagtcccca ttaccttgtc 11400taaaacaatt ttttaatgtg ttctgtggcc gttttactga cagtaactgc catttcgtgt 11460ctgtggtaac aaagtgactt gtaaaatggt ggatgtttcc ctcactgtgt tctcttcgtg 11520ggttgtttcc ttgtgggtca tagtcatacc ttctgatgag gtggagccaa caccagcaaa 11580gtatgatggc cctgtagcct ctgactagtc ctgaaacaga aggctgcact ctaggctgaa 11640ccatgctaaa agcccatgct taaataaaaa atg 11673243461PRTMus musculus 24Met Glu Arg Gly Cys Trp Ala Pro Arg Ala Leu Val Leu Ala Val Leu1 5 10 15Leu Leu Leu Ala Thr Leu Arg Ala Arg Ala Ala Thr Gly Tyr Tyr Pro 20 25 30Arg Phe Ser Pro Phe Phe Phe Leu Cys Thr His His Gly Glu Leu Glu 35 40 45Gly Asp Gly Glu Gln Gly Glu Val Leu Ile Ser Leu His Ile Ala Gly 50 55 60Asn Pro Thr Tyr Tyr Val Pro Gly Gln Glu Tyr His Val Thr Ile Ser65 70 75 80Thr Ser Thr Phe Phe Asp Gly Leu Leu Val Thr Gly Leu Tyr Thr Ser 85 90 95Thr Ser Ile Gln Ser Ser Gln Ser Ile Gly Gly Ser Ser Ala Phe Gly 100 105 110Phe Gly Ile Met Ser Asp His Gln Phe Gly Asn Gln Phe Met Cys Ser 115 120 125Val Val Ala Ser His Val Ser His Leu Pro Thr Thr Asn Leu Ser Phe 130 135 140Val Trp Ile Ala Pro Pro Ala Gly Thr Gly Cys Val Asn Phe Met Ala145 150 155 160Thr Ala Thr His Arg Gly Gln Val Ile Phe Lys Asp Ala Leu Ala Gln 165 170 175Gln Leu Cys Glu Gln Gly Ala Pro Thr Glu Ala Thr Ala Tyr Ser His 180 185 190Leu Ala Glu Ile His Ser Asp Ser Val Ile Leu Arg Asp Asp Phe Asp 195 200 205Ser Tyr Gln Gln Leu Glu Leu Asn Pro Asn Ile Trp Val Glu Cys Ser 210 215 220Asn Cys Glu Met Gly Glu Gln Cys Gly Thr Ile Met His Gly Asn Ala225 230 235 240Val Thr Phe Cys Glu Pro Tyr Gly Pro Arg Glu Leu Thr Thr Thr Cys 245 250 255Leu Asn Thr Thr Thr Ala Ser Val Leu Gln Phe Ser Ile Gly Ser Gly 260 265 270Ser Cys Arg Phe Ser Tyr Ser Asp Pro Ser Ile Thr Val Ser Tyr Ala 275 280 285Lys Asn Asn Thr Ala Asp Trp Ile Gln Leu Glu Lys Ile Arg Ala Pro 290 295 300Ser Asn Val Ser Thr Val Ile His Ile Leu Tyr Leu Pro Glu Glu Ala305 310 315 320Lys Gly Glu Ser Val Gln Phe Gln Trp Lys Gln Asp Ser Leu Arg Val 325 330 335Gly Glu Val Tyr Glu Ala Cys Trp Ala Leu Asp Asn Ile Leu Val Ile 340 345 350Asn Ser Ala His Arg Glu Val Val Leu Glu Asp Asn Leu Asp Pro Val 355 360 365Asp Thr Gly Asn Trp Leu Phe Phe Pro Gly Ala Thr Val Lys His Ser 370 375 380Cys Gln Ser Asp Gly Asn Ser Ile Tyr Phe His Gly Asn Glu Gly Ser385 390 395 400Glu Phe Asn Phe Ala Thr Thr Arg Asp Val Asp Leu Ser Thr Glu Asp 405 410 415Ile Gln Glu Gln Trp Ser Glu Glu Phe Glu Ser Gln Pro Thr Gly Trp 420 425 430Asp Ile Leu Gly Ala Val Val Gly Ala Asp Cys Gly Thr Val Glu Ser 435 440 445Gly Leu Ser Leu Val Phe Leu Lys Asp Gly Glu Arg Lys Leu Cys Thr 450 455 460Pro Tyr Met Asp Thr Thr Gly Tyr Gly Asn Leu Arg Phe Tyr Phe Val465 470 475 480Met Gly Gly Ile Cys Asp Pro Gly Val Ser His Glu Asn Asp Ile Ile 485 490 495Leu Tyr Ala Lys Ile Glu Gly Arg Lys Glu His Ile Ala Leu Asp Thr 500 505 510Leu Thr Tyr Ser Ser Tyr Lys Val Pro Ser Leu Val Ser Val Val Ile 515 520 525Asn Pro Glu Leu Gln Thr Pro Ala Thr Lys Phe Cys Leu Arg Gln Lys 530 535 540Ser His Gln Gly Tyr Asn Arg Asn Val Trp Ala Val Asp Phe Phe His545 550 555 560Val Leu Pro Val Leu Pro Ser Thr Met Ser His Met Ile Gln Phe Ser 565 570 575Ile Asn Leu Gly Cys Gly Thr His Gln Pro Gly Asn Ser Val Ser Leu 580 585 590Glu Phe Ser Thr Asn His Gly Arg Ser Trp Ser Leu Leu His Thr Glu 595 600 605Cys Leu Pro Glu Ile Cys Ala Gly Pro His Leu Pro His Ser Thr Val 610 615 620Tyr Ser Ser Glu Asn Tyr Ser Gly Trp Asn Arg Ile Thr Ile Pro Leu625 630 635 640Pro Asn Ala Ala Leu Thr Arg Asp Thr Arg Ile Arg Trp Arg Gln Thr 645 650 655Gly Pro Ile Leu Gly Asn Met Trp Ala Ile Asp Asn Val Tyr Ile Gly 660 665 670Pro Ser Cys Leu Lys Phe Cys Ser Gly Arg Gly Gln Cys Thr Arg His 675 680 685Gly Cys Lys Cys Asp Pro Gly Phe Ser Gly Pro Ala Cys Glu Met Ala 690 695 700Ser Gln Thr Phe Pro Met Phe Ile Ser Glu Ser Phe Gly Ser Ala Arg705 710 715 720Leu Ser Ser Tyr His Asn Phe Tyr Ser Ile Arg Gly Ala Glu Val Ser 725 730 735Phe Gly Cys Gly Val Leu Ala Ser Gly Lys Ala Leu Val Phe Asn Lys 740 745 750Asp Gly Arg Arg Gln Leu Ile Thr Ser Phe Leu Asp Ser Ser Gln Ser 755 760 765Arg Phe Leu Gln Phe Thr Leu Arg Leu Gly Ser Lys Ser Val Leu Ser 770 775 780Thr Cys Arg Ala Pro Asp Gln Pro Gly Glu Gly Val Leu Leu His Tyr785 790 795 800Ser Tyr Asp Asn Gly Ile Thr Trp Lys Leu Leu Glu His Tyr Ser Tyr 805 810 815Val Asn Tyr His Glu Pro Arg Ile Ile Ser Val Glu Leu Pro Asp Asp 820 825 830Ala Arg Gln Phe Gly Ile Gln Phe Arg Trp Trp Gln Pro Tyr His Ser 835 840 845Ser Gln Gly Glu Asp Val Trp Ala Ile Asp Glu Ile Val Met Thr Ser 850 855 860Val Leu Phe Asn Ser Ile Ser Leu Asp Phe Thr Asn Leu Val Glu Val865 870 875 880Thr Gln Ser Leu Gly Phe Tyr Leu Gly Asn Val Gln Pro Tyr Cys Gly 885 890 895His Asp Trp Thr Leu Cys Phe Thr Gly Asp Ser Lys Leu Ala Ser Ser 900 905 910Met Arg Tyr Val Glu Thr Gln Ser Met Gln Ile Gly Ala Ser Tyr Met 915 920 925Ile Gln Phe Ser Leu Val Met Gly Cys Gly Gln Lys Tyr Thr Pro His 930 935 940Met Asp Asn Gln Val Lys Leu Glu Tyr Ser Ala Asn His Gly Leu Thr945 950 955 960Trp His Leu Val Gln Glu Glu Cys Leu Pro Ser Met Pro Ser Cys Gln 965 970 975Glu Phe Thr Ser Ala Ser Ile Tyr His Ala Ser Glu Phe Thr Gln Trp 980 985 990Arg Arg Val Thr Val Val Leu Pro Gln Lys Thr Trp Ser Gly Ala Thr 995 1000 1005Arg Phe Arg Trp Ser Gln Ser Tyr Tyr Thr Ala Gln Asp Glu Trp 1010 1015 1020Ala Leu Asp Asn Ile Tyr Ile Gly Gln Gln Cys Pro Asn Met Cys 1025 1030 1035Ser Gly His Gly Ser Cys Asp His Gly Val Cys Arg Cys Asp Gln 1040 1045 1050Gly Tyr Gln Gly Thr Glu Cys His Pro Glu Ala Ala Leu Pro Ser 1055 1060 1065Thr Ile Met Ser Asp Phe Glu Asn Pro Ser Ser Trp Glu Ser Asp 1070 1075 1080Trp Gln Glu Val Ile Gly Gly Glu Val Val Lys Pro Glu Gln Gly 1085 1090 1095Cys Gly Val Val Ser Ser Gly Ser Ser Leu Tyr Phe Ser Lys Ala 1100 1105 1110Gly Lys Arg Gln Leu Val Ser Trp Asp Leu Asp Thr Ser Trp Val 1115 1120 1125Asp Phe Val Gln Phe Tyr Ile Gln Ile Gly Gly Glu Ser Ala Ala 1130 1135 1140Cys Asn Lys Pro Asp Ser Arg Glu Glu Gly Ile Leu Leu Gln Tyr 1145 1150 1155Ser Asn Asn Gly Gly Ile Gln Trp His Leu Leu Ala Glu Met Tyr 1160 1165 1170Phe Ser Asp Phe Ser Lys Pro Arg Phe Val Tyr Leu Glu Leu Pro 1175 1180 1185Ala Ala Gly Lys Thr Pro Cys Thr Arg Phe Arg Trp Trp Lys Pro 1190 1195 1200Val Phe Ser Gly Glu Asp Tyr Asp Gln Trp Ala Val Asp Asp Ile 1205 1210 1215Ile Ile Leu Ser Glu Lys Gln Lys Gln Val Ile Pro Val Val Asn 1220 1225 1230Pro Thr Leu Pro Gln Asn Phe Tyr Glu Lys Pro Ala Phe Asp Tyr 1235 1240 1245Pro Met Asn Gln Met Ser Val Trp Leu Met Leu Ala Asn Glu Gly 1250 1255 1260Met Ala Lys Asn Asp Ser Phe Cys Ala Thr Thr Pro Ser Ala Met 1265 1270 1275Val Phe Gly Lys Ser Asp Gly Asp Arg Phe Ala Val Thr Arg Asp 1280 1285 1290Leu Thr Leu Lys Pro Gly Tyr Val Leu Gln Phe Lys Leu Asn Ile 1295 1300 1305Gly Cys Thr Ser Gln Phe Ser Ser Thr Ala Pro Val Leu Leu Gln 1310 1315 1320Tyr Ser His Asp Ala Gly Met Ser Trp Phe Leu Leu Lys Glu Gly 1325 1330 1335Cys Phe Pro Ala Ser Ala Ala Lys Gly Cys Glu Gly Asn Ser Arg 1340 1345 1350Glu Leu Ser Glu Pro Thr Val Tyr Tyr Thr Gly Asp Phe Glu Glu 1355 1360 1365Trp Thr Arg Ile Thr Ile Ala Ile Pro Arg Ser Leu Ala Ser Ser 1370 1375 1380Lys Thr Arg Phe Arg Trp Ile Gln Glu Ser Ser Ser Gln Lys Asn 1385 1390 1395Val Pro Pro Phe Gly Leu Asp Gly Val Tyr Ile Ser Glu Pro Cys 1400 1405 1410Pro Ser Tyr Cys Ser Gly His Gly Asp Cys Ile Ser Gly Val Cys 1415 1420 1425Phe Cys Asp Leu Gly Tyr Thr Ala Ala Gln Gly Thr Cys Val Ser 1430 1435 1440Asn Thr Pro Asn His Ser Glu Met Phe Asp Arg Phe Glu Gly Lys 1445 1450 1455Leu Ser Pro Leu Trp Tyr Lys Ile Thr Gly Gly Gln Val Gly Thr 1460 1465 1470Gly Cys Gly Thr Leu Asn Asp Gly Arg Ser Leu Tyr Phe Asn Gly 1475 1480 1485Leu Gly Lys Arg Glu Ala Arg Thr Val Pro Leu Asp Thr Arg Asn 1490 1495 1500Ile Ser Leu Val Gln Phe Tyr Ile Gln Ile Gly Ser Lys Thr Ser 1505 1510 1515Gly Ile Thr Tyr Ile Thr Pro Arg Ala Arg Tyr Glu Gly Leu Val 1520 1525 1530Val Gln Tyr Ser Asn Asp Asn Gly Ile Leu Trp His Leu Leu Arg 1535 1540 1545Glu Leu Asp Phe Met Ser Phe Leu Glu Pro Gln Ile Ile Ser Ile 1550 1555 1560Asp Leu Pro Arg Glu Ala Lys Thr Pro Ala Thr Ala Phe Arg Trp 1565 1570 1575Trp Gln Pro Gln His Gly Lys His Ser Ala Gln Trp Ala Leu Gly 1580 1585 1590Asp Val Leu Ile Gly Val Asn Asp Ser Ser Gln Thr Gly Phe Gln 1595 1600 1605Asp Lys Leu Asp Gly Ser Ile Asp Leu Gln Ala Asn Trp Tyr Arg 1610 1615 1620Ile Gln Gly Gly Gln Val Asp Ile Asp Cys Leu Ser Met Asp Thr 1625 1630 1635Ala Leu Ile Phe Thr Glu Asn Ile Gly Asn Pro Arg Tyr Ala Glu 1640 1645 1650Thr Trp Asp Phe His Val Ser Glu Ser Ser Phe Leu Gln Trp Glu 1655 1660 1665Met Asn Met Gly Cys Ser Lys Pro Phe Ser Gly Ala His Gly Ile 1670 1675 1680Gln Leu Gln Tyr Ser Leu Asn Asn Gly Lys Asp Trp Gln Leu Val 1685 1690 1695Thr Glu Glu Cys Val Pro Pro Thr Ile Gly Cys Val His Tyr Thr 1700 1705 1710Glu Ser Ser Thr Tyr Thr Ser Glu Arg Phe Gln Asn Trp Arg Arg 1715 1720 1725Val Thr Val Tyr Leu Pro Leu Ala Thr Asn Ser Pro Arg Thr Arg 1730 1735 1740Phe Arg Trp Ile Gln Thr Asn Tyr Thr Val Gly Ala Asp Ser Trp 1745 1750 1755Ala Ile Asp Asn Val Ile Leu Ala Ser Gly Cys Pro Trp Met Cys 1760 1765 1770Ser Gly Arg Gly Ile Cys Asp Ser Gly Arg Cys Val Cys Asp Arg 1775 1780 1785Gly Phe Gly Gly Pro Phe Cys Val Pro Val Val Pro Leu Pro Ser 1790 1795 1800Ile Leu Lys Asp Asp Phe Asn Gly Asn Leu His Pro Asp Leu Trp 1805 1810 1815Pro Glu Val Tyr Gly Ala Glu Arg Gly Asn Leu Asn Gly Glu Thr 1820 1825 1830Ile Lys Ser Gly Thr Cys Leu Ile Phe Lys Gly Glu Gly Leu Arg 1835 1840 1845Met Leu Ile Ser Arg Asp Leu Asp Cys Thr Asn Thr Met Tyr Val 1850 1855 1860Gln Phe Ser Leu Arg Phe Ile Ala Lys Gly Thr Pro Glu Arg Ser 1865 1870 1875His Ser Ile Leu Leu Gln Phe Ser Val Ser Gly Gly Val Thr Trp 1880 1885 1890His Leu Met Asp Glu Phe Tyr Phe Pro Gln Thr Thr Ser Ile Leu 1895 1900 1905Phe Ile Asn Val Pro Leu Pro Tyr Gly Ala Gln Thr Asn Ala Thr 1910 1915 1920Arg Phe Arg Leu Trp Gln Pro Tyr Asn Asn Gly Lys Lys Glu Glu 1925 1930 1935Ile Trp Ile Ile Asp Asp Phe Ile Ile Asp Gly Asn Asn Leu Asn 1940 1945 1950Asn Pro Val Leu Leu Leu Asp Thr Phe Asp Phe Gly Pro Arg Glu 1955 1960 1965Asp Asn Trp Phe Phe Tyr Pro Gly Gly Asn Ile Gly Leu Tyr Cys 1970 1975 1980Pro Tyr Ser Ser Lys Gly Ala Pro Glu Glu Asp Ser Ala Met Val 1985 1990 1995Phe Val Ser Asn Glu Val Gly Glu His Ser Ile Thr Thr Arg Asp 2000 2005 2010Leu Ser Val Asn Glu Asn Thr Ile Ile Gln Phe Glu Ile Asn Val 2015 2020 2025Gly Cys Ser Thr Asp Ser Ser Ser Ala Asp Pro Val Arg Leu Glu 2030 2035 2040Phe Ser Arg Asp Phe Gly Ala Thr Trp His Leu Leu Leu Pro Leu 2045 2050 2055Cys Tyr His Ser Ser Ser Leu Val Ser Ser Leu Cys Ser Thr Glu 2060 2065 2070His His Pro Ser Ser Thr Tyr Tyr Ala Gly Thr Thr Gln Gly Trp 2075 2080 2085Arg Arg Glu Val Val His Phe Gly Lys Leu His Leu Cys Gly Ser 2090 2095 2100Val Arg Phe Arg Trp Tyr Gln Gly Phe Tyr Pro Ala Gly Ser Gln 2105 2110 2115Pro Val Thr Trp Ala Ile Asp Asn Val Tyr Ile Gly Pro Gln Cys 2120 2125 2130Glu Glu Met Cys Tyr Gly His Gly Ser Cys Ile Asn Gly Thr

Lys 2135 2140 2145Cys Ile Cys Asp Pro Gly Tyr Ser Gly Pro Thr Cys Lys Ile Ser 2150 2155 2160Thr Lys Asn Pro Asp Phe Leu Lys Asp Asp Phe Glu Gly Gln Leu 2165 2170 2175Glu Ser Asp Arg Phe Leu Leu Met Ser Gly Gly Lys Pro Ser Arg 2180 2185 2190Lys Cys Gly Ile Leu Ser Ser Gly Asn Asn Leu Phe Phe Asn Glu 2195 2200 2205Asp Gly Leu Arg Met Leu Val Thr Arg Asp Leu Asp Leu Ser His 2210 2215 2220Ala Arg Phe Val Gln Phe Phe Met Arg Leu Gly Cys Gly Lys Gly 2225 2230 2235Val Pro Asp Pro Arg Ser Gln Pro Val Leu Leu Gln Tyr Ser Leu 2240 2245 2250Asn Gly Gly Leu Ser Trp Ser Leu Leu Gln Glu Phe Leu Phe Ser 2255 2260 2265Asn Ser Ser Asn Val Gly Arg Tyr Ile Ala Leu Glu Met Pro Leu 2270 2275 2280Lys Ala Arg Ser Gly Ser Thr Arg Leu Arg Trp Trp Gln Pro Ser 2285 2290 2295Glu Asn Gly His Phe Tyr Ser Pro Trp Val Ile Asp Gln Ile Leu 2300 2305 2310Ile Gly Gly Asn Ile Ser Gly Asn Thr Val Leu Glu Asp Asp Phe 2315 2320 2325Ser Thr Leu Asp Ser Arg Lys Trp Leu Leu His Pro Gly Gly Thr 2330 2335 2340Lys Met Pro Val Cys Gly Ser Thr Gly Asp Ala Leu Val Phe Ile 2345 2350 2355Glu Lys Ala Ser Thr Arg Tyr Val Val Thr Thr Asp Ile Ala Val 2360 2365 2370Asn Glu Asp Ser Phe Leu Gln Ile Asp Phe Ala Ala Ser Cys Ser 2375 2380 2385Val Thr Asp Ser Cys Tyr Ala Ile Glu Leu Glu Tyr Ser Val Asp 2390 2395 2400Leu Gly Leu Ser Trp His Pro Leu Val Arg Asp Cys Leu Pro Thr 2405 2410 2415Asn Val Glu Cys Ser Arg Tyr His Leu Gln Arg Ile Leu Val Ser 2420 2425 2430Asp Thr Phe Asn Lys Trp Thr Arg Ile Thr Leu Pro Leu Pro Ser 2435 2440 2445Tyr Thr Arg Ser Gln Ala Thr Arg Phe Arg Trp His Gln Pro Ala 2450 2455 2460Pro Phe Asp Lys Gln Gln Thr Trp Ala Ile Asp Asn Val Tyr Ile 2465 2470 2475Gly Asp Gly Cys Leu Asp Met Cys Ser Gly His Gly Arg Cys Val 2480 2485 2490Gln Gly Ser Cys Val Cys Asp Glu Gln Trp Gly Gly Leu Tyr Cys 2495 2500 2505Asp Glu Pro Glu Thr Ser Leu Pro Thr Gln Leu Lys Asp Asn Phe 2510 2515 2520Asn Arg Ala Pro Ser Asn Gln Asn Trp Leu Thr Val Ser Gly Gly 2525 2530 2535Lys Leu Ser Thr Val Cys Gly Ala Val Ala Ser Gly Leu Ala Leu 2540 2545 2550His Phe Ser Gly Gly Cys Ser Arg Leu Leu Val Thr Val Asp Leu 2555 2560 2565Asn Leu Thr Asn Ala Glu Phe Ile Gln Phe Tyr Phe Met Tyr Gly 2570 2575 2580Cys Leu Ile Thr Pro Ser Asn Arg Asn Gln Gly Val Leu Leu Glu 2585 2590 2595Tyr Ser Val Asn Gly Gly Ile Thr Trp Asn Leu Leu Met Glu Ile 2600 2605 2610Phe Tyr Asp Gln Tyr Ser Lys Pro Gly Phe Val Asn Ile Leu Leu 2615 2620 2625Pro Pro Asp Ala Lys Glu Ile Ala Thr Arg Phe Arg Trp Trp Gln 2630 2635 2640Pro Arg His Asp Gly Leu Asp Gln Asn Asp Trp Ala Ile Asp Asn 2645 2650 2655Val Leu Ile Ser Gly Ser Ala Asp Gln Arg Thr Val Met Leu Asp 2660 2665 2670Thr Phe Ser Ser Ala Pro Val Pro Gln His Glu Arg Ser Pro Ala 2675 2680 2685Asp Ala Gly Pro Val Gly Arg Ile Ala Phe Glu Met Phe Leu Glu 2690 2695 2700Asp Lys Thr Ser Val Asn Glu Asn Trp Leu Phe His Asp Asp Cys 2705 2710 2715Thr Val Glu Arg Phe Cys Asp Ser Pro Asp Gly Val Met Leu Cys 2720 2725 2730Gly Ser His Asp Gly Arg Glu Val Tyr Ala Val Thr His Asp Leu 2735 2740 2745Thr Pro Thr Glu Asn Trp Ile Met Gln Phe Lys Ile Ser Val Gly 2750 2755 2760Cys Lys Val Pro Glu Lys Ile Ala Gln Asn Gln Ile His Val Gln 2765 2770 2775Phe Ser Thr Asp Phe Gly Val Ser Trp Ser Tyr Leu Val Pro Gln 2780 2785 2790Cys Leu Pro Ala Asp Pro Lys Cys Ser Gly Ser Val Ser Gln Pro 2795 2800 2805Ser Val Phe Phe Pro Thr Glu Gly Trp Lys Arg Ile Thr Tyr Pro 2810 2815 2820Leu Pro Glu Ser Leu Thr Gly Asn Pro Val Arg Phe Arg Phe Tyr 2825 2830 2835Gln Lys Tyr Ser Asp Val Gln Trp Ala Ile Asp Asn Phe Tyr Leu 2840 2845 2850Gly Pro Gly Cys Leu Asp Asn Cys Gly Gly His Gly Asp Cys Leu 2855 2860 2865Lys Glu Gln Cys Ile Cys Asp Pro Gly Tyr Ser Gly Pro Asn Cys 2870 2875 2880Tyr Leu Thr His Ser Leu Lys Thr Phe Leu Lys Glu Arg Phe Asp 2885 2890 2895Ser Glu Glu Ile Lys Pro Asp Leu Trp Met Ser Leu Glu Gly Gly 2900 2905 2910Ser Thr Cys Thr Glu Cys Gly Val Leu Ala Glu Asn Thr Ala Leu 2915 2920 2925Tyr Phe Gly Gly Ser Thr Val Arg Gln Ala Ile Thr Gln Asp Leu 2930 2935 2940Asp Leu Arg Gly Ala Lys Phe Leu Gln Tyr Trp Gly Arg Ile Gly 2945 2950 2955Ser Glu Asn Asn Met Thr Ser Cys His Arg Pro Val Cys Arg Lys 2960 2965 2970Glu Gly Val Leu Leu Asp Phe Ser Thr Asp Gly Gly Ile Thr Trp 2975 2980 2985Thr Leu Leu His Glu Met Asp Phe Gln Lys Tyr Ile Ser Val Arg 2990 2995 3000His Asp Tyr Ile Leu Leu Pro Glu Gly Ala Leu Thr Asn Thr Thr 3005 3010 3015Arg Leu Arg Trp Trp Gln Pro Phe Val Ile Ser Asn Gly Leu Val 3020 3025 3030Val Ser Gly Val Glu Arg Ala Gln Trp Ala Leu Asp Asn Ile Leu 3035 3040 3045Ile Gly Gly Ala Glu Ile Asn Pro Ser Gln Leu Val Asp Thr Phe 3050 3055 3060Asp Asp Glu Gly Ser Ser His Glu Glu Asn Trp Ser Phe Tyr Pro 3065 3070 3075Asn Ala Val Arg Thr Ala Gly Phe Cys Gly Asn Pro Ser Phe His 3080 3085 3090Leu Tyr Trp Pro Asn Lys Lys Lys Asp Lys Thr His Asn Ala Leu 3095 3100 3105Ser Ser Arg Glu Leu Ile Ile Gln Pro Gly Tyr Met Met Gln Phe 3110 3115 3120Lys Ile Val Val Gly Cys Glu Ala Thr Ser Cys Gly Asp Leu His 3125 3130 3135Ser Val Met Leu Glu Tyr Thr Lys Asp Ala Arg Ser Asp Ser Trp 3140 3145 3150Gln Leu Val Gln Thr Gln Cys Leu Pro Ser Ser Ser Asn Ser Ile 3155 3160 3165Gly Cys Ser Pro Phe Gln Phe His Glu Ala Thr Ile Tyr Asn Ala 3170 3175 3180Val Asn Ser Ser Ser Trp Lys Arg Ile Thr Ile Gln Leu Pro Asp 3185 3190 3195His Val Ser Ser Ser Ala Thr Gln Phe Arg Trp Ile Gln Lys Gly 3200 3205 3210Glu Glu Thr Glu Lys Gln Ser Trp Ala Ile Asp His Val Tyr Ile 3215 3220 3225Gly Glu Ala Cys Pro Lys Leu Cys Ser Gly His Gly Tyr Cys Thr 3230 3235 3240Thr Gly Ala Val Cys Ile Cys Asp Glu Ser Phe Gln Gly Asp Asp 3245 3250 3255Cys Ser Val Phe Ser His Glu Leu Pro Ser Tyr Ile Lys Asp Asn 3260 3265 3270Phe Glu Ser Ala Arg Val Thr Glu Ala Asn Trp Glu Thr Ile Gln 3275 3280 3285Gly Gly Val Ile Gly Ser Gly Cys Gly Gln Leu Ala Pro Tyr Ala 3290 3295 3300His Gly Asp Ser Leu Tyr Phe Asn Gly Cys Gln Ile Arg Gln Ala 3305 3310 3315Ala Thr Lys Pro Leu Asp Leu Thr Arg Ala Ser Lys Ile Met Phe 3320 3325 3330Val Leu Gln Ile Gly Ser Pro Ala Gln Thr Asp Ser Cys Asn Ser 3335 3340 3345Asp Leu Ser Gly Pro His Thr Val Asp Lys Ala Val Leu Leu Gln 3350 3355 3360Tyr Ser Val Asn Asn Gly Ile Thr Trp His Val Ile Ala Gln His 3365 3370 3375Gln Pro Lys Asp Phe Thr Gln Ala Gln Arg Val Ser Tyr Asn Val 3380 3385 3390Pro Leu Glu Ala Arg Met Lys Gly Val Leu Leu Arg Trp Trp Gln 3395 3400 3405Pro Arg His Asn Gly Thr Gly His Asp Gln Trp Ala Leu Asp His 3410 3415 3420Val Glu Val Val Leu Val Ser Thr Arg Lys Gln Asn Tyr Met Met 3425 3430 3435Asn Phe Ser Arg Gln His Gly Leu Arg His Phe Tyr Asn Arg Arg 3440 3445 3450Arg Arg Ser Leu Arg Arg Tyr Pro 3455 3460

Claims

1. A method for increasing reelin (RELN) levels in the brain of a subject in need thereof, said method comprising: administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

2. The method of claim 1, further comprising a step of: measuring an initial RELN level of the subject and comparing the measured initial RELN level to a healthy RELN level, wherein the subject is identified as having a reduced RELN level in need of increase when the measured initial RELN level is less than the healthy RELN level.

3. The method of claim 1 or 2, wherein the subject in need thereof suffers from a neuropsychiatric or neurodegenerative disorder.

4. The method of claim 3, wherein the subject in need thereof suffers from schizophrenia.

5. The method of claim 3, wherein the subject in need thereof suffers from Alzheimer's disease (AD).

6. The method of claim 3, wherein the subject in need thereof suffers from a neurological disorder characterized by enhanced expression of heme oxygenase-1 and/or increased oxidative stress in brain such as Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis, other motor neuron disorders, Down's syndrome, Creutzfeldt-Jakob disease, other prion diseases, multiple sclerosis, cerebral ischemia, cerebral hemorrhage, traumatic brain injury, spinal cord injury, cerebral malaria, schizophrenia, bipolar disease with psychosis, or autism.

7. A method for treating, preventing, or ameliorating symptoms of a neuropsychiatric disease or a neurodegenerative disorder in a subject in need thereof, said method comprising: administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

8. The method according to claim 7, wherein the composition comprising whey protein isolate and/or whey protein concentrate is administered to increase reelin (RELN) levels in the subject.

9. The method of claim 8, further comprising a step of: measuring an initial RELN level of the subject and comparing the measured initial RELN level to a healthy RELN level, wherein the subject is identified as being particularly susceptible to treatment when the measured initial RELN level is less than the healthy RELN level.

10. The method of any one of claims 7-9, wherein the neuropsychiatric disease or neurodegenerative disorder is schizophrenia.

11. The method of any one of claims 7-9, wherein the neuropsychiatric disease or neurodegenerative disorder is Alzheimer's disease (AD).

12. The method of any one of claims 7-9, wherein the neuropsychiatric disease or neurodegenerative disorder is a neurological disorder characterized by enhanced expression of heme oxygenase-1 and/or increased oxidative stress in brain such as Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis, other motor neuron disorders, Down's syndrome, Creutzfeldt-Jakob disease, other prion diseases, multiple sclerosis, cerebral ischemia, cerebral hemorrhage, traumatic brain injury, spinal cord injury, cerebral malaria, schizophrenia, bipolar disease with psychosis, or autism.

13. Use of a composition comprising whey protein isolate and/or whey protein concentrate for increasing reelin (RELN) levels in a subject in need thereof.

14. Use of a composition comprising whey protein isolate and/or whey protein concentrate in the manufacture of a medicament for increasing reelin (RELN) levels in a subject in need thereof.

15. Use of a composition comprising whey protein isolate and/or whey protein concentrate for treating, preventing, or ameliorating symptoms of a neuropsychiatric disease or a neurodegenerative disorder in a subject in need thereof.

16. Use of a composition comprising whey protein isolate and/or whey protein concentrate in the manufacture of a medicament for treating, preventing, or ameliorating symptoms of a neuropsychiatric disease or a neurodegenerative disorder in a subject in need thereof.

17. A method for restoring serotonin levels; normalizing MnSOD mRNA levels; augmenting glutathione (GSH) levels in brain cells; augmenting whole brain GSH/glutathione disulfide (GSSG) ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; improving HMOX1-related changes in brain dopamine, dopamine metabolites, serotonin, norephinephrine, and epinephrine; restoring deficient DOPAC/DA ratios; augmenting norepinephrine and dopamine concentrations in the prefrontal cortex; and/or correcting prefrontal cortex hypodopaminergia in a subject in need thereof, said method comprising: administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

18. A method for augmenting GSH reserves in the brain; normalizing brain MnSOD mRNA levels; restoration of redox homeostasis; normalization of brain dopamine, serotonin, norepinephrine, and/or epinephrine levels; normalization of brain DOPAC/DA ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; and/or ameliorating hyperlocomotion and/or stereotypic behaviour; or any combination thereof; in a subject in need thereof, said method comprising: administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

19. The method according to claim 17 or 18, wherein the subject suffers from, or is a risk of developing, a neuropsychiatric or neurodegenerative disorder.

20. The method according to claim 19, wherein the neuropsychiatric or neurodegenerative disorder is schizophrenia.

21. The method according to claim 19, wherein the neuropsychiatric or neurodegenerative disorder is Alzheimer's disease (AD).

22. The method according to claim 19, wherein the neuropsychiatric or neurodegenerative disorder is a neurological disorder characterized by enhanced expression of heme oxygenase-1 and/or increased oxidative stress in brain such as Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis, other motor neuron disorders, Down's syndrome, Creutzfeldt-Jakob disease, other prion diseases, multiple sclerosis, cerebral ischemia, cerebral hemorrhage, traumatic brain injury, spinal cord injury, cerebral malaria, schizophrenia, bipolar disease with psychosis, or autism.

23. The method according to claim 7, wherein the composition comprising whey protein isolate and/or whey protein concentrate is administered for restoring serotonin levels; normalizing MnSOD mRNA levels; augmenting glutathione (GSH) levels in brain cells; augmenting whole brain GSH/glutathione disulfide (GSSG) ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; improving HMOX1-related changes in brain dopamine, dopamine metabolites, serotonin, norephinephrine, and epinephrine; restoring deficient DOPAC/DA ratios; augmenting norepinephrine and dopamine concentrations in the prefrontal cortex; and/or correcting prefrontal cortex hypodopaminergia in the subject.

24. The method according to claim 7, wherein the composition comprising whey protein isolate and/or whey protein concentrate is administered for augmenting GSH reserves in the brain; normalizing brain MnSOD mRNA levels; restoration of redox homeostasis; normalization of brain dopamine, serotonin, norepinephrine, and/or epinephrine levels; normalization of brain DOPAC/DA ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; and/or ameliorating hyperlocomotion and/or stereotypic behaviour; or any combination thereof; in the subject.

25. Use of a composition comprising whey protein isolate and/or whey protein concentrate for restoring serotonin levels; normalizing MnSOD mRNA levels; augmenting glutathione (GSH) levels in brain cells; augmenting whole brain GSH/glutathione disulfide (GSSG) ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; improving HMOX1-related changes in brain dopamine, dopamine metabolites, serotonin, norephinephrine, and epinephrine; restoring deficient DOPAC/DA ratios; augmenting norepinephrine and dopamine concentrations in the prefrontal cortex; and/or correcting prefrontal cortex hypodopaminergia in a subject in need thereof.

26. Use of a composition comprising whey protein isolate and/or whey protein concentrate for augmenting GSH reserves in the brain; normalizing brain MnSOD mRNA levels; restoration of redox homeostasis; normalization of brain dopamine, serotonin, norepinephrine, and/or epinephrine levels; normalization of brain DOPAC/DA ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; and/or ameliorating hyperlocomotion and/or stereotypic behaviour; or any combination thereof; in a subject in need thereof.

27. Use of a composition comprising whey protein isolate and/or whey protein concentrate in the manufacture of a medicament for restoring serotonin levels; normalizing MnSOD mRNA levels; augmenting glutathione (GSH) levels in brain cells; augmenting whole brain GSH/glutathione disulfide (GSSG) ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; improving HMOX1-related changes in brain dopamine, dopamine metabolites, serotonin, norephinephrine, and epinephrine; restoring deficient DOPAC/DA ratios; augmenting norepinephrine and dopamine concentrations in the prefrontal cortex; and/or correcting prefrontal cortex hypodopaminergia in a subject in need thereof.

28. Use of a composition comprising whey protein isolate and/or whey protein concentrate in the manufacture of a medicament for augmenting GSH reserves in the brain; normalizing brain MnSOD mRNA levels; restoration of redox homeostasis; normalization of brain dopamine, serotonin, norepinephrine, and/or epinephrine levels; normalization of brain DOPAC/DA ratios; restoring GAD67 levels; reversing reduction of Nrxn1; reversing reduction of Nlgn2; preventing HMOX1-related dysregulation of miR-128 expression; and/or ameliorating hyperlocomotion and/or stereotypic behaviour; or any combination thereof; in a subject in need thereof.

29. The method according to any one of claim 1-6, 8-11, or 12, wherein the RELN levels are neuronal RELN levels.

30. The use according to claim 13 or 14, wherein the RELN levels are neuronal RELN levels.

31. The method or use according to claim 29 or 30, wherein the RELN levels are levels in the prefrontal cortex of the brain.

32. The method or use according to any one of claims 1-31, wherein the subject is a mammal.

33. The method or use according to any one of claims 1-32, wherein the subject is a human.

34. A method for inducing or restoring RELN expression and/or signalling in the entorhinal cortex and/or hippocampus of a subject in need thereof, said method comprising: administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

35. The method according to claim 34, wherein the subject has or is at risk of developing schizophrenia or Alzheimer's Disease.

36. Use of a composition comprising whey protein isolate and/or whey protein concentrate for inducing or restoring RELN expression and/or signalling in the entorhinal cortex and/or hippocampus of a subject in need thereof.

37. Use of a composition comprising whey protein isolate and/or whey protein concentrate in the manufacture of a medicament for inducing or restoring RELN expression and/or signalling in the entorhinal cortex and/or hippocampus of a subject in need thereof.

38. The use according to claim 36 or 37, wherein the subject has or is at risk of developing schizophrenia or Alzheimer's Disease.

39. A method for treating, preventing, or ameliorating symptoms of Alzheimer's Disease in a subject in need thereof, said method comprising: administering a composition comprising whey protein isolate and/or whey protein concentrate to the subject.

40. Use of a composition comprising whey protein isolate and/or whey protein concentrate for treating, preventing, or ameliorating symptoms of Alzheimer's Disease in a subject in need thereof.

41. Use of a composition comprising whey protein isolate and/or whey protein concentrate in the manufacture of a medicament for treating, preventing, or ameliorating symptoms of Alzheimer's Disease in a subject in need thereof.

42. The method or use according to any one of claims 39-41, wherein the composition is for preserving or restoring cognitive function in the subject.

43. The method or use according to any one of claims 34-42, wherein the composition is administered in an amount sufficient to provide at least one of: an increase in RELN levels in the entorhinal cortex; an increase in RELN levels in the dentate gyrus; an increase in RELN levels in the CA1 region; an increase in RELN levels in the CA3 region; a correction in a deficit in cortical GSH levels; a restoration of GAD67 expression in the hippocampal-entorhinal cortex sub-region; a restoration of p-CREB levels in the hippocampal-entorhinal cortex sub-region; an increase in RELN levels in layer II of the entorhinal cortex; a prevention of loss of RELN positive neurons in the entorhinal cortex; or a restoration of GAD67 expression in the dentate gyrus and/or CA3 region of the hippocampus.

44. The method or use according to any one of claims 34-43, wherein the subject is a mammal.

45. The method or use according to any one of claims 34-44, wherein the subject is a human.