We have conducted several investigations using postmortem human brain specimens focused primarily on understanding the pathophysiology of SZ in addition to other complex neuropsychiatric disorders. In addition to our own studies, the Section continues to provide postmortem human brain tissues to researchers and labs within and outside NIH. Given the difficulties identifying functional mutations with small effects for complex genetic disorders, our lab continues to partner validated genetic associations with biological data of specific genetic variations that affect aspects of brain function and molecular biology. Assuming complex psychiatric disorder are a combination of traits that interact with other traits, genes, and environment give rise to complex clinical phenotypes, we posit that by uncovering mechanisms of disease allows for identification of novel therapeutic targets from the association of genetic variation with relevant biological intermediate phenotypes. Our research continues to strengthen the validity of this approach as evidence in our study of NRG1 (Law et al, PNAS 2012) which combined patient lymphoblast (LCLs), postmortem human brain, and human molecular and clinical genetics with pharmacological studies in rodent to test whether abnormal PIK3 signaling is related to SZ-associated variation in ErbB4. We replicated the results in LCLs of increased PIK3R3 expression in SZ and decreased ErbB4 mRNA in DLPFC. Rats treated with an antipsychotic showed reduction in only PIK3CD gene expression in brain. PIK3R3, ErbB4 and ErbB3 mRNA expression remained unaffected however these transcripts showed alterations in brain and LCLs in SZ. These data suggest that reduction of PIK3CD activity may be relevant for the actions of antipsychotic drugs. Tao et al (J Neurosci 2012) furthered our study of genetic variation in the neuron specific cotransporter KCC2, which helps mediate the electrophysiological effects of GABA. We are the first to show that the pattern of KCC2 expression during early brain development suggests its upregulation drives the postsynaptic switch of GABA from excitatory to inhibitory. Recent studies implicate KCC2 in both the genetic and neurodevelopmental etiologies of SZ. This is consistent with studies showing that GABA receptor activity regulates expression of KCC2. The GAD1 allelic variation associated with GAD1 expression and SZ-risk predicts the level of KCC2 expression in human brain. These data suggest that the development aspects of GABA dysfunction linked with illnesses may involve regulation of KCC2 activity. SLC12A5, the gene for KCC2, shows linkage with SZ and bipolar. We previously reported decreased expression of full-length KCC2 in the hippocampus of SZ, but not in the DLPFC. Closer molecular biological examination revealed several unidentified alternative KCC2 transcripts in human adult and fetal brain. Expression levels were measured in four relatively abundant truncated splice variants, including 3 novel transcripts (exon 6, 2B and 6B) and one known transcript AK098371 in a large cohort of controls across the lifespan, SZ and effective disorders. Expression patterns across lifespan showed Exon 6 transcript was highest in the fetal period and declined during development while the others showed an inverse profile pattern. Of all 4 transcripts, only Exon6B mRNA was increased in DLPFC of SZ but reduced in MDD vs. controls. We then tested the effect of the GAD1 SZ-risk SNP on expression of the 4 variants. AK098371, previously associated with GAD1 SZ-risk SNP (rs3749034 G/G), predicted decreased expression of mRNA in DLPFC in SZ and controls. These data bring to light the complexity of KCC2 mRNA variants in both development and illness. Functionally, novel transcripts may interfere with expression and translation of other KCC2 transcripts (including full-length). Further study will be needed to better characterize their role in brain development and illness. With the development and public release of braincloud (gene expression and DNA methylation interface), we furthered our study of brain development of the PFC. Colantuoni et al (Nature 2011) combined transcriptional and genetic analyses in human neural tissue to gain a global molecular perspective on the role of the human genome in cortical development, function and ageing. We explored temporal dynamics and genetic control of transcription in human PFC in an extensive series of postmortem brains from fetal through ageing. We discovered a wave of gene expression changes occur during fetal development that are reversed in early postnatal life. This pattern of reversals is mirrored 50yrs later in ageing and neurodegeneration. We found thousands of robust associations of individual genetic polymorphisms with gene expression and also showed that there is no association between the total extent of genetic differences between subjects and the global similarity of their transcriptional profiles seemingly due to a consistent molecular architecture in the PFC produced by the human genome across the lifespan. We surmise that aberrant traits and combinations in this architecture are selected against and would not appear in studies of normal human brain development. Notwithstanding the lack of a macroscale relationship between global genetic and transcriptome profiles, we have shown the ability to analyze microscale genetic effects. Given this, perhaps individual complete genomes are better thought of as a combination of variants which are acted upon (by evolution and environment) and in which acts (in development, biological function and disease) as a whole rather than individual genetic traits alone. Lastly, in another study looking at development and ageing showed that DNA methylation in the 5 promoter region is a highly dynamic process modified by genetic variants and regulating gene transcription. Numata et al (Am J Hum Genet 2012) examined 14,500 genes at 27,000 CpG sites focused on promoters across the lifespan (fetal to 80yrs). The PFC is one of the last brain regions to mature and is critical in complex cognitive behaviors, personality, decision making, and composition of thoughts and actions, so we studied the role of epigenetics in its development and highlight some of our findings. First, the fastest changes in methylation occurred during the fetal period, and the transition from fetal to early childhood was associated with a reversal of direction of methylation. The causes of age-related changes remains unclear, increases may reflect the accumulation of random methylation events over time, and decreases might be related to altered fidelity of methyltransferases. This suggests that epigenetic factors might play a role in the pathogenesis of disease and mechanisms associated with aging and add to methylation errors which might be essential to understand pathogenesis of age-related brain disorders. We also found that age-related changes occurred outside CpGIs more than in CpGIs however the inside CpGIs changes gained methylation with age. This study found altered methylation in utero or around birth might be critical for the pathogenesis of developmental brain disorders. Sex-biased autosomal genes are methylated in normal DLPFC, may help to examine sex differences in brain disorders, code for a glutamate metabolic enzyme, and are implicated in SZ and cognition. Lastly, methylation is associated with genetic variance in that the number of cis-SNP associations is consistent with the possibility that some disease risk SNPs might affect gene expression through methylation, and direct effects on mRNA expression. Future studies will be needed to reveal how genetic and epigenetic variation, together and independently are involved in the pathophysiology of neuropsychiatric diseases.
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