Christian Neri et al. (2007) International Worm Meeting "Longevity modulators and protection against the cytotoxicity of degenerative disease proteins: of simple models and translational research."

Cendrine Tourette, J. Alex Parker, Salima Abderhamane, Emmanuel Lambert, Sabine Cleophax, Alexandre Heissat, Beate Gerstbrein, Christian Neri

[International Worm Meeting, 2007]

Genes and gene networks at the interface between longevity/survival and neuroprotection are currently the subject of intense research. Recent reports based on the use of simple model systems have emphasized a role for these genes in protection against the effects of proteins and polypeptides associated to neurodegenerative diseases. We will present a short overview of recent data in the field. We will also present our most recent data on the study of longevity modulators in simple models of Huntingtons disease (HD) pathogenesis. HD is a neurodegenerative disease caused by expanded polyglutamines (polyQs) in huntingtin (htt). Although HD is inherited, its age-at-onset shows a significant level of variability not fully explained by expanded htt alleles, suggesting a role for modifier genes. We hypothesized HD modifier genes may belong to longevity pathways that modulate the neuronal cell response to mutant htt. To study how the neuronal cell may respond to mutant htt, we use translational research primarily based on the genetic manipulation of C. elegans transgenics at the neuronal cell level and biological assays in cellular and mouse models of HD. We found sirtuin activation is a neuroprotective mechanism against the effects of mutant htt, an effect mediated by daf-16/FOXO (Parker et al., Nature Genetics 2005), providing the first direct evidence for longevity modulators like Sir2 and FOXO to modify the neurotoxic properties of neurodegenerative disease-associated proteins like mutant htt, and defining a new strategy with therapeutic potential. DAF-16/FOXO has numerous upstream modulators and downstream targets, not all of which are likely to be important to neuronal protection. Using RNAi, we identified a subset of daf-16 targets that modify the neuronal dysfunction induced by mutant polyQs. We are extending this approach to include the microarray analysis of gene expression profiles in cell-sorted polyQ neurons from C. elegans transgenics. Data integration and classification using gene networks emphasize gene classes, pathways and biological processes that are conserved in humans and may be essential to neuronal cell activity and survival in HD, providing new information to best understand the mechanisms underlying the disease pathogenesis and residual age-at-onset. Drug discovery aspects will also be commented.

Melentijevic, Ilija et al. (2013) International Worm Meeting "Neuronal Exophers: a Novel Mechanism for Removal of Neurotoxic Cytoplasm Components."

Driscoll, Monica, Melentijevic, Ilija, Toth, Marton, Neri, Christian

[International Worm Meeting, 2013]

We discovered a new, previously unknown feature of young adult C. elegans neurons-neurons can extrude substantial packets of cellular contents, which can include aggregated human neurodegenerative disease proteins or mitochondria, but no nuclear DNA. We currently call these extrusions "exophers". The ability to jettison cell contents appears to change with age, and extrusion is increased when protein turnover or autophagy is inhibited. Thus, this pathway may constitute a novel neuronal protection mechanism that serves to maintain protein/organelle homeostasis when other systems are compromised. We propose that the neuronal extrusion phenomenon constitutes a significant but currently unknown conserved pathway by which healthy neurons maintain their functions, and speculate that, in disease, this pathway may malfunction to promote spread of pathology. We will present the basic characterization of neuronal exopher production and our latest data on genetic influences on exopher generation.

Alex J Parker et al. (2003) International Worm Meeting "C. elegans as a model to study the pathogenesis and therapy of Huntingtons disease"

Abderrahmane Salima, Neri Christian, Holbert Sebastien, Alex J Parker, Lambert Emmanuel

[International Worm Meeting, 2003]

C. elegans amenability to genetic analyses and pharmacological screens may uncover aspects of the toxicity of human disease-associated proteins difficult to manipulate in vivo by other means. We have validated a worm-based approach to the study of Huntington's disease pathogenesis. We have observed that huntingtin, the HD protein, is able to interact with C. elegans proteins, and that human homologs of huntingtin interactors in worms may be involved in HD pathogenesis, as shown for the transcriptional regulator CA150, a candidate modifier gene in HD. We have described a transgenic C. elegans model of polyglutamine-dependent neuronal dysfunction without cell death. In this model, polyQ-expanded N-terminal huntingtin fused to fluorescent reporter proteins produces a significant mechanosensory defect at the tail. This phenotype partially correlates with aggregation of fusion proteins in neuronal processes and abnormal morphology of neuronal cell axons. This phenotype can be genetically suppressed with or without a reduction of aggregation, and genetic suppressors are being characterized. As part of a multi-assay neurodegeneration drug screening program coordinated by the NINDS, we have tested a collection of 1040 drugs in our transgenic animals. We have detected a manageable number of confirmed hits that show a dose-dependent restoration of touch sensitivity at the tail. One of these compounds appeared to significantly reduce aggregation in neuronal processes and abnormal morphology of axons in a dose-dependent manner. Our drug screening data suggest that the use of worm mechanosensation allows for a sensitive detection and validation of active compounds, which may be instructive in selecting drug leads.

Farina, Francesca et al. (2011) International Worm Meeting "Automated sub-cellular imaging for drug screening in C. elegans."

Vernet, Remi, Neri, Christian, Farina, Francesca, Peignelin, Betty, Meiller, Clement, Parmentier, Frederic

[International Worm Meeting, 2011]

Screening for drugs at the sub-cellular level may enhance drug discovery and development for degenerative disease. The nematode C. elegans has potential for drug screening at the sub-cellular level and in vivo as it is transparent at all stages of development and can be grown in multi-well plates. To develop drug screening at the sub-cellular level in nematodes, we took advantage of the Plate Runner HD (Trophos, France), a 96/384-well device that collects fluorescence at resolutions ranging from 1024x1024 (1 px is 7.4 mm) to 8192x8192 (1 px is 1 mm). This device has high depth-of-field (about 40 mm at resolution of 7.4 mm; 8 mm at resolution of 1 mm), thus allowing fluorescent signals to be quantified from whole animals after paralysis. This device also has a wide-field objective that allows a single image of the whole well to be acquired at once. We developed a drug screening assay at the sub-cellular level to search for compounds that may protect from the early-stage cytotoxicity of mutant PABPN1, the oculopharyngeal muscular dystrophy (OPMD) protein. Transgenic nematodes co-expressing nuclear GFP and mutant PABPN1 (PABPNA-A13) in body wall muscles show defective motility, a phenotype that is accompanied by a progressive loss of nuclear GFP signals. These phenotypes are aggravated by sirtuin (sir-2.1/SIRT1) activation and ameliorated by sir-2.1 inhibition, and they can be also manipulated by pharmacological means [1,2]. The loss of GFP nuclei in mutant PABPN1-A13 animals provide an easily-assayable phenotype to screen for drugs that may rescue mutant PABPN1 toxicity. Results from screening greater than 2000 compounds indicated that our screen was robust and selective. As part of a drug discovery program on neuromuscular diseases, we are also exploring the development of a drug screening assay at the sub-cellular level to search for compounds that protect from defective axonal transport. To this end, we generated transgenic nematodes that express extrachromosomal arrays encoding a fluorescent pre-synaptic reporter in GABAergic motor neurons and that carry a temperature-sensitive loss-of-function allele in a gene encoding an axon motor gene. Preliminary results suggest that, at the restrictive temperature, this mutant allele induces a significant change in the expression pattern of pre-synaptic signals. This effect is unrelated to a change in transgene expression and can be quantified by the Plate Runner HD after paralysis of the animals in the 96-well plate. Results from the two approaches abovementioned will be presented and discussed. [1] Catoire, H. et al. (2008) Hum Mol Genet 17, 2108-2117. [2] Pasco, M.Y. et al. (2010) J Med Chem 53, 1407-1411.

Nichols, Courtney Rose et al. (2013) International Worm Meeting "A role for the insulin signaling pathway in development of neuronal aging markers in a polyglutamine protein aggregation C. elegans model."

Parker, J. Alex, Nichols, Courtney Rose, Driscoll, Monica, Neri, Christian, Vayndorf, Elena, Taylor, Barbara

[International Worm Meeting, 2013]

Neurodegenerative diseases, such as Huntington's, Alzheimer's, and Parkinson's disease, result in the progressive loss of neuronal structure and function with age. Many neurodegenerative disorders are caused by genetic mutations and characterized by toxic aggregation of proteins within neurons. We explored the mechanism of accelerated neuronal aging in a polyglutamine (polyQ) protein aggregation model through genetic manipulation. Caenorhabditis elegans expressing the first 57 amino acids of human huntingtin protein with a toxic polyglutamine chain (polyQ128) fluorescently labeled with CFP in YFP-labeled mechanosensory neurons (Parker et al, 2001) were used to monitor neuronal aging phenotypes. We found an age-associated increase in aberrant neuronal morphology, protein aggregation, and functional impairment of the mechanosensory neurons expressing toxic 128 polyQ. We also tested the hypothesis that the insulin signaling pathway is involved in morphological and functional health of aging mechanosensory neurons using neuron-specific RNA interference (RNAi). Furthermore, we measured levels of endogenous oxidative stress and lifespan of aging animals that contain toxic polyQ128 repeats following RNAi manipulation of the insulin signaling pathway. Overall, we found that DAF-16/FOXO is neuroprotective in this accelerated aging model, which corroborates previous findings on the role of DAF-16/FOXO in polyQ128 young adults (Parker et al. 2005). To further characterize overall muscle and neuronal health, we measured action potentials of pharyngeal muscle contractions and neurons that innervate the pharynx using microfluidic electropharyngeography (EPG). In total, our observations support that insulin signaling via DAF-16/FOXO is a mechanism through which accelerated aging and neurodegeneration occurs in this model.

Lejeune, Francois-Xavier et al. (2011) International Worm Meeting "The Biogemix knowledge base project: cross-species and network-based data integration for Huntington's disease research."

Vert, Jean-Philippe, Parmentier, Frederic, Lejeune, Francois-Xavier, Neri, Christian, Bicep, Cedric, Mesrob, Lilia

[International Worm Meeting, 2011]

The identification and validation of neuroprotective targets is of primary importance in research on neurodegenerative diseases such as Huntington's disease (HD). The development of genetically tractable models of disease and their use in genome-wide screens has generated a large amount of data in several species. A current challenge is the unbiased integration of these data sets in order to prioritize candidate target genes. The Biogemix knowledge base project has been developed with the European HD network (Euro-HD) to integrate 'omics' data from models of HD pathogenesis as available in several species (invertebrates, mammalian cells, mice, human samples). This project relies on the combination of network-based and cross-species procedures to unlock the biological information buried into disease data sets. The Biogemix procedure is a method that relies on the use of molecular networks for the unbiased integration of 'omics' data across different species. This method is particularly suited to the analysis of data sets for which the number of genes analyzed clearly exceeds the number of conditions tested. Single data sets are firstly processed with respect to a reference molecular network (for instance, use of MouseNet to analyze mouse data) to extract clusters (modules) that are made of highly interconnected genes, enriched in HD-relevant information and automatically annotated for their biological role and biomedical potential. In a second step, cross-species clusters (meta-modules) are calculated by balancing gene/protein connectivity with protein sequence similarities. In a third step, all of the Biogemix products are ranked according to topological and biological features of interest, which is part of a larger prioritisation system that uses several criteria to classify modules and genes of high interest. A user-friendly graphical interface and query system is being developed to allow the users to browse and select Biogemix information of interest. Further developments will aim at fine-tuning data analysis and information display in view of making the Biogemix knowledge base v 1.0 publically available on-line. Preliminary results will be shown to illustrate how the Biogemix system might be useful for basic research and disease research.

Melentijevic, Ilija et al. (2015) International Worm Meeting "Neuronal Exophers: a novel mechanism for the removal of neurotoxic cytoplasm components."

Driscoll, Monica, Arnold, Meghan, Parker, Alex, Guasp, Ryan, Toth, Marton, Neri, Christian, Harinath, Girish, Melentijevic, Ilija

[International Worm Meeting, 2015]

Combating late-onset neurodegenerative disease and age associated functional decline in brain are major health challenges of our time. For the effective design of interventions that protect the nervous system from disease-induced and/or age-associated deterioration, we must fully understand endogenous mechanisms for neuronal protection and how they might fail to enable disease promotion. Recently, it has come to be appreciated that neurodegenerative disease proteins/aggregates can be found outside of mammalian neurons, and when outside, can actually be taken up by neighboring cells. Transfer of offending molecules has been suggested to be a mechanism of pathogenesis spread for multiple neurodegenerative diseases, including the prevalent Alzheimer's and Parkinson's diseases.We discovered a novel capacity of young adult C. elegans neurons - neurons can extrude substantial packets of cellular contents, which can include aggregated human neurodegenerative disease proteins, mitochondria, or lysosomes, but no nuclear DNA. We currently call these extrusions "exophers". The ability to jettison cell contents appears to change with age, and extrusion is increased when protein turnover is impaired, autophagy is inhibited, or mitochondria are compromised. Moreover, exophers can selectively incorporate aggregation-prone proteins and mitochondria with elevated levels of an oxidized reporter. Thus, exopher-mediated extrusion may constitute a novel neuronal protection mechanism that serves to maintain protein/organelle homeostasis when other systems are compromised or overloaded. We propose that the neuronal extrusion phenomenon constitutes a significant but currently unknown conserved pathway by which healthy neurons maintain their functions, and speculate that, in neurodegenerative diseases, this pathway may malfunction to promote spread of pathology. We will present the basic characterization of neuronal exopher production and our latest data on genetic influences on exopher generation.

Vazquez-manrique, Rafael et al. (2011) International Worm Meeting "AMPK activation is protective in models of neuron dysfunction and death in Huntington's disease."

Deglon, Nicole, Neri, Christian, Offner, Nicolas, Vazquez-manrique, Rafael, Cambon, Karine, Orfila, Anne-marie, Darbois, Aurelie

[International Worm Meeting, 2011]

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine (polyQ) expansion in the huntingtin protein (Htt). Expression of the first exon of Htt containing an expanded polyQ produces neuronal dysfunction and axonal dystrophy in mechanosensory neurons in C. elegans (Parker et al., 2001). Genetic pathway analysis using this nematode model has emphasized a role for the longevity-promoting factor daf-16/FoxO in the protection of neurons from expanded polyQ toxicity (Parker et al., 2005). Here, we investigated the role of AMP-activated protein kinase (AMPK), a well-known energy sensor involved in lifespan and health span extension, on neuronal dysfunction in expanded-polyQ nematodes and vulnerability to cell death in mutant Htt striatal cells (HdhQ111 knock-in mice). In C. elegans, activating this enzyme (aak-2/AMPK) with metformin reduces the neuronal dysfunction caused by expanded polyQ expression. In contrast, aak-2 mutants show enhanced neuronal impairment. In striatal cells from HdhQ111 knock-in mice, reducing AMPK levels by siRNA treatment enhances the susceptibility to cell death of mutant htt cells, and metformin treatment has the opposite effect. Additionally, overexpressing AMPK reduced striatal neurodegeneration in a rat lentiviral-based fragment model of HD pathogenesis. Collectively, our results suggest that AMPK activation has therapeutic potential to protect from neuron dysfunction and degeneration in HD.

Vayndorf, Elena et al. (2015) International Worm Meeting "Protein homeostasis dysregulation is associated with aberrant morphology and reduced function of aging mechanosensory neurons."

Hunter, Skyler, Driscoll, Monica, Toth, Marton, Scerbak, Courtney, Taylor, Barbara, Neri, Christian, Parker, J. Alex, Vayndorf, Elena

[International Worm Meeting, 2015]

In both C. elegans and humans, the aging nervous system is characterized by decreased synaptic activity, deteriorating short-term and long-term memory, and altered neuronal morphology. Given the overwhelming evidence for proteostasis disruption in neuronal aging, we sought to explain the accumulation of neuronal morphological abnormalities by focusing on protein homeostasis in 6 mechanosensory neurons of aging C. elegans nematodes. We examined the effects of disrupted proteostasis on the integrity of neuronal cytoarchitecture using a transgenic model with an excessively high neuronal protein load, and RNAi knock down of specific genes involved in protein turnover. We found that animals expressing the first 57 amino acids of the human huntingtin gene and an expanded polyglutamine CAG tract (Q128) in mechanosensory neurons accumulate more aberrations that are distinct from those found in animals that express the non-toxic (Q19) number of repeats, or those that express no repeats. We scored and tallied these changes in both the soma and processes and found that they are sometimes associated with improved or reduced function. Next, we used an RNAi candidate gene approach to target genes involved in the maintenance of protein homeostasis in wild-type animals. We found that genes involved in protein turnover play an important role in maintaining the integrity of healthy neurons, and that their knockdown leads to distinct morphological changes in both the process and the soma of wild-type mechanosensory neurons. Taken together, these results suggest that protein homeostasis is critical for maintaining neuronal integrity and function, and that disrupted proteostasis contributes to morphological abnormalities that occur more frequently with advanced age.

Vayndorf, Elena et al. (2013) International Worm Meeting "Protein homeostasis dysregulation drives aberrant morphology of aging mechanosensory neurons."

Neri, Christian, Parker, J. Alex, Toth, Marton, Nichols, Courtney, Vayndorf, Elena, Taylor, Barbara, Driscoll, Monica, Hunter, Skyler, Parker, Cyrena

[International Worm Meeting, 2013]

In both C. elegans and mammals, the aging nervous system is characterized by decreased synaptic activity, deteriorating short-term and long-term memory, and altered neuronal morphology. We sought to elucidate the functional consequences of altered neuronal morphology by focusing on protein homeostasis in individual neurons. We examined the effects of disrupting proteostasis on the integrity of neuronal cytoarchitecture using a transgenic model with an excessively high neuronal protein load. We found that animals expressing the first 57 amino acids of the human huntingtin gene and an expanded polyglutamine CAG tract (128Q) in mechanosensory neurons accumulate significantly more neuronal aberrations and more protein aggregates, and have a significantly greater decline in function with age, compared to animals that express the non-toxic (19Q) number of repeats, or those that express no repeats. We identified specific morphological alterations, in particular extreme outgrowths in the soma of ALM mechanosensory neurons, as well as a wavy phenotype in processes of PLM neurons, as the major aberrant morphological types in this transgenic background. Our RNAi studies suggest that targeting genes expressed in organelles associated with the maintenance of proteostasis, in particular the proteosome, lysosome and endoplasmic reticulum, alters neuronal morphology and accelerates aging in wild-type animals. Taken together, these results suggest that protein homeostasis is critical for maintaining neuronal integrity and that disrupted proteostasis contributes to morphological abnormalities that increase in frequency with age.