Impact of phosphorylation of alpha-synuclein in Parkinson's disease

Parkinson's disease (PD) is characterized by the degeneration of dopamineric neurons of the substantia nigra pars compacta projecting to the striatum, and by the deposition of cytoplasmic protein inclusions. It is further defined by the persistent presence of the α-synuclein (α-syn) protein in these inclusions. Moreover, mutations of α-syn as well as a triplication of the α-syn gene have been identified as a cause of inherited PD. These rare familial cases indicate that modifications in the sequence and in the level of expression have a tremendous impact on the protein's properties. In both familial and sporadic PD, post-translational modifications of α-syn have been reported. In particular, phosphorylation of α-syn Serine at position 129 is found to be a major modification, tightly associated with pathogenesis in human PD. Whether it be through changes in the protein's folding properties or through aberrant interactions with other proteins and lipids, these modifications transform α-syn into toxic species. In the present work, we unilaterally injected one hemisphere of the substantia nigra of rats with recombinant adeno-associated vectors (rAAV) mediating the expression of human wild-type α-syn or of human α-syn bearing the familial A30P mutation. In both cases, we engineered the human α-syn gene to replace its Serine 129 either with an Alanine (S129A), in order to prevent the attachment of a phosphate, or with an Aspartate (S129D) with the objective of mimicking the negative charge of a bound phosphate at this site. In our experimental design, rAAV-mediated human α-syn expression levels in the substantia nigra were roughly equivalent to those of the rat endogenous protein. Eight weeks after injection, we measured a significant, dose-dependent decline in the number of dopaminergic neurons injected with the wild-type form or with A30P, as compared to the non-injected side. Thus a single amino-acid modification at position 129 caused marked differences in the patterns of toxicity. Notably, expression of the S129A variant dramatically increased the pathology in rats, while expression of the S129D appeared to protect against neuronal loss. This response was the exact opposite of that obtained following expression of the S129A and S129D variants in transgenic Drosophila. We then showed that the enhanced dopaminergic loss triggered by S129A was accompanied by extensive presence of α-syn- and β-pleated sheet-positive, proteinase K-resistant small aggregates. Immuno-electron microscopy revealed that these electro-dense cytoplasmic aggregates were frequently associated with lysosomes and with a phagosome-like glassy body with vesicular content. α-syn S129A was also extensively present in intracytoplasmic tubulo-membranous systems, in the outer membrane of mitochondria, in the nucleus, and was associated with neurofilaments within neuritic fibers. The nuclei of cells exhibiting these aggregates were lobulated, and aggregates were particularly present in the indented regions of the nucleus. In addition to the morphological characteristics of apoptosis, such as nucleus condensation and lobulation, markers of apoptotic pathways were activated. Strikingly, despite the absence of dopaminergic cells loss, aggregates were present also in the S129D-injected brains, although to a much lesser extent, and apoptotic markers were also activated in these rats. The present study did not report any substantial differences that can be attributed to the expression of wild-type α-syn versus that of the A30P mutant, whether these were mutated at Serine 129 or not. However, the divergence of our results from those of the Drosophila raises the question of the adequacy of the fly as a model for PD, in view of its nervous system and its lack of any endogenous homologue to α-syn. The fact that cellular mechanisms of death were triggered by the expression of all variants, despite their significant differences in terms of induced nigral degeneration, indicates that discrepancies between variants mostly reside in the dynamics of the pathogenesis and/or in the toxic potential of the aggregated proteins. Therefore this work also brings new perspective to the current efforts to modulate kinases/phosphatases activity directed towards α-syn as a therapeutic tool.