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Genetic mutations and aging-associated oxidative damage underlie the onset and progression of neurodegenerative diseases, like Parkinson's disease (PD) and Machado-Joseph disease (MJD). Natural products derived from plants have been regarded as important sources of novel bioactive compounds to counteract neurodegeneration. Here, we tested the neuroprotective effect of an ethanolic extract of rapeseed pomace (RSP), a rapeseed (canola) oil production by-product, in C. elegans models of MJD and PD. The extract, containing sinapine and other phenolics, restored motor function of mutant ataxin-3 (ATXN3) animals (MJD) and prevented degeneration of dopaminergic neurons in one toxin-induced and two genetic models of PD. Whole-organism sensors of antioxidant and xenobiotic response activation revealed the induction of phase II detoxification enzymes, including glutathione S- transferase (GST-4) upon RSP extract supplementation. Furthermore in vivo pharmacogenetic studies confirmed gst-4 is required for the therapeutic effect of RSP extract in the two disease models. The results suggest that GST-4-mediated antioxidant pathways may constitute promising therapeutic co-targets for neurodegenerative diseases and confirm the utility of searching for bioactive compounds in novel sources, including food and agricultural waste/by-products, such as RSP.Genetic mutations and aging-associated oxidative damage underlie the onset and progression of neurodegenerative diseases, like Parkinson's disease (PD) and Machado-Joseph disease (MJD). Natural products derived from plants have been regarded as important sources of novel bioactive compounds to counteract neurodegeneration. Here, we tested the neuroprotective effect of an ethanolic extract of rapeseed pomace (RSP), a rapeseed (canola) oil production by-product, in C. elegans models of MJD and PD. The extract, containing sinapine and other phenolics, restored motor function of mutant ataxin-3 (ATXN3) animals (MJD) and prevented degeneration of dopaminergic neurons in one toxin-induced and two genetic models of PD. Whole-organism sensors of antioxidant and xenobiotic response activation revealed the induction of phase II detoxification enzymes, including glutathione S- transferase (GST-4) upon RSP extract supplementation. Furthermore in vivo pharmacogenetic studies confirmed gst-4 is required for the therapeutic effect of RSP extract in the two disease models. The results suggest that GST-4-mediated antioxidant pathways may constitute promising therapeutic co-targets for neurodegenerative diseases and confirm the utility of searching for bioactive compounds in novel sources, including food and agricultural waste/by-products, such as RSP.

Microglia have been increasingly implicated in neurodegenerative diseases (NDs), and specific disease associated microglia (DAM) profiles have been defined for several of these NDs. Yet, the microglial profile in Machado–Joseph disease (MJD) remains unexplored. Here, we characterized the profile of microglia in the CMVMJD135 mouse model of MJD. This characterization was performed using primary microglial cultures and microglial cells obtained from disease-relevant brain regions of neonatal and adult CMVMJD135 mice, respectively. Machine learning models were implemented to identify potential clusters of microglia based on their morphological features, and an RNA-sequencing analysis was performed to identify molecular perturbations and potential therapeutic targets. Our findings reveal morphological alterations that point to an increased activation state of microglia in CMVMJD135 mice and a disease-specific transcriptional profile of MJD microglia, encompassing a total of 101 differentially expressed genes, with enrichment in molecular pathways related to oxidative stress, immune response, cell proliferation, cell death, and lipid metabolism. Overall, these results allowed us to define the cellular and molecular profile of MJD-associated microglia and to identify genes and pathways that might represent potential therapeutic targets for this disorder.

The study of C. elegans has led to ground-breaking discoveries in gene-function, neuronal circuits, and physiological responses. Subtle behavioral phenotypes, however, are often difficult to measure reproducibly. We have developed an experimental and computational infrastructure to simultaneously record and analyze the physical characteristics, movement, and social behaviors of dozens of interacting free-moving nematodes. Our algorithm implements a directed acyclic network that reconstructs the complex behavioral trajectories generated by individual C. elegans in a free moving population by chaining hundreds to thousands of short tracks into long contiguous trails. This technique allows for the high-throughput quantification of behavioral characteristics that require long-term observation of individual animals. The graphical interface we developed will enable researchers to uncover, in a reproducible manner, subtle time-dependent behavioral phenotypes that will allow dissection of the molecular mechanisms that give rise to organism-level behavior.

Aim: Experimental models are a powerful aid in visualizing molecular phenomena. This work reports how the worm Caenorhabditis elegans (C. elegans) can be effectively explored for students to learn how molecular cues dramatically condition axonal guidance and define nervous system structure and behavior at the organism level. Summary of work: A loosely oriented observational activity preceded detailed discussions on molecules implied in axonal migration. C. elegans mutants were used to introduce second-year medical students to the deleterious effects of gene malfunctioning in neuron response to extracellular biochemical cues and to establish links between molecular function, nervous system structure, and animal behavior. Students observed C. elegans cultures and associated animal behavior alterations with the lack of function of specific axon guidance molecules (the soluble cue netrin/UNC-6 or two receptors, DCC/UNC-40 and UNC-5H). Microscopical observations of these strains, in combination with pan-neuronal GFP expression, allowed optimal visualization of severely affected neurons. Once the list of mutated genes in each strain was displayed, students could also relate abnormal patterns in axon migration/ventral and dorsal nerve cord neuron formation in C. elegans with mutated molecular components homologous to those in humans. Summary of results: Students rated the importance and effectiveness of the activity very highly. Ninety-three percent found it helpful to grasp human axonal migration, and all students were surprised with the power of the model in helping to visualize the phenomenon.

The low regeneration potential of the central nervous system (CNS) represents a challenge for the development of new therapeutic strategies for neurodegenerative diseases, including spinocerebellar ataxias. Spinocerebellar ataxia type 3 (SCA3)—or Machado–Joseph disease (MJD)—is the most common dominant ataxia, being mainly characterized by motor deficits; however, SCA3/MJD has a complex and heterogeneous pathophysiology, involving many CNS brain regions, contributing to the lack of effective therapies. Mesenchymal stem cells (MSCs) have been proposed as a potential therapeutic tool for CNS disorders. Beyond their differentiation potential, MSCs secrete a broad range of neuroregulatory factors that can promote relevant neuroprotective and immunomodulatory actions in different pathophysiological contexts. The objective of this work was to study the effects of (1) human MSC transplantation and (2) human MSC secretome (CM) administration on disease progression in vivo, using the CMVMJD135 mouse model of SCA3/MJD. Our results showed that a single CM administration was more beneficial than MSC transplantation—particularly in the cerebellum and basal ganglia—while no motor improvement was observed when these cell-based therapeutic approaches were applied in the spinal cord. However, the effects observed were mild and transient, suggesting that continuous or repeated administration would be needed, which should be further tested.

The atypical antipsychotic aripiprazole is a Food and Drug Administration-approved drug for the treatment of psychotic, mood, and other psychiatric disorders. Previous drug discovery efforts pinpointed aripiprazole as an effective suppressor of Machado–Joseph disease (MJD) pathogenesis, as its administration resulted in a reduced abundance and aggregation of mutant Ataxin-3 (ATXN3) proteins. Dopamine partial agonism and functional selectivity have been proposed as the main pharmacological mechanism of action of aripiprazole in the treatment of psychosis; however, this mechanism remains to be determined in the context of MJD. Here, we focus on confirming the efficacy of aripiprazole to reduce motor dysfunction in vivo, using a Caenorhabditis elegans (C. elegans) model of MJD, and on unveiling the drug targets required for its positive action against mutant ATXN3 pathogenesis. We employed pharmacogenetics and pharmacological approaches to identify which dopamine and serotonin receptors are critical for aripiprazole-mediated improvements in motor function. We demonstrated that dopamine D2-like and serotonin 5-HT1A and 5-HT2A receptors play important roles in this process. Our findings strengthen the relevance of dopaminergic and serotoninergic signaling modulation against mutant ATXN3-mediated pathogenesis. The identification of aripiprazole’s cellular targets, relevant for MJD and perhaps other neurodegenerative diseases, may pave the way for prospective drug discovery and development campaigns aiming to improve the features of this prototypical compound and reduce side effects not negligible in the case of aripiprazole.

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 thermotolerance in 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.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado–Joseph disease (MJD), is a neurodegenerative disorder caused by a polyglutamine expansion in the ATXN3 gene. In spite of the identification of a clear monogenic cause 25 years ago, the pathological process still puzzles researchers, impairing prospects for an effective therapy. Here, we propose the disruption of protein homeostasis as the hub of SCA3 pathogenesis, being the molecular mechanisms and cellular pathways that are deregulated in SCA3 downstream consequences of the misfolding and aggregation of ATXN3. Moreover, we attempt to provide a realistic perspective on how the translational/clinical research in SCA3 should evolve. This was based on molecular findings, clinical and epidemiological characteristics, studies of proposed treatments in other conditions, and how that information is essential for their (re-)application in SCA3. This review thus aims i) to critically evaluate the current state of research on SCA3, from fundamental to translational and clinical perspectives; ii) to bring up the current key questions that remain unanswered in this disorder; and iii) to provide a frame on how those answers should be pursued.

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.

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.