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Functional Characterization of NOS1AP and microRNAs in Schizophrenia Susceptibility

by
Anna M. Dulencin
B.A., Rutgers University - 2003


Thesis Advisor: James H. Millonig, Ph.D.
Graduate Program in Neuroscience


CABM - Room 010
Piscataway

Monday, December 16, 2013
3:00 p.m.


Abstract

Schizophrenia (SCZ) and bipolar disorder (BPD) are common and debilitating neuropsychiatric disorders with a strong genetic basis. Although the etiology of SCZ and BPD is unknown, inherited genetic polymorphisms as well as aberrant microRNA expression contribute to risk.
Earlier reports identified Nitric Oxide Synthase 1 Adaptor Protein 1 (NOS1AP) as a SZ susceptibility gene. Recent experiments have identified three SNPs in intron 2 associated with schizophrenia: rs12742393, rs1415263, and rs4145621.
Luciferase assays determined that schizophrenia-associated alleles of all three SNPs are functional and increase gene expression. EMSAs demonstrated that the over-transmitted alleles bind nuclear factors more avidly than their non-associated counterparts. Furthermore, bioinformatics and mass spectrometry analysis identified greater amounts of heterogenous nuclear ribonucleoprotein (hnRNPA3) binding rs12742393 schizophrenia-associate allele than to non-associated allele.
Subsequent experiments validated that hnRNPA3 mediates the effect of the associated allele. Firstly, correlation analysis revealed that changes in hnRNPA3 mRNA levels are highly likely to result in changes in NOS1AP mRNA, and secondly, knock-down of hnRNPA3 diminished increased levels of SCZ-associated A allele in luciferase assays.
Another level of gene regulation implicated in neuropsychiatric disorder etiologies is microRNAs. Previous research has reported misexpression of 24 miRNAs in schizophrenia and bipolar disorder affected individuals. We chose to focus on four miRNAs: miR-22, miR-138, miR-151, and miR-338.
Overexpression analysis in primary neurons demonstrated that only miR-22 and miR-138 resulted in disease-relevant phenotypic changes in vitro. Bioinformatics analysis identified target genes predicted to be regulated by miR-22 and miR-138. Preliminary experiments have investigated whether these predicted targets, previously implicated in SCZ etiology, mediate the observed phenotypic effects.
In summary, we have provided evidence for two different modes of increasing schizophrenia susceptibility: firstly, we identified hnRNPA3 as the mediator that binds rs12742393 schizophrenia-associated allele and, in turn, increases NOS1AP gene levels, and secondly, we demonstrated that aberrant expression of miR-22 and miR-138 affects cell biological phenotypes and levels of downstream targets known to play a role in SCZ risk.


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