Functional characterization of AC5 gain-of-function variants: Impact on the molecular basis of ADCY5-related dyskinesia.

Adenylyl cyclases are key points for the integration of stimulatory and inhibitory G protein-coupled receptor (GPCR) signals. Adenylyl cyclase type 5 (AC5) is highly expressed in striatal medium spiny neurons (MSNs), and is known to play an important role in mediating striatal dopaminergic signaling. Dopaminergic signaling from the D expressing MSNs of the direct pathway, as well as the D expressing MSNs of the indirect pathway both function through the regulation of AC5 activity, controlling the production of the 2nd messenger cAMP, and subsequently the downstream effectors. Here, we used a newly developed cell line that used Crispr-Cas9 to eliminate the predominant adenylyl cyclase isoforms to more accurately characterize a series of AC5 gain-of-function mutations which have been identified in ADCY5-related dyskinesias. Our results demonstrate that these AC5 mutants exhibit enhanced activity to Gα-mediated stimulation in both cell and membrane-based assays. We further show that the increased cAMP response at the membrane effectively translates into increased downstream gene transcription in a neuronal model. Subsequent analysis of inhibitory pathways show that the AC5 mutants exhibit significantly reduced inhibition following D dopamine receptor activation. Finally, we demonstrate that an adenylyl cyclase "P-site" inhibitor, SQ22536 may represent an effective future therapeutic mechanism by preferentially inhibiting the overactive AC5 gain-of-function mutants.