Limits...
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.

Show MeSH

Related in: MedlinePlus

Efficient allele-specific suppression of mutant human ataxin-3 in the rat brain at an early time point (3 weeks) mediates striatal neuroprotection.A–L) Laser confocal microscopy showing the effects of recombinant lentiviral vectors encoding shAtaxMUT or shAtaxWT and MUT ATX3 in the rat brain striatum at an early time point (3 weeks). β-galactosidase, expressed from a separate PGK-lacZ cassette in the vectors, allows identification of infected neurons (A and D). shAtaxMUT specifically silences MUT ATX3, promoting the clearance of MUT ATX3-positive aggregates (E and J), whereas shAtaxWT has almost no effect on MUT ATX3 expression (B and G). A considerable loss of DARPP-32-immunoreactivity is observed in rat striatum co-infected with MUT ATX3 and shAtaxWT (H and the merged image I), whereas no DARPP-32 downregulation is observed in rat striatum co-infected with MUT ATX3 and shAtaxMUT (K and the merged image L), suggesting neuroprotection. The adult rats were co-injected bilaterally in the striatum with MUT ATX3 and the shAtaxWT or shAtaxMUT vectors (n = 2) and were killed three weeks later. All the pictures were taken around the injection site.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2553199&req=5

pone-0003341-g004: Efficient allele-specific suppression of mutant human ataxin-3 in the rat brain at an early time point (3 weeks) mediates striatal neuroprotection.A–L) Laser confocal microscopy showing the effects of recombinant lentiviral vectors encoding shAtaxMUT or shAtaxWT and MUT ATX3 in the rat brain striatum at an early time point (3 weeks). β-galactosidase, expressed from a separate PGK-lacZ cassette in the vectors, allows identification of infected neurons (A and D). shAtaxMUT specifically silences MUT ATX3, promoting the clearance of MUT ATX3-positive aggregates (E and J), whereas shAtaxWT has almost no effect on MUT ATX3 expression (B and G). A considerable loss of DARPP-32-immunoreactivity is observed in rat striatum co-infected with MUT ATX3 and shAtaxWT (H and the merged image I), whereas no DARPP-32 downregulation is observed in rat striatum co-infected with MUT ATX3 and shAtaxMUT (K and the merged image L), suggesting neuroprotection. The adult rats were co-injected bilaterally in the striatum with MUT ATX3 and the shAtaxWT or shAtaxMUT vectors (n = 2) and were killed three weeks later. All the pictures were taken around the injection site.

Mentions: In animals infected with lentiviral vectors encoding mutant ataxin-3(C) and the non specific shAtaxWT(G) or the shGFP control, immunohistochemical analysis of coronal rat brain sections with anti-ataxin-3 (1H9) and β-galactosidase antibodies showed that many neurons expressed both transgenes (Fig. 4A–C, Fig 5A–L). In animals expressing mutant ataxin-3 and shAtaxMUT(C), far fewer neurons produced the pathogenic protein (Fig. 4E, Fig. 5M). The merged images (Fig. 4F, Fig. 5O) indicate that only a few mutant ataxin-3-positive-cells did not express the lacZ reporter gene present in the shAtaxMUT(C) vector (high magnification Fig. 5R). These cells corresponded to neurons not co-infected with both vectors, which were therefore not treated with the siRNA.


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)

Efficient allele-specific suppression of mutant human ataxin-3 in the rat brain at an early time point (3 weeks) mediates striatal neuroprotection.A–L) Laser confocal microscopy showing the effects of recombinant lentiviral vectors encoding shAtaxMUT or shAtaxWT and MUT ATX3 in the rat brain striatum at an early time point (3 weeks). β-galactosidase, expressed from a separate PGK-lacZ cassette in the vectors, allows identification of infected neurons (A and D). shAtaxMUT specifically silences MUT ATX3, promoting the clearance of MUT ATX3-positive aggregates (E and J), whereas shAtaxWT has almost no effect on MUT ATX3 expression (B and G). A considerable loss of DARPP-32-immunoreactivity is observed in rat striatum co-infected with MUT ATX3 and shAtaxWT (H and the merged image I), whereas no DARPP-32 downregulation is observed in rat striatum co-infected with MUT ATX3 and shAtaxMUT (K and the merged image L), suggesting neuroprotection. The adult rats were co-injected bilaterally in the striatum with MUT ATX3 and the shAtaxWT or shAtaxMUT vectors (n = 2) and were killed three weeks later. All the pictures were taken around the injection site.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003341-g004: Efficient allele-specific suppression of mutant human ataxin-3 in the rat brain at an early time point (3 weeks) mediates striatal neuroprotection.A–L) Laser confocal microscopy showing the effects of recombinant lentiviral vectors encoding shAtaxMUT or shAtaxWT and MUT ATX3 in the rat brain striatum at an early time point (3 weeks). β-galactosidase, expressed from a separate PGK-lacZ cassette in the vectors, allows identification of infected neurons (A and D). shAtaxMUT specifically silences MUT ATX3, promoting the clearance of MUT ATX3-positive aggregates (E and J), whereas shAtaxWT has almost no effect on MUT ATX3 expression (B and G). A considerable loss of DARPP-32-immunoreactivity is observed in rat striatum co-infected with MUT ATX3 and shAtaxWT (H and the merged image I), whereas no DARPP-32 downregulation is observed in rat striatum co-infected with MUT ATX3 and shAtaxMUT (K and the merged image L), suggesting neuroprotection. The adult rats were co-injected bilaterally in the striatum with MUT ATX3 and the shAtaxWT or shAtaxMUT vectors (n = 2) and were killed three weeks later. All the pictures were taken around the injection site.
Mentions: In animals infected with lentiviral vectors encoding mutant ataxin-3(C) and the non specific shAtaxWT(G) or the shGFP control, immunohistochemical analysis of coronal rat brain sections with anti-ataxin-3 (1H9) and β-galactosidase antibodies showed that many neurons expressed both transgenes (Fig. 4A–C, Fig 5A–L). In animals expressing mutant ataxin-3 and shAtaxMUT(C), far fewer neurons produced the pathogenic protein (Fig. 4E, Fig. 5M). The merged images (Fig. 4F, Fig. 5O) indicate that only a few mutant ataxin-3-positive-cells did not express the lacZ reporter gene present in the shAtaxMUT(C) vector (high magnification Fig. 5R). These cells corresponded to neurons not co-infected with both vectors, which were therefore not treated with the siRNA.

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