Examining the role of the ADCY5 point mutation p.R418W on cellular cAMP levels and how cAMP affects neuronal differentiation of murine stem cells

ADCY5-related Dyskinesia is a rare movement disorder, with early onset in childhood and adolescence. Previous studies have linked this disease to various point mutations in the ADCY5 gene; one study has demonstrated two of these mutations cause an increase in cyclic adenosine monophosphate (cAMP). However, the molecular basis of this disease phenotype has yet to be fully understood. Our study seeks to characterize the effects of one specific point mutation, p.R418W, on cellular cAMP levels using HEK293T cells, and to determine if increased cAMP levels affect neuronal differentiation using mouse embryonic stem cells (mESCs). Our initial experiments demonstrated successful differentiation of mESCs into a neuronal lineage, with 1-2% dopaminergic neuron production, detected by Immunocytochemistry (ICC). This preliminary data showed that treatment with 1400 uM cAMP did affect neuronal differentiation.The most striking finding was the presence of contracting neuronal clusters in the treated neurons, versus non-treated. Current experiments include creating a CRISPR model: a HEK293T cell line containing the p.R418W mutation. We have designed the appropriate guide RNA, confirmed a high transfection efficiency of the CRISPR components using fluorescence microscopy, and are currently screening colonies by sequence analysis. Once the mutant cell line is verified, it will be treated with pharmacological agonists to stimulate cAMP production in mutated cells as well as control cells. The amount of cAMP produced in mutated cells versus wild-type will be quantified using enzyme-linked immunosorbent assay (ELISA). Once we characterize the amount of cAMP produced in the ACDY5 p.R418W 293T cells, we will see if this level of cAMP affects differentiation of mESCs to dopaminergic neurons using our optimized mESC differentiation protocol. This data will not only help us better understand the role of ADCY5 in neuronal development, it will be beneficial to our collaborators, in choosing appropriate drugs for screening of induced pluripotent stem cells (iPSCs), collected from ADCY5 patients.https://digitalcommons.humboldt.edu/inrsep_posters/1020/thumbnail.jp