PROJECT SUMMARY Despite acutely enhancing monoamine function, chronic administration of monoamine antidepressants is required for clinical response, suggesting that adaptations downstream of enhanced monoaminergic signaling are central to their therapeutic efficacy. One such downstream adaption is the epigenetic upregulation of brain- derived neurotrophic factor (BDNF), a key protein in the neurotrophic process. This is in line with the neurotrophic hypothesis of depression, which posits that insufficient neuronal growth contributes to the pathogenesis of MDD, as evidenced by reduced hippocampal volume in the postmortem brains of depressed patients, and thus the restoration of neurotrophy is central to the efficacy of antidepressants. In addition to requiring chronic treatment, traditional antidepressants drugs are also ineffective in a significant proportion of depressed patients and produce a wide variety of undesirable side effects. On the other hand, ketamine, a N-methyl-D-aspartate receptor (NMDAR) antagonist and anesthetic, a single dosage of which has been shown to relieve symptoms in less than an hour and for at least one week in patients who failed to response to at least two typical antidepressants. Unlike traditional antidepressants, ketamine appears to promote BDNF-mediated neurotrophy rapidly and directly but optimizing this anesthetic for antidepressant application and designing novel drugs its image requires further elucidation of its targets and the mechanisms underlying its therapeutic effects. We hypothesize that rather than rather than indirectly altering neurotrophic genes, as with chronic administration of typical antidepressants, ketamine bypasses this prolonged process of transcriptional regulation and instead induces RNA modifications that rapidly upregulates the translation of neurotrophic proteins. stress-induced changes in levels of N6,2’-O-dimethyladenosine (m6A), the most abundant RNA modification, have been observed in MDD patients and single-nucleotide polymorphisms in the m6A demethylase, fat mass and obesity associated protein (FTO), are associated with increased risk of MDD. Furthermore, inhibition of glycogen synthase kinase 3 (GSK- 3), which ketamine does through NMDAR antagonism, increases FTO concentrations which promotes conversion of the pro-apoptotic proBDNF to BDNF through demethylation of matrix metalloprotease 9 (MMP- 9). Taken together, these findings present a plausible epitranscriptomic mechanism for the rapid neurotrophic and antidepressant effects of ketamine. Because m6A methylation varies between species, and the methylation sites of relevant genes, such as MMP-9, differ between mice and humans, we will use human induced pluripotent stem cell (hiPSC)-derived cortical glutamatergic neurons to delineate the effects of ketamine on the epitranscriptome, m6A machinery, as well as neuronal structure and function. The successful completion of these aims will elucidate the mechanisms underlying the rapid and robust antidepressant effects of ketamine thus enabling the identification and optimization of novel antidepressants.

PROJECT NARRATIVE The proposed studies will provide critical insight into the targets and mechanisms that underlie ketamine’s rapid and robust therapeutic benefits for cases of depression in which traditional antidepressants are ineffective. By delineating how modification of RNA encoding key neurotrophic proteins these studies may help to guide the optimization existing antidepressants and to design novel fast-acting pharmacotherapies for treatment resistant depression.