Characterization of Synphilin-1/alpha-Synuclein Double Transgenic Mice

Abstract

Parkinson's disease (PD) is a sporadic and progressive neurodegenerative disease characterized by dopaminergic neuronal cell death in the substantia nigra pars compacta and by the presence of characteristic Lewy bodies (LB) and Lewy neuritic (LN) inclusions. These inclusions are mainly constituted of the alpha-synuclein (a-syn) protein, suggesting a role of a-syn in the neuropathophysiology of PD. Synphilin-1 (sph1) is described as an interacting partner of a-syn, present in LBs and sharing the same intracellular compartments as a-syn. Sph1 was shown to induce the formation of a-syn positive inclusions in vivo and in vitro as well as to promote clearance of protein inclusions by autophagy. But the impact of sph1 in a-syn-mediated toxicity is inconsistent, presumably due to the variety of models used to model synucleinopathy, the variety of toxicity markers analyzed and the poor description of aggregates found in these studies. Furthermore, the normal function of sph1 needs still to be explored. The overall aim of this study is to characterize the effects of sph1 on a-syn aggregation and on the development of synucleinopathy in vivo. Therefore, we cross-bred mice overexpressing the human sph1 under the mouse PrP promoter with a synucleinopathy mouse model overexpressing the human A30P mutated a-syn under the Thy-1 promoter. Using different fractionation protocols of brain lysates, we did not observe in mice coexpressing sph1 an increased amount of soluble a-syn nor detergent-insoluble a-syn. At the histological level, we observed that the coexpression of sph1 drastically decreased levels of proteinase K resistant a-syn inclusions as well as thioflavin S positive structures. These changes were accompanied by the formation of aggresome-like structures positive for a-syn, K63 polyubiquitinated proteins and gamma-tubulin in double transgenic animals. We also observed in double transgenic mice a reduced neuropathology reflected by a decreased neuroinflammation and suggesting a protective role of sph1 in synucleinopathy. Accordingly, we demonstrated that sph1 reduces the accumulation of soluble full-length and truncated species of a-syn with aging. Similarly, we also shown that sph1 decreases levels of fibrillar forms of a-syn. Moreover, we also reported increased levels of ubiquitinated proteins in double transgenic mice without measuring an impairment of the ubiquitin-proteasome system activity. Interestingly, we observed an increased autophagic flux in double transgenic mice, suggesting a possible role of the autophagy-lysosome pathway in the reduction of a-syn species. We tracked the evolution of neuropathology and associated behavioral alterations over 18 months. We found that sph1 coexpression in transgenic mice overexpressing the human mutated A30P alpha-synuclein led to a delayed motor phenotype in rotarod when compared to single transgenic mice overexpressing A30P a-syn. A motor phenotype was also observed in single transgenic sph1 mice when using challenging beam walk. Interestingly, mouse gait analysis suggested a different origin in motor impairment in sph1 and A30P single transgenic mice.