Summary Aggregated alpha-synuclein inclusions are found where cell death occurs in several diseases, including Parkinson’s Disease, Dementia with Lewy Bodies and Multiple System Atrophy. However, the relationship between inclusion formation and an individual cell’s fate has been difficult to study with conventional techniques. We developed a system that allows for in vivo imaging of the same neurons over months. We show that intracerebral injection of preformed fibrils of recombinant alpha-synuclein can seed aggregation of transgenically-expressed and endogenous alpha-synuclein in neurons. Somatic inclusions undergo a stage-like maturation, with progressive compaction coinciding with decreased soluble somatic and nuclear alpha-synuclein. Mature inclusions bear the post-translational hallmarks of human Lewy pathology. Long-term imaging of inclusion-bearing neurons and neighboring neurons without inclusions demonstrates selective degeneration of inclusion-bearing cells. Our results indicate that inclusion formation is tightly correlated with cellular toxicity and that seeding may be a pathologically relevant mechanism of progressive neurodegeneration in many synucleinopathies.

Alpha-synuclein is a presynaptic protein that forms abnormal cytoplasmic aggregates in Lewy body disorders. Although nuclear alpha-synuclein localization has been described, its function in the nucleus is not well understood. We demonstrate that alpha-synuclein modulates DNA repair. First, alpha-synuclein colocalizes with DNA damage response components within discrete foci in human cells and mouse brain. Removal of alpha-synuclein in human cells leads to increased DNA double-strand break (DSB) levels after bleomycin treatment and a reduced ability to repair these DSBs. Similarly, alpha-synuclein knock-out mice show increased neuronal DSBs that can be rescued by transgenic reintroduction of human alpha-synuclein. Alpha-synuclein binds double-stranded DNA and helps to facilitate the non-homologous end-joining reaction. Using a new, in vivo imaging approach that we developed, we find that serine-129-phosphorylated alpha-synuclein is rapidly recruited to DNA damage sites in living mouse cortex. We find that Lewy inclusion-containing neurons in both mouse model and human-derived patient tissue demonstrate increased DSB levels. Based on these data, we propose a model whereby cytoplasmic aggregation of alpha-synuclein reduces its nuclear levels, increases DSBs, and may contribute to programmed cell death via nuclear loss-of-function. This model could inform development of new treatments for Lewy body disorders by targeting alpha-synuclein-mediated DNA repair mechanisms.

Parkinson's disease and dementia with Lewy bodies are associated with abnormal neuronal aggregation of ␣-synuclein. However, the mechanisms of aggregation and their relationship to disease are poorly understood. We developed an in vivo multiphoton imaging paradigm to study ␣-synuclein aggregation in mouse cortex with subcellular resolution. We used a green fluorescent protein-tagged human ␣-synuclein mouse line that has moderate overexpression levels mimicking human disease. Fluorescence recovery after photobleaching (FRAP) of labeled protein demonstrated that somatic ␣-synuclein existed primarily in an unbound, soluble pool. In contrast, ␣-synuclein in presynaptic terminals was in at least three different pools: (1) as unbound, soluble protein; (2) bound to presynaptic vesicles; and (3) as microaggregates. Serial imaging of microaggregates over 1 week demonstrated a heterogeneous population with differing ␣-synuclein exchange rates. The microaggregate species were resistant to proteinase K, phosphorylated at serine-129, oxidized, and associated with a decrease in the presynaptic vesicle protein synapsin and glutamate immunogold labeling. Multiphoton FRAP provided the specific binding constants for ␣-synuclein's binding to synaptic vesicles and its effective diffusion coefficient in the soma and axon, setting the stage for future studies targeting synuclein modifications and their effects. Our in vivo results suggest that, under moderate overexpression conditions, ␣-synuclein aggregates are selectively found in presynaptic terminals.