Novel Androgen Receptor Protein in Brain: Implication for Parkinson's Disease

Objective: Men have a two-fold increased risk for Parkinson’s disease (PD) than women. Testosterone, the major male sex hormone, can increase calcium influx and cell death in dopamine neurons via a putative membrane androgen receptor (mAR). The mAR induced calcium increase may be due to activation of Gaq protein-coupled receptor (GPCR) that is involved in calcium mobilization. Currently, the mAR remains unidentified. Recent studies only found miniscule levels of androgen receptors (AR) in the substantia nigra (SN). This low AR expression in the SN may be due to absence of full length classical AR that contains an N terminus domain (NTD), especially as these studies used an antibody targeting the AR NTD. It is possible that ARs in the SN consist of a splice variant that does not possess a NTD, such as AR45. AR45 is not able to be assayed using an NTD antibody, and thus we used a C-terminus domain (CTD) antibody. Therefore, we hypothesize that the putative mAR is the AR45 splice variant that acts through a Gaq GPCR. Materials and Methods: We examined the expression of classical full length AR and AR45 in a dopaminergic N27 cell line and rat SN. Protein expression of AR and AR45 was quantified by Western blot analysis and immunohistochemistry (IHC). We used antibodies targeting either the NTD or CTD of the AR, along with antibodies targeting Gαq, Gαs and Gαo GPCRs. To determine the association between mAR and GPCR subunits we performed co-immunoprecipitation using AR-CTD and Gaq antibodies. Results: Our results showed that the SN and the N27 cells express very low AR-NTD positive cells, indicative of low full length classical AR expression. However, both N27 cells and SN showed very high levels of AR-CTD positive cells. Furthermore, protein expression of AR-CTD was observed at 45 kDa molecular weight, which is consistent with the AR splice variant, AR45. This AR45 splice variant was found to be associated with Gaq in both N27 cells and SN. Conclusions: Our data indicates that the mAR in dopaminergic neurons is the AR45 splice variant, which is associated with a Gaq subunit. These results provide a mechanism for our prior studies wherein testosterone increased intracellular calcium levels. This is the first observation of an AR splice variant in neuronal tissue. Further characterization of this protein may provide a novel therapeutic target to slow the progression of PD in men.