-
Ferrara, Lorenzo, Callaly, Frank, Morgan, Fearghal, Mc Ginley, Brian, Leskovsky, Peter, Blau, Axel, Machado, Pedro, Krewer, Finn, McGinnity, Martin, Epelde, Gorka, Petrushin, Alexey, Mujika, Andoni
[
International Worm Meeting,
2015]
Despite being one of the five best-characterized model organisms, there is still only sparse knowledge on how C. elegans' nervous system codes for its rich behavioral repertoire. The European Si elegans project aims at unravelling C. elegans' nervous system function (hermaphrodite) by its emulation with 302 parallelly interconnected field-programmable gate array (FPGA) neurons and by embodying this hardware nervous system with a biophysically accurate soft-body representation in a virtual behavioral arena. We critically discuss past and present neurocomputational approaches and pitfalls of simulating nervous system function. We then present the first Si elegans implementation and system integration steps with focus on its key components and challenges. The biophysics-based simulation platform shall allow scientists to test neural processing hypotheses and (un)published behavioral paradigms. Users will have access to all currently known and biologically relevant variables for the reverse-engineering of nervous system function to confirm or even anticipate some of the underlying principles. This contribution aims at opening a scientific discourse on the requirements, possibilities and limits of the faithful biomimetic emulation of an organism from both the biological and engineering perspectives.Acknowledgements: This project has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement ndeg 601215, FET Proactive, call ICT-2011.9.11: Neuro-Bio-Inspired Systems (NBIS). www.si-elegans.eu.
-
[
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.
-
[
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.
-
[
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.
-
Jalles, A., Miranda, A., Araujo, M., Morimoto, R., Maciel, P., Bessa, C., Teixeira-Castro, 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.
-
[
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.
-
[
European Worm Meeting,
2006]
The ubiquitin-proteasome system is responsible for selective proteolysis of intracellular proteins in eukaryotic cells. In this system, protein substrates are marked for degradation by attachment of multiubiquitin chains. The process of multiubiquitylation requires a cascade of enzymes, additional proteins ensure correct targeting to the 26S proteasome. As has been shown for a variety of genes of the ubiquitin pathway, also the disease pathology of spinocerebellar ataxia type 3/Machado-Joseph Disease (SCA3/MJD) is attributed with alterations in ubiquitin-pathway functions. This disease is one of at least nine neurodegenerative polyQ (polyglutamine) diseases including Huntingtons disease caused by the pathological expansion of a polyQ region in the disease protein, which leads to a misfolding process and subsequent accumulation of insoluble protein aggregates. One protein implicated in SCA3/MJD is Ataxin-3/ATX-3, which comprises two distinct catalytic activities functionally related to the ubiquitin system: a deubiquitinating Josephin domain at the N-terminus and C-terminal UIM domains which mediate binding to ubiquitin.. Currently, we are interested in the molecular function of the C. elegans homolog of mammalian Ataxin-3. We were able to show the de-ubiquitylation activity with recombinantly expressed protein in vitro and are now investigating the binding properties of Ataxin-3/ATX-3 to ubiquitin or to ubiquitylated proteins. With the yeast two hybrid assay we identified new interactors of Ataxin-3/ATX-3. One of the identified interactors shows an overlapping expression pattern with Ataxin-3/ATX-3 in the spermatheca. Moreover, our genetic analysis with two
atx-3 loss of function mutants revealed a genetic interaction: brood size defects of a mutant allele for this interactor is suppressed in the
atx-3 mutant background. Our current model suggests, that Ataxin-3/ATX-3 in conjunction with additional components of the ubiquitin-proteasome system modulates ubiquitin-dependent pathways which might play a role throughout sex-determination.
-
Maciel, Patricia, da Silva, Jorge Diogo, Teixeira-Castro, Andreia, Almeida, Dulce, Costa, Marta Daniela Araujo, Pereira-Sousa, Joana
[
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.
-
[
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.