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Allele-specific RNA silencing of mutant ataxin-3 mediates neuroprotection in a rat model of Machado-Joseph disease.

Alves S, Nascimento-Ferreira I, Auregan G, Hassig R, Dufour N, Brouillet E, Pedroso de Lima MC, Hantraye P, Pereira de Almeida L, Déglon N - PLoS ONE (2008)

Bottom Line: Lentiviral-mediated silencing of mutant human ataxin-3 was demonstrated in vitro and in a rat model of MJD in vivo.The allele-specific silencing of ataxin-3 significantly decreased the severity of the neuropathological abnormalities associated with MJD.These data demonstrate that RNAi has potential for use in MJD treatment and constitute the first proof-of-principle for allele-specific silencing in the central nervous system.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurosciences and Cell Biology, University of Coimbra, Coimbra, Portugal.

ABSTRACT
Recent studies have demonstrated that RNAi is a promising approach for treating autosomal dominant disorders. However, discrimination between wild-type and mutant transcripts is essential, to preserve wild-type expression and function. A single nucleotide polymorphism (SNP) is present in more than 70% of patients with Machado-Joseph disease (MJD). We investigated whether this SNP could be used to inactivate mutant ataxin-3 selectively. Lentiviral-mediated silencing of mutant human ataxin-3 was demonstrated in vitro and in a rat model of MJD in vivo. The allele-specific silencing of ataxin-3 significantly decreased the severity of the neuropathological abnormalities associated with MJD. These data demonstrate that RNAi has potential for use in MJD treatment and constitute the first proof-of-principle for allele-specific silencing in the central nervous system.

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Reduction of ubiquitin-positive inclusions in the striatum of adult rats as result of mutant human ataxin-3 knock-down.Animals infected with MUT ATX3 and the control shGFP (left; n = 4) or shAtaxWT (right, n = 8) show the accumulation of ubiquitin-positive inclusions, typical biomarkers of neuropathology, whereas no such accumulation is observed in animals co-infected with MUT ATX3 and the selective shAtaxMUT (middle, n = 7). The figure shows representative images of ubiquitin immunohistochemical stainings that were reproducible among the different groups.
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pone-0003341-g007: Reduction of ubiquitin-positive inclusions in the striatum of adult rats as result of mutant human ataxin-3 knock-down.Animals infected with MUT ATX3 and the control shGFP (left; n = 4) or shAtaxWT (right, n = 8) show the accumulation of ubiquitin-positive inclusions, typical biomarkers of neuropathology, whereas no such accumulation is observed in animals co-infected with MUT ATX3 and the selective shAtaxMUT (middle, n = 7). The figure shows representative images of ubiquitin immunohistochemical stainings that were reproducible among the different groups.

Mentions: We assessed the efficacy of this approach further, by carrying out a, histological evaluation at two months, at which time MJD pathology was severe [4]. β-galactosidase staining indicated a similar transduction efficiency for all groups eight weeks post-injection (Fig. 6A–C). In animals injected with mutant ataxin-3, we observed a typical accumulation of inclusions (Fig. 6D, G, P), with a mean size of 34.3±1.4 µm2 (Fig. 6Q) and a loss of DARPP-32 production (Fig. 6R). Comparison between this group and that including animals treated with the non selective siRNA (shAtaxWT(G); Fig. 6C, F, I, L, O) revealed no statistically significant differences in terms of the formation of inclusions (Fig. 6P, Q) and the DARPP-32-depleted area (Fig. 6R). Co-transduction with mutant ataxin-3(C) and shAtaxMUT(C) significantly decreased the number of inclusions (48.2±10.8 % considering shGFP as the control; 55.5%±9.3% considering shAtaxWT as the control) and the apparent size of the remaining inclusions (by 12.7±4.3% if compared with shGFP and 9.92±4.4 % if compared with shAtaxWT(G); Fig. 6P–R). Similar results were obtained with ubiquitin staining, which also showed important reduction in the number of inclusions upon mutant ataxin-3 silencing with shAtaxMUT (Fig. 7). Double staining for DARPP-32 and ataxin-3 showed co-localization between ataxin-3 inclusions and DARPP-32 loss of immunoreactivity in control animals (Fig. 8A–F), and rescue of DARPP-32 immunoreactivity co-localizing with reduction of inclusion number upon shAtaxMUT expression (Fig. 8G–I).


Allele-specific RNA silencing of mutant ataxin-3 mediates neuroprotection in a rat model of Machado-Joseph disease.

Alves S, Nascimento-Ferreira I, Auregan G, Hassig R, Dufour N, Brouillet E, Pedroso de Lima MC, Hantraye P, Pereira de Almeida L, Déglon N - PLoS ONE (2008)

Reduction of ubiquitin-positive inclusions in the striatum of adult rats as result of mutant human ataxin-3 knock-down.Animals infected with MUT ATX3 and the control shGFP (left; n = 4) or shAtaxWT (right, n = 8) show the accumulation of ubiquitin-positive inclusions, typical biomarkers of neuropathology, whereas no such accumulation is observed in animals co-infected with MUT ATX3 and the selective shAtaxMUT (middle, n = 7). The figure shows representative images of ubiquitin immunohistochemical stainings that were reproducible among the different groups.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2553199&req=5

pone-0003341-g007: Reduction of ubiquitin-positive inclusions in the striatum of adult rats as result of mutant human ataxin-3 knock-down.Animals infected with MUT ATX3 and the control shGFP (left; n = 4) or shAtaxWT (right, n = 8) show the accumulation of ubiquitin-positive inclusions, typical biomarkers of neuropathology, whereas no such accumulation is observed in animals co-infected with MUT ATX3 and the selective shAtaxMUT (middle, n = 7). The figure shows representative images of ubiquitin immunohistochemical stainings that were reproducible among the different groups.
Mentions: We assessed the efficacy of this approach further, by carrying out a, histological evaluation at two months, at which time MJD pathology was severe [4]. β-galactosidase staining indicated a similar transduction efficiency for all groups eight weeks post-injection (Fig. 6A–C). In animals injected with mutant ataxin-3, we observed a typical accumulation of inclusions (Fig. 6D, G, P), with a mean size of 34.3±1.4 µm2 (Fig. 6Q) and a loss of DARPP-32 production (Fig. 6R). Comparison between this group and that including animals treated with the non selective siRNA (shAtaxWT(G); Fig. 6C, F, I, L, O) revealed no statistically significant differences in terms of the formation of inclusions (Fig. 6P, Q) and the DARPP-32-depleted area (Fig. 6R). Co-transduction with mutant ataxin-3(C) and shAtaxMUT(C) significantly decreased the number of inclusions (48.2±10.8 % considering shGFP as the control; 55.5%±9.3% considering shAtaxWT as the control) and the apparent size of the remaining inclusions (by 12.7±4.3% if compared with shGFP and 9.92±4.4 % if compared with shAtaxWT(G); Fig. 6P–R). Similar results were obtained with ubiquitin staining, which also showed important reduction in the number of inclusions upon mutant ataxin-3 silencing with shAtaxMUT (Fig. 7). Double staining for DARPP-32 and ataxin-3 showed co-localization between ataxin-3 inclusions and DARPP-32 loss of immunoreactivity in control animals (Fig. 8A–F), and rescue of DARPP-32 immunoreactivity co-localizing with reduction of inclusion number upon shAtaxMUT expression (Fig. 8G–I).

Bottom Line: Lentiviral-mediated silencing of mutant human ataxin-3 was demonstrated in vitro and in a rat model of MJD in vivo.The allele-specific silencing of ataxin-3 significantly decreased the severity of the neuropathological abnormalities associated with MJD.These data demonstrate that RNAi has potential for use in MJD treatment and constitute the first proof-of-principle for allele-specific silencing in the central nervous system.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurosciences and Cell Biology, University of Coimbra, Coimbra, Portugal.

ABSTRACT
Recent studies have demonstrated that RNAi is a promising approach for treating autosomal dominant disorders. However, discrimination between wild-type and mutant transcripts is essential, to preserve wild-type expression and function. A single nucleotide polymorphism (SNP) is present in more than 70% of patients with Machado-Joseph disease (MJD). We investigated whether this SNP could be used to inactivate mutant ataxin-3 selectively. Lentiviral-mediated silencing of mutant human ataxin-3 was demonstrated in vitro and in a rat model of MJD in vivo. The allele-specific silencing of ataxin-3 significantly decreased the severity of the neuropathological abnormalities associated with MJD. These data demonstrate that RNAi has potential for use in MJD treatment and constitute the first proof-of-principle for allele-specific silencing in the central nervous system.

Show MeSH
Related in: MedlinePlus