771 research outputs found
Wild-type HTT modulates the enzymatic activity of the neuronal palmitoyl transferase HIP14
No full textHuntington disease (HD) is caused by polyglutamine expansion in the huntingtin (HTT) protein. Huntingtin-interacting protein 14 (HIP14), one of 23 DHHC domain-containing palmitoyl acyl transferases (PATs), binds to HTT and robustly palmitoylates HTT at cysteine 214. Mutant HTT exhibits reduced palmitoylation and interaction with HIP14, contributing to the neuronal dysfunction associated with HD. In this study, we confirmed that, among 23 DHHC PATs, HIP14 and its homolog DHHC-13 (HIP14L) are the two major PATs that palmitoylate HTT. Wild-type HTT, in addition to serving as a palmitoylation substrate, also modulates the palmitoylation of HIP14 itself. In vivo , HIP14 palmitoylation is decreased in the brains of mice lacking one HTT allele ( hdh +/−) and is further reduced in mouse cortical neurons treated with HTT antisense oligos (HTT-ASO) that knockdown HTT expression by ∼95%. Previously, it has been shown that palmitoylation of DHHC proteins may affect their enzymatic activity. Indeed, palmitoylation of SNAP25 by HIP14 is potentiated in vitro in the presence of wild-type HTT. This influence of HTT on HIP14 activity is lost in the presence of CAG expansion. Furthermore, in both brains of hdh +/− mice and neurons treated with HTT-ASO, we observe a significant reduction in palmitoylation of endogenous SNAP25 and GluR1, synaptic proteins that are substrates of HIP14, suggesting wild-type HTT also influences HIP14 enzymatic activity in vivo . This study describes an important biochemical function for wild-type HTT modulation of HIP14 palmitoylation and its enzymatic activity.Peer reviewedfinal article publishe
DEVELOPMENT OF AN AAV-BASED GENE EDITING SYSTEM FOR NEURONAL HTT INACTIVATION IN THE MOUSE BRAIN
No full textHuntington's disease (HD) is a fatal neurodegenerative disorder, caused by a dominant genetic mutation within the first exon of the huntingtin gene (HTT). HD patients go through a progressive and severe motor and cognitive decline, associated with the selective degeneration of striatal and cortical neurons in the brain. Currently, no curative treatment is available, and the pathology management is mainly focused on symptoms reduction. Among the therapeutic strategies under development, CRISPR/Cas9-mediated inactivation of the HTT gene represents a promising approach. However, most studies to date have focused exclusively on the treatment of the striatum with less regard to the involvement of cortical neurons in the HD pathogenesis.
ln the present project, we aim to improve this approach, by developing an innovative and powerful gene editing tool, to simultaneously target cortical and striatal projection neurons in the mouse brain, leading to consistent HTT inactivation in both cell types. In order to simplify the assessment of technical readouts in vivo, we took advantage of wild-type mice and a sgRNA targeting the mouse HTT to establish and characterize the system. We used CRISPR/Cas9 to selectively inactivate the mouse HTT by targeting a region close the translation start site of the gene, using either the spCas9 or the saCas9 nuclease variant. The latter, because of its small size, was used to develop a single AAV vector driving the expression of both the nuclease and the sgRNA, in order to potentially increase the efficiency of our approach, with ail transduced cells expressing both elements. A first, in vitro screening, demonstrated promising results using both systems. However, the dual vector spCas9 showed higher efficiency in vivo compared to the single vector saCas9 and was therefore selected for further studies. We combined the efficiency of the spCas9 gene editing system with the retrograde transport feature of the newly characterized AAV2-retro variant, and the broad local diffusion promoted by the AAV2/10 serotype, reaching consistent and simultaneous HTT inactivation in both striatal and cortical projection neurons within the mouse brain, following a single injection procedure. The efficiency of our system in vivo was confirmed by a substantial reduction of the HTT protein expression observed in the mouse striatum two month after the surgery.
Finally, we improved the biosafety of our approach by developing an optimized AAV version of the
self-inactivating KamiCas9 system previously described, showing transient Cas9 expression while maintaining high on-target efficiency both in vitro and in vivo.
Based on this promising results, our system represents an innovative and powerful tool for the future development of new and efficient genome editing therapeutic approaches for HD, potentially maximizing their therapeutic effect by promoting the simultaneous HTT inactivation in both striatal and cortical projection neurons within the brain.
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La maladie de Huntington (MH) est une maladie neurodégénérative mortelle, causée par une mutation génétique dominante dans le premier exon du gène huntingtin (HTT). Les patients atteints de la MH connaissent un déclin moteur et cognitif progressif et sévère, associé à la dégénérescence sélective des neurones striataux et corticaux du cerveau. Actuellement, aucun traitement curatif n'est disponible, et la prise en charge de la pathologie est principalement axée sur la réduction des symptômes. Parmi les stratégies thérapeutiques en cours de développement, l'inactivation du gène HTT par CRISPR/Cas9 représente une approche prometteuse. Cependant, la plupart des études menées jusqu'à présent se sont concentrées exclusivement sur le traitement du striatum, sans tenir compte de l'implication des neurones corticaux dans la pathogenèse de la MH.
Dans le présent projet, nous visons à améliorer cette approche, en développant un outil d'édition génétique innovant et puissant, pour cibler simultanément les neurones de projection corticaux et striataux dans le cerveau de la souris, conduisant à une inactivation HTT cohérente dans les deux types de cellules. Afin de simplifier l'évaluation des résultats techniques in vivo, nous avons utilisé des souris de type sauvage et un ARNg ciblant la HTT de la souris pour établir et caractériser le système. Nous avons utilisé CRISPR/Cas9 pour inactiver sélectivement la HTT de la souris en ciblant une région proche du site de début de traduction du gène, en utilisant la variante de nucléase spCas9 ou saCas9. Cette dernière, en raison de sa petite taille, a été utilisée pour développer un vecteur AAV unique permettant l'expression à la fois de la nucléase et du sgRNA, afin d'augmenter potentiellement l'efficacité de notre approche, toutes les cellules transduites exprimant les deux éléments. Un premier criblage in vitro a montré des résultats prometteurs en utilisant les deux systèmes. Cependant, le vecteur double spCas9 a montré une plus grande efficacité in vivo par rapport au vecteur simple saCas9 et a donc été sélectionné pour des études supplémentaires. Nous avons combiné l'efficacité du système d'édition de gènes spCas9 avec la caractéristique de transport rétrograde de la variante AAV2-retro nouvellement caractérisée, et la large diffusion locale favorisée par le sérotype AAV2/l 0, pour atteindre une inactivation cohérente et simultanée des HTT dans les neurones de projection striataux et corticaux du cerveau de la souris, après une seule procédure d'injection. L'efficacité de notre système in vivo a été confirmée par une réduction substantielle de l'expression de la protéine HTT observée dans le striatum de la souris deux mois après la chirurgie.
Enfin, nous avons amélioré la biosécurité de notre approche en développant une version AAV optimisée du système KamiCas9 auto-inactivant précédemment décrit, montrant une expression transitoire de Cas9 tout en maintenant une efficacité élevée sur la cible à la fois in vitro et in vivo.
Sur la base de ces résultats prometteurs, notre système représente un outil innovant et puissant pour le développement futur de nouvelles approches thérapeutiques efficaces d'édition du génome pour la MH, maximisant potentiellement leur effet thérapeutique en favorisant l'inactivation simultanée de la HTT dans les neurones de projection striataux et corticaux du cerveau
Data Compensation with Gaussian Processes Regression: Application in Smart Building’s Sensor Network
No full textData play an essential role in the optimal control of smart buildings’ operation, especially in building energy-management for the target of nearly zero buildings. The building monitoring system is in charge of collecting and managing building data. However, device imperfections and failures of the monitoring system are likely to produce low-quality data, such as data loss and inconsistent data, which then seriously affect the control quality of the buildings. This paper proposes a new approach based on Gaussian process regression for data-quality monitoring and sensor network data compensation in smart buildings. The proposed method is proven to effectively detect and compensate for low-quality data thanks to the application of data analysis to the energy management monitoring system of a building model in Viet Nam. The research results provide a good opportunity to improve the efficiency of building energy-management systems and support the development of low-cost smart buildings
Identification of binding sites in huntingtin for the huntingtin interacting proteins HIP14 and HIP14L
No full textHuntington disease is an adult onset neurodegenerative disease characterized by motor, cognitive, and psychiatric dysfunction, caused by a CAG expansion in the HTT gene. Huntingtin Interacting Protein 14 (HIP14) and Huntingtin Interacting Protein 14-like (HIP14L) are palmitoyl acyltransferases (PATs), enzymes that mediate the post-translational addition of long chain fatty acids to proteins in a process called palmitoylation. HIP14 and HIP14L interact with and palmitoylate HTT and are unique among PATs as they are the only two that have an ankyrin repeat domain, which mediates the interaction between HIP14 and HTT. These enzymes show reduced interaction with and palmitoylation of mutant HTT, leading to increased mutant HTT inclusion formation and toxicity. The interaction between HIP14 and HTT goes beyond that of only an enzyme–substrate interaction as HTT is essential for the full enzymatic activity of HIP14. It is important to further understand and characterize the interactions of HTT with HIP14 and HIP14L to guide future efforts to target and enhance this interaction and increase enzyme activity to remediate palmitoylation of HTT and their substrates, as well as to understand the relationship between the three proteins. HIP14 and HIP14L have been previously shown to interact with HTT amino acids 1–548. Here the interaction of HIP14 and HIP14L with N- and C-terminal HTT 1–548 deletion mutations was assessed. We show that HTT amino acids 1–548 were sufficient for full interaction of HTT with HIP14 and HIP14L, but partial interaction was also possible with HTT 1–427 and HTT 224–548. To further characterize the binding domain we assessed the interaction of HIP14-GFP and HIP14L-GFP with 15Q HTT 1-548D257-315. Both enzymes showed reduced but not abolished interaction with 15Q HTT 1-548D257-315. This suggests that two potential binding domains exist, one around residues 224 and the other around 427, for the PAT enzymes HIP14 and HIP14L.Peer reviewedfinal article publishe
Questioning the causality of HTT CAG-repeat expansions in FTD/ALS: Response to Dewan et al., short HTT CAG-repeat expansions cause Huntington's disease with frontotemporal dementia-like phenotype.
No full textInternational audienceQuestioning the causality of HTT CAG-repeat expansions in FTD/ALS You can't judge a frontal dementia by its cover Response to Dewan et al., short HTT CAG-repeat expansions cause Huntington's disease with frontotemporal dementia-like phenotype. Dear Madam, Sir, We read the article written by Dewan and collaborators entitled "Pathogenic Huntingtin Repeat Expansions in Patients with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis" published in Neuron (Dewan et al., 2020) with great interest. In this work, the authors report whole-genome analyses in 4 different large cohorts of i) 2,442 patients diagnosed with frontotemporal dementia (FTD) and/or amyotrophic lateral sclerosis (ALS), ii) 2,599 patients diagnosed with Lewy body dementia (LBD), iii) 3,158 neurologically healthy individuals and iv) 3,674 individuals diagnosed with FTD/ALS from an additional independent "replication" cohort. They identified a total of 8 individuals carrying pathogenic HTT CAG-expansion repeats, 3 in the initial FTD/ALS cohort and 5 more in the independent "replication" cohort, but none in either the LBD cohort or among healthy controls. They showed that this overall 0.13% carrier rate (8/6,116) is 4.4 times higher tha
Huntingtin interacting proteins 14 and 14-like are required for chorioallantoic fusion during early placental development
No full textHuntington disease (HD) is an adult-onset neurodegenerative disease characterized by motor, cognitive, and psychiatric symptoms that is caused by a CAG expansion in the HTT gene. Palmitoylation is the addition of saturated fatty acids to proteins by DHHC palmitoylacyl transferases. HTT is palmitoylated by huntingtin interacting proteins 14 and 14-like (HIP14 and HIP14L or ZDHHC17 and 13 respectively). Mutant HTT is less palmitoylated and this reduction of palmitoylation accelerates its aggregation and increases cellular toxicity. Mouse models deficient in either Hip14 (Hip14−/−) or Hip14l (Hip14l−/−) develop HD-like phenotypes. The biological function of HTT palmitoylation and the role that loss of HTT palmitoylation plays in the pathogenesis of HD are unknown. To address these questions mice deficient for both genes were created. Loss of Hip14 and Hip14l leads to early embryonic lethality at day embryonic day 10–11 due to failed chorioallantoic fusion. The chorion is thickened and disorganized and the allantois does not fuse correctly with the chorion and forms a balloon-like shape compared to Hip14l−/−; Hip14+/+ littermate control embryos. Interestingly, the Hip14−/− ; Hip14−/− embryos share many features with the Htt−/− embryos, including folding of the yolk sac, a bulb shaped allantois, and a thickened and disorganized chorion. This may be due to a decrease in HTT palmitoylation. In Hip14−/−; Hip14l−/− mouse embryonic fibroblasts show a 25% decrease in HTT palmitoylation compared to wild type cells. This is the first description of a double PAT deficient mouse model where loss of a PAT or multiple PATs results in embryonic lethality in mammals. These results reinforce the physiological importance of palmitoylation during embryogenesis.Peer reviewedfinal article publishedHuntington diseasePlacentChorioallantoic fusionHIP14L or ZDHHC13HIP14 or ZDHHC17Palmitoylatio
Analysis of short tandem repeats linked to polyglutamine diseases from whole-genome sequencing reveals intermediate alleles of HTT associated with an early disease onset in C9orf72 carriers
No full textCarriers of the GGGGCC pathogenic expansion in C9orf72 can develop symptoms of frontotemporal dementia and/or amyotrophic lateral sclerosis, with variable and unpredictable ages at onset. Previous studies aiming to decipher the genetic bases of the clinical variability in this rare disease included bi-allelic polymorphisms, excluding short tandem repeats. Whole-genome sequencing data of 195 C9orf72 patients were used to consider all short tandem repeats linked to polyglutamine disorders as potential genetic modifiers given the existing links between C9orf72 and polyglutamine diseases. Intermediate alleles of HTT encoding huntingtin were associated with an earlier age at onset among C9orf72 carriers in the discovery cohort (n = 195, P = 0.0003) and in a European replication cohort (n = 145, P = 0.006). In the merged cohort (n = 340), the average difference of age at disease onset was 9.42 ± 2.14 years (P = 1.3 × 10e-5) between carriers and non-carriers of HTT-intermediate alleles. Neuropathology of one C9orf72 case heterozygous for HTT-intermediate allele showed typical TDP-43 inclusions related to the C9orf72 pathogenic expansion and was negative for polyglutamine inclusion. No somatic expansion of HTT was detected in blood of all C9orf72exp/HTT-intermediate carriers. If this study reinforces potential biological links between huntingtin and C9orf72 that remain to be explored, the results also illustrate the interest of considering short tandem repeats from whole-genome data in association studies which paves the way to more exhaustive approaches to explore the trait heritability due to short-tandem-repeats still hidden in the genome
Serotonin transporter gene (5-HTT): association analysis with temporal lobe epilepsy.
No full textTwo functional polymorphisms, a 44 bp insertion/deletion polymorphism in the 5' regulatory region and a variable number of tandem repeat polymorphisms in the second intron of the serotonin transporter gene (5-HTT), were previously identified and suggested to modulate transcription. The current study was designed to determine the contribution of these polymorphisms in the 5-HTT gene to susceptibility to temporal lobe epilepsy (TLE). Two hundred and seventy six patients with TLE, and 309 age- and sex-matched healthy controls from Calabria (Southern Italy) were studied. Patients and controls were genotyped using the WAVE TM DNA Fragment Analysis System for the insertion/deletion polymorphism in the promoter region (5-HTTLPR), and the GENESCAN TM System for the variable number tandem repeat (VNTR) in the second intron of the 5-HTT gene (5-HTTVNTR). The program UNPHASED was used to compare genotype, allele and haplotype frequencies between cases and controls, including age and gender as covariates in the model. No significant differences between cases and controls were observed for 5-HTTLPR, but a significant association was obtained for the 5-HTTVNTR polymorphism, both modeling genotypes (P-value = 0.0 145) or alleles (P-value = 0.0086). Patients with TLE showed lower frequencies of the 10 repeat at 5-HTTVNTR than the controls (26.2% in patients versus 40.8% in controls). The frequency of homozygous individuals for the 10 allele was observed to be lower among patients than the controls (5.2% of patients were 10/10 versus 18.8% of controls). Haplotype analysis did not increase the evidence for association. These results suggest that the serotonin transporter gene may play a role in the etiology of TLE. (c) 2007 Elsevier Ireland Ltd. All rights reserve
Huntingtin and cortical development
No full textLa maladie de Huntington (MH) est un trouble neurologique transmis selon un mode autosomique dominant qui conduit à l’apparition de symptômes moteurs, psychiatriques et cognitifs chez l’adulte. La MH est caractérisée par une neurodégénérescence massive des neurones striataux et corticaux. La MH est causée par une mutation de la séquence codante de la protéine Huntingtine (HTT) conduisant à la production d’une protéine mutée (mHTT). La mHTT gagne de nouvelles fonctions toxiques mais perd aussi certaines fonctions normales. L’étude de ces deux aspects (à la fois gain et perte de fonction) est donc indispensable à la compréhension du processus pathologique de la MH.La HTT et mHTT participent au développement des structures cérébrales. Notre hypothèse est donc que les défauts développementaux induits par la mHTT contribuent à la progression physiopathologique de la MH. Notre équipe s’intéresse tout particulièrement au développement du cortex cérébral, largement atteint dans la MH. Nos précédentes études ont démontré le rôle de la HTT et l’effet de sa mutation dans la prolifération des précurseurs neuronaux du cortex cérébral. Cependant, les fonctions de la HTT et de la mHTT lors des étapes plus avancées du développement cortical, restent à ce jour inconnues.Mon projet de thèse se décompose en deux axes principaux : (i) l’étude des fonctions de la HTT dans les neurones nouvellement produits dans le cortex en développement, notamment au cours de leur migration et de leur maturation dendritique et (ii), la caractérisation de la neurogenèse corticale dans un modèle génétique de la MH : zQ175.Huntington disease (HD) is an autosomal dominant inherited neurological disorder conducting to the appearance of motors, psychiatrics and cognitives symptoms during mid-adulthood. HD is characterised by a massive neurodegenerescence of both striatal and cortical neurons. HD is caused by a mutation in coding region of the protein Huntingtin (HTT) leading to the production of a mutated form (mHTT). mHTT gain new toxic function but also loss some of normal function of HTT. Therefore, studying both gain and loss of function is mandatory to better understand the physiopathological progression of HD.HTT and mHTT both contribute to development of cerebral structures. Our hypothesis is that developmental defects induced by mHTT could contribute at least in part to the physiological progression of HD. Our work focuses on cerebral cortex development a structure which is largely impacted in HD. Our previous studies demonstrated roles of HTT and the effect of mHTT in neuronal precursor proliferation during neurogenesis. However, roles and functions of HTT and mHTT during later step of cortical neurogenesis remain elusive.My PhD project has focused on two main aspects: (i) study the function of HTT in newborn post-mitotic neurons in cerebral cortex, notably during their migration and maturation, and (ii), characterising cortical neurogenesis in genetically integrated mouse model of HD: zQ175
Huntingtine et développement cortical
No full textHuntington disease (HD) is an autosomal dominant inherited neurological disorder conducting to the appearance of motors, psychiatrics and cognitives symptoms during mid-adulthood. HD is characterised by a massive neurodegenerescence of both striatal and cortical neurons. HD is caused by a mutation in coding region of the protein Huntingtin (HTT) leading to the production of a mutated form (mHTT). mHTT gain new toxic function but also loss some of normal function of HTT. Therefore, studying both gain and loss of function is mandatory to better understand the physiopathological progression of HD.HTT and mHTT both contribute to development of cerebral structures. Our hypothesis is that developmental defects induced by mHTT could contribute at least in part to the physiological progression of HD. Our work focuses on cerebral cortex development a structure which is largely impacted in HD. Our previous studies demonstrated roles of HTT and the effect of mHTT in neuronal precursor proliferation during neurogenesis. However, roles and functions of HTT and mHTT during later step of cortical neurogenesis remain elusive.My PhD project has focused on two main aspects: (i) study the function of HTT in newborn post-mitotic neurons in cerebral cortex, notably during their migration and maturation, and (ii), characterising cortical neurogenesis in genetically integrated mouse model of HD: zQ175.La maladie de Huntington (MH) est un trouble neurologique transmis selon un mode autosomique dominant qui conduit à l’apparition de symptômes moteurs, psychiatriques et cognitifs chez l’adulte. La MH est caractérisée par une neurodégénérescence massive des neurones striataux et corticaux. La MH est causée par une mutation de la séquence codante de la protéine Huntingtine (HTT) conduisant à la production d’une protéine mutée (mHTT). La mHTT gagne de nouvelles fonctions toxiques mais perd aussi certaines fonctions normales. L’étude de ces deux aspects (à la fois gain et perte de fonction) est donc indispensable à la compréhension du processus pathologique de la MH.La HTT et mHTT participent au développement des structures cérébrales. Notre hypothèse est donc que les défauts développementaux induits par la mHTT contribuent à la progression physiopathologique de la MH. Notre équipe s’intéresse tout particulièrement au développement du cortex cérébral, largement atteint dans la MH. Nos précédentes études ont démontré le rôle de la HTT et l’effet de sa mutation dans la prolifération des précurseurs neuronaux du cortex cérébral. Cependant, les fonctions de la HTT et de la mHTT lors des étapes plus avancées du développement cortical, restent à ce jour inconnues.Mon projet de thèse se décompose en deux axes principaux : (i) l’étude des fonctions de la HTT dans les neurones nouvellement produits dans le cortex en développement, notamment au cours de leur migration et de leur maturation dendritique et (ii), la caractérisation de la neurogenèse corticale dans un modèle génétique de la MH : zQ175
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