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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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Allele-specific silencing of mutant human ataxin-3 mediates robust reduction of the number of ataxin-3 inclusions and preservation of DARPP-32 immunoreactivity in the rat striatum.A–R) Co-overexpression of MUT ATX3 and various shRNAs (shAtaxWT, n = 8; shAtaxMUT, n = 7 and shGFP, n = 4) in the striatum of adult rats, 2 months post-injection. The vectors encoding the shRNA cassette and the lacZ reporter gene infect an extensive region of the rat striatum, as shown by β-galactosidase immunoreactivity (A, B and C). shAtaxMUT specifically downregulates MUT ATX3, promoting a significant decrease in the number of MUT ATX3-positive aggregates (E and H ), whereas shAtaxWT has almost no effect on MUT ATX3 expression (F and I), as shown by comparison with the results obtained with the mistargeted shGFP (D and G). A major loss of DARPP-32 immunoreactivity is observed in the striatum infected with MUT ATX3 and shAtaxWT (L and O) or shGFP (J and M), whereas minor DARPP-32 is observed in the striatum infected with shAtaxMUT (K and N), this downregulation being limited to the needle track area. P–R) Quantification of the effect of the different shRNAs on the absolute number (P) and mean size/surface* (Q) of MUT ATX3-positive cells (*p<0,05). R) Quantitative analysis of the DARPP-32-depleted region in the brains of rats in which the striatum was injected with MUT ATX3 and various shRNAs. The lesion volume in brains infected with shAtaxMUT and MUT ATX3 is much smaller than that in brains infected with shAtaxWT or shGFP, indicative of a neuroprotective effect conferred by the selective shAtaxMUT. Statistical significance was evaluated with Fisher's test. (* the mean size of the objects was estimated taking into account the pixels with a gray-scale level for intensity below the mean value, used as a threshold).
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pone-0003341-g006: Allele-specific silencing of mutant human ataxin-3 mediates robust reduction of the number of ataxin-3 inclusions and preservation of DARPP-32 immunoreactivity in the rat striatum.A–R) Co-overexpression of MUT ATX3 and various shRNAs (shAtaxWT, n = 8; shAtaxMUT, n = 7 and shGFP, n = 4) in the striatum of adult rats, 2 months post-injection. The vectors encoding the shRNA cassette and the lacZ reporter gene infect an extensive region of the rat striatum, as shown by β-galactosidase immunoreactivity (A, B and C). shAtaxMUT specifically downregulates MUT ATX3, promoting a significant decrease in the number of MUT ATX3-positive aggregates (E and H ), whereas shAtaxWT has almost no effect on MUT ATX3 expression (F and I), as shown by comparison with the results obtained with the mistargeted shGFP (D and G). A major loss of DARPP-32 immunoreactivity is observed in the striatum infected with MUT ATX3 and shAtaxWT (L and O) or shGFP (J and M), whereas minor DARPP-32 is observed in the striatum infected with shAtaxMUT (K and N), this downregulation being limited to the needle track area. P–R) Quantification of the effect of the different shRNAs on the absolute number (P) and mean size/surface* (Q) of MUT ATX3-positive cells (*p<0,05). R) Quantitative analysis of the DARPP-32-depleted region in the brains of rats in which the striatum was injected with MUT ATX3 and various shRNAs. The lesion volume in brains infected with shAtaxMUT and MUT ATX3 is much smaller than that in brains infected with shAtaxWT or shGFP, indicative of a neuroprotective effect conferred by the selective shAtaxMUT. Statistical significance was evaluated with Fisher's test. (* the mean size of the objects was estimated taking into account the pixels with a gray-scale level for intensity below the mean value, used as a threshold).

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)

Allele-specific silencing of mutant human ataxin-3 mediates robust reduction of the number of ataxin-3 inclusions and preservation of DARPP-32 immunoreactivity in the rat striatum.A–R) Co-overexpression of MUT ATX3 and various shRNAs (shAtaxWT, n = 8; shAtaxMUT, n = 7 and shGFP, n = 4) in the striatum of adult rats, 2 months post-injection. The vectors encoding the shRNA cassette and the lacZ reporter gene infect an extensive region of the rat striatum, as shown by β-galactosidase immunoreactivity (A, B and C). shAtaxMUT specifically downregulates MUT ATX3, promoting a significant decrease in the number of MUT ATX3-positive aggregates (E and H ), whereas shAtaxWT has almost no effect on MUT ATX3 expression (F and I), as shown by comparison with the results obtained with the mistargeted shGFP (D and G). A major loss of DARPP-32 immunoreactivity is observed in the striatum infected with MUT ATX3 and shAtaxWT (L and O) or shGFP (J and M), whereas minor DARPP-32 is observed in the striatum infected with shAtaxMUT (K and N), this downregulation being limited to the needle track area. P–R) Quantification of the effect of the different shRNAs on the absolute number (P) and mean size/surface* (Q) of MUT ATX3-positive cells (*p<0,05). R) Quantitative analysis of the DARPP-32-depleted region in the brains of rats in which the striatum was injected with MUT ATX3 and various shRNAs. The lesion volume in brains infected with shAtaxMUT and MUT ATX3 is much smaller than that in brains infected with shAtaxWT or shGFP, indicative of a neuroprotective effect conferred by the selective shAtaxMUT. Statistical significance was evaluated with Fisher's test. (* the mean size of the objects was estimated taking into account the pixels with a gray-scale level for intensity below the mean value, used as a threshold).
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Related In: Results  -  Collection

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

pone-0003341-g006: Allele-specific silencing of mutant human ataxin-3 mediates robust reduction of the number of ataxin-3 inclusions and preservation of DARPP-32 immunoreactivity in the rat striatum.A–R) Co-overexpression of MUT ATX3 and various shRNAs (shAtaxWT, n = 8; shAtaxMUT, n = 7 and shGFP, n = 4) in the striatum of adult rats, 2 months post-injection. The vectors encoding the shRNA cassette and the lacZ reporter gene infect an extensive region of the rat striatum, as shown by β-galactosidase immunoreactivity (A, B and C). shAtaxMUT specifically downregulates MUT ATX3, promoting a significant decrease in the number of MUT ATX3-positive aggregates (E and H ), whereas shAtaxWT has almost no effect on MUT ATX3 expression (F and I), as shown by comparison with the results obtained with the mistargeted shGFP (D and G). A major loss of DARPP-32 immunoreactivity is observed in the striatum infected with MUT ATX3 and shAtaxWT (L and O) or shGFP (J and M), whereas minor DARPP-32 is observed in the striatum infected with shAtaxMUT (K and N), this downregulation being limited to the needle track area. P–R) Quantification of the effect of the different shRNAs on the absolute number (P) and mean size/surface* (Q) of MUT ATX3-positive cells (*p<0,05). R) Quantitative analysis of the DARPP-32-depleted region in the brains of rats in which the striatum was injected with MUT ATX3 and various shRNAs. The lesion volume in brains infected with shAtaxMUT and MUT ATX3 is much smaller than that in brains infected with shAtaxWT or shGFP, indicative of a neuroprotective effect conferred by the selective shAtaxMUT. Statistical significance was evaluated with Fisher's test. (* the mean size of the objects was estimated taking into account the pixels with a gray-scale level for intensity below the mean value, used as a threshold).
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