Abstract
Alpha synuclein (αsyn) aggregates are associated with the pathogenesis of Parkinson's disease and others related disorders. Although modulation of αsyn aggregation is an attractive therapeutic target, new powerful methodologies are desperately needed to facilitate in vivo screening of novel therapeutics. Here, we describe an in vivo rodent model with the unique ability to rapidly track αsyn-αsyn interactions and thus oligomerization using a bioluminescent protein complementation strategy that monitors spatial and temporal αsyn oligomerization ex vivo. We find that αsyn forms oligomers in vivo as early as 1 week after stereotactic AAV injection into rat substantia nigra. Strikingly, although abundant αsyn expression is also detected in striatum at 1 week, no αsyn oligomers are detected at this time point. By 4 weeks, oligomerization of αsyn is detected in both striatum and substantia nigra homogenates. Moreover, in a proof-of-principle experiment, the effect of a previously described Hsp90 inhibitor known to prevent αsyn oligomer formation, demonstrates the utility of this rapid and sensitive animal model to monitor αsyn oligomerization status in the rat brain.
Highlights
Under pathological conditions alpha-synuclein can misfold and aggregate into insoluble deposits that accumulate in cells to toxic levels
The bioluminescent αsyn protein fragment complementation assay (PCA) strategy has been widely used by our lab and others (Outeiro et al, 2008; Putcha et al, 2010; Danzer et al, 2011; Moussaud et al, 2015) to investigate αsyn oligomerization in living cells
Two hαsyn proteins fused to N- or C-terminal halves of a reporter protein can reconstitute the enzymatic activity of the reporter when αsynαsyn interactions occur, providing a readout of αsynαsyn interactions and oligomerization
Summary
Under pathological conditions alpha-synuclein (αsyn) can misfold and aggregate into insoluble deposits that accumulate in cells to toxic levels. The conversion of αsyn from its functional conformation into a misfolded and toxic conformation constitutes the basis of a group of diseases known as synucleinopathies which include Parkinson’s disease (PD), multiple system atrophy, and dementia with Lewy bodies (Goedert and Spillantini, 1998; Kim et al, 2014). Fibrillar forms of αsyn are the major component of glial cytoplasmic inclusions, Lewy bodies (LBs), and Lewy neurites, defined as intracytoplasmatic inclusions and considered the pathological hallmarks of synucleopathies (Forno, 1996; Spillantini et al, 1997; Halliday et al, 2011). The processes that lead to pathological aggregate formation occur through
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