In Vivo RNAi-Mediated α-Synuclein Silencing Induces Nigrostriatal Degeneration

Abstract

Two small-interfering RNAs (siRNAs) targeting alpha-synuclein (alpha-syn) and three control siRNAs were cloned in an adeno-associated virus (AAV) vector and unilaterally injected into rat substantia nigra pars compacta (SNc). Reduction of alpha-syn resulted in a rapid (4 week) reduction in the number of tyrosine hydroxylase (TH) positive cells and striatal dopamine (DA) on the injected side. The level of neurodegeneration induced by the different siRNAs correlated with their ability to downregulate alpha-syn protein and mRNA in tissue culture and in vivo. Examination of various SNc neuronal markers indicated that neurodegeneration was due to cell loss and not just downregulation of DA synthesis. Reduction of alpha-syn also resulted in a pronounced amphetamine induced behavioral asymmetry consistent with the level of neurodegeneration. In contrast, none of the three control siRNAs, which targeted genes not normally expressed in SNc, showed evidence of neurodegeneration or behavioral asymmetry, even at longer survival times. Moreover, co-expression of both rat alpha-syn and alpha-syn siRNA partially reversed the neurodegenerative and behavioral effects of alpha-syn siRNA alone. Our data show that alpha-syn plays an important role in the rat SNc and suggest that both up- and downregulation of wild-type alpha-syn expression increase the risk of nigrostriatal pathology.

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

“…This may have altered the relative expression levels of the shRNA in dopaminergic neurons. In combination, these methodological differences may account for the reported disparity in the level of α-synuclein knockdown: 70%-85% loss of nigral α-synuclein expression was found at 4 weeks after transduction in the previous study (64), compared with ≈35% knockdown at 6 weeks in the present work. Consequently, it is possible that the degree of acute α-synuclein knockdown determines whether toxicity occurs.…”

“…In contrast, a single previous study unexpectedly found rapidly progressive neurodegeneration in rats following AAVmediated delivery of shRNA directed at the Snca transcript (64). TH-immunoreactive neurons were depleted in the nigra by more than 90% at 4 weeks after transduction, and this was associated with loss of striatal TH expression and asymmetric evoked motor function.…”

“…However, acute knockdown of Snca in the adult brain could potentially cause unwanted effects by circumventing developmental compensatory mechanisms that occur in germline null mutants. There are four previous reports of RNAi-mediated knockdown of endogenous α-synuclein in the adult CNS in vivo (63)(64)(65)(66). Naked siRNA infused into the mouse hippocampus gave rise to a significant 60%-80% reduction in Snca mRNA and a qualitative decrease in α-synuclein protein levels, but no overt cell loss or microgliosis (65).…”

RNAi Therapeutic Potentials and Prospects in CNS Disease

“…To date, conflicting results were reported. Regarding the effectiveness and tolerability, there is a report that nigrostriatal degeneration was detected after depleting the α-syn level in the brain [138]. It can be inferred that RNAi approaches can be used to validate them in genetic and sporadic models of PD.…”

Genetic inactivation of alpha-synuclein affects embryonic development of dopaminergic neurons of the substantia nigra, but not the ventral tegmental area, in mouse brain

“…Alpha-36 synuclein exerts a modulating effect on the formation of DA neurons in the SN and 37 has no effect on the formation of DA neurons in VTA, the structure that is much less 38 susceptible to degeneration in PD brain, suggesting a potential role of alpha-39 synuclein in the development of the population of DA neurons in substantia nigra. (Gorbatyuk et al 2010;Robertson et al 2004), and sensitivity to neurotoxins [9]. 55 The key mechanism of pathology of PD, severe loss of DA neurons of the SN, 56 remains a major focus of research on the way to revealing specific molecular targets 57 for developing the disease-modifying drugs.…”