C-terminal part of α-synuclein mediates its activity in promoting proliferation of dopaminergic cells

Although abnormal aggregation of alpha-synuclein (alpha-syn) is involved in several neurodegenerative diseases, its biological functions remain poorly understood, which limits our understanding of its pathogenic mechanisms. alpha-Syn exhibits MAP-like activity and promotes the assembly of microtubules. Since microtubules play a pivotal role in proliferative cell division, it is possible that alpha-syn affects cell proliferation by facilitating microtubule assembly. The role of alpha-syn in promoting cell proliferation was reported previously in PC12 dopaminergic cells overexpressing alpha-syn. Here, we extended this study aiming at finding the association between the cell proliferation effect of alpha-syn and its microtubule assembly activity, and identifying the potential active domain for the effect of alpha-syn on cell proliferation. By exploiting the property that the 11-mer repeats of synuclein molecules are able to mediate a rapid intracellular translocation of these proteins across the plasma membrane without being degraded by the cellular proteolytic system, we added recombinant full-length alpha-syn (wild type and A53T and A30P mutants) and beta-syn to the culture medium of MES23.5 dopaminergic cells, and observed their intracellular translocation, subcellular distribution and effects on cell proliferation. We found that all the synuclein molecules could enter the cells where they were localized in both the cytoplasm and nucleus. However, only the wild-type alpha-syn, which had been shown to have microtubule assembly activity, was able to promote proliferation of the MES23.5 cells. The A53T and A30P mutant alpha-syn as well as beta-syn, which had been proved not to possess microtubule assembly activity, did not exhibit any effect on cell proliferation. Since the alpha-syn activity in microtubule assembly was shown to be related to its specific functional domain, we then generated different functional fragments (N-terminal aa1-65, NAC aa61-95 and C-terminal aa96-140) and tested their activities in cell proliferation. We showed that all the alpha-syn fragments could enter the cells, but with different subcellular localizations. The N-terminal and NAC fragments were localized in the cytoplasm and the C-terminal fragment mainly in the nucleus. In accordance with the activity for the C-terminal part of alpha-syn in microtubule assembly, only the NAC and C-terminal fragments exhibited the activity in cell proliferation. The N-terminal fragment without microtubule assembly activity did not promote cell proliferation. The above results suggest that the alpha-syn function in promoting cell proliferation is associated with its microtubule assembly activity with the functional domain localized in its C-terminal part.