Silencing mutant ataxin-3 rescues motor deficits and neuropathology in machado-joseph disease transgenic mice.

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly-inherited neurodegenerative disorder caused by the over-repetition of a CAG codon in the MJD1 gene. This expansion translates into a polyglutamine tract that confers a toxic gain-of-function to the mutant protein - ataxin-3, leading to neurodegeneration in specific brain regions, with particular severity in the cerebellum. No treatment able to modify the disease progression is available. However, gene silencing by RNA interference has shown promising results. Therefore, in this study we investigated whether lentiviral-mediated allele-specific silencing of the mutant ataxin-3 gene, after disease onset, would rescue the motor behavior deficits and neuropathological features in a severely impaired transgenic mouse model of MJD. For this purpose, we injected lentiviral vectors encoding allele-specific silencing-sequences (shAtx3) into the cerebellum of diseased transgenic mice expressing the targeted C-variant of mutant ataxin-3 present in 70% of MJD patients. This variation permits to discriminate between the wild-type and mutant forms, maintaining the normal function of the wild-type allele and silencing only the mutant form. Quantitative analysis of rotarod performance, footprint and activity patterns revealed significant and robust alleviation of gait, balance (average 3-fold increase of rotarod test time), locomotor and exploratory activity impairments in shAtx3-injected mice, as compared to control ones injected with shGFP. An important improvement of neuropathology was also observed, regarding the number of intranuclear inclusions, calbindin and DARPP-32 immunoreactivity, fluorojade B and Golgi staining and molecular and granular layers thickness. These data demonstrate for the first time the efficacy of gene silencing in blocking the MJD-associated motor-behavior and neuropathological abnormalities after the onset of the disease, supporting the use of this strategy for therapy of MJD.

Calpastatin-mediated inhibition of calpains in the mouse brain prevents mutant ataxin 3 proteolysis, nuclear localization and aggregation, relieving Machado-Joseph disease.

Machado-Joseph disease is the most frequently found dominantly-inherited cerebellar ataxia. Over-repetition of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within the ataxin 3 protein, which upon proteolysis may trigger Machado-Joseph disease. We investigated the role of calpains in the generation of toxic ataxin 3 fragments and pathogenesis of Machado-Joseph disease. For this purpose, we inhibited calpain activity in mouse models of Machado-Joseph disease by overexpressing the endogenous calpain-inhibitor calpastatin. Calpain blockage reduced the size and number of mutant ataxin 3 inclusions, neuronal dysfunction and neurodegeneration. By reducing fragmentation of ataxin 3, calpastatin overexpression modified the subcellular localization of mutant ataxin 3 restraining the protein in the cytoplasm, reducing aggregation and nuclear toxicity and overcoming calpastatin depletion observed upon mutant ataxin 3 expression. Our findings are the first in vivo proof that mutant ataxin 3 proteolysis by calpains mediates its translocation to the nucleus, aggregation and toxicity and that inhibition of calpains may provide an effective therapy for Machado-Joseph disease.

A prospective molecular surveillance study evaluating the clinical impact of community-acquired respiratory viruses in lung transplant recipients.

BACKGROUND: Community-acquired respiratory viral infections (RVIs) are common in lung transplant patients and may be associated with acute rejection and bronchiolitis obliterans syndrome (BOS). The use of sensitive molecular methods that can simultaneously detect a large panel of respiratory viruses may help better define their effects. METHODS: Lung transplant recipients undergoing serial surveillance and diagnostic bronchoalveolar lavages (BALs) during a period of 3 years were enrolled. BAL samples underwent multiplex testing for a panel of 19 respiratory viral types/subtypes using the Luminex xTAG respiratory virus panel assay. RESULTS: Demographics, symptoms, and forced expiratory volume in 1 sec were prospectively collected for 93 lung transplant recipients enrolled. Mean number of BAL samples was 6.2+/-3.1 per patient. A respiratory virus was isolated in 48 of 93 (51.6%) patients on at least one BAL sample. Of 81 positive samples, the viruses isolated included rhinovirus (n=46), parainfluenza 1 to 4 (n=17), coronavirus (n=11), influenza (n=4), metapneumovirus (n=4), and respiratory syncytial virus (n=2). Biopsy-proven acute rejection (> or =grade 2) or decline in forced expiratory volume in 1 sec > or =20% occurred in 16 of 48 (33.3%) patients within 3 months of RVI when compared with 3 of 45 (6.7%) RVI-negative patients within a comparable time frame (P=0.001). No significant difference was seen in incidence of acute rejection between symptomatic and asymptomatic patients. Biopsy-proven obliterative bronchiolitis or BOS was diagnosed in 10 of 16 (62.5%) patients within 1 year of infection. CONCLUSION: Community-acquired RVIs are frequently detected in BAL samples from lung transplant patients. In a significant percentage of patients, symptomatic or asymptomatic viral infection is a trigger for acute rejection and obliterative bronchiolitis/BOS.

RNA interference mitigates motor and neuropathological deficits in a cerebellar mouse model of machado-joseph disease.

Machado-Joseph disease or Spinocerebellar ataxia type 3 is a progressive fatal neurodegenerative disorder caused by the polyglutamine-expanded protein ataxin-3. Recent studies demonstrate that RNA interference is a promising approach for the treatment of Machado-Joseph disease. However, whether gene silencing at an early time-point is able to prevent the appearance of motor behavior deficits typical of the disease when initiated before onset of the disease had not been explored. Here, using a lentiviral-mediated allele-specific silencing of mutant ataxin-3 in an early pre-symptomatic cerebellar mouse model of Machado-Joseph disease we show that this strategy hampers the development of the motor and neuropathological phenotypic characteristics of the disease. At the histological level, the RNA-specific silencing of mutant ataxin-3 decreased formation of mutant ataxin-3 aggregates, preserved Purkinje cell morphology and expression of neuronal markers while reducing cell death. Importantly, gene silencing prevented the development of impairments in balance, motor coordination, gait and hyperactivity observed in control mice. These data support the therapeutic potential of RNA interference for Machado-Joseph disease and constitute a proof of principle of the beneficial effects of early allele-specific silencing for therapy of this disease.