Abstract
Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly-inherited neurodegenerative disorder caused by the over-repetition of a CAG codon in the MJD1 gene. This expansion translates into a polyglutamine tract that confers a toxic gain-of-function to the mutant protein – ataxin-3, leading to neurodegeneration in specific brain regions, with particular severity in the cerebellum. No treatment able to modify the disease progression is available. However, gene silencing by RNA interference has shown promising results. Therefore, in this study we investigated whether lentiviral-mediated allele-specific silencing of the mutant ataxin-3 gene, after disease onset, would rescue the motor behavior deficits and neuropathological features in a severely impaired transgenic mouse model of MJD. For this purpose, we injected lentiviral vectors encoding allele-specific silencing-sequences (shAtx3) into the cerebellum of diseased transgenic mice expressing the targeted C-variant of mutant ataxin-3 present in 70% of MJD patients. This variation permits to discriminate between the wild-type and mutant forms, maintaining the normal function of the wild-type allele and silencing only the mutant form. Quantitative analysis of rotarod performance, footprint and activity patterns revealed significant and robust alleviation of gait, balance (average 3-fold increase of rotarod test time), locomotor and exploratory activity impairments in shAtx3-injected mice, as compared to control ones injected with shGFP. An important improvement of neuropathology was also observed, regarding the number of intranuclear inclusions, calbindin and DARPP-32 immunoreactivity, fluorojade B and Golgi staining and molecular and granular layers thickness. These data demonstrate for the first time the efficacy of gene silencing in blocking the MJD-associated motor-behavior and neuropathological abnormalities after the onset of the disease, supporting the use of this strategy for therapy of MJD.
Highlights
Machado-Joseph disease (MJD), designated spinocerebellar ataxia type 3 (SCA3), is the most common dominantly-inherited cerebellar ataxia worldwide [1,2,3,4]
We investigated if specific silencing would reduce the number of aggregates in the cerebellum, a region that is involved in MJD, in a transgenic mouse model of MJD with particular expression of mutant ataxin-3 in Purkinje cells [15]
Further analysis revealed that mice injected with the control vectors encoding the short-hairpin targeting GFP exhibit ataxin-3 aggregates (HA tag) that co-localize with the co-expressed LacZ reporter gene; whereas aggregates from mice injected with vectors encoding the shorthairpin against mutant ataxin-3 do not co-localize with LacZ and correspond to non-transduced cells (Figure S4)
Summary
Machado-Joseph disease (MJD), designated spinocerebellar ataxia type 3 (SCA3), is the most common dominantly-inherited cerebellar ataxia worldwide [1,2,3,4]. It is part of a group of nine known polyglutamine (polyQ) disorders which share expanded CAG repeat mutations that translate into polyQ tracts [5,6]. The signs and symptoms of MJD include progressive postural instability, gait and limb ataxia, weight loss and, in severe cases, premature death [2,7,8]. The polyQ expansion confers a toxic gain-offunction to the mutant protein, leading to the formation of neuronal intranuclear inclusions, neuronal dysfunction and degeneration [13]
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