Striatal and nigral pathology in a lentiviral rat model of Machado-Joseph disease

Machado-Joseph disease (MJD) is a fatal, dominant neurodegenerative disorder. MJD results from polyglutamine repeat expansion in the MJD-1 gene, conferring a toxic gain of function to the ataxin-3 protein. In this study, we aimed at overexpressing ataxin-3 in the rat brain using lentiviral vectors (LV), to generate an in vivo MJD genetic model and, to study the disorder in defined brain regions: substantia nigra, an area affected in MJD, cortex and striatum, regions not previously reported to be affected in MJD. LV encoding mutant or wild-type human ataxin-3 was injected in the brain of adult rats and the animals were tested for behavioral deficits and neuropathological abnormalities. Striatal pathology was confirmed in transgenic mice and human tissue. In substantia nigra, unilateral overexpression of mutant ataxin-3 led to: apomorphine-induced turning behavior; formation of ubiquitinated ataxin-3 aggregates; α-synuclein immunoreactivity; and loss of dopaminergic markers (TH and VMAT2). No neuropathological changes were observed upon wild-type ataxin-3 overexpression. Mutant ataxin-3 expression in striatum and cortex, resulted in accumulation of misfolded ataxin-3, and within striatum, loss of neuronal markers. Striatal pathology was confirmed by observation in MJD transgenic mice of ataxin-3 aggregates and substantial reduction of DARPP-32 immunoreactivity and, in human striata, by ataxin-3 inclusions, immunoreactive for ubiquitin and α-synuclein. This study demonstrates the use of LV encoding mutant ataxin-3 to produce a model of MJD and brings evidence of striatal pathology, suggesting that this region may contribute to dystonia and chorea observed in some MJD patients and may represent a target for therapie

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

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 syste

Neuroprotection by Hsp104 and Hsp27 in lentiviral-based rat models of Huntington's disease

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expansion of glutamine repeats in the huntingtin (htt) protein. Abnormal protein folding and the accumulation of mutated htt are hallmarks of HD neuropathology. Heat-shock proteins (hsps), which refold denatured proteins, might therefore mitigate HD. We show here that hsp104 and hsp27 rescue striatal dysfunction in primary neuronal cultures and HD rat models based on lentiviral-mediated overexpression of a mutated htt fragment. In primary rat striatal cultures, production of hsp104 or hsp27 with htt171-82Q restored neuronal nuclei (NeuN)-positive cell density to that measured after infection with vector expressing the wild-type htt fragment (htt171-19Q). In vivo, both chaperones significantly reduced mutated-htt-related loss of DARPP-32 expression. Furthermore, hsps affected the distribution and size of htt inclusions, with the density of neuritic aggregates being remarkably increased in striatal neurons overexpressing hsps. We also found that htt171-82Q induced the up-regulation of endogenous hsp70 that was co-localized with htt inclusions, and that the overexpression of hsp104 and hsp27 modified the subcellular localization of hsp70 that became cytoplasmic. Finally, hsp104 induced the production of endogenous hsp27. These data demonstrate the protective effects of chaperones in mammalian models of HD

Sustained Effects of Nonallele-Specific Huntingtin Silencing

Objective: Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin (htt) protein. No cure is available to date to alleviate neurodegeneration. Recent studies have demonstrated that RNA interference represents a promising approach for the treatment of autosomal dominant disorders. But whether an allele-specific silencing of mutant htt or a nonallele-specific silencing should be considered has not been addressed

Allele-specific silencing of mutant huntingtin in rodent brain and human stem cells.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder resulting from polyglutamine expansion in the huntingtin (HTT) protein and for which there is no cure. Although suppression of both wild type and mutant HTT expression by RNA interference is a promising therapeutic strategy, a selective silencing of mutant HTT represents the safest approach preserving WT HTT expression and functions. We developed small hairpin RNAs (shRNAs) targeting single nucleotide polymorphisms (SNP) present in the HTT gene to selectively target the disease HTT isoform. Most of these shRNAs silenced, efficiently and selectively, mutant HTT in vitro. Lentiviral-mediated infection with the shRNAs led to selective degradation of mutant HTT mRNA and prevented the apparition of neuropathology in HD rat's striatum expressing mutant HTT containing the various SNPs. In transgenic BACHD mice, the mutant HTT allele was also silenced by this approach, further demonstrating the potential for allele-specific silencing. Finally, the allele-specific silencing of mutant HTT in human embryonic stem cells was accompanied by functional recovery of the vesicular transport of BDNF along microtubules. These findings provide evidence of the therapeutic potential of allele-specific RNA interference for HD

Reduction of ubiquitin-positive inclusions in the striatum of adult rats as result of mutant human ataxin-3 knock-down.

<p>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.</p

The single nucleotide polymorphism strategy used for the specific elimination of mutant or wild-type human ataxin-3 (ATX3) by RNA interference.

<p>A) Schematic representation of the lentiviral constructs encoding wild-type human ataxin-3 (27 CAG repeats) or mutant human ataxin-3 (72 CAG repeats) under control of the phosphoglycerate kinase-1 (PGK-1) promoter. Immediately after the last CAG repeat in the 3′ end, there is a linked single nucleotide polymorphism (SNP) (<u>G</u>⁹⁸⁷GG→<u>C</u>⁹⁸⁷GG) between wild-type and mutant human ataxin-3. B) Diagram of the shAtax vectors used to downregulate human ataxin-3: shRNA cassette under control of the H1 promoter (pol III) and a separate cassette containing the <i>lacZ</i> reporter gene under control of the PGK-1 promoter, making it possible to follow the expression of infected neurons. These shRNAs were designed to silence wild-type (shAtaxWT) or mutant human ataxin-3 (shAtaxMUT) selectively, making use of the (<u>G</u>⁹⁸⁷GG→<u>C</u>⁹⁸⁷GG) SNP.</p

Allele-specific silencing of mutant human ataxin-3 in rat brain.

<p>A) Laser confocal microscopy, showing neuronal transduction 2 months after injection in the rat striatum with recombinant lentiviral vectors encoding shAtaxMUT (n = 7), shAtaxWT (n = 8) or shGFP (n = 4) and mutant human ataxin-3 (MUT ATX3). The viral vectors also contained a separate PGK-<i>LacZ</i> cassette encoding β-galactosidase, to facilitate the detection of infected neurons (B, H, N and E, K, Q, high magnification). In adult rats expressing MUT ATX3 and shAtaxMUT (n = 7), the number of neurons containing MUT ATX3-positive aggregates was much smaller (M) and the high magnification merged image (R) indicates that the few cells positive for MUT ATX3 did not express the <i>lacZ</i> reporter gene present in the shAtaxMUT vector. These cells were therefore not transduced with the vectors encoding the silencing sequences. By contrast, in animals expressing MUT ATX3 and shGFP (n = 4) or the control shAtaxWT (n = 8) (A and G, respectively) high magnification merged images show many MUT ATX3-positive cells simultaneously expressing the <i>lacZ</i> reporter gene present in both shAtaxWT or shGFP (F and L, respectively). The figure shows representative images of immunohistochemical stainings that were reproducible among the different groups.</p

shRNAs mediate the <i>in vitro</i> allele-specific suppression of mutant or wild-type human ataxin-3 by RNAi.

<p>A–F) shAtaxMUT- or shAtaxWT-encoding plasmids selectively targeting mutant ataxin-3 and wild-type ataxin-3, respectively, resulted in much lower levels of these proteins than the mistargeted control (shGFP) or the non allele-specific shRNA. Quantitative real-time PCR analysis showing the silencing of human ATX3 mRNA in 293T cells co-expressing mutant human ataxin-3 (MUT ATX3) (A, top left) or wild-type human ataxin-3 (WT ATX3) (B, top right) and shAtaxWT, shAtaxMUT, or shGFP. Endogenous ß-actin mRNA was used as an internal control for the normalization and quantitative analysis of the ataxin-3 mRNA levels. Results are expressed as the mean elative mRNA level±SEM. C and D) Western-blot analysis of lysates of 293T cells co-transfected with the plasmid constructs encoding MUT ATX3 (C, middle left) or WT ATX3 (D, middle right) and the shAtax vectors (48 hours after calcium phosphate-mediated transfection; ratio ATX3/shRNA 1∶5). Tubulin staining is shown as a loading control. E and F) Optical densitometry was normalized according to the amount of tubulin loaded in the corresponding lane. A partition ratio was calculated and expressed as a percentage (bottom). All western blots and RT-PCRs shown are representative of three or four independent experiments. Statistical significance was evaluated using Fisher's test (*p<0.05).</p