1 Scientific RepoRts | 6:28220 | DOI: 10.1038/srep28220 www.nature.com/scientificreports Fibroblasts of Machado Joseph Disease patients reveal autophagy impairment Isabel onofre 1,2,* , Nuno Mendonça 1,3,4,* , sara Lopes 1,5 , Rui Nobre 1,5 , Joana Barbosa de Melo 1,4,6 , Isabel Marques Carreira 1,4,6 , Cristina Januário 3,4 , António Freire Gonçalves 3,4 & Luis pereira de Almeida 1,2 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 fbroblasts, a common somatic cell type used in the derivation of induced pluripotent stem cells and subsequent diferentiation into neurons, for in vitro disease modeling. We generated and studied adult dermal fbroblasts from 5 MJD patients and 4 healthy individuals and we found that early passage MJD fbroblasts exhibited autophagy impairment with an underlying mechanism of decreased autophagosome production. The overexpression of beclin-1 on MJD fbroblasts reverted partially autophagy impairment by increasing the autophagic fux but failed to increase the levels of autophagosome production. Overall, our results provide a well-characterized MJD fbroblast resource for neurodegenerative disease research and contribute for the understanding of mutant ataxin-3 biology and its molecular consequences. Machado Joseph Disease (MJD) also known as Spinocerebellar Ataxia Type 3 (SCA3) is an autosomal domi- nant inherited cerebellar ataxia and a progressive, adult-onset neurodegenerative disease 1,2 . SCA3 is caused by a CAG-repeat expansion in the ATXN3 gene on chromosome 14q24.3–q32.2, which results in an abnormally long polyglutamine tract in the ataxin-3 protein 3 . Tere is strong evidence that proteins with an overlong mutant polyglutamine tract are inefciently degraded by the ubiquitin-proteosome system (UPS) but may be cleared by macroautophagy (hereafer referred to as auto- phagy), an intracellular degradation pathway with a crucial role in degradation of insoluble aggregate-prone proteins 4 such as the polyglutamine proteins involved in neurodegenerative diseases 5 . Our group previously pro- vided 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 autopha- gosome formation, in human fbroblasts from two MJD patients 6 . Adult dermal fbroblasts are an accessible source of patient cells, easy to grow in culture and currently the most suitable somatic cell type for reprogramming giving an efcient yield of induced pluripotent stem cells (iPSCs) 7 . Studying patient-derived fbroblasts, as somatic cell type of origin can give new insights in the establishment of diseased phenotype of patient-derived neurons resulting from iPSCs diferentiation, taking in account that fbro- blasts hold the native genetic background of the patient without further genetic manipulation 8,9 . Terefore, in this work we collected and studied a cohort of human primary fbroblast 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) 1 cnc-center for neuroscience and cell Biology, coimbra, Portugal. 2 faculty of Pharmacy, University of coimbra, coimbra, Portugal. 3 neurology Department, coimbra University Hospital center, coimbra, Portugal. 4 faculty of Medicine, University of coimbra, Portugal. 5 iiiUc- institute for interdisciplinary Research, University of coimbra, coimbra, Portugal. 6 cytogenetics and Genomics Laboratory, faculty of Medicine, University of coimbra, coimbra, Portugal. * these authors contributed equally to this work. correspondence and requests for materials should be addressed to L.P.d.A. (email: luispa@cnc.uc.pt) Received: 29 December 2015 Accepted: 24 May 2016 Published: 22 June 2016 opeN