It is well known there are difficulties in identifying functional mutations with small effects for complex genetic disorders, so we continue to partner validated genetic associations with biological data of specific genetic variations that affect aspects of brain function and molecular biology. Assuming schizophrenia is a combination of traits that interact with other traits, genes, and environment giving rise to complex clinical phenotypes, we posit that uncovering mechanisms of disease will allow for the identification of novel therapeutic targets from the association of genetic variation with relevant biological intermediate phenotypes. Our research continues to strengthened the validity of this approach as evidenced in our studies of GABA signaling molecules (Hyde, J Neurosci 2011), DISC1 (Newburn , Transl Psych 2011), as well as our lab's contributions on a myriad of other studies. From our studies of susceptibility genes for schizophrenia (SZ) (Wong, Neuroscience 2010) determined which of 4 major BDNF alternate transcripts are altered in brain regions in SZ. BDNF is critical for neural survival, development and maturation. We showed reductions in cortical BDNF expression via reduced expression of at least one transcript. However, antidepressants are shown to up-regulate specific BDNF transcripts with regional effects. Future work will study the molecular control of BDNF regulation as a therapeutic target for mental illness. A study of DISC1, (Eastwood, Hum Mol Genet 2010) explored the impact of genetic variants in DISC1 on centrosomal PCM1 localization in glia. DISC1, ser704cys and leu60phe were shown to affect PCM1 localization which effects glial function;however there was no difference in localization between controls and SZ. These 2 variants were significant in African Americans and may have a cumulative effect. While DISC1 remains one of the most promising schizophrenia-susceptibility genes, we also report finding a large number of DISC1 alternative splice variants in human brain. Newburn (Transl Psych 2011) demonstrated that DISC1 short isoforms, ∆3, ∆7∆8, and Esv1 were overexpressed in SZ brains and in risk-associated allele carriers. Future examination of protein-protein interactions of these isoforms may inform about functional effects of overexpression in mental illness. We proceeded to examine their binding ability with related proteins and role in the neurobiology of schizophrenia. It is plausible that short variant binding with DISC1 partners could have a negative impact when compared with binding to full-length DISC1. Moreover, we posit that if short variant binding is attainable, overexpression in SZ brains may present deleterious effects such as inappropriately activating signaling processes or interference with full-length DISC 1 dimerization. We showed that all short variants bind to full-length DISC1, FEZ1, and GSKbeta. However, interactions with NDEL1, and PDE4B were weak. Previous reports suggested that coexpression of short variants with full-length DISC1 results in aberrant function. We further suggest that this may be the mechanism by which short variants bind full-length DISC1, making it unavailable for dimerization and partner binding, and thereby exerting a pathological role in psychiatric disorders. We have yet to determine the functionality of these interactions however we speculate that short variant binding of GSK3beta may impact the GSK3beta signaling pathway and may serve as a binding target for short variants overexpressed in the brains of patients. We had also reported that DISC1 expression is higher in fetal life relative to postnatal, perhaps indicating importance in neurodevelopment. In summary, our findings suggest that DISC1 short variants may form unique protein complexes which could suggest mechanisms that exert pathological effects to confer risk for mental illness. Our study in Hyde et al, J of Neurosci 2011, is the first to report on the developmental trajectory of both GAD67 and GAD25 expression in human brain. We examined the expression of transcripts derived from 3 genes related to GABA signaling GAD1 (GAD67 and GAD25), SLC12A2 (NKCC1), and SLC12A5 (KCC2) in PFC and hippocampal formation (HF) in healthy brains across the lifespan and in patients with schizophrenia (SZ). GABA signaling molecules are important for human brain development and pathophysiology of SZ. We found a SZ risk-associated promoter SNP in GAD1 (rs3749034) related to the expression of these transcripts. We also revealed that development and maturation of both the PFC and HF are characterized by progressive switches in expression from GAD25 to GAD67 and from NKCC1 to KCC2. The former leads to synthesis of GABA and the latter leads to GABA switching from an excitatory to an inhibitory neurotransmitter. Ratios of GAD25/GAD67 and NKCC1/KCC2 reflect the maturational state of GABA function in the human PFC and HF. In SZ hippocampus GAD25/GAD67 and NKCC1/KCC2 ratios are increased while KCC2 levels are decreased, reflecting a potentially immature state of the GABA system. GAD1 risk alleles are also associated with GAD25, GAD67 and KCC2 expression reflecting a less mature pattern of development. These findings suggest that abnormalities in GABA signaling, critical for brain development and maturation, may also contribute to increased genetic risk for SZ. These findings also implicate a genetic mechanism associated with clinical diagnosis and provide a developmental context to these associations. Studies involving these brains and neuroleptic treated rats suggest that the changes we have seen in these postmortem human brains are not related to neuroleptic treatment confounds. Developmental patterns of human PFC and HF KCC2 expression during development found in our study agreed with and expanded on previous reports. Of note is that prefrontal neuropsychological test performance improves during childhood and adolescence in conjunction with increased KCC2 expression. KCC2 mRNA expression is both brain-specific and neuron-specific. The progressive increase of KCC2 expression in DLPFC may be a molecular correlate of maturation and is consistent with other evidence that GABA tuning of cortical circuitry is critical for higher order PFC cognition. Expression patterns of NKCC1 in PFC and HF are consistent with animal studies that describe a rise in NKCC1 mRNA expression in both the neocortex and HF after birth, but this rise is less steep than KCC2. GAD25 is also involved in the early phases of brain development, and the ratio of GAD25/GAD67 mRNA expression decreases as a brain structure matures. The early decline of GAD25 expression and the changes in GAD25/GAD67 ratio suggest there may be a developmental switch in presynaptic GABA signaling. This study looks at both the switch from GAD25 to GAD67 which leads to GABA synthesis and the switch of GABA from excitatory to inhibitory which is mediated by relative abundance of KCC2 and NKCC1.

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Morita, Yukitaka; Callicott, Joseph H; Testa, Lauren R et al. (2014) Characteristics of the cation cotransporter NKCC1 in human brain: alternate transcripts, expression in development, and potential relationships to brain function and schizophrenia. J Neurosci 34:4929-40
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