Andreia Neves-Carvalho

Neuron-specific proteotoxicity of mutant ataxin-3 in C. elegans: rescue by the DAF-16 and HSF-1 pathways

The risk of developing neurodegenerative diseases increases with age. Although many of the molecular pathways regulating proteotoxic stress and longevity are well characterized, their contribution to disease susceptibility remains unclear. In this study, we describe a new Caenorhabditis elegans model of Machado-Joseph disease pathogenesis. Pan-neuronal expression of mutant ATXN3 leads to a polyQ-length dependent, neuron subtype-specific aggregation and neuronal dysfunction. Analysis of different neurons revealed a pattern of dorsal nerve cord and sensory neuron susceptibility to mutant ataxin-3 that was distinct from the aggregation and toxicity profiles of polyQ-alone proteins. This reveals that the sequences flanking the polyQ-stretch in ATXN3 have a dominant influence on cell-intrinsic neuronal factors that modulate polyQ-mediated pathogenesis. Aging influences the ATXN3 phenotypes which can be suppressed by the downregulation of the insulin/insulin growth factor-1-like signaling pathway and activation of heat shock factor-1.

Chronic Treatment with 17-DMAG Improves Balance and Coordination in A New Mouse Model of Machado-Joseph Disease

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disease currently with no treatment. We describe a novel mouse model of MJD which expresses mutant human ataxin-3 at near endogenous levels and manifests MJD-like motor symptoms that appear gradually and progress over time. CMVMJD135 mice show ataxin-3 intranuclear inclusions in the CNS and neurodegenerative changes in key disease regions, such as the pontine and dentate nuclei. Hsp90 inhibition has shown promising outcomes in some neurodegenerative diseases, but nothing is known about its effects in MJD. Chronic treatment of CMVMJD mice with Hsp90 inhibitor 17-DMAG resulted in a delay in the progression of their motor coordination deficits and, at 22 and 24 weeks of age, was able to rescue the uncoordination phenotype to wild-type levels; in parallel, a reduction in neuropathology was observed in treated animals. We observed limited induction of heat-shock proteins with treatment, but found evidence that 17-DMAG may be acting through autophagy, as LC3-II (both at mRNA and protein levels) and beclin-1 were induced in the brain of treated animals. This resulted in decreased levels of the mutant ataxin-3 and reduced intranuclear aggregation of this protein. Our data validate this novel mouse model as a relevant tool for the study of MJD pathogenesis and for pre-clinical studies, and show that Hsp90 inhibition is a promising therapeutic strategy for MJD.

The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3’-P and 5’-OH, are processed by mammalian polynucleotide kinase 3’-phosphatase (PNKP), a bifunctional enzyme with 3’-phosphatase and 5’-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14–41 to 55–82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP’s 3’ phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3’-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients’ brain. Finally, long amplicon quantitative PCR analysis of human MJD patients’ brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.

Serotonergic signalling suppresses ataxin 3 aggregation and neurotoxicity in animal models of Machado-Joseph disease

Polyglutamine diseases are a class of dominantly inherited neurodegenerative disorders for which there is no effective treatment. Here we provide evidence that activation of serotonergic signalling is beneficial in animal models of Machado-Joseph disease. We identified citalopram, a selective serotonin reuptake inhibitor, in a small molecule screen of FDA-approved drugs that rescued neuronal dysfunction and reduced aggregation using a Caenorhabditis elegans model of mutant ataxin 3-induced neurotoxicity. MOD-5, the C. elegans orthologue of the serotonin transporter and cellular target of citalopram, and the serotonin receptors SER-1 and SER-4 were strong genetic modifiers of ataxin 3 neurotoxicity and necessary for therapeutic efficacy. Moreover, chronic treatment of CMVMJD135 mice with citalopram significantly reduced ataxin 3 neuronal inclusions and astrogliosis, rescued diminished body weight and strikingly ameliorated motor symptoms. These results suggest that small molecule modulation of serotonergic signalling represents a promising therapeutic target for Machado-Joseph disease.

The Secretome of Bone Marrow and Wharton Jelly Derived Mesenchymal Stem Cells Induces Differentiation and Neurite Outgrowth in SH-SY5Y Cells

The goal of this study was to determine and compare the effects of the secretome of mesenchymal stem cells (MSCs) isolated from human bone-marrow (BMSCs) and the Wharton jelly surrounding the vein and arteries of the umbilical cord (human umbilical cord perivascular cells (HUCPVCs)) on the survival and differentiation of a human neuroblastoma cell line (SH-SY5Y). For this purpose, SH-SY5Y cells were differentiated with conditioned media (CM) from the MSCs populations referred above. Retinoic acid cultured cells were used as control for neuronal differentiated SH-SY5Y cells. SH-SY5Y cells viability assessment revealed that the secretome of BMSCs and HUCPVCs, in the form of CM, was able to induce their survival. Moreover, immunocytochemical experiments showed that CM from both MSCs was capable of inducing neuronal differentiation of SH-SY5Y cells. Finally, neurite lengths assessment and quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis demonstrated that CM from BMSCs and HUCPVCs differently induced neurite outgrowth and mRNA levels of neuronal markers exhibited by SH-SY5Y cells. Overall, our results show that the secretome of both BMSCs and HUCPVCs was capable of supporting SH-SY5Y cells survival and promoting their differentiation towards a neuronal phenotype.

Absence of ataxin-3 leads to enhanced stress response in C. elegans.

Ataxin-3, the protein involved in Machado-Joseph disease, is able to bind ubiquitylated substrates and act as a deubiquitylating enzyme in vitro, and it has been involved in the modulation of protein degradation by the ubiquitin-proteasome pathway. C. elegans and mouse ataxin-3 knockout models are viable and without any obvious phenotype in a basal condition however their phenotype in stress situations has never been described. Considering the role of ataxin-3 in the protein degradation pathway, we analyzed the effects of heat shock, a known protein homeostasis stressor, in C. elegans ataxin-3 (ATX-3) knockout animals. We found that ATX-3 mutants have an exacerbated stress response and survive significantly better than wild type animals when subjected to a noxious heat shock stimulus. This increased thermotolerance of mutants was further enhanced by pre-exposure to a mild heat shock. At a molecular level, ATX-3 mutants have a distinct transcriptomic and proteomic profile with several molecular chaperones abnormally up-regulated during heat shock and recovery, consistent with the observed resistance phenotype. The improved thermotolerancein ATX-3 mutants is independent of heat shock factor 1, the maestro of the heat shock response, but fully dependent on DAF-16, a critical stress responsive transcription factor involved in longevity and stress resistance. We also show that the increased thermotolerance of ATX-3 mutants is mainly due to HSP-16.2, C12C8.1 and F44E5.5 given that the knockdown of these heat shock proteins using RNA interference causes the phenotype to revert. This report suggests that the absence of ATX-3 activates the DAF-16 pathway leading to an overexpression of molecular chaperones, which yields knockout animals with an improved capacity for dealing with deleterious stimuli.

ATX-3, CDC-48 and UBXN-5: a new trimolecular complex in Caenorhabditis elegans.

Ataxin-3 is the protein involved in Machado-Joseph disease, a neurodegenerative disorder caused by a polyglutamine expansion. Ataxin-3 binds ubiquitylated proteins and acts as a deubiquitylating enzyme in vitro. It was previously proposed that ataxin-3, along with the VCP/p97 protein, escorts ubiquitylated substrates for proteasomal degradation, although other players of this escort complex were not identified yet. In this work, we show that the Caenorhabditis elegans ataxin-3 protein (ATX-3) interacts with both VCP/p97 worm homologs, CDC-48.1 and CDC-48.2 and we map the interaction domains. We describe a motility defect in both ATX-3 and CDC-48.1 mutants and, in addition, we identify a new protein interactor, UBXN-5, potentially an adaptor of the CDC-48-ATX-3 escort complex. CDC-48 binds to both ATX-3 and UBXN-5 in a non-competitive manner, suggesting the formation of a trimolecular complex. Both CDC-48 and ATX-3, but not UBXN-5, were able to bind K-48 polyubiquitin chains, the standard signal for proteasomal degradation. Additionally, we describe several common interactors of ATX-3 and UBXN-5, some of which can be in vivo targets of this complex.

Combined therapy with m-TOR-dependent and -independent autophagy inducers causes neurotoxicity in a mouse model of Machado–Joseph disease

A major pathological hallmark in several neurodegenerative disorders, like polyglutamine disorders (polyQ), including Machado-Joseph disease (MJD), is the formation of protein aggregates. MJD is caused by a CAG repeat expansion in the ATXN3 gene, resulting in an abnormal protein, which is prone to misfolding and forms cytoplasmic and nuclear aggregates within neurons, ultimately inducing neurodegeneration. Treatment of proteinopathies with drugs that up-regulate autophagy has shown promising results in models of polyQ diseases. Temsirolimus (CCI-779) inhibits the mammalian target of rapamycin (m-TOR), while lithium chloride (LiCl) acts by inhibiting inositol monophosphatase, both being able to induce autophagy. We have previously shown that chronic treatment with LiCl (10.4 mg/kg) had limited effects in a transgenic MJD mouse model. Also, others have shown that CCI-779 had mild positive effects in a different mouse model of the disease. It has been suggested that the combination of mTOR-dependent and -independent autophagy inducers could be a more effective therapeutic approach. To further explore this avenue toward therapy, we treated CMVMJD135 transgenic mice with a conjugation of CCI-779 and LiCl, both at concentrations known to induce autophagy and not to be toxic. Surprisingly, this combined treatment proved to be deleterious to both wild-type (wt) and transgenic animals, failing to rescue their neurological symptoms and actually exerting neurotoxic effects. These results highlight the possible dangers of manipulating autophagy in the nervous system and suggest that a better understanding of the potential disruption in the autophagy pathway in MJD is required before successful long-term autophagy modulating therapies can be developed.

Limited effect of chronic valproic acid treatment in a mouse model of Machado-Joseph disease

Machado-Joseph disease (MJD) is an inherited neurodegenerative disease, caused by a CAG repeat expansion within the coding region of ATXN3 gene, and which currently lacks effective treatment. In this work we tested the therapeutic efficacy of chronic treatment with valproic acid (VPA) (200mg/kg), a compound with known neuroprotection activity, and previously shown to be effective in cell, fly and nematode models of MJD. We show that chronic VPA treatment in the CMVMJD135 mouse model had limited effects in the motor deficits of these mice, seen mostly at late stages in the motor swimming, beam walk, rotarod and spontaneous locomotor activity tests, and did not modify the ATXN3 inclusion load and astrogliosis in affected brain regions. However, VPA chronic treatment was able to increase GRP78 protein levels at 30 weeks of age, one of its known neuroprotective effects, confirming target engagement. In spite of limited results, the use of another dosage of VPA or of VPA in a combined therapy with molecules targeting other pathways, cannot be excluded as potential strategies for MJD therapeutics.

Dominant negative effect of polyglutamine expansion perturbs normal function of ataxin-3 in neuronal cells

The physiological function of Ataxin-3 (ATXN3), a deubiquitylase (DUB) involved in Machado-Joseph Disease (MJD), remains elusive. In this study, we demonstrate that ATXN3 is required for neuronal differentiation and for normal cell morphology, cytoskeletal organization, proliferation and survival of SH-SY5Y and PC12 cells. This cellular phenotype is associated with increased proteasomal degradation of α5 integrin subunit (ITGA5) and reduced activation of integrin signalling and is rescued by ITGA5 overexpression. Interestingly, silencing of ATXN3, overexpression of mutant versions of ATXN3 lacking catalytic activity or bearing an expanded polyglutamine (polyQ) tract led to partially overlapping phenotypes. In vivo analysis showed that both Atxn3 knockout and MJD transgenic mice had decreased levels of ITGA5 in the brain. Furthermore, abnormal morphology and reduced branching were observed both in cultured neurons expressing shRNA for ATXN3 and in those obtained from MJD mice. Our results show that ATXN3 rescues ITGA5 from proteasomal degradation in neurons and that polyQ expansion causes a partial loss of this cellular function, resulting in reduced integrin signalling and neuronal cytoskeleton modifications, which may be contributing to neurodegeneration.