Characterization and Regulation of Alpha-Synuclein Phosphorylation in Enteric Neurons

Alpha-synuclein is a 140 aa neuronal protein pathologically and genetically linked to Parkinson's disease (PD). In PD, the major protein modification of alpha-synuclein is phosphorylation at serine 129. Alpha-synuclein phosphorylation also occurs at low levels in normal brains and cultured CNS neurons, but its regulation and role are only beginning to be investigated. The enteric nervous system is now recognized as a second brain in its own right, and therefore, we set out to examine the regulation of alpha-synuclein phosphorylation in enteric neurons. To this end, primary cultures of rat enteric nervous system (ENS) and enteric neurons, which both express alpha-synuclein, were either depolarized or treated with forskolin and analyzed by western blot. We found that membrane depolarization and forskolin induced alpha-synuclein phosphorylation via a Ca²⁺-calmodulin-dependent protein kinase and cAMP/exchange protein directly activated by cyclic AMP (EPAC) signaling pathway, respectively. Both pathways converged on Polo-like kinase 2 (PLK2) to phosphorylate alpha-synuclein. PLK2 inhibition increased the amount of alpha-synuclein secretion while reducing its intracellular phosphorylation level in both cytoplasmic and membranous fractions. To investigate alpha-synuclein phosphorylation in the ENS further, specimens of human colon were analyzed to show that the distribution of phosphorylated alpha-synuclein in the ENS was highly variable and that the amount of soluble phosphorylated alpha-synuclein did not differ between PD and control subjects. Our study is the first to show that alpha-synuclein phosphorylation can be regulated in enteric neurons, providing a basis to unravel the functions of alpha-synuclein and its phosphorylation in the ENS.