Manganese-induced alpha-synuclein overexpression impairs synaptic vesicle fusion by disrupting the Rab3 cycle in primary cultured neurons.

Overexposure to Manganese (Mn) has been known to disrupt neurotransmitter release in the brain. However, the underlying mechanisms of Mn exposure on neurotransmitter vesicle release are still unclear. The current study investigated whether Mn-induced alpha-synuclein protein overexpression could disrupt the Rab3 cycle leading to synaptic vesicle fusion dysfunction. After the neurons were exposed to Mn (100 μM) for 0, 6, 12, 24 h, [Ca2+]i, alpha-synuclein and Rab3A-GTP protein expression increased gradually. However, the interaction of synaptotagmin/Rab3-GAP and Rab3A-GTP/Rab3-GAP decreased significantly in response to Mn treatment for 12-24 h. Remarkably, the treatment with Mn caused an increase in the interaction of alpha-synuclein/Rab3A-GTP. To further validate that Mn-induced alpha-synuclein disrupted the proteins interactions of Rab3A-GTP/Rab3-GAP, the lentivirus vector of alpha-synuclein/negative shRNA was transfected in primary cultured neurons to knockdown the expression of alpha-synuclein. Our results showed that the interaction of Rab3A-GTP/Rab3-GAP in alpha-synuclein knockdown neurons treated with Mn for 24 h had a significant recovery. These results suggested that Mn-induced alpha-synuclein protein overexpression, which bound to Rab3A-GTP and inhibited the GTP hydrolysis of Rab3 protein, disrupted the Rab3 cycle leading to synaptic vesicle fusion dysfunction.