Yoan Arribat

La Maladie de Huntington (MH) reste à ce jour une pathologie neurodégénérative dévastatrice pour laquelle aucun traitement n'est disponible. L'agrégation de la Huntingtine Mutante (Htt PolyQ) joue un rôle majeur dans les processus pathologiques. Dans ce contexte, des études récentes ont démontré que la partie N-terminale de la Huntingtine Humaine (Htt wt) ou de son Homologue drosophile (dHtt) sont toutes deux capables de réduire l'agrégation et la toxicité de Htt PolyQ. En se basant sur cette observation, le travail de thèse décrit dans ce manuscrit a mis au point un découpage du fragment N-terminal de Htt wt de manière à isoler en son sein, une courte séquence de 23 acides aminés (nommée P42), capable d'inhiber spécifiquement l'agrégation de Htt PolyQ en modèle cellulaire. L'effet protecteur de ce peptide a été confirmé in vivo, sur un modèle drosophile de la MH. Le potentiel thérapeutique que représente P42 a servi de point de départ à une étude menée sur le modèle murin R6/2 de la MH. L'effet de P42 a été potentialisé par l'ajout du peptide de transduction TAT de manière à faciliter son entrée dans les cellules cibles. Puis, la protéine fusion P42-TAT a été vectorisée sous forme de microémulsion de manière à assurer à la fois une administration simple de la molécule, et un accès au système nerveux central. Ce protocole original a permis d'observer des bénéfices sans précédent sur les phénotypes comportementaux, histologiques et moléculaires que présentent les souris R6/2.Au-delà de son aspect thérapeutique, P42 est avant tout une séquence méconnue située dans une région cruciale de la Huntingtine. L'étude du rôle physiologique de ce site, a mené à une meilleure compréhension de la fonction sauvage de la protéine toute entière. En outre, une analyse biochimique a montré la capacité du fragment N-terminal de Htt wt à se lier aux microtubules. Cette interaction avec le cytosquelette dépend de plusieurs processus (clivages, dimérisation) et semble affilier la Huntingtine à la grande famille des MAP.L'identification de P42 ouvre donc une voie nouvelle vers la compréhension du rôle physiologique de la Huntingtine, mais représente surtout un espoir thérapeutique captivant.

Metastasis is largely controlled by Natural Killer (NK) cell-mediated immune surveillance. To colonize new environments, cancer cells undergo epithelial to mesenchymal transition (EMT), which allows them to detach and migrate. EMT is fueled by fatty acid oxidation (FAO), which partially replaces glycolytic-based tumor metabolism. Whether metabolic rewiring affects the targeting of cancer cells by NK cells remains unknown. Here, we show that forcing solid cancer cells to perform FAO by inhibiting pyruvate dehydrogenase kinase 1 with dichloroacetate (DCA) activates extracellular signal-regulated kinase-5 (ERK5), triggering EMT and tumor cell migration and invasion. Concomitantly, FAO induces the expression of ligands that mediate NK cell recognition. Consequently, NK cells better infiltrated DCA-treated 3D tumor spheroids, where they exerted their cytotoxic effects. DCA-treated cells showed increased migration in a zebrafish model, whereas metastasis from mammary cancer cells grafted into immune-deficient mice was enhanced by DCA. These migrating/metastatic cells are preferentially killed by NK cells, which strongly limit their invasive potential. Hence, FAO promotes both metastasis and NK-mediated tumor surveillance, highlighting the Achilles’ heel of metastatic cells, which may offer new therapeutic opportunities. Teaser: Metastasis recognition and killing by immune cells, such as NK cells, requires a metabolic shift that relies on lipid metabolism.

Significance: The hypocretin (Hcrt, also known as orexin) neuropeptides regulate sleep and wake stability, and disturbances of Hcrt can lead to sleep disorders. MicroRNAs (miRNAs) are short noncoding RNAs that fine-tune protein expression levels, and miRNA-based therapeutics are emerging. We report a functional interaction between miRNA (miR-137) and Hcrt . We demonstrate that intracellular miR-137 levels in Hcrt neurons regulate Hcrt expression with downstream effects on wakefulness. Specifically, lowering of miR-137 levels increased wakefulness in mice. We further show that the miR-137:Hcrt interaction is conserved across mice and humans, that miR-137 also regulates sleep–wake balance in zebrafish, and that the MIR137 locus is genetically associated with sleep duration in humans. Together, our findings reveal an evolutionarily conserved sleep–wake regulatory role of miR-137.

Cell-penetrating peptides (CPPs) allow intracellular delivery of bioactive cargo molecules. The mechanisms allowing CPPs to enter cells are ill-defined. Using a CRISPR/Cas9-based screening, we discovered that KCNQ5, KCNN4, and KCNK5 potassium channels positively modulate cationic CPP direct translocation into cells by decreasing the transmembrane potential (V_m). These findings provide the first unbiased genetic validation of the role of V_m in CPP translocation in cells. In silico modeling and live cell experiments indicate that CPPs, by bringing positive charges on the outer surface of the plasma membrane, decrease the V_m to very low values (–150 mV or less), a situation we have coined megapolarization that then triggers formation of water pores used by CPPs to enter cells. Megapolarization lowers the free energy barrier associated with CPP membrane translocation. Using dyes of varying dimensions in CPP co-entry experiments, the diameter of the water pores in living cells was estimated to be 2 (–5) nm, in accordance with the structural characteristics of the pores predicted by in silico modeling. Pharmacological manipulation to lower transmembrane potential boosted CPP cellular internalization in zebrafish and mouse models. Besides identifying the first proteins that regulate CPP translocation, this work characterized key mechanistic steps used by CPPs to cross cellular membranes. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.

Lipid droplets (LD) are affected in multiple human disorders. These highly dynamic organelles are involved in many cellular roles. While their intracellular dispersion is crucial to ensure their function and other organelles-contact, underlying mechanisms are still unclear. Here we show that Spastin, one of the major proteins involved in Hereditary Spastic Paraplegia (HSP), controls LD dispersion. Spastin depletion in zebrafish affects metabolic properties and organelle dynamics. These functions are ensured by a conserved complex set of splice variants. M1 isoforms determine LD dispersion in the cell by orchestrating endoplasmic reticulum (ER) shape along microtubules (MTs). To further impact LD fate, Spastin modulates transcripts levels and subcellular location of other HSP key players, notably Seipin and REEP1. In pathological conditions, mutations in human Spastin M1 disrupt this mechanism and impacts LD network. Spastin depletion influences not only other key proteins but also modulates specific neutral lipids and phospholipids, revealing an impact on membrane and organelle components. Altogether our results show that Spastin and its partners converge in a common machinery that coordinates LD dispersion and ER shape along MTs. Any alteration of this system results in HSP clinical features and impacts lipids profile, thus opening new avenues for novel biomarkers of HSP. Hereditary spastic paraplegia is a group of inherited diseases characterized by spasticity and gait disorder. Spastin mutations represent the most common cause of hereditary spastic paraplegia. While Spastin was initially defined as a microtubule severing protein, alternative isoforms impact organelles. In the present work, we identify how Spastin combines the specificity of different splice variants to coordinate endoplasmic reticulum shape and lipid droplet dispersion. When mutated, Spastin impairs this synchronization between organelle dynamics, and affects other proteins involved in hereditary spastic paraplegia, notably REEP1 and Seipin. The coordination of lipid droplet dispersion and reticulum shaping under the control of Spastin is a unique trafficking model, affected in the different forms of hereditary spastic paraplegia. The impact of Spastin on this machinery results in the alteration of the lipid profile, particularly phospholipids and neutral lipids in muscle and brain. These potential biomarkers open new therapeutic avenues in hereditary spastic paraplegia.

Cell-penetrating peptides (CPPs) allow intracellular delivery of cargo molecules. They provide efficient methodology to transfer bioactive molecules in cells, in particular in conditions when transcription or translation of cargo-encoding sequences is not desirable or achievable. The mechanisms allowing CPPs to enter cells are ill-defined. Using a CRISPR/Cas9-based screening, we discovered that KCNQ5, KCNN4, and KCNK5 potassium channels positively modulate cationic CPP direct translocation into cells by decreasing the transmembrane potential (V _m ). These findings provide the first unbiased genetic validation of the role of Vm in CPP translocation in cells. In silico modeling and live cell experiments indicate that CPPs, by bringing positive charges on the outer surface of the plasma membrane, decrease the V _m to very low values (−150 mV or less), a situation we have coined megapolarization that then triggers formation of water pores used by CPPs to enter cells. Megapolarization lowers the free energy barrier associated with CPP membrane translocation. Using dyes of varying sizes, we assessed the diameter of the water pores in living cells and found that they readily accommodated the passage of 2 nm-wide molecules, in accordance with the structural characteristics of the pores predicted by in silico modeling. Pharmacological manipulation to lower transmembrane potential boosted CPPs cellular internalization in zebrafish and mouse models. Besides identifying the first proteins that regulate CPP translocation, this work characterized key mechanistic steps used by CPPs to cross cellular membrane. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo .

Organogenesis is well characterized in vertebrates. However, the anatomical and functional development of intracellular compartments during this phase of development remains unknown. Taking an organellogenesis point of view, we characterize the spatiotemporal adaptations of the mitochondrial network during zebrafish embryogenesis. Using state of the art microscopy approaches, we find that mitochondrial network follows three distinct distribution patterns during embryonic development. Despite of this constant morphological change of the mitochondrial network, electron transport chain supercomplexes occur at early stages of embryonic development and conserve a stable organization throughout development. The remodeling of the mitochondrial network and the conservation of its structural components go hand-in-hand with somite maturation; for example, genetic disruption of myoblast fusion impairs mitochondrial network maturation. Reciprocally, mitochondria quality represents a key factor to determine embryonic progression. Alteration of mitochondrial polarization and electron transport chain halts embryonic development in a reversible manner suggesting developmental checkpoints that depend on mitochondrial integrity. Our findings establish the subtle dialogue and co-dependence between organogenesis and mitochondria in early vertebrate development. They also suggest the importance of adopting subcellular perspectives to understand organelle-organ communications during embryogenesis.

Growing evidence shows that alterations occurring at early developmental stages contribute to symptoms manifested in adulthood in the setting of neurodegenerative diseases. Here, we studied the molecular mechanisms causing giant axonal neuropathy (GAN), a severe neurodegenerative disease due to loss-of-function of the gigaxonin-E3 ligase. We showed that gigaxonin governs Sonic Hedgehog (Shh) induction, the developmental pathway patterning the dorso-ventral axis of the neural tube and muscles, by controlling the degradation of the Shh-bound Patched receptor. Similarly to Shh inhibition, repression of gigaxonin in zebrafish impaired motor neuron specification and somitogenesis and abolished neuromuscular junction formation and locomotion. Shh signaling was impaired in gigaxonin null zebrafish and was corrected by both pharmacological activation of the Shh pathway and human gigaxonin, pointing to an evolutionary-conserved mechanism regulating Shh signaling. Gigaxonin-dependent inhibition of Shh activation was also demonstrated in primary fibroblasts from GAN patients and in a Shh activity reporter line depleted in gigaxonin. Our findings establish gigaxonin as a key E3 ligase that positively controls the initiation of Shh transduction, reveal the causal role of Shh dysfunction in motor deficits, thus highlighting the developmental origin of GAN.

Hypocretin/orexin (HCRT) and melanin concentrating hormone (MCH) neuropeptides are exclusively produced by the lateral hypothalamus and play important roles in sleep, metabolism, reward, and motivation. Loss of HCRT (ligands or receptors) causes the sleep disorder narcolepsy with cataplexy in humans and in animal models. How these neuropeptides are produced and involved in diverse functions remain unknown. Here, we developed methods to sort and purify HCRT and MCH neurons from the mouse late embryonic hypothalamus. RNA sequencing revealed key factors of fate determination for HCRT ( Peg3 , Ahr1 , Six6 , Nr2f2 , and Prrx1 ) and MCH ( Lmx1 , Gbx2 , and Peg3 ) neurons. Loss of Peg3 in mice significantly reduces HCRT and MCH cell numbers, while knock-down of a Peg3 ortholog in zebrafish completely abolishes their expression, resulting in a 2-fold increase in sleep amount. We also found that loss of HCRT neurons in Hcrt-ataxin-3 mice results in a specific 50% decrease in another orexigenic neuropeptide, QRFP, that might explain the metabolic syndrome in narcolepsy. The transcriptome results were used to develop protocols for the production of HCRT and MCH neurons from induced pluripotent stem cells and ascorbic acid was found necessary for HCRT and BMP7 for MCH cell differentiation. Our results provide a platform to understand the development and expression of HCRT and MCH and their multiple functions in health and disease.