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 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 3

AAV-sh[SNCA] reduces α-synuclein expression in substantia nigra dopaminergic neurons.

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AAV-sh[SNCA] reduces α-synuclein expression in substantia nigra dopamine...
Two groups of 5 rats received either AAV-sh[SNCA] or AAV-sh[control] unilaterally in the substantia nigra (cohort 2). Forty-two days later, midbrain sections were immunolabeled for TH (green), α-synuclein (red), and DAPI (blue). Measurements of α-synuclein immunoreactivity were taken from nontransduced (white triangles), AAV-sh[control]–transduced (gray circles), or AAV-sh[SNCA]–transduced (black squares) substantia nigra dopaminergic neurons. (A and B) High-magnification confocal images of the substantia nigra from (A) no-vector and (B) AAV-sh[SNCA]–transduced sides of a single section. The positions of dopaminergic neuronal outlines revealed by TH labeling are shown as white dotted lines in the α-synuclein channel. Scale bars: 10 μm. (C) Cytoplasmic α-synuclein immunoreactivity was quantified in 60–100 nigral dopaminergic neurons on each side of each section by confocal imaging. The small markers show α-synuclein immunofluorescence signal for each cell on the vector-transduced side expressed as percentage of the mean value for dopaminergic neurons on the nontransduced side of the same section. The large markers show mean ± SEM for each animal. (D) Mean dopaminergic neuron α-synuclein immunofluorescence is shown for each side of the brain in each animal (+, vector side; –, nontransduced side; lines join the means for the two sides of each brain). The left graph shows measurements from animals that were transduced with AAV-sh[control] and the right AAV-sh[SNCA]. Large markers show mean ± SEM for all five animals in each group.*P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed paired t test, vector transduced side versus control side.