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.

Rafael Vazquez-Manrique et al. (2002) European Worm Meeting "Characterisation of the Caenorhabditis elegans orthologue of the Friedreich's ataxia gene"

Rafael Vazquez-Manrique, Howard Baylis, Pilar Gonzalez-Cabo, Francesc Palau

[European Worm Meeting, 2002]

Friedreich's ataxia is an autosomal recessive neurodegenerative disease caused by loss of function mutations in the FRDA gene. The most frequent mutation in the expansion of a GAA trinucleotide repeat in the first intron of the gene. Deficiency of frataxin, the product of the FRDA gene, results in ataxic movements, sensory axonal neuropathy, cardiomyopathy, skeletal deformities, and diabetes mellitus or glucose intolerance. This protein has been localized in the inner membrane of mitochondria. Lack of yeast frataxin, Yfh1p, produces an increase in mitochondrial iron, increased sensitivity to oxidative stress, and respiration deficiency. Some other experiments, in yeasts and mammalian cells point towards the possibility that the protein is implicated in ATP production or in assembling sulfur-iron cluster proteins. Our aim is to establish a C. elegans model to study the function of the frataxin and the pathophysiology of the disease.

Rafael P Vazquez-Manrique et al. (2003) International Worm Meeting "Caenorhabditis elegans as a model organism to study Friedreichs ataxia"

Pilar Gonzalez-Cabo, Howard A Baylis, Francesc Palau, Rafael P Vazquez-Manrique

[International Worm Meeting, 2003]

C. elegans has many orthologues of human genes including many genes involved in human disease. This feature allows the use of this worm as a model organism for biomedical research. Our aim in the present work is to establish a C. elegans model to study the pathophysiology of the Friedreichs ataxia. Friedreich's ataxia is an autosomal recessive neurodegenerative disease caused by mutations in the FRDA gene. The most frequent mutation is the expansion of a GAA triplet, which prevents correct transcription. This leads to a decrease in the level of frataxin, the product of this gene. This results in ataxic movements, sensory axonal neuropathy, cardiomyopathy, skeletal deformities, and diabetes mellitus or glucose intolerance. Frataxin is localized in the inner membrane of mitochondria. Lack of yeast frataxin, Yfh1p, produces an increase in mitochondrial iron, increased sensitivity to oxidative stress, and respiration deficiency. Iron-sulphur cluster biogenesis also seems to be affected.We describe the expression pattern of the C. elegans FRDA orthologue, frh-1 (F59G1.7). To elucidate the expression pattern of the nematode frataxin we generated transgenic lines by injecting a number of frh-1::gfp constructs differing in the size of the 5' fragments of genomic DNA wich they contained. frh-1 is expressed in specific head neurons, muscles, pharynx and intestine. This tissue pattern shows some analogy to human histological expression of frataxin.Transient knock-down experiments by RNAi produce a specific phenotype in worms of the F1 generation. The worms are: thin and short, Egl, have slow and arrhythmic pharynx pumping, and an increase in the period of the defecation rhythm. This model may be useful not only for studying pathogenic mechanisms of frataxin deficiency but also for drug screening.

Rafael P Vazquez-Manrique et al. (2008) European Worm Meeting "Phospholipase C-epsilon Regulates Epidermal Morphogenesis in Caenorhabditis elegans"

Sung Ly, Rafael P Vazquez-Manrique, Aniko I Nagy, James C Legg, Olivia A M Bales, Howard A Baylis

[European Worm Meeting, 2008]

Calcium is a ubiquitous intracellular signalling molecule that controls a. wide range of cellular functions. An important means of regulating calcium. signals is through the inositol 1,4,5-trisphosphate (IP3) pathway. IP3 is. produced by the action of the enzyme phospholipase C (PLC). IP3 acts on its. ion-channel receptor, the IP3-receptor, to release Ca2+ from the. endoplasmic reticulum. It has been shown that disruption of this pathway. results in defects in several aspects of embryogenesis in Caenorhabditis. elegans (1,2). Previous work has demonstrated a role for IP3-mediated. calcium signalling in the migration of epidermal cells during embryonic. morphogenesis (1). However, the mechanism by which IP3 production is. regulated in this process is unknown. By surveying the PLC family of C.. elegans using RNAi and mutant strains, we discovered that depletion of PLC-. 1/PLC-epsilon produced substantial embryonic lethality (3). Using the epithelial. cell marker ajm-1::GFP, to follow the behaviour of epidermal cells, we. found that 96% of these arrested embryos show morphogenetic defects. These. defects include defective ventral migration and aberrant dorsal. intercalation. Using time-lapse confocal microscopy we show that the. migration of the ventral epidermal cells, especially of the leading cells,. is slower and often fails in plc-1(tm753) embryos. As a consequence plc-1. loss of function results in ruptured embryos with a Gex phenotype (gut in. the exterior) and lumpy larvae. Thus PLC-1 is involved in the regulation of. morphogenesis. Genetic studies using gain- and loss-of-function alleles of. itr-1, the gene encoding the IP3 receptor in C. elegans, demonstrate that. PLC-1 acts through ITR-1. Using RNAi and double mutants to deplete the. other PLCs in a plc-1 background, we show that PLC-3/PLC-v and EGL-8/PLC-beta. can compensate for reduced PLC-1 activity. Our work places PLC-epsilon into a. pathway controlling epidermal cell migration, thus establishing a novel. role for PLC (3).. 1.Thomas-Virnig CL, Sims PA, Simske JS, Hardin J. (2004) The inositol. 1,4,5-trisphosphate receptor regulates epidermal cell migration in. Caenorhabditis elegans. Curr Biol.14:1882-7.. 2.Walker DS, Gower NJ, Ly S, Bradley GL, Baylis HA. (2002) Regulated. disruption of inositol 1,4,5-trisphosphate signaling in Caenorhabditis. elegans reveals new functions in feeding and embryogenesis. Mol Biol. Cell. 13:1329-37.. 3.Vazquez-Manrique RP, Nagy AI, Legg JC, Bales O AM, Ly S, Baylis HA.. (2008) Phospholipase C-epsilon Regulates Epidermal Morphogenesis in. Caenorhabditis elegans.PLOS Genetics, in press

Rafael P Vazquez-Manrique et al. (2007) International Worm Meeting "PLC-1/Phospholipase C epsilon regulates embryonic morphogenesis through the IP3 receptor in Caenorhabditis elegans."

Sung Ly, Howard A Baylis, Olivia Bales, Rafael P Vazquez-Manrique, James C Legg

[International Worm Meeting, 2007]

Signalling through the second messenger IP₃ is required at several stages of embryonic development in C. elegans. Previous work has demonstrated a role for IP₃ signalling in the rearrangement of epidermal cells during morphogenesis. However little is known about the molecules acting in this pathway. IP₃ is produced by phospholipase C enzymes (PLC). In order to identify signalling pathways upstream of IP₃ production, in epidermal morphogenesis, we sought to identify which of the five active PLCs in C. elegans is important for this process. We tested the function of PLC genes using RNAi and mutant strains, and we found that depletion of PLC-1/PLC-<font face=symbol>e</font> produced substantial embryonic lethality. 96% of these arrested embryos show morphogenetic defects, which include aberrant dorsal intercalation and defective ventral migration. As a consequence plc-1 loss of function results in ruptured embryos with Gex phenotype (gut in the exterior) and lumpy larvae. Thus PLC-1 is involved in the regulation of morphogenesis. However null or severe loss of function plc-1 mutants do not show the same level of embryonic lethality as some itr-1 mutants. We therefore tested if other PLCs may act redundantly with PLC-1. Using double mutants of PLCs together with plc-1, we show that PLC-3/PLC-<font face=symbol>g</font> and EGL-8/PLC-<font face=symbol>b</font> can compensate for the lack of PLC-1 activity. Finally, we demonstrate that PLC-1 acts through the IP₃ receptor of C. elegans (ITR-1), a molecule known to be involved in the regulation of C. elegans morphogenesis.

Tortajada Perez*, Julia et al. (2021) International Worm Meeting "Downstream effectors of the synergic activation of AMPK by metformin and salicylate to reduce polyQ aggregation"

Gomez Escribano, Ana Pilar, Vazquez Manrique, Rafael, Tortajada Perez*, Julia, Trujillo del Rio*, Cristina

[International Worm Meeting, 2021]

Huntington disease (HD) is a rare neurodegenerative disorder of autosomal dominant inheritance, characterized by an expansion in the number of CAG triplets in the huntingtin gene's (HTT) first exon. No therapy has been proven effective for HD treatment thus-far, but some compounds, such as metformin, have been reported as potentially beneficial disease-modifiers of this pathology. Previous findings have shown that the synergistic activation of AMP-activated protein kinase (AMPK), with metformin and salicylate, reduces aggregation and neuronal damage in Caenorhabditis elegans models of toxicity induced by polyglutamines (polyQs). Furthermore, strong evidence suggests that activation of AMPK, with just metformin, is neuroprotective in mouse models of HD. AMPK is a master regulator of healthspan and lifespan modulating energy metabolism, stress resistance and cellular proteostasis; via pathways such as autophagy, oxidative stress, and lipid metabolism. For instance, AMPK activates Nrf2/SKN-1, FoxO/DAF-16, and SIRT1/SIR-2.1 signalling pathways; and inhibits the signalling of CRTC-1/CRTC-1, and mTOR/LET-363. An activation of autophagy has been detected in metformin/salicylate-treated worms promoting proteostasis in a poliQs-induced toxicity context, however, preliminary results in our laboratory indicate that this activation may be independent of AMPK. In this work, we have studied several targets downstream of AMPK, potentially responsible for the protective effect dependent on salicylate and metformin synergistic AMPK activation, and show their relationship with this signalling pathway.

James Legg et al. (2006) European Worm Meeting "Testing the in vivo Importance of Putative Regulatory Sites in IP3 Receptors"

Rafael Vazquez-Manrique, Howard Baylis, James Legg

[European Worm Meeting, 2006]

James Legg, Rafael Vazquez-Manrique and Howard Baylis. Signalling through the second messenger inositol trisphosphate (IP3) and its receptor (IP3R) is a central mechanism by which extracellular signals regulate intracellular calcium signals. IP3 signalling plays roles in a wide range of processes in C. elegans. For example, IP3 signalling is widely involved in non-neuronal ultradian rhythmic processes; in particular feeding, defecation and ovulation (see 1 and references therein). IP3 signalling is also involved in developmental processes; embryos with disrupted IP3 signalling have defects in differentiation and in morphogenesis (2,3). The ability of IP3Rs to function in such a diverse range of processes is likely to require complex differential regulation. This is likely to be achieved, in part, through modulation of IP3R activity by other signalling molecules. A substantial number of potential and putative regulatory interactions have been identified in IP3Rs from a range of sources. These include interactions with other proteins and with small ligands such as ATP. Although much of this work has been performed with mammalian IP3Rs, the binding sites for many of these interactors are conserved in the C. elegans receptor. We have recently established a system that allows us to modify the itr-1 genomic DNA using homologous recombination in E. coli and then reintroduce modified itr-1 genes into C. elegans, in particular into itr-1 l.o.f mutants. This allows us to test the effects of modifying particular binding sites in the IP3R on its ability to rescue particular functions. We have used this system to test the importance of two sites (a) a putative ATP binding site and (b) the predicted binding site for FKBP12. Disruption of the ATP binding site appears to have modest effects on itr-1 function whereas disruption of the FKBP12 site appears to have severe effects on function. Therefore this system allows us to test the importance of regulatory sites, identified using biochemical and molecular approaches, in whole animals.. 1. Baylis HA (2005) Cell 123, 5-7. 2. Walker DS et al. (2002) Mol Biol Cell 13, 1329-1337. 3. Thomas-Virnig CL et al. (2004) Curr Biol 14,1882-1887

Gomez Escribano, Ana Pilar et al. (2021) International Worm Meeting "Neurohormonal signalling modulates polyQ aggregation by controlling fat metabolism"

Mora, Carlos, Vazquez Manrique, Rafael, Panadero, Joaquin, Burton, Nick, Schafer, William, Canizares, Joaquin, Sequedo, Maria Dolores, Saini, Ratni, Trujillo del Rio, Cristina, Blanca, Jose, Lahoz, Agustin, Roca, Marta, Burguera, Juan Andres, Tortajada Perez, Julia, Gomez Escribano, Ana Pilar, Knolker, Hans-Joachim, Walker, Denise, Millan, Jose Maria

[International Worm Meeting, 2021]

The maintenance of protein homeostasis is essential for organism survival. Misfolded proteins may alter this balance because they are susceptible to form toxic aggregates. This pathological context is common in many neurodegenerative disorders such as Huntington disease, Alzheimer, Parkinson and ALS. Aggregation dynamics may be monitored in vivo using C. elegans that expresses prone-to-aggregation molecules, tagged with fluorescent proteins. We did a random mutagenesis screening to isolate modifier genes that modulates protein aggregation of polyglutamines (polyQs). We isolated a mutant allele (vlt10), of the electrical synapse modulator unc-1/Stomatin Like protein 3. This allele enhances polyQ aggregation in muscle cells in a neuron-specific manner. We show that vlt10 induces an excess of neurohormonal signalling from the cytosolic sulfotransferase SSU-1, which functions in ASJ neurons. This hormonal signal targets into the nuclear hormone receptor 1 (NHR-1), which appear to regulate genes of the lipid metabolism, which in turns modulate polyQ aggregation. Additionally, we have identified an opposite modulator role from another hormonal pathway which leads to DAF-12. Our findings suggest that steroid hormones induce changes in fat metabolism that impact over protein homeostasis. Some components of this pathways are enzymes or receptors susceptible to be modulated by compounds, and therefore they are potential druggable targets to treat neurodegenerative diseases.