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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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Rescue of DARPP-32 immunoreactivity and reduced accumulation of ataxin-3 inclusions upon shAtaxMUT expression.A–I) Laser confocal microscopy showing the expression of recombinant lentiviral vectors expressing MUT ATX3 and shGFP (n = 4), shAtaxWT (n = 8) or shMUT (n = 7) and its effects on DARPP-32 expression in the rat striatum 2 months post-injection. Slight DARPP-32 downregulation is observed in striatum infected with MUT ATX3 and the selective shAtaxMUT (H and the merged image I), whereas a significant loss of DARPP-32-immunoreactive neurons is observed in rat striatum co-infected with MUT ATX3 and shAtaxWT (E and the merged image F) or MUT ATX3 and the control shGFP (B and the merged image C). The figure shows representative images of immunohistochemical stainings that were reproducible among the different groups.
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pone-0003341-g008: Rescue of DARPP-32 immunoreactivity and reduced accumulation of ataxin-3 inclusions upon shAtaxMUT expression.A–I) Laser confocal microscopy showing the expression of recombinant lentiviral vectors expressing MUT ATX3 and shGFP (n = 4), shAtaxWT (n = 8) or shMUT (n = 7) and its effects on DARPP-32 expression in the rat striatum 2 months post-injection. Slight DARPP-32 downregulation is observed in striatum infected with MUT ATX3 and the selective shAtaxMUT (H and the merged image I), whereas a significant loss of DARPP-32-immunoreactive neurons is observed in rat striatum co-infected with MUT ATX3 and shAtaxWT (E and the merged image F) or MUT ATX3 and the control shGFP (B and the merged image C). The figure shows representative images of 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)

Rescue of DARPP-32 immunoreactivity and reduced accumulation of ataxin-3 inclusions upon shAtaxMUT expression.A–I) Laser confocal microscopy showing the expression of recombinant lentiviral vectors expressing MUT ATX3 and shGFP (n = 4), shAtaxWT (n = 8) or shMUT (n = 7) and its effects on DARPP-32 expression in the rat striatum 2 months post-injection. Slight DARPP-32 downregulation is observed in striatum infected with MUT ATX3 and the selective shAtaxMUT (H and the merged image I), whereas a significant loss of DARPP-32-immunoreactive neurons is observed in rat striatum co-infected with MUT ATX3 and shAtaxWT (E and the merged image F) or MUT ATX3 and the control shGFP (B and the merged image C). The figure shows representative images of immunohistochemical stainings that were reproducible among the different groups.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553199&req=5

pone-0003341-g008: Rescue of DARPP-32 immunoreactivity and reduced accumulation of ataxin-3 inclusions upon shAtaxMUT expression.A–I) Laser confocal microscopy showing the expression of recombinant lentiviral vectors expressing MUT ATX3 and shGFP (n = 4), shAtaxWT (n = 8) or shMUT (n = 7) and its effects on DARPP-32 expression in the rat striatum 2 months post-injection. Slight DARPP-32 downregulation is observed in striatum infected with MUT ATX3 and the selective shAtaxMUT (H and the merged image I), whereas a significant loss of DARPP-32-immunoreactive neurons is observed in rat striatum co-infected with MUT ATX3 and shAtaxWT (E and the merged image F) or MUT ATX3 and the control shGFP (B and the merged image C). The figure shows representative images of 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