Identification et validation de cibles thérapeutiques pour traiter le syndrome de Wolfram

Wolfram Syndrome (WS) is a neurodegenerative disease that combines diabetes mellitus, diabetes insipidus, optic atrophy, sensorineural hearing loss and various neurological symptoms. Patients die early, most often from respiratory distress or miscarriage, and to date no effective treatment is available. WS is caused by mutations in the WFS1 gene that encodes an endoplasmic reticulum (ER) transmembrane protein, Wolframine. Mutations generally reduce the stability of the protein, altering its homeostasis, decreasing calcium transfer, increasing the activation of the Unfolded Protein Response (UPR) and leading to mitochondrial dysfunction and cell death. The objective of my thesis was to explore original therapeutic approaches in this indication. For this purpose, several models were used and characterized: patient fibroblasts in vitro, and in vivo, wfs1aC825X, wfs1bW493X and wfs1abKO mutant zebrafish lines, and Wfs1∆Exon8 mice. We showed that: (1) These different models develop cellular alterations, such as mitochondrial deficits, impaired mitochondrial calcium transfer, activation of UPR pathways, and autophagy, associated with behavioral deficits, such as visual, locomotor, anxiety, and cognitive impairments. (2) Overexpression of NCS1, a partner of wolframine, in vivo restored the behavioral and cellular deficits developed by the zebrafish line wfs1abKO confirming the in vitro study previously conducted on patient fibroblasts. (3) The chaperone protein sigma-1 (S1R) was a relevant target to restore functional ER-mitochondria communication in WS. Our in vitro and in vivo studies showed that activation of S1R by an agonist or by overexpression of its mRNA reversed behavioral and cellular alterations. (4) Based on the motor response of wfs1abKO zebrafish larvae, we conducted a phenotypic screening of 371 molecules potentially affine to S1R which led to the identification of 8 hits allowing to fully restore the locomotor deficit of mutant larvae. Some of them will be valorised by an optimization approach, but among the repositionable molecules, we have identified MED0092 which acts as a positive modulator of S1R and whose beneficial effects have been shown on behavioral deficits in two mouse models of neurodegenerative diseases, SW and Alzheimer's disease. To conclude, this thesis work has not only allowed the phenotyping of new mouse and zebrafish models of WS, but also the validation of two therapeutic targets allowing a functional recovery of MAM: NCS1, which can be directly overexpressed by gene therapy, and S1R, which can be targeted by small molecules and for which new very promising chemical series of synthetic or natural origin have been highlighted.Le syndrome de Wolfram (SW) est une maladie neurodégénérative qui associe un diabète sucré, un diabète insipide, une atrophie optique, une perte auditive neurosensorielle et divers symptômes neurologiques. Les patients décèdent précocement le plus souvent d’une détresse respiratoire ou de fausse route et à ce jour aucun traitement efficace n’est disponible. Le SW est causé par des mutations du gène WFS1 qui code une protéine transmembranaire du réticulum endoplasmique (RE), la Wolframine. Les mutations réduisent généralement la stabilité de la protéine, modifiant son homéostasie, diminuant le transfert calcique, augmentant l’activation de la réponse aux protéines mal repliées (Unfolded Protein Response, UPR) et conduisant à un dysfonctionnement mitochondrial et à la mort cellulaire. L’objectif de ma thèse a été d'explorer des voies thérapeutiques originales dans cette indication. Pour cela, plusieurs modèles d’étude ont été utilisés et caractérisés : des fibroblastes de patients in vitro, et in vivo, des lignées de poissons zèbres mutantes wfs1aC825X, wfs1bW493X et wfs1abKO, et des souris Wfs1∆Exon8. Nous avons montré que : (1) Ces différents modèles développent des altérations cellulaires, telles que des déficits mitochondriaux, l’altération du transfert calcique mitochondrial, l’activation des voies UPR et l’autophagie, associés à des déficits comportementaux, tels que des troubles visuels, locomoteurs, de l’anxiété et des altérations cognitives. (2) La surexpression de NCS1, partenaire de la wolframine, in vivo a permis de restaurer les déficits comportementaux et cellulaires développés par la lignée poisson zèbre wfs1abKO confirmant l’étude in vitro précédemment menée sur des fibroblastes de patients. (3) La protéine chaperonne sigma-1 (S1R) était une cible pertinente pour rétablir une communication RE-mitochondrie fonctionnelle dans le SW. Nos études in vitro et in vivo ont montré que l’activation de S1R par un agoniste ou par surexpression de son ARNm permettait de renverser les altérations comportementales et cellulaires. (4) À partir de la réponse motrice des larves de poisson zèbre wfs1abKO, nous avons mené un criblage phénotypique de 371 molécules potentiellement affines pour S1R qui a mené à identifier 8 hits permettant de restaurer totalement le déficit locomoteur des larves mutantes. Certaines seront valorisées par une démarche d'optimisation, mais parmi les molécules dites repositionnables, nous avons identifié le MED0092 qui agit comme un modulateur positif de S1R et dont les effets bénéfiques ont été montrés sur les déficits comportementaux de deux modèles murins de maladies neurodégénératives, le SW et la maladie d’Alzheimer. Pour conclure, ce travail de thèse a permis non seulement de phénotyper de nouveaux modèles murins et poissons-zèbres du SW, mais également de valider deux cibles thérapeutiques permettant une récupération fonctionnelle des MAM : NCS1, qui peut être directement surexprimé par thérapie génique, et S1R, qui peut être ciblé par de petites molécules et pour lequel de nouvelles séries chimiques très prometteuses d’origine synthétique ou naturelle ont été mises en évidence

Identification et validation de cibles thérapeutiques pour traiter le syndrome de Wolfram

Wolfram Syndrome (WS) is a neurodegenerative disease that combines diabetes mellitus, diabetes insipidus, optic atrophy, sensorineural hearing loss and various neurological symptoms. Patients die early, most often from respiratory distress or miscarriage, and to date no effective treatment is available. WS is caused by mutations in the WFS1 gene that encodes an endoplasmic reticulum (ER) transmembrane protein, Wolframine. Mutations generally reduce the stability of the protein, altering its homeostasis, decreasing calcium transfer, increasing the activation of the Unfolded Protein Response (UPR) and leading to mitochondrial dysfunction and cell death. The objective of my thesis was to explore original therapeutic approaches in this indication. For this purpose, several models were used and characterized: patient fibroblasts in vitro, and in vivo, wfs1aC825X, wfs1bW493X and wfs1abKO mutant zebrafish lines, and Wfs1∆Exon8 mice. We showed that: (1) These different models develop cellular alterations, such as mitochondrial deficits, impaired mitochondrial calcium transfer, activation of UPR pathways, and autophagy, associated with behavioral deficits, such as visual, locomotor, anxiety, and cognitive impairments. (2) Overexpression of NCS1, a partner of wolframine, in vivo restored the behavioral and cellular deficits developed by the zebrafish line wfs1abKO confirming the in vitro study previously conducted on patient fibroblasts. (3) The chaperone protein sigma-1 (S1R) was a relevant target to restore functional ER-mitochondria communication in WS. Our in vitro and in vivo studies showed that activation of S1R by an agonist or by overexpression of its mRNA reversed behavioral and cellular alterations. (4) Based on the motor response of wfs1abKO zebrafish larvae, we conducted a phenotypic screening of 371 molecules potentially affine to S1R which led to the identification of 8 hits allowing to fully restore the locomotor deficit of mutant larvae. Some of them will be valorised by an optimization approach, but among the repositionable molecules, we have identified MED0092 which acts as a positive modulator of S1R and whose beneficial effects have been shown on behavioral deficits in two mouse models of neurodegenerative diseases, SW and Alzheimer's disease. To conclude, this thesis work has not only allowed the phenotyping of new mouse and zebrafish models of WS, but also the validation of two therapeutic targets allowing a functional recovery of MAM: NCS1, which can be directly overexpressed by gene therapy, and S1R, which can be targeted by small molecules and for which new very promising chemical series of synthetic or natural origin have been highlighted.Le syndrome de Wolfram (SW) est une maladie neurodégénérative qui associe un diabète sucré, un diabète insipide, une atrophie optique, une perte auditive neurosensorielle et divers symptômes neurologiques. Les patients décèdent précocement le plus souvent d’une détresse respiratoire ou de fausse route et à ce jour aucun traitement efficace n’est disponible. Le SW est causé par des mutations du gène WFS1 qui code une protéine transmembranaire du réticulum endoplasmique (RE), la Wolframine. Les mutations réduisent généralement la stabilité de la protéine, modifiant son homéostasie, diminuant le transfert calcique, augmentant l’activation de la réponse aux protéines mal repliées (Unfolded Protein Response, UPR) et conduisant à un dysfonctionnement mitochondrial et à la mort cellulaire. L’objectif de ma thèse a été d'explorer des voies thérapeutiques originales dans cette indication. Pour cela, plusieurs modèles d’étude ont été utilisés et caractérisés : des fibroblastes de patients in vitro, et in vivo, des lignées de poissons zèbres mutantes wfs1aC825X, wfs1bW493X et wfs1abKO, et des souris Wfs1∆Exon8. Nous avons montré que : (1) Ces différents modèles développent des altérations cellulaires, telles que des déficits mitochondriaux, l’altération du transfert calcique mitochondrial, l’activation des voies UPR et l’autophagie, associés à des déficits comportementaux, tels que des troubles visuels, locomoteurs, de l’anxiété et des altérations cognitives. (2) La surexpression de NCS1, partenaire de la wolframine, in vivo a permis de restaurer les déficits comportementaux et cellulaires développés par la lignée poisson zèbre wfs1abKO confirmant l’étude in vitro précédemment menée sur des fibroblastes de patients. (3) La protéine chaperonne sigma-1 (S1R) était une cible pertinente pour rétablir une communication RE-mitochondrie fonctionnelle dans le SW. Nos études in vitro et in vivo ont montré que l’activation de S1R par un agoniste ou par surexpression de son ARNm permettait de renverser les altérations comportementales et cellulaires. (4) À partir de la réponse motrice des larves de poisson zèbre wfs1abKO, nous avons mené un criblage phénotypique de 371 molécules potentiellement affines pour S1R qui a mené à identifier 8 hits permettant de restaurer totalement le déficit locomoteur des larves mutantes. Certaines seront valorisées par une démarche d'optimisation, mais parmi les molécules dites repositionnables, nous avons identifié le MED0092 qui agit comme un modulateur positif de S1R et dont les effets bénéfiques ont été montrés sur les déficits comportementaux de deux modèles murins de maladies neurodégénératives, le SW et la maladie d’Alzheimer. Pour conclure, ce travail de thèse a permis non seulement de phénotyper de nouveaux modèles murins et poissons-zèbres du SW, mais également de valider deux cibles thérapeutiques permettant une récupération fonctionnelle des MAM : NCS1, qui peut être directement surexprimé par thérapie génique, et S1R, qui peut être ciblé par de petites molécules et pour lequel de nouvelles séries chimiques très prometteuses d’origine synthétique ou naturelle ont été mises en évidence

Identification and validation of therapeutic targets to treat Wolfram syndrome

Le syndrome de Wolfram (SW) est une maladie neurodégénérative qui associe un diabète sucré, un diabète insipide, une atrophie optique, une perte auditive neurosensorielle et divers symptômes neurologiques. Les patients décèdent précocement le plus souvent d’une détresse respiratoire ou de fausse route et à ce jour aucun traitement efficace n’est disponible. Le SW est causé par des mutations du gène WFS1 qui code une protéine transmembranaire du réticulum endoplasmique (RE), la Wolframine. Les mutations réduisent généralement la stabilité de la protéine, modifiant son homéostasie, diminuant le transfert calcique, augmentant l’activation de la réponse aux protéines mal repliées (Unfolded Protein Response, UPR) et conduisant à un dysfonctionnement mitochondrial et à la mort cellulaire. L’objectif de ma thèse a été d'explorer des voies thérapeutiques originales dans cette indication. Pour cela, plusieurs modèles d’étude ont été utilisés et caractérisés : des fibroblastes de patients in vitro, et in vivo, des lignées de poissons zèbres mutantes wfs1aC825X, wfs1bW493X et wfs1abKO, et des souris Wfs1∆Exon8. Nous avons montré que : (1) Ces différents modèles développent des altérations cellulaires, telles que des déficits mitochondriaux, l’altération du transfert calcique mitochondrial, l’activation des voies UPR et l’autophagie, associés à des déficits comportementaux, tels que des troubles visuels, locomoteurs, de l’anxiété et des altérations cognitives. (2) La surexpression de NCS1, partenaire de la wolframine, in vivo a permis de restaurer les déficits comportementaux et cellulaires développés par la lignée poisson zèbre wfs1abKO confirmant l’étude in vitro précédemment menée sur des fibroblastes de patients. (3) La protéine chaperonne sigma-1 (S1R) était une cible pertinente pour rétablir une communication RE-mitochondrie fonctionnelle dans le SW. Nos études in vitro et in vivo ont montré que l’activation de S1R par un agoniste ou par surexpression de son ARNm permettait de renverser les altérations comportementales et cellulaires. (4) À partir de la réponse motrice des larves de poisson zèbre wfs1abKO, nous avons mené un criblage phénotypique de 371 molécules potentiellement affines pour S1R qui a mené à identifier 8 hits permettant de restaurer totalement le déficit locomoteur des larves mutantes. Certaines seront valorisées par une démarche d'optimisation, mais parmi les molécules dites repositionnables, nous avons identifié le MED0092 qui agit comme un modulateur positif de S1R et dont les effets bénéfiques ont été montrés sur les déficits comportementaux de deux modèles murins de maladies neurodégénératives, le SW et la maladie d’Alzheimer. Pour conclure, ce travail de thèse a permis non seulement de phénotyper de nouveaux modèles murins et poissons-zèbres du SW, mais également de valider deux cibles thérapeutiques permettant une récupération fonctionnelle des MAM : NCS1, qui peut être directement surexprimé par thérapie génique, et S1R, qui peut être ciblé par de petites molécules et pour lequel de nouvelles séries chimiques très prometteuses d’origine synthétique ou naturelle ont été mises en évidence.Wolfram Syndrome (WS) is a neurodegenerative disease that combines diabetes mellitus, diabetes insipidus, optic atrophy, sensorineural hearing loss and various neurological symptoms. Patients die early, most often from respiratory distress or miscarriage, and to date no effective treatment is available. WS is caused by mutations in the WFS1 gene that encodes an endoplasmic reticulum (ER) transmembrane protein, Wolframine. Mutations generally reduce the stability of the protein, altering its homeostasis, decreasing calcium transfer, increasing the activation of the Unfolded Protein Response (UPR) and leading to mitochondrial dysfunction and cell death. The objective of my thesis was to explore original therapeutic approaches in this indication. For this purpose, several models were used and characterized: patient fibroblasts in vitro, and in vivo, wfs1aC825X, wfs1bW493X and wfs1abKO mutant zebrafish lines, and Wfs1∆Exon8 mice. We showed that: (1) These different models develop cellular alterations, such as mitochondrial deficits, impaired mitochondrial calcium transfer, activation of UPR pathways, and autophagy, associated with behavioral deficits, such as visual, locomotor, anxiety, and cognitive impairments. (2) Overexpression of NCS1, a partner of wolframine, in vivo restored the behavioral and cellular deficits developed by the zebrafish line wfs1abKO confirming the in vitro study previously conducted on patient fibroblasts. (3) The chaperone protein sigma-1 (S1R) was a relevant target to restore functional ER-mitochondria communication in WS. Our in vitro and in vivo studies showed that activation of S1R by an agonist or by overexpression of its mRNA reversed behavioral and cellular alterations. (4) Based on the motor response of wfs1abKO zebrafish larvae, we conducted a phenotypic screening of 371 molecules potentially affine to S1R which led to the identification of 8 hits allowing to fully restore the locomotor deficit of mutant larvae. Some of them will be valorised by an optimization approach, but among the repositionable molecules, we have identified MED0092 which acts as a positive modulator of S1R and whose beneficial effects have been shown on behavioral deficits in two mouse models of neurodegenerative diseases, SW and Alzheimer's disease. To conclude, this thesis work has not only allowed the phenotyping of new mouse and zebrafish models of WS, but also the validation of two therapeutic targets allowing a functional recovery of MAM: NCS1, which can be directly overexpressed by gene therapy, and S1R, which can be targeted by small molecules and for which new very promising chemical series of synthetic or natural origin have been highlighted

Assessment of Topographic Memory in Mice in a Complex Environment Using the Hamlet Test

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Use of Zebrafish Models to Boost Research in Rare Genetic Diseases

International audienceRare genetic diseases are a group of pathologies with often unmet clinical needs. Even if rare by a single genetic disease (from 1/2000 to 1/more than 1,000,000), the total number of patients concerned account for approximatively 400 million peoples worldwide. Finding treatments remains challenging due to the complexity of these diseases, the small number of patients and the challenge in conducting clinical trials. Therefore, innovative preclinical research strategies are required. The zebrafish has emerged as a powerful animal model for investigating rare diseases. Zebrafish combines conserved vertebrate characteristics with high rate of breeding, limited housing requirements and low costs. More than 84% of human genes responsible for diseases present an orthologue, suggesting that the majority of genetic diseases could be modelized in zebrafish. In this review, we emphasize the unique advantages of zebrafish models over other in vivo models, particularly underlining the high throughput phenotypic capacity for therapeutic screening. We briefly introduce how the generation of zebrafish transgenic lines by gene-modulating technologies can be used to model rare genetic diseases. Then, we describe how zebrafish could be phenotyped using state-of-the-art technologies. Two prototypic examples of rare diseases illustrate how zebrafish models could play a critical role in deciphering the underlying mechanisms of rare genetic diseases and their use to identify innovative therapeutic solutions

Knockdown of the CXCL12/CXCR7 chemokine pathway results in learning deficits and neural progenitor maturation impairment in mice

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Sigma-1 Receptor Is Critical for Mitochondrial Activity and Unfolded Protein Response in Larval Zebrafish

International audienceThe sigma-1 receptor (S1R) is a highly conserved transmembrane protein highly enriched in mitochondria-associated endoplasmic reticulum (ER) membranes, where it interacts with several partners involved in ER-mitochondria Ca2+ transfer, activation of the ER stress pathways, and mitochondria function. We characterized a new S1R deficient zebrafish line and analyzed the impact of S1R deficiency on visual, auditory and locomotor functions. The s1r+25/+25 mutant line showed impairments in visual and locomotor functions compared to s1rWT. The locomotion of the s1r+25/+25 larvae, at 5 days post fertilization, was increased in the light and dark phases of the visual motor response. No deficit was observed in acoustic startle response. A critical role of S1R was shown in ER stress pathways and mitochondrial activity. Using qPCR to analyze the unfolded protein response genes, we observed that loss of S1R led to decreased levels of IRE1 and PERK-related effectors and increased over-expression of most of the effectors after a tunicamycin challenge. Finally, S1R deficiency led to alterations in mitochondria bioenergetics with decreased in basal, ATP-linked and non-mitochondrial respiration and following tunicamycin challenge. In conclusion, this new zebrafish model confirmed the importance of S1R activity on ER-mitochondria communication. It will be a useful tool to further analyze the physiopathological roles of S1R

Neuroprotection in non-transgenic and transgenic mouse models of Alzheimer's disease by positive modulation of σ1 receptors

International audienceThe sigma-1 (σ1) receptor is an endoplasmic reticulum (ER) chaperone protein, enriched in mitochondria-associated membranes. Its activation triggers physiological responses to ER stress and modulate Ca2+ mobilization in mitochondria. Small σ1 agonist molecules activate the protein and act behaviorally as antidepressant, anti-amnesic and neuroprotective agents. Recently, several chemically unrelated molecules were shown to be σ1 receptor positive modulators (PMs), with some of them a clear demonstration of their allostericity. We here examined whether a σ1 PM also shows neuroprotective potentials in pharmacological and genetic models of Alzheimer's disease (AD). For this aim, we describe (±)-2-(3-chlorophenyl)-3,3,5,5-tetramethyl-2-oxo-[1,4,2]-oxazaphosphinane (OZP002) as a novel σ1 PM. OZP002 does not bind σ1 sites but induces σ1 effects in vivo and boosts σ1 agonist activity. OZP002 was antidepressant in the forced swim test and its effect was blocked by the σ1 antagonist NE-100 or in σ1 receptor knockout mice. It potentiated the antidepressant effect of the σ1 agonist igmesine. In mice tested for Y-maze alternation or passive avoidance, OZP002 prevented scopolamine-induced learning deficits, in a NE-100 sensitive manner. Pre-administered IP before an ICV injection of amyloid Aβ25-35 peptide, a pharmacological model of Alzheimer's disease, OZP002 prevented the learning deficits induced by the peptide after one week in the Y-maze, passive avoidance and novel object tests. Biochemical analyses of the mouse hippocampi showed that OZP002 significantly decreased Aβ25-35-induced increases in reactive oxygen species, lipid peroxidation, and increases in Bax, TNFα and IL-6 levels. Immunohistochemically, OZP002 prevented Aβ25-35-induced reactive astrogliosis and microgliosis in the hippocampus. It also alleviated Aβ25-35-induced decreases in synaptophysin level and choline acetyltransferase activity. Moreover, chronically administered in APPswe mice during 2 months, OZP002 prevented learning deficits (in all tests plus place learning in the water-maze) and increased biochemical markers. This study shows that σ1 PM with high neuropotective potential can be identified, combining pharmacological efficacy, selectivity and therapeutic safety, and identifies a novel promising compound, OZP002

Loss of Pde6a Induces Rod Outer Segment Shrinkage and Visual Alterations in pde6aQ70X Mutant Zebrafish, a Relevant Model of Retinal Dystrophy

International audienceRetinitis pigmentosa (RP) is one of the most common forms of inherited retinal degeneration with 1/4,000 people being affected. The vision alteration primarily begins with rod photoreceptor degeneration, then the degenerative process continues with cone photoreceptor death. Variants in 71 genes have been linked to RP. One of these genes, PDE6a is responsible for RP43. To date no treatment is available and patients suffer from pronounced visual impairment in early childhood. We used the novel zebrafish pde6a Q70X mutant, generated by N-ethyl-N-nitrosourea at the European Zebrafish Resource Centre, to better understand how PDE6a loss of function leads to photoreceptor alteration. Interestingly, zebrafish pde6a Q70X mutants exhibited impaired visual function at 5 dpf as evidenced by the decrease in their visual motor response (VMR) compared to pde6a WT larvae. This impaired visual function progressed with time and was more severe at 21 dpf. These modifications were associated with an alteration of rod outer segment length at 5 and 21 dpf. In summary, these findings suggest that rod outer segment shrinkage due to Pde6a deficiency begins very early in zebrafish, progresses with time. The zebrafish pde6a Q70X mutant represents an ideal model of RP to screen relevant active small molecules that will block the progression of the disease

Sigma-1 (σ1) receptor activity is necessary for physiological brain plasticity in mice

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