shRNA targeting α-synuclein prevents neurodegeneration in a Parkinson’s disease model
Alevtina D. Zharikov, … , J. Timothy Greenamyre, Edward A. Burton
Alevtina D. Zharikov, … , J. Timothy Greenamyre, Edward A. Burton
Published July 1, 2015; First published June 15, 2015
Citation Information: J Clin Invest. 2015;125(7):2721-2735. https://doi.org/10.1172/JCI64502.
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Research Article Neuroscience

shRNA targeting α-synuclein prevents neurodegeneration in a Parkinson’s disease model

Abstract

Multiple convergent lines of evidence implicate both α-synuclein (encoded by SCNA) and mitochondrial dysfunction in the pathogenesis of sporadic Parkinson’s disease (PD). Occupational exposure to the mitochondrial complex I inhibitor rotenone increases PD risk; rotenone-exposed rats show systemic mitochondrial defects but develop specific neuropathology, including α-synuclein aggregation and degeneration of substantia nigra dopaminergic neurons. Here, we inhibited expression of endogenous α-synuclein in the adult rat substantia nigra by adeno-associated virus–mediated delivery of a short hairpin RNA (shRNA) targeting the endogenous rat Snca transcript. Knockdown of α-synuclein by ~35% did not affect motor function or cause degeneration of nigral dopaminergic neurons in control rats. However, in rotenone-exposed rats, progressive motor deficits were substantially attenuated contralateral to α-synuclein knockdown. Correspondingly, rotenone-induced degeneration of nigral dopaminergic neurons, their dendrites, and their striatal terminals was decreased ipsilateral to α-synuclein knockdown. These data show that α-synuclein knockdown is neuroprotective in the rotenone model of PD and indicate that endogenous α-synuclein contributes to the specific vulnerability of dopaminergic neurons to systemic mitochondrial inhibition. Our findings are consistent with a model in which genetic variants influencing α-synuclein expression modulate cellular susceptibility to environmental exposures in PD patients. shRNA targeting the SNCA transcript should be further evaluated as a possible neuroprotective therapy in PD.

Authors

Alevtina D. Zharikov, Jason R. Cannon, Victor Tapias, Qing Bai, Max P. Horowitz, Vipul Shah, Amina El Ayadi, Teresa G. Hastings, J. Timothy Greenamyre, Edward A. Burton

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Figure 1

Generation of a viral vector to target endogenous α-synuclein in vivo.

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Generation of a viral vector to target endogenous α-synuclein in vivo. (...
(A) siRNAs targeting Snca were identified and tested in vitro (Supplemental Figures 1–3). The most effective of these, siRNA526, is shown in red. Multiple base mismatches between siRNA526 and the corresponding sequences of Sncb and Sncg (encoding β- and γ-synucleins, respectively) are colored green. (B) A variety of viral vectors was evaluated for in vivo gene transfer to the rat substantia nigra (Supplemental Figure 4). An adeno-associated virus serotype 2 (AAV2) vector expressing a GFP reporter gene showed extensive transduction of TH+ nigral dopaminergic neurons (upper panels), with resulting GFP expression in their striatal terminals (lower panels). Scale bars: 100 μm. (C) An AAV2 vector, AAV-sh[SNCA], was constructed to express shRNA526 targeting Snca from the U6 promoter and a GFP reporter from a separate expression cassette. The control vector AAV-sh[control] was isogenic to AAV-sh[SNCA], except that it expressed a nontargeting shRNA instead of shRNA526.