ABSTRACT: Allopregnanolone (AP?), as a functional neurosteroid, exhibits the neuroprotective effect on neurodegenerative diseases such as Parkinson's disease (PD) through ?-aminobutyric acid A receptor (GABAAR), but it has not been completely understood about its molecular mechanisms. In order to investigate the neuroprotective effect of AP?, as well as to clarify its possible molecular mechanisms, SH-SY5Y neuronal cell lines were incubated with 6-hydroxydopamine (6-OHDA), which has been widely used as an in vitro model for PD, along with AP? alone or in combination with GABAAR antagonist (bicuculline, Bic), intracellular Ca2+ chelator (EGTA) and voltage-gated L-type Ca2+ channel blocker (Nifedipine). The viability, proliferation, and differentiation of SH-SY5Y cells, the expression levels of calmodulin (CaM), Ca2+/calmodulin-dependent protein kinase II ?3 (CaMKII?3), cyclin-dependent kinase-1 (CDK1) and brain-derived neurotrophic factor (BDNF), as well as the interaction between CaMKII?3 and CDK1 or BDNF, were detected by morphological and molecular biological methodology. Our results found that the cell viability and the number of tyrosine hydroxylase (TH), bromodeoxyuridine (BrdU) and TH/BrdU-positive cells in 6-OHDA-treated SH-SY5Y cells were significantly decreased with the concomitant reduction in the expression levels of aforementioned proteins, which were ameliorated following AP? administration. In addition, Bic could further increase the number of TH or BrdU-positive cells as well as the expression levels of aforementioned proteins except for TH/BrdU-double positive cells, while EGTA and Nifedipine could attenuate the expression levels of CaM, CaMKII?3 and BDNF. Moreover, there existed a direct interaction between CaMKII?3 and CDK1 or BDNF. As a result, AP?-induced an increase in the number of TH-positive SH-SY5Y cells might be mediated through GABAAR via Ca2+/CaM/CaMKII?3/BDNF (CDK1) signaling pathway, which would ultimately facilitate to elucidate PD pathogenesis and hold a promise as an alternative therapeutic target for PD.
