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[
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.
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[
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.
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[
International Worm Meeting,
2005]
Medical students are often fascinated with the nervous system and with organismal impacts of its dynamics and destruction. The study of molecular cues underlying phenomena such as axonal migration, if properly explored, could catch the students motivation and commitment to learn biochemistry. C. elegans is a suitable, low cost, laboratory model to let moderately skilled students explore the consequences of molecular events at the whole organism level. This work reports on the use of C. elegans to introduce 2nd year medical students to the deleterious effects of gene Knockouts upon response of neurons to extracellular biochemical cues. The main goals of this learning activity were for the students to (1) understand at a basic level the mechanisms of axon guidance and (2) propose possible applications for the usage of knowledge on axon guidance molecules in the treatment of medical pathologies. We divided the activity in three moments. First, students performed behavioral assays to detect movement alterations in C. elegans mutants lacking specific axon guidance molecules. At this moment, students were not aware which genes were mutated. Microscopy observation of abnormal axonal migration patterns of dorsal and ventral cord neurons was also performed. For optimal visualization of neurons, these mutants had been crossed with a pan-neuronal GFP expression strain (Unc-119::GFP). Identification of the specific gene mutated in each strain was then provided to students: Unc-6; Unc-5 and Unc-40. The students were asked to relate abnormal patterns in axon migration/nerve formation with the absent molecular components and the phenotype of mutant strains. Analysis and discussion of a review article about extracellular guidance cues allowed students to further acquire background and consolidate knowledge on this topic (Dynamic regulation of axon guidance. Yu T.W. and Bargmann C.I.; 2001; Nature Neuroscience 4:1169-1176). Finally, students proposed some clinical applications of the recent knowledge concerning the molecular guidance cues and their role in neuronal migration.
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[
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.
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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.