Teixeira-Castro, A. et al. (2009) International Worm Meeting "Ataxin-3 protein context and cell-specific factors modulate polyQ-mediated neuronal aggregation in a C. elegans model of Machado-Joseph disease."

Morimoto, R., Teixeira-Castro, A., Maciel, P.

[International Worm Meeting, 2009]

Understanding the basis for neuronal subtype-specific protein aggregation is of central importance for several human neurodegenerative diseases, including Machado-Joseph disease. In this study, we developed a novel Ataxin-3 (ATXN3) pathogenesis model in Caenorhabditis elegans and examined the aggregation profile of human ATXN3 by performing FRAP analysis, in live neuronal cells. We found that full-length ATXN3 aggregates only at high Q-length, not found in human patients, whereas C-terminal ATXN3 causes aggregation and neurotoxicity at a threshold length of 75 glutamines. Analysis of specific neurons in C. elegans, reveals that the ventral nerve cord motor neurons are highly affected. Interestingly, certain sensory neurons of the head contain aggregated foci only when the polyQ-stretch is expressed within ATXN3 protein flanking sequences. Moreover, co-expression of full-length human pathological ATXN3 (below aggregation threshold) with an aggregated species capable of initiating the nucleation events, aggravates the aggregation phenotype and new ATXN3-polyQ co-aggregates are formed also in the sensory neurons of these animals, which are not affected when the two species are expressed alone. These results provide direct evidence that protein context and cell-specific factors are major modifiers of polyQ pathogenesis.

Andreia Teixeira-Castro et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "In vivo dynamics of human ataxin-3 aggregation and neurotoxicity in C. elegans neuronal cells is modulated by Q-length and age"

Andreia Teixeira-Castro, Patricia Maciel, Richard Morimoto

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Machado-Joseph disease, like other polyglutamine (polyQ) diseases, is a late onset neurological disorder characterized by the appearance of misfolded protein species, aggregates, neuronal dysfunction and cell death. Although the mechanism(s) underlying the formation of ataxin-3 (AT3) neuronal inclusions are poorly understood, it is becoming increasingly evident that proteolysis of full-length AT3 is a biological relevant event in the disease since it occurs and affects aggregation both in vitro and in vivo. In this study, we developed a new model for AT3 pathogenesis in Caenorhabditis elegans, in which we observed that expression of the full-length human pathogenic AT3 alone did not cause aggregation in live neuronal cells. In contrast, expression of a C-terminal fragment of mutant AT3 resulted in protein aggregation, suggesting that the aggregation-prone fragment was behaving as seed capable of initiating the nucleation events. Moreover, we studied the dynamics of the sequestration process of full-length pathogenic and wild-type AT3 into polyQ aggregates and observed that this process occurs in an age-dependent manner and that there is a tight correlation between aggregation and neuronal toxicity onset. We are currently using this model to address the molecular mechanisms of the ageing-dependence of the aggregation and neurological phenotypes, which could provide clues to the late onset of the human disease.

Andreia Teixeira-Castro et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Pathological ataxin-3 misfolding in C. elegans neurons"

Patricia Maciel, Richard Morimoto, Andreia Teixeira-Castro

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Expansion of polyglutamine (polyQ) tracts has been identified as the basis of at least nine neurodegenerative diseases, including Machado-Joseph disease (MJD). MJD is a hereditary ataxia of adult onset caused by expansion of a polyQ tract in ataxin-3 (AT3). AT3 is widely expressed and consists of an N-terminal globular domain with significant helical content, which spans the Josephin domain (JD), and a flexible C-terminal tail containing up to three Ubiquitin interacting motifs (UIM) and the polyQ tract. AT3-induced neurodegeneration affects a specific subset of neurons and is characterized by the presence of AT3- containing protein aggregates. Mutant AT3 forms mainly intranuclear inclusions in diseased human brain as well as in cell culture. Studies suggest that the pathological form of AT3 undergoes a conformational change leading to an alteration in protein homeostasis, misfolding and toxicity. To identify the factors involved in cell-specific pathogenesis observed for MJD, we generated pan-neural Caenorhabditis elegans models expressing chimeric fusion proteins of AT3, with normal and expanded polyQ lengths, tagged on the C-terminus with YFP. We are currently performing the behavioral analysis and looking at the aggregation properties of these models with particular emphasis on polyQ length-dependent aggregation and neurotoxicity. Once we have characterized our model, we will search for genetic modulators of AT3 pathogenesis thus revealing a subset of regulating genes uniquely relevant for mutant AT3 misfolding and toxicity in a metazoan. The comparison to the existing C. elegans polyQ models will contribute significantly in identifying the importance of protein context in cell-specific pathogenesis, providing a better understanding of the disease mechanisms.

Teixeira-Castro, A. et al. (2013) International Worm Meeting "Searching for therapeutic compounds for Machado-Joseph disease: a C. elegans-based screening."

Morimoto, R., Teixeira-Castro, A., Bessa, C., Maciel, P., Jalles, A., Araujo, M., Miranda, A.

[International Worm Meeting, 2013]

Despite the many efforts that are under way to develop therapeutic strategies, no preventive treatment is yet available for any of the polyglutamine diseases. Machado-Joseph disease (MJD) is one of the polyQ disorders caused by the expansion of a polyQ tract within the C-terminal of the ataxin-3 (ATXN3) protein. Mutant ATXN3 acquires the ability to self-associate and enter an aggregation process, which is associated with several pathophysiological consequences for neurons. The lack of therapeutic strategies that effectively prevent neurodegeneration in MJD patients prompted us to search for compounds that modulate mutant ATXN3-related pathogenesis. Recent data from our lab have shown that many aspects of MJD can be properly modeled in the round worm Caenorhabditis elegans. This study is based on the idea that our C. elegans MJD model can be used to perform large-scale drug screenings, in which the identification of effective drugs can be accomplished by looking simultaneously at protein aggregation in the live neuronal cells, and on its impact on neuron-regulated behavior of the whole-animal. Our goal was to screen a library of ~1200 mainly FDA-approved out-of-patent compounds for their ability to prevent or delay the formation of fluorescent mutant ATXN3 aggregates and neurological dysfunction. We excluded the small molecules that were found to be toxic or cause developmental delay to the C. elegans. Ten percent of the non-toxic compounds significantly reduced the locomotion deficits of the animals, three of which made mutant ATXN3 expressing worms perform like wild-type animals in the motility assay. The hits are FDA-approved compounds or are currently in clinical trials for other neurological disorders. We should be able to identify efficacious compounds that can be tested in higher organisms and eventually enter clinical development.

Jerome Reboul et al. (2001) International C. elegans Meeting "Open-reading-frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans"

Philippe Vaglio, Tadasu SHIN-I, Jerome Reboul, James L Hartley, Danielle Thierry-Mieg, Jean Vandenhaute, Troy Moore, Yuji KOHARA, Joseph Hitti, Lynn Doucette-Stamm, Jean Thierry-Mieg, Michael A Brasch, David E Hill, Gary F Temple, Cindy Jackson, Nicolas Thierry-Mieg, Hongmei Lee, Jean Francois Rual, Philippe E Lamesch, Marc Vidal

[International C. elegans Meeting, 2001]

The genome sequences of Caenorhabditis elegans , Drosophila melanogaster and Arabidopsis thaliana have been predicted to contain 19,000, 13,600 and 25,500 genes, respectively. Before this information can be fully used for evolutionary and functional studies, several issues need to be addressed. First, the gene number estimates obtained in silico and not yet supported by any experimental data need to be verified. For example, it seems biologically paradoxical that C. elegans would have 50% more genes than D. melanogaster . Second, intron/exon predictions need to be experimentally tested. Third, complete sets of open reading frames (ORFs), or ORFeomes, need to be cloned into various expression vectors. To simultaneously address these issues, we have designed and applied to C. elegans the following strategy. Predicted ORFs are amplified by PCR from a highly representative cDNA library using ORF-specific primers, cloned by Gateway recombination cloning, and then sequenced to generate ORF sequence tags (OSTs), as a way to verify identity and splicing. In a sample (n=1,222) of the nearly 10,000 genes predicted ab initio (that is, for which no expressed sequence tag (EST) is available so far), at least 70% were verified by OSTs. We also observed that 27% of these experimentally confirmed genes have a different structure from that predicted by GeneFinder. We now have experimental evidence that supports the existence of at least 17,300 genes in C. elegans . Hence we suggest that gene counts based primarily upon ESTs may underestimate the number of genes, in human and in other organisms.

Joseph Gallagher et al. (2006) European Worm Meeting "Development and Optimisation of Mos1 for Gene Tagging in C. elegans"

Patricia Kuwabara, Joseph Gallagher

[European Worm Meeting, 2006]

Joseph Gallagher and Patricia Kuwabara. The Drosophila transposon, Mos1, has been used successfully to generate insertional mutants in C. elegans1. Mos1 can be mobilised in C. elegans worms, which carry two independent extrachromosal arrays, one encoding the Mos1 transposase under the control of a heat shock promoter, and the other carrying copies of the Mos1 transposase substrate. Heat induction of the transposase leads to the mobilisation of the transposon and its integration within the C.elegans genome. Stable Mos1 insertions are detected by PCR after animals have been clonally passaged for a number of generations. As a member of the NemaGENETAG Consortium, which is funded through EU fp6, we are working with our partners to optimise the already existing Mos1 tools and to develop new strains to facilitate the high-throughput screening of C. elegans Mos1 insertional mutants. The generation of C. elegans transposon tagged genes will enhance our understanding of gene function, especially those that are associated with human disease, and provide an invaluable resource to the research community.. Here, we present our preliminary results, which were obtained after screening a library consisting of almost 1000 strains. So far, from this and subsequent screens, over 300 Mos1 strains have been identified by PCR. Each of these strains has been frozen at 80?C in triplicate, test thawed and re-tested to validate the presence of a Mos1 insertion. Many of the characterised strains carry multiple copies of Mos1 inserted at different sites, so the actual number of tagged loci is greater than the number of strains frozen. Steps are now being taken to improve the rate of transposition and the recovery of mutants. 1.. Bessereau, J. L. et al. Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line. Nature 413, 70-4 (2001).

Soltesz, Z. et al. (2011) International Worm Meeting "Carbon dioxide avoidance is mediated by a diverse set of sensory neurons in C. elegans."

Bretscher, A. J., de Bono, M., Soltesz, Z.

[International Worm Meeting, 2011]

Monitoring carbon dioxide levels has a twofold importance for many living organisms: CO2 can act as a sensory cue for food or other animals, while regulating internal CO2 levels is an important part of homeostasis. C. elegans relies on diffusion for gas exchange, and avoids CO2 levels as low as 1%. We are interested in the neural and molecular mechanisms underlying the C. elegans CO2 avoidance behaviour. Mutants defective in the tax-4 or tax-2 genes, which encode the a and b subunits, respectively, of a cGMP-gated ion channel, showed reduced CO2 avoidance in behavioural assays1,2. By expressing tax-2 cDNA from neuron-specific promoters in tax-2 mutants to rescue the avoidance behaviour, and by imaging neurons using the genetically encoded calcium indicator YC3.60, we have shown that sensory neurons previously implicated in oxygen, temperature, and salt-sensing, including BAG, AFD and ASE, are CO2 sensors as well3. We have observed both persistent and transient cell-intrinsic calcium-responses in several sensory neurons, suggesting that CO2 stimuli could modulate neural activity in C. elegans in a complex manner. We are therefore investigating how CO2 stimuli can affect neural processing in downstream neurons. 1 Andrew Jonathan Bretscher, Karl Emanuel Busch, and Mario de Bono, A carbon dioxide avoidance behavior is integrated with responses to ambient oxygen and food in Caenorhabditis elegans. PNAS 105(23):8044-8049 2 Elissa A. Hallem and Paul W. Sternberg, Acute carbon dioxide avoidance in Caenorhabditis elegans. PNAS 105(23):8038-8043 3 Andrew Jonathan Bretscher, Eiji Kodama-Namba, Karl Emanuel Busch, Robin Joseph Murphy, Zoltan Soltesz, Patrick Laurent and Mario de Bono, Temperature, Oxygen, and Salt-Sensing Neurons in C. elegans Are Carbon Dioxide Sensors that Control Avoidance Behavior. Neuron 69(6):1099-1113.

Andreia Teixeira-Castro et al. (2007) International Worm Meeting "In vivo dynamics of ataxin-3 aggregation in C. elegans neurons."

Patricia Maciel, Richard Morimoto, Andreia Teixeira-Castro

[International Worm Meeting, 2007]

At least nine human neurodegenerative diseases are caused by the expansion of CAG repeats within otherwise unrelated genes. In these diseases, including Machado-Joseph disease (MJD), polyglutamine (polyQ) expansions cause the appearance of misfolded protein species, aggregates, neuronal dysfunction and cell death. Along with the pathogenic motif, all these diseases have in common the fact that the associated gene products are widely expressed but affect only specific subsets of neurons. This specificity suggests that protein misfolding and its toxic outcomes may be determined by the amino acid sequence of the particular protein. Ataxin-3 (AT3) is a polyQ protein and expansion of its repetitive glutamine tract causes MJD. MJD, like other polyQ diseases, is characterized by the formation of intraneuronal inclusions but the mechanism underlying their formation is poorly understood. Caenorhabditis elegans offers unique advantages for examining the aggregation behavior and toxic effects of polyQ proteins on individual neurons, since the transparency of all 959 cells allows easy detection of fluorescent proteins in live animals. Here, we used high-end imaging techniques, such as Fluorescence Recovery after photobleaching (FRAP) and Fluorescence Resonance Energy Transfer (FRET), to analyze the biophysical properties of YFP-tagged AT3, in live C. elegans neurons. In our novel pan-neuronal C. elegans model of AT3 aggregation, we show that expression of human pathogenic full-length AT3 alone did not cause aggregation, assessed by FRAP, but was dependent on the presence of an aggregated seed capable of initiating the nucleation events. FRAP analysis showed that when full-length AT3 is sequestered into aggregated polyQ-alone proteins, it acquires properties of immobile, aggregated protein. FRET results suggested that AT3 does not orderly interact with polyQ-only protein within these co-aggregates. Moreover, the study of the dynamics of the sequestration process of pathogenic and non-pathogenic wild-type AT3 showed that this process may occur in an ageing-dependent manner.

Fard Ghassemi, Yasmin et al. (2017) International Worm Meeting "Rescue of ATXN3 Neuronal Toxicity in C. elegans by Chemical Modification of ER Stress."

Gosselin, Sarah, Tauffenberger, Arnaud, Parker, Alex, Fard Ghassemi, Yasmin

[International Worm Meeting, 2017]

Polyglutamine expansion diseases are a class of dominantly inherited neurodegenerative disorders that develop when a CAG repeat in the causative genes is unstably expanded above a certain threshold. The expansion of trinucleotide CAG repeats causes hereditary adult-onset neurodegenerative disorders such as multiple forms of spinocerebellar ataxia (SCA). The most common dominantly inherited spinocerebellar ataxia is the type 3 (SCA3) also known as Machado-Joseph disease (MJD), an autosomal dominant, progressive neurological disorder. The gene causing MJD is ATXN3 (ATAXIN-3). The prevalence of MJD is increasing and there are no pharmacological therapies available that successfully treat this disease. Therefore, the development of novel therapeutics for MJD is urgently needed. In this study, we generated transgenic C. elegans strains expressing wild type or mutant human ATXN3 genes and tested them for recovery of motility defects, decreased lifespan, and neurodegeneration phenotypes upon treatment with compounds known to modulate ER stress and having neuroprotective roles. We observed differences between both transgenic lines and found that the motility defects, the reduced lifespan and neurodegeneration were rescued by compounds that have been previously identified in our laboratory. These compounds were also able to prevent the oxidative stress and the ER stress response induced by mutant ATXN3 in transgenic worms. These promising results prompted us to expand our approach perform to a comprehensive, blind drug screen of ~3600 compounds in our transgenic ATXN3 lines. The aim of this screen is to identify new compounds that allow us to directly gain insights into the mechanisms underlying MJD. We introduce novel C. elegans models for MJD based on the expression of full-length ATXN3 in GABAergic motor neurons. Using these models we discovered that chemical modulation of the ER unfolded protein response reduced neurodegeneration and could be a new therapeutic approach for the treatment of MJD. Also, using C. elegans to study MJD in conjunction with well-characterized compounds, we may identify underlying mechanisms that could also be used to develop novel therapeutic approaches.

Costa, Marta Daniela Araujo et al. (2021) International Worm Meeting "Balancing aging, proteostasis and nervous system function: differential effects of multiple lifespan-extending genetic manipulations in MJD/SCA3."

da Silva, Jorge Diogo, Costa, Marta Daniela Araujo, Pereira-Sousa, Joana, Teixeira-Castro, Andreia, Almeida, Dulce, Maciel, Patricia

[International Worm Meeting, 2021]

Over the past few years evidence that contradicted aging as an inevitable phenomenon has surged, leading the scientific community to concentrate efforts to test drugs that effectively tackle aging. In parallel, this approach aimed to decrease the prevalence of a number of different disorders, such as neurodegenerative diseases, for which aging is a key risk factor. Here, the hypothesis that delaying aging is neuroprotective was assessed in a C. elegans model of Spinocerebellar Ataxia (SCA) Type 3, also known as Machado-Joseph disease (SCA3/MJD), the most common SCA worldwide. This neurodegenerative disease has a clear genetic cause, the abnormal expansion of a CAG triplet in the ataxin-3 gene. However, the contribution of additional genetic/environmental factors have been proposed to explain the variable disease phenotype. Lifespan-increasing mutations that are representative of well-known and conserved aging regulator mechanisms (insulin/IGF-1 signaling, dietary restriction, germline ablation and mitochondrial dysfunction) were introduced in the genetic background of the SCA3 nematode model. Their impact in key aspects of the disease was then assessed. Lifespan-extension improved the SCA3 motor phenotype if induced by altered nutrient sensing pathways, as is the case of the insulin/IGF-1 and mTOR signaling, but not when associated with other pathways, such as mitochondrial dysfunction and germline ablation. This challenges the idea that delaying aging is by itself beneficial and regarded a guaranteed therapy for these diseases. Additional experiments pointed to significant transcriptomic alterations in the proteostasis network caused by the downregulation of IGF-1/insulin signaling. However, not all insulin/IGF-1-dependent transcriptional responses seemed disease-modifying, suggesting that neuroprotective effects of aging can be restricted to more specific aging factors. Finally, chronic treatment of the C. elegans SCA3 model with insulin/IGF-1 signaling inhibitors also improved the motor phenotype, further demonstrating the therapeutic value of insulin/IGF-1 downregulation for the disease, increasing prospects for additional drug repurposing centered in this pathway. These results provide key insights to guide future therapeutic strategies for neurodegenerative diseases based on the manipulation of the aging process.