Characterization and regulation of alpha-synuclein phosphorylation in enteric neurons under physiological conditions

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 is also phosphorylated at low levels under physiological conditions but its regulation and role are still largely unknown. Mounting evidence suggests a role for the gut-brain axis in CNS homeostasis, and therefore we set out to examine the regulation of alpha-synuclein phosphorylation in enteric neurons under physiological conditions. 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 kinases 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. PLK 2 inhibition increased the amount of alpha-synuclein secretion while reducing its intracellular phosphorylation level in both cytoplasmic and membranous fraction. 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 controls subjects. Our study is the first to show that alpha-synuclein phosphorylation can be physiologically regulated in enteric neurons, providing a basis to unravel the physiological functions of alpha-synuclein and its phosphorylation in the ENS.