630. EPITRANSCRIPTOMIC REGULATION OF STRIATAL FUNCTION: A NEW NEUROBIOLOGY OF SCHIZOPHRENIA?

Abstract Background The EDiPS model (Enhanced Dopamine in Prodromal Schizophrenia) reiterates the key discovery regarding the development of schizophrenia, that patients experience increased dopamine synthesis and release in the dorsal striatum. The EDiPS model displays structural adaptations such as an increase in dopamine release sites and dopamine synapse numbers. RNA methylation involves the attachment of a methyl group at the N6 position of the adenosine to form N6-methyladenosine (m6A), and is essential for brain function, plasticity, and enabling appropriate adaption to external challenges. Aims & Objectives The aim of this project is to determine whether RNA methylation is the underlying mechanism through which synaptic adaptation occurs in response to enhanced dopamine release in the striatum of EDiPS models. Method To generate EDiPS animals, we injected a construct coding for tyrosine hydroxylase and GTP cyclohydrolase 1 into the substantia nigra of juvenile postnatal day 35 rats. These enzymes are transported to the dorsal striatum, where they increase dopamine synthesis capacity. Six weeks following construct delivery, we collected the dorsal striatum, extracted RNA and performed methylated RNA(m6A) immunoprecipitation (meRIP) sequencing. Western blot was conducted to assess the expression of RNA modifiers that regulate RNA methylation. RNA modifiers include m6A methyltransferases Mettl3 and Mettl14(writers), demethylase FTO(erasers), and m6A readers that facilities m6A-modified RNA translation such as YT521-B homology domain family (YTHDF) proteins. Hypermethylated RNAs were targeted and demethylated using a CRISPR-Cas-inspired RNA targeting system (CIRTS). Results RIP-sequencing results showed that 80% of mRNAs in the EDiPS dorsal striatum were significantly hypermethylated (95 out of 119 mRNAs differentially modified). KEGG pathway analysis revealed that altered m6A RNAs were enriched in synaptic vesicle cycling, glutamate, dopamine synapses and long-term potentiation. These results suggest that enhanced dopamine release induces RNA hypermethylation, which may lead to the increased dopamine synaptic number in EDiPS. Consistent with enhanced RNA methylation, there was increased expression of Mettl14 writer protein and the m6A reader YTHDF1 mRNA in EDiPS dorsal striatum. Enhanced m6A accumulation on phospholipase C (PLC)-β1 RNA, linked to schizophrenia, resulted in elevated protein levels in EDiPS striatum. In vitro experiments using primary striatal neuronal cultures showed that demethylation of PLC-β1 RNA via CIRTS reduced protein translation, indicating that RNA methylation promotes protein synthesis. Functionally, removing m6A from PLC-β1 reduced calcium responses to dopamine stimulation. Discussion & Conclusions Hyper-dopaminergia in the dorsal striatum is a robust marker that provides a highly plausible neurochemical prophylactic target. The EDiPS model provides the opportunity to examine a possible interaction between this clinical finding, and potentially crucial changes to the epitranscriptome. We have provided the first evidence that increased dopamine synthesis capacity in the dorsal striatum enhances RNA methylation, which in turn regulates synaptic structure and plasticity. Further exploration of this finding will not only provide etiological insight, but also help to identify novel treatment targets for schizophrenia.