THE ROLE OF NRG3 IN MENTAL ILLNESS AND NEURODEVELOPMENT: PREDISPOSITION TO “DELUSION” PHENOTYPE
Zeledon, Mariela Angela
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Background Schizophrenia (SZ) is a chronic, common, disabling neuropsychiatric disease that affects about 1% of the population. Despite evidence for a prominent genetic contribution to risk for SZ, there are relatively few replicated positive results. Among the genes that have been identified as possible SZ susceptibility genes are ErbB4, NRG1, and the topic of my dissertation, NRG3. In 2003, Fallin et al. reported a linkage signal in chromosome 10q22 in a rigorously-phenotyped homogeneous Ashkenazi Jewish (AJ) population, and a follow-up association study across the linkage peak identified a strong association of several intronic SNPs in NRG3 (rs60827755, rs10883866 and rs10748842) with the a factor designated “Delusion.” This was one of 9 factors derived from a principal component analysis of 73 phenotypic features. Methods We used next-generation sequencing in a population of patients that ranked at the extremes of the delusion score distribution in order to identify those variants in the 162 Kb that were most associated with delusion score. Of the top-ranking SNPs, five were chosen for dual-luciferase reporter assays to test for regulatory potential. In order to test for nuclear protein binding, we used electrophoretic mobility shift assays (EMSAs). We compared binding patterns between alleles and determined relative binding strength by competing with unlabeled probe. Using an in silico approach, we predicted which known proteins may bind our regions of interest, and proceeded to test one by supershift. We also characterized a Nrg3 knockout mouse developed by our laboratory. We performed a battery of behavioral tests relevant to models of schizophrenia. In order to test whether our mouse has the interneuron migration deficits predicted by previous publications, we assayed neuronal migration by labeling cells born at E12.5 with EdU, and then immunostaining for age-appropriate GABAergic interneuron markers at various developmental and postnatal ages. Results and Conclusions We identified three SNPs that exhibit regulatory potential and are most-highly associated with our delusion phenotype. We show that all three SNPs bind to nuclear proteins and that two of them do so differentially. We then show evidence for possible binding of CNOT4 to a 21-bp region of DNA centered around rs60827755. Further experiments are required to confirm this observation, but we believe that elucidating the pathway whereby NRG3 mediates its effect on the SZ phenotype will be beneficial in understanding the neuronal basis of delusions. The Nrg3-/- animals exhibit a pervasive hyperactivity phenotype, with more subtle deficits in pre-pulse inhibition and startle response. Additional findings include decreased anxiety, pre-pulse inhibition and startle response deficits, fear and/or emotional- based learning or memory deficits, and perturbed working memory. Since these findings are consistent and comparable to those of other mouse models for schizophrenia developed by knocking out other genes in the NRG/ErbB4 pathway, we conclude that this is a useful model for schizophrenia research. Migration assays revealed that the absence of Nrg3 does not lead to a depletion of interneurons in several regions of the cortex, but there does appear to be a depletion of interneurons in at least one other region (striatum). We surmise that a migratory change concentrates GABAergic interneurons in the cortex at the expense of other regions of the brain.