Abstract
Machado Joseph Disease (MJD) is the most frequent autosomal dominantly inherited cerebellar ataxia caused by the over-repetition of a CAG trinucleotide in the ATXN3 gene. This expansion translates into a polyglutamine tract within the ataxin-3 protein that confers a toxic gain-of-function to the mutant protein ataxin-3, contributing to protein misfolding and intracellular accumulation of aggregates and neuronal degeneration. Autophagy impairment has been shown to be one of the mechanisms that contribute for the MJD phenotype. Here we investigated whether this phenotype was present in patient-derived fibroblasts, a common somatic cell type used in the derivation of induced pluripotent stem cells and subsequent differentiation into neurons, for in vitro disease modeling. We generated and studied adult dermal fibroblasts from 5 MJD patients and 4 healthy individuals and we found that early passage MJD fibroblasts exhibited autophagy impairment with an underlying mechanism of decreased autophagosome production. The overexpression of beclin-1 on MJD fibroblasts reverted partially autophagy impairment by increasing the autophagic flux but failed to increase the levels of autophagosome production. Overall, our results provide a well-characterized MJD fibroblast resource for neurodegenerative disease research and contribute for the understanding of mutant ataxin-3 biology and its molecular consequences.
Highlights
Machado Joseph Disease (MJD) known as Spinocerebellar Ataxia Type 3 (SCA3) is an autosomal dominant inherited cerebellar ataxia and a progressive, adult-onset neurodegenerative disease[1,2]
Skin explants were obtained from healthy individuals and MJD patients followed at the Coimbra University Hospital Centre and cultured as previously described[15,16,20] taking advantage of the outgrowing property of fibroblasts from skin, which enabled a high cell yield in a short period of time
As expected we found that the sum of wild-type and mutant ataxin-3 protein levels in MJD fibroblasts was similar to the levels of wild-type ataxin-3 in healthy controls (Fig. 3E) and that the levels of wild-type ataxin-3 in patient cells were half those found in controls. qPCR analysis of mRNA levels of ataxin-3 further confirmed that the levels of ataxin-3 were similar in both groups (Fig. 3G)
Summary
Machado Joseph Disease (MJD) known as Spinocerebellar Ataxia Type 3 (SCA3) is an autosomal dominant inherited cerebellar ataxia and a progressive, adult-onset neurodegenerative disease[1,2]. Our group previously provided evidence of an impairment of the autophagy pathway in a MJD rodent model and decreased levels of Beclin-1/ATG6, a component of the class III PI3 kinase complex required for autophagy initiation and autophagosome formation, in human fibroblasts from two MJD patients[6]. In this work we collected and studied a cohort of human primary fibroblast cultures obtained from MJD patients and healthy controls in order to elucidate whether this type of peripheral cells presents a MJD related phenotype, at molecular, cellular or functional level. For this purpose, we examined the levels of i) www.nature.com/scientificreports/. To clarify whether a) autophagosome formation is impaired or b) the autophagic flux (rate of autophagosome delivery to lysosomes) is compromised we investigated the levels of LC3-II11
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