Atp6v1b2

Amina Dulac

Abdul-Raouf Issa

Jun Sun

Giorgio Matassi

Baya Chérif- Zahar

Daniel Cattaert

Serge Birman
email: serge.birman@espci.fr

Keywords: ATP6AP1/ATP6AP1L, Drosophila melanogaster, neuron, neuromuscular junction, synaptic transmission, synaptic vesicle acidification, V-ATPase complex, VhaAC45L

Cette thèse n'aurait pas pu voir le jour sans le soutien et l'aide de nombreuses personnes. De l'obtention de la bourse jusqu'à la rédaction du manuscrit, ces années n'auront pas été un long fleuve tranquille, et c'est tant mieux, car j'en ressors grandie et riche de plein belles rencontres.

J'aimerais en premier exprimer ma plus profonde gratitude au Dr. Serge Birman, mon directeur de thèse, sans qui rien de tout cela n'aurait été possible. Merci d'abord de m'avoir donné ma chance en m'acceptant dans votre équipe pour mon stage de Master, malgré mes lacunes évidentes en biologie. C'est la passion contagieuse que vous déployez dans chacun de vos projets qui m'a convaincue de poursuivre par une thèse. Ce sont ensuite vos encouragements, votre patience et vos conseils, qui m'ont permis d'obtenir une bourse de thèse. Enfin, c'est votre optimisme à toute épreuve, et nos nombreux échanges scientifiques, qui m'ont portée jusqu'à l'aboutissement de ce beau projet. J'adresse également mes plus vifs remerciements aux Drs. Alice Carrier at Bassem Hassan pour avoir suivi ce projet, et aider à son avancement grâce aux suggestions et conseils prodigués lors de mes comités de suivi de thèse. Je suis également très reconnaissante aux membres de mon jury de thèse, les Drs. Florence Besse, Brigitte Grima, Bassem Hassan et Nicolas Vitale, pour avoir accepté de donner de leur temps en tant que rapporteur et examinateur, et au Dr. Iris Salecker pour s'être engagée à présider ce jury. C'est bien entourée que j'ai eu la chance de vivre ces années riches en émotions. Un grand merci à toute l'équipe GCRN pour tous les bon moments, scientifiques ou non. Les ciné-clubs, les déjeuners ensemble, les sorties pour aller acheter des glaces les aprèsmidis d'été, et puis plus tard, Covid oblige, les rendez-vous Zoom du dimanche. Passés ou présents, tous les membres de l'équipe m'ont aidé à évoluer sur le plan scientifique. Si nous avons démarré comme collègues, c'est comme amis que nous nous quittons. Donc merci à vous tous, et plus précisément, dans un ordre absolument arbitraire : Xiaojing, merci pour ta gentillesse au quotidien. J'aurais été bien seule sans toi pendant tous ces week-ends passés au labo. Je te souhaite le meilleur pour la suite de ton projet. Jun, merci d'avoir toujours trouvé le temps de répondre à mes questions et de m'avoir mis le pied à l'étrier pour mes premiers pas au labo. Baya, seul autre membre restant du clan des frileux, merci pour ta bonne humeur au quotidien et ton expertise technique qui m'aura sauvée bien des fois. Zohra, merci pour tes conseils, en particulier pendant la rédaction de mon article, ton pragmatisme m'a aidé à y voir plus clair à de nombreuses reprises. Mathilde, merci pour ta rigueur scientifique et pour avoir veillé au bon fonctionnement de l'équipe quotidiennement afin que nous puissions tous travailler dans un environnement agréable. Céline, merci pour ta pédagogie et ta patience lors de mes premières manipes. Merci pour les nombreuses conversations partagées sur tout et rien, qui ont rythmé nos journées de travail. Amélie, merci pour la bonne humeur que tu apportes au labo, et les anecdotes sur Elia qui sont toujours une bonne façon d'égayer une journée. Marion, merci pour ton soutien sans faille pendant toute la durée de mon stage de Master. Merci aussi pour tes conseils toujours d'une grande aide, et ton esprit scientifique intransigeant.

Raouf, sans qui ce projet n'aurait pas existé puisque tu l'as initié. Merci pour ton encadrement au début de mon Master, et pour tes conseils même après ton départ, tout au long de ma thèse. Je te souhaite une belle carrière dans la recherche.

Alexandra, bien que tu aies quitté le labo avant mon arrivée, j'ai quand même l'impression que nous avons travaillé ensemble ! En plus de tes bons conseils, nos discussions ne manquent jamais de me remonter le moral, donc merci à toi. Khadija, merci pour ton accueil, en particulier pendant mon premier mois au laboratoire. Ta bonne humeur et ton écoute m'auront permis d'être rapidement à l'aise dans ce nouvel environnement.

Bien sûr, il n'y a pas que les membres de l'équipe qui ont embelli ces années, mais aussi tous ceux du laboratoire. Un grand merci surtout aux étudiants pour tous leurs précieux conseils pendant les Student Meetings. Merci également à Audrey, notre valeureuse technicienne pour son aide au quotidien.

, and ultimately led to the identification of V-ATPase genes in other species by homology, thanks to the high conservation of the complex across species.

In higher eukaryotes, multiple subunits are coded by several genes.

Je souhaite également remercier le Dr. Daniel Cattaert, qui m'a accueillie dans son laboratoire à Bordeaux dans le cadre d'une collaboration. Cette expérience a été non seulement très enrichissante, mais a également grandement influencé mes plans d'avenir puisqu'elle m'a fait découvrir, et adoré, le monde de l'électrophysiologie, une technique que j'espère maintenant pouvoir apprendre et pratiquer dans le futur.

Un grand merci à tous mes amis en dehors du laboratoire. Les années ont passé, les chemins ont divergé, chacun atterrissant dans un domaine complètement différent des autres, mais l'amitié est restée. Merci à tous pour vos encouragements, et pour m'avoir souvent rappelé qu'il n'y a pas que la recherche dans la vie (un fait qu'on a parfois tendance à oublié en thèse).

Enfin, mes derniers remerciements sont adressés aux personnes les plus importantes de ma vie : ma famille. Merci pour votre amour et votre soutien inconditionnel. En particulier à celle qui a probablement été l'inspiration inconsciente de mon désir de faire une thèse : ma mère. Tes encouragements et ta foi en moi sans faille sont ma plus grande force, et il faudrait un roman entier pour pouvoir t'exprimer ma gratitude.

Merci aussi à ma tante, Djahida, toujours rassurante et encourageante. Merci de croire autant en moi. Je n'oublie pas non plus la famille de l'autre côté de la méditerranée.

Même à distance, vous avez su me soutenir à chaque étape de cette aventure. Loin des yeux mais jamais loin du coeur.

Merci également à ma soeur Gaëlle, la touche artistique de ma vie. Tes encouragements et ta persévérance à mieux comprendre ce que je fais, malgré l'éloignement de nos deux mondes, m'ont toujours beaucoup touché.

Ces remerciements s'étendent également à tous les amis de la famille, eux-mêmes famille par extension, toujours bienveillants et encourageants.

Pour finir, je ne remercierai pas les mouches, victimes silencieuses de ce projet.

A la place, je préfère leur présenter mes excuses. Bien que la vie de ces petits insectes semble valoir moins que d'autres, à mes yeux elle est aussi précieuse et j'espère faire de mon mieux pour que leur sacrifice ne soit pas vain. La complexité et l'ingéniosité de ces organismes à échelle réduite ne cesse de m'impressionner et de me fasciner chaque jour, et elles ont pour cela tout mon respect.

Summary in French

De nombreux processus biologiques sont étroitement liés à l'environnement électrochimique du compartiment cellulaire dans lequel ils se déroulent : de la modification post-traductionnelle des protéines nouvellement synthétisées dans le réticulum endoplasmique et l'appareil de Golgi, jusqu'à leur dégradation finale dans les lysosomes. Ainsi, la plupart des organites, tels que les lysosomes ou les vésicules synaptiques, doivent maintenir une valeur de pH intraluminal spécifique et contrôlée pour garantir un fonctionnement optimal. Cela se fait principalement grâce à un complexe enzymatique essentiel, capable de transporter des protons à travers les membranes, appelé H+-ATPase vacuolaire ou V-ATPase. La V-ATPase est composé de deux parties : un domaine associé à la membrane nommé V 0 , et un domaine cytosolique nommé V 1 [START_REF] Vasanthakumar | Structure and Roles of V-type ATPases[END_REF]. Le domaine V 0 est constitué de 5 sous-unités : a, c, c'', d, and e, et le domaine V 1 est quant à lui composé de 8 sous-unités : A, B, C, D, E, F, G et H. L'association de V 1 à V 0 est nécessaire au fonctionnement de la pompe à protons. V 1 possède des sous-unités catalytiques capables d'hydrolyser l'ATP en ADP.

L'énergie produite par cette réaction est utilisée par le domaine V 0 , via le changement de conformation de plusieurs sous-unités du complexe, pour transporter des protons à travers une membrane. Cette structure unique est extrêmement bien conservée chez tous les Eucaryotes.

La V-ATPase est un complexe ubiquitaire. Cependant, tous les organites des différents tissus ou types cellulaires ne nécessitent pas les mêmes niveaux d'acidification.

Cela soulève une question importante : comment la machinerie générale du complexe estelle régulée pour répondre à des besoins spécifiques ? Les mécanismes permettant d'adapter la V-ATPase à des contextes différents sont loin d'être entièrement élucidés, bien que plusieurs processus influençant son activité aient été mis en évidence. L'un d'eux consiste en l'assemblage réversible des domaines V 0 et V 1 . Lorsque les deux domaines sont séparés, l'activité d'hydrolyse par V 1 , ainsi que la capacité de V 0 à transporter des protons, sont toutes deux bloquées, rendant le complexe entièrement inactif. Un autre paramètre modulant la V-ATPase est sa composition en différents isoformes de certaines sous-unités. Si la structure et le mécanisme général restent les mêmes, des V-ATPases présentes dans différents types cellulaires ou associées à des organites distincts peuvent varier dans leur composition. En effet, chez la plupart des espèces, chaque sous-unité peut être codée par plusieurs gènes, et chaque gène peut produire plusieurs isoformes. Cela permet de nombreuses combinaisons possibles pour construire le complexe V-ATPase. Il a ainsi été montré que différentes isoformes de la sous-unité a de V 0 correspondent à différentes localisations de la pompe à protons chez plusieurs espèces (Manolson et al., 1994 ;Morel et al., 2003). Enfin, un autre mécanisme de régulation étudié au cours des dernières décennies est l'interaction de la V-ATPase avec des protéines accessoires capables de cibler le complexe vers une localisation spécifique, ou d'influencer l'association/dissociation de V 1 et V 0 [START_REF] Jansen | Accessory subunit Ac45 controls the V-ATPase in the regulated secretory pathway[END_REF]. On sait encore très peu de choses sur les acteurs précis impliqués dans ces voies et sur les détails de ces mécanismes de régulation.

Les mutations et les dérèglements de la V-ATPase conduisent à de nombreuses pathologies, ce qui n'est pas surprenant compte tenu de sa nature ubiquitaire. Récemment, la V-ATPase est ainsi devenu un centre d'intérêt dans le contexte des maladies neurodégénératives (Colacurcio and Nixon, 2016). Cependant, l'implication générale de la V-ATPase dans diverses fonctions cellulaires constitue également un obstacle à son utilisation thérapeutique, rendant les effets secondaires des médicaments qui la ciblent difficilement prédictibles. Une meilleure compréhension des gènes impliqués dans la régulation de la V-ATPase permettrait potentiellement de surmonter cet obstacle, et les gènes identifiés pourraient également constituer de nouvelles cibles thérapeutiques.

Mon projet de doctorat a porté sur l'étude d'une protéine de drosophile appelée Lome (prononcé Lomé), dont les bases de donnée protéiques telles qu'InterPro (Hunter et al., 2009) prédisent qu'elle joue un rôle dans le complexe V-ATPase, en raison de similitudes de séquence avec d'autres gènes connus. De manière intéressante, selon les bases de données d'expression génique chez la drosophile (Chintapalli et al., 2007 ;[START_REF] Davie | A single-cell transcriptome atlas of the aging Drosophila brain[END_REF], Lome semble être une protéine spécifique du système nerveux et présente uniquement dans les neurones, ce qui en fait un candidat potentiel pour la régulation de la V-ATPase neuronale. Notre objectif a donc été d'étudier la fonction de Lome, et plus particulièrement son implication possible dans la physiologie synaptique. Tout d'abord, nous avons examiné plus en détail le domaine d'expression de Lome.

La base de données FlyAtlas Affymetrix (Chintapalli et al., 2007) indique que ce gène est fortement enrichi dans le système nerveux. Les données RNAseq unicellulaires disponibles [START_REF] Davie | A single-cell transcriptome atlas of the aging Drosophila brain[END_REF] suggèrent également que Lome est exprimé dans l'ensemble des neurones et non pas dans la glie. Pour vérifier cela, nous avons réalisé une analyse qPCR sur différentes parties du corps de la mouche : la tête, le thorax et l'abdomen. La tête de drosophile contient le cerveau composé d'environ 130 000 neurones. Le thorax contient également environ 20 000 neurones dans la corde nerveuse ventrale, l'équivalent chez la mouche de la moelle épinière des vertébrés. L'abdomen, quant à lui, contient très peu de neurones. Les résultats de la PCR quantitative (qPCR) ont montré que les transcrits de Lome pouvaient être détectés principalement dans la tête, en moindre quantité dans le thorax et non dans l'abdomen, suivant ainsi la répartition du système nerveux central (SNC) des mouches. En utilisant trois ARN interférents (ARNi) différents, nous avons ensuite diminué l'expression de Lome spécifiquement dans les neurones. L'expression pan-neuronale durant toute la durée du développement a conduit à un phénotype létal chez la drosophile pour deux des ARNi. De plus, l'expression du troisième ARNi, le seul donnant des adultes viables, uniquement dans les neurones, est suffisante pour réduire le niveau d'ARN messager (ARNm) de Lome d'environ 80% dans la tête, confirmant que l'expression de Lome est prédominante dans ce type de cellule. En revanche, l'expression des constructions ARNi de Lome dans les cellules gliales n'a eu aucun effet sur son niveau ARMm, ou sur la viabilité et la santé des mouches. Pour finir, le mutant knock-out de Lome est létal au stade embryonnaire, en accord avec les résultats des constructions ARNi. Cette létalité a pu être corrigée en ré-exprimant Lome uniquement dans les neurones, montrant ainsi que son expression n'est requise que dans ce type cellulaire.

Dans le but de mieux comprendre le rôle de Lome dans les neurones, nous avions besoin de connaître sa localisation subcellulaire. Pour cela, nous avons utilisé la technique CRISPR-Cas9 qui nous a permis d'insérer par mutagenèse génomique une étiquette (tag) V5 à l'extrémité C-terminale de la protéine endogène. Les mouches possédant cette mutation sont viables à l'état homozygote et ne présentent pas de défaut comportemental évident, ce qui suggère que la protéine marquée est encore au moins en partie fonctionnelle. De manière intéressante, des expériences d'immunomarquage sur cette souche nous ont permis de constater que la protéine Lome se trouve principalement localisée dans le neuropile, où sont situées la plupart des synapses du système nerveux central, aussi bien chez les larves que chez les mouches adultes. Le fait d'avoir à disposition la protéine Lome-V5 nous a aussi permis d'effectuer des expériences de coimmunoprécipitation, qui ont montré que Lome interagit avec des sous-unités du domaine V 0 de la V-ATPase, connues pour être présente dans les neurones, ce qui suggère que cette protéine s'associe effectivement de façon permanente ou transitoire avec le complexe de la V-ATPase neuronale.

Pour étudier la fonction synaptique de Lome, nous nous sommes concentrés sur les effets de sa perte de fonction partielle en l'inactivant par ARNi (knockdown). Dans les neurones, et plus spécifiquement dans les terminaisons nerveuses, la V-ATPase est essentielle pour acidifier les vésicules synaptiques, permettant ainsi aux transporteurs vésiculaires de neurotransmetteurs de les remplir correctement (Moriyama, et al., 1992).

Puisque Lome semble être spécifique des neurones et enrichi aux synapses, il était possible que son activité soit nécessaire au bon fonctionnement de la V-ATPase associée aux vésicules synaptiques, et donc à leur acidification. Afin de tester cette hypothèse, nous avons analysé le rôle de Lome à la jonction neuromusculaire de la larve de drosophile, un modèle qui a souvent été utilisé pour analyser les mécanismes synaptiques (Ruiz-Cañada and Budnik, 2006). Afin de mesurer le pH relatif des vésicules synaptiques dans le contexte du knockdown de Lome par rapport aux contrôles, nous avons utilisé une sonde pH exprimée génétiquement ciblant les vésicules synaptiques, la VMAT-pHluorine. Cette sonde est fluorescente uniquement en pH neutre, le signal étant aboli en milieu acide. Les vésicules synaptiques ont normalement un pH d'environ 5,5, donc suffisamment bas pour abolir entièrement le signal de la sonde, comme nous avons pu le confirmer chez les drosophiles de type sauvage. Cependant, dans les motoneurones exprimant un ARNi de Lome, un signal résiduel de fluorescence était détectable, ce qui suggère que le pH des vésicules synaptiques est plus élevé que la normale chez ce mutant.

Ce résultat a pu être confirmé en utilisant deux ARNi différents.

Pour déterminer si ce défaut d'acidification des vésicules synaptiques avait des conséquences phénotypiques, nous avons testé le comportement de locomotion des larves knockdown en utilisant l'imagerie par « réflexion interne totale frustrée » (FTIR) (système FIM) et le logiciel FIMtrack (Risse et al., 2013). Les larves déficientes en Lome se sont effectivement révélées avoir une locomotion réduite par rapport aux larves contrôles : elles n'étaient capables de couvrir que la moitié de la distance parcourue par ces dernières dans un même intervalle de temps. Ce résultat a aussi été confirmé avec le second ARNi de Lome.

Un défaut dans l'acidification des vésicules synaptiques devrait conduire à un moindre remplissage en neurotransmetteurs, ce qui signifie que la taille du quanta synaptique, qui représente la réponse post-synaptique à la libération d'une seule vésicule, pourrait être diminuée. Nous avons collaboré avec Daniel Cattaert (INCIA, CNRS, Université de Bordeaux), pour réaliser des expériences d'électrophysiologie afin de tester cette hypothèse. Nous avons enregistré les événements spontanés à l'intérieur de muscle de larves présentant un niveau réduit de Lome dans les motoneurones, afin de mesurer la taille des quanta synaptiques. Les résultats ont révélé que les larves déficientes en Lome ont un quanta synaptique réduit par rapport aux animaux contrôles. Là encore, des effets similaires ont été obtenus avec deux ARNi différents pour cette expérience. Dans l'ensemble, les résultats obtenus au cours de mon travail de thèse ont permis de caractériser une nouvelle sous-unité accessoire du complexe de la V-ATPase neuronale et de mettre en évidence la contribution essentielle de cette protéine dans l'acidification des vésicules synaptiques et la libération des neurotransmetteurs chez la drosophile. Les perspectives pour l'avenir seront d'analyser le rôle régulateur précis de Lome au sein de la V-ATPase, qui pourrait concerner la localisation, l'assemblage ou l'activité de ce complexe, et de déterminer si cette protéine aurait ou non d'autres fonctions dans les neurones que l'acidification des vésicules synaptiques, et si elle pourrait constituer une nouvelle cible thérapeutique dans un contexte neuropathologique.

List of abbreviation

B. Results

Article submitted

A. Introduction

In this first part of the introduction, I will describe the core mechanism and structure of the V-ATPase, before attempting to paint a general, but non-exhaustive, picture of the vast V-ATPase functional roles and regulations in health and disease.

Finally, the last section will focus on neuronal V-ATPase.

V-ATPase structure and core mechanism

The V-ATPase is a beautiful example of nature's incredible talent for ingenious design. This cylinder shaped nanoscale motor, of about 28 nm length and 12 nm diameter (Figure A2.A) [START_REF] Wilkens | A structural model of the vacuolar ATPase from transmission electron microscopy[END_REF], is capable of generating a proton gradient of more than 4 order of magnitudes, according to a mechanochemical model of its functioning [START_REF] Grabe | The mechanochemistry of V-ATPase proton pumps[END_REF]. For that, it produces energy through ATP hydrolysis to power a rotational motor translocating protons from one side of a membrane to the other.

Functioning of the pump requires the presence of magnesium ions (Mg2+) [START_REF] Carlsson | A Mg++-ATP dependent storage mechanism in the amine granules of the adrenal medulla[END_REF][START_REF] Kakinuma | Properties of H+-translocating adenosine triphosphatase in vacuolar membranes of SAccharomyces cerevisiae[END_REF], and produces adenosine diphosphate (ADP) and phosphate (P i ) (Figure A2.B). Even if some questions are still unresolved [START_REF] Rawson | Rotating with the brakes on and other unresolved features of the vacuolar ATPase[END_REF], high resolution reconstruction of the complex combined to functional study of individual subunits have contributed to the progressive construction of the now wellaccepted model for the V-ATPase general structure, and the core mechanism of the rotary motor.

composed of a soluble domain, responsible for ATP hydrolysis (or synthesis), and a membrane-bound domain, responsible for proton translocation. The number of different subunits in each complex varies, but most subunits possess functional equivalent in the other two complexes (Figure A3). With its 8 different subunits, the smallest amount of the three, the F-ATPase is often referred to as the "simplest" complex of the family. The V-ATPase, and its 14 different subunits, is by opposition usually considered the most complicated one. Despise this difference, the core mechanism seems to be well conserved between the three groups, and most experiments shedding light on V-ATPases functioning were based on previous studies of F-ATPases. The rest of the manuscript will focus only on the V-ATPase.

V-ATPase structure

Cryo-electron microscopy reconstructions of the complex in different conformational states, and with higher and higher resolution over the years, have shed light on the structural properties of the V-ATPase [START_REF] Radermacher | Molecular architecture of Manduca sexta midgut V1 ATPase visualized by electron microscopy[END_REF][START_REF] Rizzo | Resolution of the V1 ATPase from Manduca sexta into subcomplexes and visualization of an ATPase-active A3B3EG complex by electron microscopy[END_REF][START_REF] Zhang | Yeast V1-ATPase: affinity purification and structural features by electron microscopy[END_REF][START_REF] Wilkens | Three-dimensional structure of the vacuolar ATPase: localization of subunit H by difference imaging and chemical cross-linking[END_REF][START_REF] Muench | Cryo-electron microscopy of the vacuolar ATPase motor reveals its mechanical and regulatory complexity[END_REF]Mazhab-Jafari and Rubinstein, 2016).

The V-ATPase is a large protein complex, containing 14 different subunits. These subunits can be partitioned in several ways, but are usually segregated according to their appartenance to either the soluble domain of the complex, called V 1 , or the membranebound domain called V 0 .

V 1 , which contains the catalytic unit responsible for ATP hydrolysis, is composed of eight different subunits: A, B, C, D, E, F, G and H. These subunits do not all have the same stoichiometry within the complex. The full complex contains three copies of subunits A, B, E and G, but only one copy of subunits C, D, F and H [START_REF] Kitagawa | Stoichiometry of the peripheral stalk subunits E and G of yeast V1-ATPase determined by mass spectrometry[END_REF][START_REF] Muench | Cryo-electron microscopy of the vacuolar ATPase motor reveals its mechanical and regulatory complexity[END_REF]. In summary, the stoichiometry of V 1 subunits is as follows:

A 3 B 3 CDE 3 FG 3 H.
The membrane-bound domain V 0 is the one containing the proton port. In most organisms, it is made of five subunits: a, c, c", d and e. All V 0 subunits are present in one copy within the full complex, with the exception of the c subunit which has the highest representation with 9 copies per V-ATPase. Yeast V-ATPases also have an additional subunit called c', which takes the place of one the the c subunit in the full complex.

Recently, a potential new subunit, named f, has been identified in a 3.9-angstrom resolution electron cryomicroscopy reconstruction of the V 0 domain of yeast V-ATPase (Mazhab-Jafari et al., 2016). Later on, in rat brain, a protein called RNAseK, that had already been shown to associate with the V-ATPase [START_REF] Perreira | RNASEK is a V-ATPase-associated factor required for endocytosis and the replication of rhinovirus, influenza A virus, and dengue virus[END_REF], was detected in the complex and suggested to be the mammalian homolog of yeast f subunit (Abbas et al., 2020). In summary, the stoichiometry of V0 subunits is likely to be as follows:

ac 9 c"def or ac 8 c'c"def for yeast.
names all start by "ATP6", indicative of the V-ATPase family, followed by either "V0"

or "V1", referring to the domain the gene is part of, and ending by the letter and the number designating which isoform of which subunit it corresponds to (for example ATP6V0A2 is the second isoform of subunit a of V 0 ). Table A1 lists all isoforms of the human V-ATPase, and their corresponding homologs in yeast.

In the rest of the manuscript, uppercase letters will always refer to subunit from the V 1 domain (i.e. A, B, C, D, E, F, G and H), while lowercase letters in italic will refer to V 0 subunits (i.e. a, b, c, d, e).

Table A1. List of human isoforms for all V-ATPase subunits and their yeast homologs.

V-ATPase domain Subunit Human genes Yeast genes

can be grouped into a rotating domain, called the rotor, and a static one called the stator (Figure A4). The rotation being relative between the two domains, the decision to designate one as the stator and the other one as the rotor was based on the molecular weight of the two domains [START_REF] Wilkens | Rotary molecular motors[END_REF], the heaviest one being labelled the stator.

Following this convention, the stator can itself be broken down into several functional parts (Figure 5A):

• three catalytic units, each capable of hydrolysing ATP (Vasilyeva and Forgac, 1996;[START_REF] Maher | Crystal structure of A3B3 complex of V-ATPase from Thermus thermophilus[END_REF]. Each catalytic unit is made of a dimer of V 1 subunit A and B, and ATP hydrolysis is believed to take place at the interface between the two subunits [START_REF] Forgac | Structure, mechanism and regulation of the clathrin-coated vesicle and yeast vacuolar H (+)-ATPases[END_REF].

• the proton port, crossing through the entire membrane, which corresponds to the V 0 a subunit. The dominant model of proton translocation by rotary ATPases infers the existence of two aqueous half channels within this port: one opening on the cytoplasm and reaching mid-way through the lipid bilayer, and the other one starting mid-way in the lipid bilayer and ending in the organelle lumen [START_REF] Angevine | Aqueous access pathways in subunit a of rotary ATP synthase extend to both sides of the membrane[END_REF]Mazhab-Jafari and Rubinstein, 2016).

• a stator stalk unit, ensuring that the catalytic units and the proton port are static relatively to each other [START_REF] Boekema | Visualization of a peripheral stalk in V-type ATPase: evidence for the stator structure essential to rotational catalysis[END_REF][START_REF] Arata | Cysteine-directed cross-linking to subunit B suggests that subunit E forms part of the peripheral stalk of the vacuolar H+-ATPase[END_REF]. This unit includes the subunits E, G, C and H of V 1 . Subunits E and G form dimers acting as peripheral stalks reffered to as EG1, EG2 and EG3 (Collins and Forgac, 2020).

The rotor is composed of two functional parts (Figure A5.A):

• a central rotating shaft, made of subunit D and F from V 1 , and subunit d from V 0 , that converts ATP-hydrolysing energy into a rotational movement.

• the c-ring, embedded in the membrane [START_REF] Yokoyama | Rotation of the proteolipid ring in the V-ATPase[END_REF], which rotation will be driven by the central shaft. As said in the name, the c-ring includes the c and c" subunits of V0 (and c' in the case of yeast). These subunits are highly hydrophobic and form together a proteolipid structure in a ring shape.

Subunits e and f of V 0 were not listed in the different functional parts of the motor.

Being the most recently discovered, their role within the complex is not yet known, but their putative localization, attached to the a subunit of V 0 (Mazhab-Jafari et al., 2016; Since the c-ring is composed of 10 proteins (9 subunits c + 1 subunit c", or 8 subunits c + 1 subunit c' + 1 subunit c" in yeast), each of them having one glutamate residue able to capture protons, one full rotation of the complex can transport a maximum of ten protons. To complete a rotation, three ATPs need to be hydrolysed, which means that the maximum V-ATPase efficiency, or maximum ratio proton:ATP, is 10:3.

enters the proton port through the cytosolic hemi-channel, and is then dragged along by the c-ring for a complete revolution, thanks to the rotational movement powered by ATP hydrolysis, before being released in the luminal hemi-channel of the proton port.

V-ATPase functions

The primary function of V-ATPases is to acidy cellular compartments or the extracellular milieu, depending on their localization. Furthermore, over the years, several pieces of evidence have emerged about possible roles of V-ATPases in other processes.

Functional study of V-ATPases was made possible because of the existence of various specific inhibitors, the most common ones being bafilomycin A and concanamycin A [START_REF] Bowman | Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells[END_REF][START_REF] Droese | Inhibitory effect of modified bafilomycins and concanamycins on P-and V-type adenosinetriphosphatases[END_REF]. Pharmacological blockade of V-ATPases permitted to identify on which membranes the complex was acting, and the physiological relevance of its activity.

Function of acidification 1.2.1.1. Intracellular V-ATPases

V-ATPases are found in several compartments of all eukaryotic cells, where they play a key role in the ubiquitous pathways of exocytosis and endocytosis.

Secretory pathway

Secretory vesicles, like chromaffin granules or synaptic vesicles, have an acidic luminal pH [START_REF] Johnson | Internal pH of isolated chromaffin vesicles[END_REF][START_REF] Mellman | Acidification of the endocytic and exocytic pathways[END_REF][START_REF] Njus | Bioenergetics of secretory vesicles[END_REF]. This proton gradient is the driving force for vesicular transporters which are able to load the vesicles by exchanging protons against secretory molecules, like neurotransmitters, peptides or hormones. Acidity is also important for peptide precursor processing [START_REF] Schoonderwoert | Inhibition of the vacuolar H+-ATPase perturbs the transport, sorting, processing and release of regulated secretory proteins[END_REF], like the conversion of proinsulin to insulin [START_REF] Orci | Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles[END_REF].

Endocytic pathway

Endocytosis allows cells to internalize molecules from the plasma membrane in order to either recycle them back to the membrane or degrade them. Its main components are early endosomes, late endosomes, and lysosomes.

Endosomes are the first player in this chain of events. Their lumen undergoes a progressive acidification controlled by V-ATPase activity during their maturation, starting at a pH of 6.8 in early endosomes and ending at a pH between 6.0 and 4.8 in late endosomes [START_REF] Maxfield | Endosome acidification and the pathways of receptor-mediated endocytosis[END_REF]Wang et al., 2017). This increasing acidity allows several processes to happen at the right step. For example, the proton concentration in early endosomes will facilitate the separation of ligands from the internalized receptors [START_REF] Maxfield | Endosome acidification and the pathways of receptor-mediated endocytosis[END_REF]. Receptors will then either be recycled and trafficked back to the plasma membrane, or be carried by the endocytic pathway to the late endosomes before ending in the lysosomes where they will be degraded.

Lysosomes are one of the most acidic cell compartment, with a pH between 4.5 and 5 [START_REF] Ohkuma | Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents[END_REF][START_REF] Anderson | A view of acidic intracellular compartments[END_REF]. One of their purposes is to degrade and recycle vesicle's cargo and cytosolic proteins. For that, they are filled with various hydrolases and lipases that can break down internalized proteins and lipids, respectively, generating re-usable building blocks for synthesis of new cellular products.

Lysosomal enzymes have low pH optimums [START_REF] Bond | Intracellular proteases[END_REF] and thus require a highly acidic environment to properly function. The V-ATPase complex is the main source of acidification of this organelle as shown by the pH increase of lysosomes exposed to the V-ATPase inhibitor bafilomycin A1 [START_REF] Yoshimori | Bafilomycin A1, a specific inhibitor of vacuolar-type H (+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells[END_REF]. In addition to degrading extracellular components, lysosomes also participate in cell maintenance, through the autophagic pathways, by degrading intracellular material such as misfolded proteins, or damaged organelles.

Along the endosomal-lysosomal axis, the V-ATPase is essential to another important organelle of the endocytic pathway: the Golgi apparatus. The Golgi apparatus is slightly acid, with distinct pH equilibrium in its different sub-compartments. The acidity increases along the cis-trans axis, from a pH of 6.7 in cis-Golgi to a pH of 6 in trans-Golgi. Blocking V-ATPase activity with an inhibitor such as Concanamycin A leads to an alkalization of the Golgi, highlighting the leading role of the complex in acidifying this compartment [START_REF] Kellokumpu | Golgi pH, Ion and Redox Homeostasis: How Much Do They Really Matter?[END_REF]. Proper acidity was shown to be required for post-translational modifications occurring in the Golgi [START_REF] Thorens | Chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion[END_REF][START_REF] Axelsson | Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins[END_REF][START_REF] Rivinoja | Elevated Golgi pH impairs terminal N-glycosylation by inducing mislocalization of Golgi glycosyltransferases[END_REF], as well as membrane trafficking between different Golgi sub-compartments [START_REF] Griffiths | Dissection of the Golgi complex. I. Monensin inhibits the transport of viral membrane proteins from medial to trans Golgi cisternae in baby hamster kidney cells infected with Semliki Forest virus[END_REF][START_REF] Palokangas | Retrograde transport from the pre-Golgi intermediate compartment and the Golgi complex is affected by the vacuolar H+-ATPase inhibitor bafilomycin A1[END_REF].

The Golgi also provides the lysosomal enzymes, essential for cargo degradation, through pH-dependent exchanges with endosomes. After being synthetized in the endoplasmic reticulum, the inactive enzymes are moved to the Golgi where they are tagged with a mannose 6-phospate (M6P) ligand [START_REF] Coutinho | Mannose-6-phosphate pathway: a review on its role in lysosomal function and dysfunction[END_REF]. This marker separates proteins targeted to the lysosomes from the ones destined for secretory vesicles.

M6P-labelled proteins are recognized and bound by M6P receptors (MPR), and the receptor-protein couples are transferred to endosomes. There, the lower pH allows the separation of M6P-labelled proteins from M6PR, followed by the dissociation of the M6P tag [START_REF] Coutinho | Mannose-6-phosphate pathway: a review on its role in lysosomal function and dysfunction[END_REF]. From the endosomes, the enzymes are finally delivered to the lysosomes where the even lower pH will permit their full maturation and activation.

acidification is a required step for moving cargo from early to late endosomes, a pHsensor must exist to feel the luminal pH and transduce the signal across the endosomal membrane. The V-ATPase has been proposed to play this role, in addition to its acidification function. Backing this theory is the discovery of pH-dependent interactions between subunit c of the complex and Arf6 [START_REF] Marshansky | The V-ATPase a2-subunit as a putative endosomal pH-sensor[END_REF], a cytosolic protein from the Arf (ADP-ribosylation factor) family, that has been implicated on endocytosis regulation [START_REF] Donaldson | ARF: a key regulatory switch in membrane traffic and organelle structure[END_REF], and between subunit a and ARNO (Arf nucleotide site opener), an activator of Arf6.

On lysosomes, it is not pH that the V-ATPase is suspected to sense but amino acid concentration. The mechanistic target of rapamycin complex 1 (mTORC1) is a well characterized hub integrating various cellular signals, like growth factor [START_REF] Efeyan | Nutrients and growth factors in mTORC1 activation[END_REF], energy levels [START_REF] Inoki | TSC2 mediates cellular energy response to control cell growth and survival[END_REF] or oxygen availability [START_REF] Brugarolas | Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex[END_REF][START_REF] Reiling | The hypoxia-induced paralogs Scylla and Charybdis inhibit growth by down-regulating S6K activity upstream of TSC in Drosophila[END_REF], to either promote cell growth or repress it. One well studied regulator of mTORC1 is amino acid concentration, reflecting the level of nutrient availability in the cell. When amino acid concentration is high, mTORC1 is recruited to the lysosomal membrane, where it is activated. This activation then triggers a transcriptional cascade leading to cell growth (Bar-Peled and Sabatini, 2014). The V-ATPase was shown to be required for mTORC1 recruitment to lysosomal membrane, through interactions dependent on amino acid levels within the lysosome lumen [START_REF] Zoncu | mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H+-ATPase[END_REF][START_REF] Bar-Peled | Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1[END_REF]. As of now, it is not clear if the amino acid concentration directly acts on the proton pump to regulate mTORC1 activation, or if the V-ATPase is just one upstream piece to mTORC1 in this sensing pathway. In the same line of idea, the V-ATPase seems to be also involved in lysosomal recruitment of mTORC1 and its subsequent activation in response to glucose availability [START_REF] Leprivier | How does mTOR sense glucose starvation? AMPK is the usual suspect[END_REF].

V-ATPases have also been proposed to play a part in the vesicle fusion machinery along with SNARE proteins. As this mechanism has mainly been investigated for synaptic vesicles or granule exocytosis in various organisms, it is discussed in section 1.5., which is dedicated to neuronal V-ATPases.

V-ATPase regulation

The ubiquitous nature of the V-ATPase, and the variety of processes and organelles requiring it, imply the existence of tight regulations in order to adapt the general machinery to specific needs. Indeed, the complex first needs to be targeted to the right cellular compartments, and then to fit the specific pH range of each organelle. In addition, even within one organelle, changes in activity level might be necessary depending on the cellular context. We can then separate the regulations of the V-ATPase in two categories:

spatial regulation, to define its localization.

activity modulation, to adapt acidification level to organelle type and environmental clues.

Isoform composition

As previously seen in section 1.1., different subunit isoforms can be used to form a complete V-ATPase. This variety of combination has been shown to influence both localization and efficiency of the complex.

In yeast, each subunit is coded only by one gene, with the exception of subunit a of V 0 coded by two genes. The two isoforms coding for V 0 subunit a, Vph1p and Stv1p [START_REF] Manolson | The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H (+)-ATPase[END_REF]Manolson et al., 1994), are able to regulate the complex localization. Both contain targeting sequence in their N-terminal addressing them to different cellular compartments: Stv1p-containing V-ATPases end up in the Golgi, while

Vph1p-containing V-ATPases are sent to vacuoles (Kawasaki-Nishi et al., 2001a).

This difference of composition does not only affect localization of the complex but also its efficiency, as it was shown that Vph1p-containing V-ATPases transport 4-5 times more protons for the same amount of ATP hydrolysis than Stv1p-containing V-ATPases (Kawasaki- Nishi et al., 2001b). The V 0 a-subunit is a key piece of the proton pump as it is believed to be the actual port allowing protons to cross the membrane, and a possible explanation for this difference of efficiency between the two isoforms could be found in the molecular composition of the cytosolic half-channel. Vph1p has reportedly a negative surface charge at the entrance of the half-channel, increasing local concentration of positively charged protons, while Stv1p presents a positive surface charge, thus potentially decreasing proton local concentration [START_REF] Vasanthakumar | Structural comparison of the vacuolar and Golgi V-ATPases from Saccharomyces cerevisiae[END_REF]. The superior efficiency of Vhp1p-containing V-ATPases is consistent with the higher acidity level required in vacuoles, where it is targeted, compared to the less acidic Golgi where Stv1p-containing V-ATPases are located.

In higher eukaryotes, several subunits are coded by multiple genes. Some subunit isoforms are expressed in an ubiquitous manner, while others present specific tissue distribution. For example, in mice, C1, d1 and G1 do not show tissue specificity, but C2, d2 and G3 are enriched in kidney (Sun-Wada et ak., 2003;[START_REF] Smith | Vacuolar H+-ATPase d2 subunit: molecular characterization, developmental regulation, and localization to specialized proton pumps in kidney and bone[END_REF]. Tissue specificity seems to be conserved between different species since C2, d2 and G3 have also been mainly found in human kidney [START_REF] Smith | Molecular cloning and characterization of novel tissue-specific isoforms of the human vacuolar H+-ATPase C, G and d subunits, and their evaluation in autosomal recessive distal renal tubular acidosis[END_REF][START_REF] Nishi | Expression and function of the mouse V-ATPase d subunit isoforms[END_REF]. Other subunits, like a1 and G2, show specificity for the brain [START_REF] Murata | Differential localization of the vacuolar H+ pump with G subunit isoforms (G1 and G2) in mouse neurons[END_REF], while a3 is enriched in osteoclasts [START_REF] Toyomura | Three subunit a isoforms of mouse vacuolar H+-ATPase: preferential expression of the a3 isoform during osteoclast differentiation[END_REF].

Within one cell type, different subunit isoforms can also show preferential localization to certain organelles. Like in yeast, the intracellular targeting seems to be controlled by the a-subunit. For example, the a1 subunit, enriched in the brain, is specifically targeted to synapses in Torpedo neurons (Morel et al., 2003). In mice tubule epithelial cells, a2-containing V-ATPases are targeted to early endosomes, while the a4 isoform localizes mainly on the plasma membrane [START_REF] Oka | a4, a unique kidney-specific isoform of mouse vacuolar H+-ATPase subunit a[END_REF][START_REF] Hurtado-Lorenzo | V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway[END_REF].

Regulated assembly of V 0 and V 1

The two domains, membrane-associated V 0 and cytosolic V 1 , of the V-ATPase can assemble and disassemble in a reversible manner. Following separation of the two domains, ATP hydrolysis by V 1 and proton translocation by V 0 are both blocked, rendering the complex inactive (Figure A8). Dissociation of V 1 from V 0 is accompanied by the release of the C subunit from the cytosolic domain, and a change of conformation of subunit H necessary for inhibition of ATP hydrolysis [START_REF] Parra | The H subunit (Vma13p) of the yeast V-ATPase inhibits the ATPase activity of cytosolic V1 complexes[END_REF][START_REF] Diab | Subunit interactions and requirements for inhibition of the yeast V1-ATPase[END_REF] could also be regulated by reversible assembly came from dendritic cells, which are professional antigen-presenting cells capable of activating T cells of the immune system.

Dendritic cells first need to go through a maturation process to present antigen on their surface, and it was shown that a required step of this evolution was a drop in lysosomal pH. This higher acidification of lysosomes was attributed to an increase in V-ATPase assembly in mature dendritic cells compared to immature ones [START_REF] Trombetta | Activation of lysosomal function during dendritic cell maturation[END_REF].

Given existing results on yeast, another good candidate for regulation of V-ATPase assembly in mammals was glucose. In line with observation in yeast, increase of glucose concentration was found to increase V-ATPase assembly on plasma membrane of pig and human proximal tubular cell lines [START_REF] Nakamura | Glucose activates H+-ATPase in kidney epithelial cells[END_REF]Sautin et al., 2005).

Other stimuli have been found to regulate V-ATPase assembly, like serotonin in insects [START_REF] Rein | Fluorescence measurements of serotonin-induced V-ATPase-dependent pH changes at the luminal surface in salivary glands of the blowfly Calliphora vicina[END_REF][START_REF] Rein | Hormone-induced assembly and activation of V-ATPase in blowfly salivary glands is mediated by protein kinase A[END_REF], osmotic stress in yeast [START_REF] Li | Vacuolar H+-ATPase works in parallel with the HOG pathway to adapt Saccharomyces cerevisiae cells to osmotic stress[END_REF], or high palmitate in mammals [START_REF] Liu | Palmitate-induced vacuolar-type H+-ATPase inhibition feeds forward into insulin resistance and contractile dysfunction[END_REF].

Assembly factors and accessory proteins 1.3.3.1. Assembly factors

The process of V-ATPase assembly was mainly investigated in yeast, where screening for the Vma -phenotype, i.e. the unability to grow on neutral pH typical of V-ATPase loss-of-function, has revealed three proteins that were not found in the purified complex: Vma12p, Vma21p and Vma22p (Figure A9). Loss-of-function of any of these three proteins specifically affects assembly of the V-ATPase V 0 domain in the endoplasmic reticulum (ER) and not other protein complexes [START_REF] Graham | Structure and assembly of the yeast V-ATPase[END_REF].

Vma12p and Vma22p localization is restricted to the ER membrane. The two proteins work together, as a complex [START_REF] Graham | Assembly of the yeast vacuolar H+-ATPase occurs in the endoplasmic reticulum and requires a Vma12p/Vma22p assembly complex[END_REF][START_REF] Hill | Vma22p is a novel endoplasmic reticulum-associated protein required for assembly of the yeast vacuolar H+-ATPase complex[END_REF] that associates transiently to the a subunit of the V-ATPase. Loss of Vma12p, which is a membrane protein, disrupts membrane localization of Vma22p, that has no transmembrane domain, suggesting that Vma12p acts as an anchor for the complex [START_REF] Hill | Vma22p is a novel endoplasmic reticulum-associated protein required for assembly of the yeast vacuolar H+-ATPase complex[END_REF].

It might be a bit confusing to see Vma21p listed here as an assembly factor as Figure A9. The V 0 domain of yeast V-ATPase is assembled in the ER, with the assistance of assembly factors Vma12, Vma22 and Vma21. Vma12 and Vma22 act as a complex that stays in the ER, while Vma21 accompanies V 0 to the Golgi before being retrieved to the ER. (Adapted from Forgac, 2007).

Accessory proteins

In addition to the 14 core subunits constituting the V-ATPase complex, two other proteins have been identified in mammalian V-ATPases. These proteins were thus designated as "accessory subunits", and named ATP6AP1 and ATP6AP2 (AP for Accessory Protein). ATP6AP1 and ATP6AP2 are both believed to have regulating effects on the proton pump.

ATP6AP1, also called Ac45, is a highly glycosylated transmembrane protein that was first discovered in V-ATPases isolated from bovine chromaffin granules [START_REF] Supek | A novel accessory subunit for vacuolar H (+)-ATPase from chromaffin granules[END_REF], and which associates with the V 0 domain, on the luminal side (Figure A10) [START_REF] Supek | A novel accessory subunit for vacuolar H (+)-ATPase from chromaffin granules[END_REF][START_REF] Feng | Cytoplasmic terminus of vacuolar type proton pump accessory subunit Ac45 is required for proper interaction with V0 domain subunits and efficient osteoclastic bone resorption[END_REF]. Its expression is ubiquitous and it seems to be enriched in neuronal and neuroendocrine cells [START_REF] Supek | A novel accessory subunit for vacuolar H (+)-ATPase from chromaffin granules[END_REF][START_REF] Holthuis | Molecular probing of the secretory pathway in peptide hormone-producing cells[END_REF].

In multicellular organisms, mutations of V-ATPase subunits often lead to a lethal phenotype at various developmental stages, whether in mice (Inoue et al., 1999;[START_REF] Sun-Wada | Acidic endomembrane organelles are required for mouse postimplantation development[END_REF], C. elegans (Lee et al., 2010a), or flies (Davies et al., 1996;Allan et al., 2005). Similarly, ATP6AP1 knock-out was found embryonic lethal in mice (Schoonderwoert and Martens, 2002). Its precise function within the complex still remains unclear but its overexpression in Xenopus melanotrope cells was shown to affect subcellular localization of V-ATPases [START_REF] Jansen | Accessory subunit Ac45 controls the V-ATPase in the regulated secretory pathway[END_REF], as well as to increase granular acidification [START_REF] Jansen | V-ATPase-mediated granular acidification is regulated by the V-ATPase accessory subunit Ac45 in POMC-producing cells[END_REF]. In osteoclasts, where ATP6AP1 was also found to be abundantly expressed [START_REF] Xu | Structure and function of V-ATPases in osteoclasts: potential therapeutic targets for the treatment of osteolysis[END_REF], mutation of the accessory subunit, or expression of an RNAi against it, led to a decrease in bone resorption capacity of the cells [START_REF] Feng | Cytoplasmic terminus of vacuolar type proton pump accessory subunit Ac45 is required for proper interaction with V0 domain subunits and efficient osteoclastic bone resorption[END_REF][START_REF] Yang | Silencing of Ac45 Simultaneously Inhibits Osteoclast-Mediated Bone Resorption and Attenuates Dendritic Cell-Mediated Inflammation through Impairing Acidification and Cathepsin K Secretion[END_REF]. For a period of time, ATP6AP1 was believed to have no homolog in yeast. However, recent work has identified Voa1p, a yeast V-ATPase assembly factor, as a potential homolog for ATP6AP1 (Jansen et al., 2016). This result was consistent with a later on cryo-electron microscopic reconstruction of the yeast V-ATPase V 0 domain, which showed that Voa1p remains in the complex after assembly, in a luminal position like ATP6AP1 [START_REF] Roh | The 3.5-Å CryoEM structure of nanodisc-reconstituted yeast vacuolar ATPase Vo proton channel[END_REF].

ATP6AP2, also called (P)RR and M8-9, was also first identified in bovine chromaffin granules where it interacts with the V 0 domain [START_REF] Ludwig | Identification and characterization of a novel 9.2-kDa membrane sectorassociated protein of vacuolar proton-ATPase from chromaffin granules[END_REF]. Four years after the identification of ATP6AP2, the (pro)renin receptor ((P)RR) was discovered [START_REF] Nguyen | Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin[END_REF] as an important player in the renin-angiotensin system (RAS) regulating blood pressure and salt balance in mammals. A few years later, it was revealed that ATP6AP2 is in fact the C-terminal fragment of (P)RR [START_REF] Bader | Spotlight on Renin: The second life of the (Pro) renin receptor[END_REF].

Interestingly, ATP6AP2 has homologs in invertebrate species, which do not have a RAS, suggesting that the function of the protein as a (pro)renin receptor might have evolved later compared to its role in the V-ATPase complex. ATP6AP2 is ubiquitously expressed, but enriched in the nervous system and kidney [START_REF] Advani | The (Pro) renin receptor: site-specific and functional linkage to the vacuolar H+-ATPase in the kidney[END_REF][START_REF] Cruciat | Requirement of prorenin receptor and vacuolar H+-ATPasemediated acidification for Wnt signaling[END_REF].

Its knock-out was shown to be lethal in C. elegans and Zebrafish [START_REF] Zima | Prorenin Receptor Homologue VHA-20 is Critical for Intestinal pH Regulation, Ion and Water Management and Larval Development in C. elegans[END_REF][START_REF] Amsterdam | A large-scale insertional mutagenesis screen in zebrafish[END_REF], highlighting the importance of its function. The precise role of ATP6AP2 in relation to the V-ATPase is still under investigation, but several evidences point toward a role in V 0 assembly. In mice cardiomyocytes, loss of ATP6AP2 led to a defect of vesicular acidification coupled with a decrease in V 0 subunits specifically, while the V 1 domain did not seem affected [START_REF] Kinouchi | The (pro) renin receptor/ATP6AP2 is essential for vacuolar H+-ATPase assembly in murine cardiomyocytes[END_REF]. Recently, a study in Drosophila wing cells demonstrated that both gain-of-function and loss-of-function of ATP6AP2 led to ER dysfunction also believed to be attributed to a defect in V 0 assembly (Guida et al., 2018). Moreover, these authors were also able to rescue the Vma - phenotype, typical of V-ATPase loss of function in yeast, of the V-ATPase assembly factor Voa1p mutant by ectopic expression of Drosophila ATP6AP2. Interestingly, the rescue was even more efficient when ATP6AP1 was co-expressed with ATP6AP2, suggesting that the two proteins could work together in V 0 assembly in higher eukaryotes.

diseases (Figure 11). Here we list a few chosen examples of pathological conditions where the proton pump has been of special interest.

V-ATPases in cancer

Cancer research has shed light on several roles played by the complex in this very broad and heterogenic pathological group. V-ATPase dysregulation has been linked to several parameters of cancer progression, such as invasiveness, angiogenesis, proliferation, tumorigenesis, and drug resistance [START_REF] Lu | The expression of V-ATPase is associated with drug resistance and pathology of non-smallcell lung cancer[END_REF][START_REF] Sennoune | Plasmalemmal vacuolar H+-ATPases in angiogenesis, diabetes and cancer[END_REF][START_REF] Santos-Pereira | Emerging insights on the role of V-ATPase in human diseases: Therapeutic challenges and opportunities[END_REF]. Several aspects of V-ATPase regulation seem impacted in tumour cells. For example, V-ATPases are found on the plasma membrane of highly metastatic and invasive cancer cells, but not on weakly metastatic ones. In these very invasive cells, V-ATPases are believed to participate in the alkalination of the cytoplasm while acidifying the extracellular tumor microenvironment [START_REF] Sennoune | Vacuolar H+-ATPase in human breast cancer cells with distinct metastatic potential: distribution and functional activity[END_REF][START_REF] Hinton | Function of a subunit isoforms of the V-ATPase in pH homeostasis and in vitro invasion of MDA-MB231 human breast cancer cells[END_REF]. The aberrant low pH generated by the proton pumps helps cancer cells to digest the extracellular matrix, thus facilitating metastatic invasion [START_REF] Chung | The vacuolar-ATPase modulates matrix metalloproteinase isoforms in human pancreatic cancer[END_REF][START_REF] Cotter | Activity of plasma membrane V-ATPases is critical for the invasion of MDA-MB231 breast cancer cells[END_REF][START_REF] Kulshrestha | Vacuolar ATPase 'a2'isoform exhibits distinct cell surface accumulation and modulates matrix metalloproteinase activity in ovarian cancer[END_REF]. In addition to its shift in localization, several V-ATPase subunits are overexpressed in different types of cancer (Cotter et al., 2016;[START_REF] Xu | Expression and functional role of vacuolar H+-ATPase in human hepatocellular carcinoma[END_REF][START_REF] Santos-Pereira | Emerging insights on the role of V-ATPase in human diseases: Therapeutic challenges and opportunities[END_REF].

If the role of the V-ATPase probably varies between different cancer types, or even different cancer cells, it seems that the aberrant activity of the complex is promoting cancer progression. Because of that, targeted V-ATPase inhibition could be a promising strategy for therapeutic development [START_REF] Pérez-Sayáns | V-ATPase inhibitors and implication in cancer treatment[END_REF]Whitton et al., 2018).

Viral infection

To infect a host, a virus must first penetrate its cells and release its genetic material into the cytoplasm. For some viruses, such as influenza virus or the Dengue virus, this internalization is achieved through endocytosis [START_REF] Dou | Influenza A virus cell entry, replication, virion assembly and movement[END_REF][START_REF] Ho | Blockade of dengue virus infection and viral cytotoxicity in neuronal cells in vitro and in vivo by targeting endocytic pathways[END_REF]. Once in endosomes, the low pH provided by V-ATPase activity facilitates the fusion of viral particles with endosomal membrane and the subsequent release of viral genetic material in the cytoplasm [START_REF] Mercer | Virus entry by endocytosis[END_REF]. Thus, V-ATPase acidification of the endocytic pathway plays a crucial part in some viral invasion, and many genes coding V-ATPase subunits have been identified in screening for essential host factors of viral infection [START_REF] Hao | Drosophila RNAi screen identifies host genes important for influenza virus replication[END_REF][START_REF] Karlas | Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication[END_REF][START_REF] König | Human host factors required for influenza virus replication[END_REF][START_REF] Chin | A genome wide RNA interference screening method to identify host factors that modulate influenza A virus replication[END_REF].

V-ATPases are therefore considered potential therapeutic targets against several viruses. Targeting the host proton pump allows persistence of the treatment effect despite frequent virus mutations. If V-ATPase inhibitors show promising results in vitro [START_REF] Guinea | Requirement for vacuolar proton-ATPase activity during entry of influenza virus into cells[END_REF][START_REF] Tscherne | Time-and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry[END_REF][START_REF] Chen | Inhibitory and combinatorial effect of diphyllin, a v-ATPase blocker, on influenza viruses[END_REF], the translation to in vivo use is complicated by the toxic side effects of such compounds [START_REF] Manabe | Inhibitors of vacuolartype H+-ATPase suppresses proliferation of cultured cells[END_REF][START_REF] Pérez-Sayáns | V-ATPase inhibitors and implication in cancer treatment[END_REF].

Mutations of tissue-specific isoforms

As described previously, several human V-ATPase subunits have multiple isoforms showing tissue distribution, and organelle, specificity. If a complete loss-offunction of a ubiquitous subunit is very likely to result in early developmental death, mutations of specific isoforms have been identified in several diseases with various clinical features.

Mutations of ATP6V0A2 are responsible for autosomal recessive cutis laxa type II A (ARCL-IIA), a rare disease characterized by skin laxity (hypo-elastic hanging skin), skeletal alterations and variable neurological defect, like mental retardation [START_REF] Berk | Cutis laxa: a review[END_REF]. Several type of mutations have been identified in ARCL-IIA patients, including missense, nonsense and frame-shift, but they are all believed to result in a loss-of-function of the protein, leading to impaired vesicular trafficking [START_REF] Kornak | Impaired glycosylation and cutis laxa caused by mutations in the vesicular H+-ATPase subunit ATP6V0A2[END_REF][START_REF] Hucthagowder | Loss-of-function mutations in ATP6V0A2 impair vesicular trafficking, tropoelastin secretion and cell survival[END_REF][START_REF] Esmail | Molecular mechanisms of cutis laxa-and distal renal tubular acidosis-causing mutations in V-ATPase a subunits, ATP6V0A2 and ATP6V0A4[END_REF]. There is no existing cure for this disease at the moment.

Mutations of another isoform of V 0 subunit a, ATP6V0A3 which is enriched in osteaoclasts, are the primary cause of infantile malignant autosomal recessive osteopetrosis [START_REF] Frattini | Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis[END_REF][START_REF] Susani | TCIRG1-dependent recessive osteopetrosis: mutation analysis, functional identification of the splicing defects, and in vitro rescue by U1 snRNA[END_REF]. In this rare congenital disease, the inability of osteoclasts to properly function leads to an imbalance between bone resorption and bone formation, resulting in excessive bone density and diminished bone strength, among other symptoms [START_REF] Wilson | Autosomal recessive osteopetrosis: diagnosis, management, and outcome[END_REF]. When diagnosed early, the disease responds well to stem cell transplantation restoring bone resorption, but this treatment is only used on the most severe cases given the dangerousness of the procedure [START_REF] Teti | Osteopetroses, emphasizing potential approaches to treatment[END_REF]. Hyper-activity of osteoclasts produces the opposite phenotype, called osteoporosis, with low bone density and deterioration of bone tissue, increasing the risk of fracture. Interestingly, mutations in ATP6V1G1 have been linked to this class of disease [START_REF] Tan | Bivariate genome-wide association study implicates ATP6V1G1 as a novel pleiotropic locus underlying osteoporosis and age at menarche[END_REF], suggesting here a gain-of-function of the protein. Inhibitors of V-ATPase isoforms specific of osteoclasts have been successfully tested in several models against osteoporosis (Visentin et al., 2000;[START_REF] Ostrov | Identification of enoxacin as an inhibitor of osteoclast formation and bone resorption by structure-based virtual screening[END_REF][START_REF] Kartner | Inhibition of osteoclast bone resorption by disrupting vacuolar H+-ATPase a3-B2 subunit interaction[END_REF][START_REF] Crasto | Luteolin inhibition of V-ATPase a3-d2 interaction decreases osteoclast resorptive activity[END_REF], but more investigation will be needed to evaluate the potential secondary effects of such compounds and develop viable therapeutic strategies [START_REF] Duan | V-ATPases and osteoclasts: ambiguous future of V-ATPases inhibitors in osteoporosis[END_REF].

Distal renal tubular acidosis (DRTA) is an autosomal recessive disorder, characterized by the failure of kidney to excrete excess acids, resulting in metabolic acidosis (acid-base imbalance of the body) and impaired urine acidification. It is caused by mutations in two V-ATPase isoforms enriched in kidney: ATP6V0A4 and ATP6V1B1 [START_REF] Smith | Mutations in ATP6N1B, encoding a new kidney vacuolar proton pump 116-kD subunit, cause recessive distal renal tubular acidosis with preserved hearing[END_REF][START_REF] Stover | Novel ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss[END_REF]. Current treatment consists in alkali administration, to fight metabolic acidosis. Investigation of the pathogenic mechanism revealed that mutations in ATP6V0A4 lead to its retention in the ER, blocking its targeting to the plasma membrane in intercalated cells [START_REF] Esmail | Molecular mechanisms of cutis laxa-and distal renal tubular acidosis-causing mutations in V-ATPase a subunits, ATP6V0A2 and ATP6V0A4[END_REF], thus preventing proper urine acidification. Mutations in ATP6V1B1 impaired V-ATPase trafficking, as well as its assembly [START_REF] Yang | Vacuolar H+-ATPase B1 subunit mutations that cause inherited distal renal tubular acidosis affect proton pump assembly and trafficking in inner medullary collecting duct cells[END_REF].

V-ATPase dysfunction has also been connected to several neuronal pathologies.

The pathogenic processes underlying V-ATPase implication in such disorders is detailed in section 1.5.

V-ATPases in neurons 1.5.1. Neuronal V-ATPase functions

In neurons, like in other cell types, V-ATPases are essential to the endocytic pathway and the secretory pathway. Alongside the endocytic pathway, neuronal V-ATPases have been of special interest in autophagy, while in the secretory pathway, they play a particularly important role in synaptic transmission through synaptic vesicle acidification.

Autophagy

As post-mitotic cells, neurons need to efficiently deal with cellular stress in order to survive. One major system for this is autophagy, from the ancient Greek autóphagos meaning "self-devouring". Autophagy is a degrading and recycling pathway, where misfolded proteins and damaged organelles are broken down into reusable pieces within the acidic lysosomes. Autophagic dysfunction has been observed in several neuropathologies, such as Parkinson Disease or Alzheimer disease, and is believed to play a crucial part in the pathogenesis ultimately leading to neurodegeneration [START_REF] Nixon | Autophagy failure in Alzheimer's disease-locating the primary defect[END_REF][START_REF] Lynch-Day | The role of autophagy in Parkinson's disease[END_REF]. As detailed previously, V-ATPase activity is required for proper lysosomal degradation, making it a key component of neuronal stress response and survival (Colacurcio and Nixon, 2016).

Secretory pathway

Neurons are specialized in communicating with each other, and most of this communication is chemical, making them experts of the secretory pathway. To release neurotransmitters and activate postsynaptic receptors, neurons must first pack

The membrane-bound domain V 0 of the V-ATPase has been proposed to play a part in membrane fusion during exocytosis, independently of its proton transport function. This theory spawned a series of studies, sometimes contradicting each other.

The widely accepted model for membrane fusion consists in SNAREs (soluble NSF-attachment protein receptors) forcing membranes into close proximity. It was demonstrated that SNAREs alone are sufficient to induce fusion in an in vitro system, but in a much longer timelapse (hours) than what is observed in physiological conditions (seconds or minutes) (Weber et al., 1998). This suggests that, while SNAREs are the minimum requirement for membrane fusion, other proteins probably participate to this process in neurons or secretory cells.

The premises of the idea of a role of V 0 in membrane fusion can be traced back to the discovery of a protein port in Torpedo presynaptic membrane capable of releasing acetylcholine [START_REF] Israël | Purification of a presynaptic membrane protein that mediates a calcium-dependent translocation of acetylcholine[END_REF]. This complex, made of several subunits was named the "mediatophore". A couple of years later, it was revealed that the mediatophore was no other than the proteolipid ring of the V-ATPase [START_REF] Birman | A 15 kDa proteolipid found in mediatophore preparations from Torpedo electric organ presents high sequence homology with the bovine chromaffin granule protonophore[END_REF]). An interesting finding giving that acetylcholine is much larger than protons, which suggested that V 0 could mediate transport of bigger molecules than expected, potentially through radial enlargement, as was confirmed in more recent studies [START_REF] Couoh-Cardel | Yeast V-ATPase proteolipid ring acts as a large-conductance transmembrane protein pore[END_REF]. This result then fuelled another theory: what if the port formed by V 0 was part of the membrane fusion process? This hypothesis was the centre of a study on yeast V-ATPase that showed that the membrane-domain could form trans-complexes, i.e. association between adjacent membranes, downstream SNARE pairing [START_REF] Peters | Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion[END_REF]. They hypothesised that these V 0 trans-complexes facilitate membrane fusion. Thus, and it is important to stress this point, V 0 would not be strictly necessary to membrane fusion but rather accelerate the kinetic of this process, consistent with previous observations. This study was met with scepticism, in particular after another paper showed that yeast mutant of the c subunit, vma3Δ, did not present vacuole fragmentation as what would be expected if fusion was impaired and as what was observed in ypt7Δ cells, a mutant for a Rab GTPase known to be required for vacuole fusion [START_REF] Graham | Structure and assembly of the yeast V-ATPase[END_REF]. However, around the same time, several other publications presented evidence that mutation in several V-ATPase subunits indeed resulted in vacuole fragmentation [START_REF] Seeley | Genomic analysis of homotypic vacuole fusion[END_REF][START_REF] Perzov | Characterization of yeast V-ATPase mutants lacking Vph1p or Stv1p and the effect on endocytosis[END_REF][START_REF] Bayer | Vacuole membrane fusion: V0 functions after trans-SNARE pairing and is coupled to the Ca2+-releasing channel[END_REF]. The debate also moved to higher eukaryotes, and more particularly to a cell type where exocytosis has been of special interest for a long time: the neurons.

In Drosophila, mutation of the neuronal specific subunit a1 resulted in decreased amplitude release, associated with an accumulation of synaptic vesicles, suggesting that the probability of release was decreased (Hiesinger et al., 2005). In addition, a1 was shown to interact with two SNARE proteins. Overall, the results suggested an action of V 0 in exocytosis downstream SNARE pairing. A few years after, a study on cortical slices and cultured neurons presented evidence of an interaction between the c subunit of V 0 and the SNARE protein VAMP2 (synaptobrevin). Disruption of this interaction also led to a decrease of the release probability, without affecting acidification of synaptic vesicles [START_REF] Di Giovanni | V-ATPase membrane sector associates with synaptobrevin to modulate neurotransmitter release[END_REF]. More recently, a publication revealed that V 0 and V 1 interact in a pH-dependent manner in secretory granules, controlling exocytosis [START_REF] Poëa-Guyon | The V-ATPase membrane domain is a sensor of granular pH that controls the exocytotic machinery[END_REF]. The same result was also demonstrated in hippocampal neurons, where high pH promoted assembly of V 0 and V 1 , while low pH increased disassembly [START_REF] Bodzęta | The presynaptic v-ATPase reversibly disassembles and thereby modulates exocytosis but is not part of the fusion machinery[END_REF]. One consequence of this is that V 1 dissociates from V 0 before exocytosis, and blocking this event actually decreases the release probability (Figure A13). Interestingly, blocked disassembly does not seem to abrogate exocytosis but rather to slow it down.

When given enough time, vesicles with full V-ATPase (associated V 0 and V 1 ) are still able to dock, which led the authors to conclude that dissociation of the two domains regulates exocytosis upstream of SNARE pairing. The fact that blocking dissociation of V 1 and V 0 only decreases exocytosis, instead of completely abrogating it, and that this regulation is believe to happen upstream of SNARE docking, instead of downstream,

were the two points used to justify the bold following conclusion: V 0 is "not part of the fusion machinery" (quote from the publication's title), a statement contradicting the previous studies. It is not clear how dissociation of the two domains upstream to docking necessary implies the impossibility for V 0 to additionally facilitate membrane fusion downstream of docking, as it seems to us that these are two distinct modes of regulation of the same pathway.

In conclusion, although the role of V 0 in exocytosis still remains unclear, it seems that all studies agree that the V-ATPase plays a part in the regulation of synaptic vesicle dynamic during neurotransmitter release.

Indirect V-ATPase dysregulation

Alzheimer disease (AD) is the most prevalent neurodegenerative disease, usually manifesting in people over 60 years old. A small percentage of the cases, however, present an early onset of the disease. Patients affected by this pathology suffer from a progressive deterioration of their cognitive functions, such as memory. Common lateonset AD is believed to be of multifactorial origin, with interaction of both environmental and genetic causes. Early-onset AD, also called the familial form, is caused by mutations in one of three main identified genes: Presenilin-1 (PS1), Presenilin-2 (PS2), and amyloid precursor protein (APP) [START_REF] Tanzi | The gene defects responsible for familial Alzheimer's disease[END_REF]. Its hallmark symptoms include intraneuronal aggregates of tau protein, called neurofibrillary tangles, and patches of extracellular βamyloid (Aβ) associated with dystrophic neurites, called neuritic plaques [START_REF] Querfurth | Alzheimer's disease[END_REF]. Together with PS2, PS1 forms the catalytic subunit of γ-secretase, an enzymatic complex responsible for processing many substrates, including APP. Cleavage of APP by γ-secretase produces Aβ [START_REF] Zhang | Biological function of Presenilin and its role in AD pathogenesis[END_REF]. Mutations of PS1 are believed to alter this process, leading to the formation of neuritic plaques, even if the molecular mechanism is still debated [START_REF] Kelleher | Presenilin-1 mutations and Alzheimer's disease[END_REF]. However, other hypotheses argue for a role of PS1 in the pathology independent of γ-secretase, notably to explain the defect in autophagy and lysosomal degradation observed in PS1 mutant neurons [START_REF] Orr | Autophagic/lysosomal dysfunction in Alzheimer's disease[END_REF]. One of these hyptheses proposes that PS1 acts as a chaperone to facilitate the traffic of the V-ATPase to lysosomes [START_REF] Lee | Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations[END_REF]Williamson and Hiesinger, 2010;[START_REF] Coffey | Lysosomal alkalization and dysfunction in human fibroblasts with the Alzheimer's disease-linked presenilin 1 A246E mutation can be reversed with cAMP[END_REF].

Another disease where the V-ATPase is suspected to be indirectly dysregulated is the Wolfram syndrome, a childhood disorder associated with diabetes, motor and intellectual disability, optic atrophy, deafness and neurodegeneration. Various treatments are available to alleviate some of the symptoms, but there is no existing cure for this pathology which often leads to premature death around the age of 30 years old [START_REF] Urano | Wolfram syndrome: diagnosis, management, and treatment[END_REF]. The disease is caused by mutations in WFS1, the gene coding for Wolframin, an ER-bound protein [START_REF] Rigoli | Wolfram syndrome 1 and Wolfram syndrome 2[END_REF]. Wolframin is necessary for stabilizing subunit A of the V-ATPase, and its loss-of-function results in a decreased protein level of this subunit, affecting compartment acidification, which could be one aspect of the pathogenesis [START_REF] Gharanei | Vacuolar-type H+-ATPase V1A subunit is a molecular partner of Wolfram syndrome 1 (WFS1) protein, which regulates its expression and stability[END_REF]. How WFS1 mutations impairs neuronal V-ATPase function still remains to be determined.

Direct V-ATPase mutation

Among the pathologies presented in section 1.4., caused by mutations of V-ATPase subunits, several include neurological defects. Osteopetrosis is associated with visual impairment [START_REF] Keith | Retinal atrophy in osteopetrosis[END_REF], while ARCL-II patients are susceptible to mental retardation, visual impairment and seizures [START_REF] Morava | Defining the phenotype in an autosomal recessive cutis laxa syndrome with a combined congenital defect of glycosylation[END_REF][START_REF] Guillard | Vacuolar H+-ATPase meets glycosylation in patients with cutis laxa[END_REF].

About one third of people suffering from autosomal recessive DRTA experience a progressive and irreversible hearing loss [START_REF] Stover | Novel ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss[END_REF][START_REF] Fry | Inherited renal acidoses[END_REF].

Interestingly, the V-ATPase isoforms mutated in this disease, ATP6V1B1 and ATP6V0A4, known to be enriched in the kidney, were also detected in the human inner ear, potentially explaining the progressive deafness [START_REF] Karet | Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness[END_REF][START_REF] Stover | Novel ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss[END_REF].

Mutations of brain specific isoform B2 of the V 1 domain are involved in two genetic disorders: the dominant deafness-onychodystrophy syndrome (DDOD) and the Zimmermann-Laband syndrome (ZL) [START_REF] Yuan | De novo mutation in ATP6V1B2 impairs lysosome acidification and causes dominant deafnessonychodystrophy syndrome[END_REF][START_REF] Kortüm | Mutations in KCNH1 and ATP6V1B2 cause Zimmermann-Laband syndrome[END_REF][START_REF] Menendez | Dominant deafness-onychodystrophy syndrome caused by an ATP6V1B2 mutation[END_REF]. Both are rare autosomal dominant pathologies, characterized by sensory deafness associated with onychodystrophy, i.e. abnormalities in nail morphology.

In addition, ZL patients present intellectual disabilities and seizures. In DDOD, the deafness is congenital and believed to be cause by lysosomal dysfunction, due to loss of acidification, leading to neurodevelopmental defects of the inner ear [START_REF] Yuan | De novo mutation in ATP6V1B2 impairs lysosome acidification and causes dominant deafnessonychodystrophy syndrome[END_REF].

Mutations in the accessory subunit ATP6AP2 have been identified in two severe congenital neurologic disorders: X-linked mental retardation and epilepsy, and X-linked

Parkinson disease with spasticity.

X-linked mental retardation and epilepsy (XMRE) was diagnosed in seven males of an American family, all suffering from seizures and mental retardation [START_REF] Hedera | Novel mental retardationepilepsy syndrome linked to Xp21. 1-p11. 4[END_REF]. They did not exhibit parkinsonism symptoms, except for two of them demonstrating mild rigidity. The gene responsible for this trouble was not identified right away, but a few years later. A silent mutation in ATP6AP2, resulting in the absence of exon 4 in about 50% of RNA transcripts, was found to be the culprit [START_REF] Ramser | A unique exonic splice enhancer mutation in a family with X-linked mental retardation and epilepsy points to a novel role of the renin receptor[END_REF].

For unknown reasons, the role of ATP6AP2 in V-ATPase activity was simply ignored in this study, which solely focused on the effect of this mutation on the RAS. The mutated receptor exhibited no difference in its capacity to increase renin catalytic activity, compared to the wild-type one, but a modest decrease in ERK1/2 activation. Since neuronal knockdown of ATP6AP2 has since been shown to induce cognitive impairment and parkinsonism, associated with defective autophagy, in Drosophila (which does not have a RAS) and mice (Dubos et al., 2015), it is likely that the symptoms observed in XMRE patients could result from V-ATPase dysfunction as well.

X-linked Parkinson disease with spasticity (XPDS) was first identified in three males of an American family that displayed typical parkinsonism symptoms such as bradykinesia (slowness of movement), tremors and rigidity [START_REF] Poorkaj | A novel X-linked four-repeat tauopathy with Parkinsonism and spasticity[END_REF]. The degree of penetrance of each symptom varied between the three subjects, but none of them exhibited any sign of intellectual disability. The pathology was later pinpointed to a mutation in the ATP6AP2 gene by the same group (Korvatska et al., 2013). More precisely, the mutation affected the splicing of the gene, leading to a predicted 50% of proteins with an internal deletion of 32 amino acids due to the absence of exon 4 in the transcripts. This mutation sounds familiar as it is similar to the one described in XMRE patients! Technically, the mutation is not the same between the two families (c.321C>T

in XMRE, and c.345C>T in XPDS), however the end result on the protein structure seems to be identical. A more recent study actually analyzed yet another mutation of ATP6AP2, this time in exon2, in two brothers [START_REF] Gupta | A splice site mutation in ATP6AP2 causes X-linked intellectual disability, epilepsy, and parkinsonism[END_REF]. Interestingly, one sibling displayed parkinsonism symptoms, like tremor, bradykinesia and rigidity, with no intellectual disability, while the other sibling presented psychomotor delay, mental retardation and epilepsy. This result highlights how one identical mutation of the accessory subunit can lead to various phenotypes in different individuals. The authors concluded by proposing that XMRE and XPDS could actually be two manifestations of the same pathology.

The key role of the V-ATPase, in health as well as in several pathological contexts, is of great interest. The complexity of the pump itself, and of the regulations necessary to finely tune its activity in different cellular environments, make it a fascinating, and somewhat endless, subject of investigation, which could greatly benefit from the use of relatively simple models. Because of the high level of conservation of the V-ATPase across species, from yeast to mammals, much can be learnt from different organisms. In the next section of the manuscript, we describe the Drosophila model, that has contributed significantly to V-ATPase studies, and that was used in our project.

The Drosophila model

Drosophila melanogaster, commonly called the fruit fly, migrated from Sub-Saharan Africa about 15,000 years ago [START_REF] Arguello | Demographic history of the human commensal Drosophila melanogaster[END_REF]. It now inhabits all continents except Antarctica, its most recent conquests being Australia and the Americas (about 200 years ago), where its career as a research model debuted in the early 20 th century. It was popularized by the success of Thomas Hunt Morgan who used it to confirm the chromosome theory, thus demonstrating the basis of heredity [START_REF] Morgan | Sex limited inheritance in Drosophila[END_REF][START_REF] Morgan | The origin of five mutations in eye color in Drosophila and their modes of inheritance[END_REF]. Since then, the fruit fly has contributed to the advance of many fundamental research questions, such as the genetic control of embryonic development [START_REF] Lewis | A gene complex controlling segmentation in Drosophila[END_REF][START_REF] Nüsslein-Volhard | Mutations affecting segment number and polarity in Drosophila[END_REF], the molecular mechanisms of circadian rhythms [START_REF] Hardin | Feedback of the Drosophila period gene product on circadian cycling of its messenger RNA levels[END_REF]Vosshall et al., 1994), or the organization of the olfactory system [START_REF] Buck | A novel multigene family may encode odorant receptors: a molecular basis for odor recognition[END_REF].

The life of a Drosophila

Drosophila owes its success in science to the many advantages it offers, the first one being its very small size. An adult measures about 3 mm long, thus allowing laboratories to keep thousands of individuals with various genotypes in an incubator no bigger than a wardrobe. Fruit flies also have a short longevity compared to murine models, as they usually live between 50 and 70 days at 25°C in laboratory conditions, against 2-3 years for mice and rats, which facilitates aging studies.

Drosophila embryogenesis lasts about 24 hours, after which small larvae hatch.

They feed and grow for about 4 days until they have stored enough energy to start their metamorphosis. For that, they will form a cocoon, called the pupa, creating a sealed environment in which their whole body will be completely remodelled to give rise to adult flies. This fascinating development cycle only takes 10 days in its entirety, from the laying of an embryo to the hatching of an adult fly, when happening at 25°C (Figure A14).

Once eclosed from the pupa, adult flies reach sexual maturity in a matter of hours. That, added to the fact that one single female can produce about one hundred eggs in the course

Genomic edition

Drosophila genes can be edited, either by deleting part of a sequence to create knock-out mutants, or by inserting a new sequence to build knock-in mutants. This was originally achieved with the help of transposable elements [START_REF] Rubin | Genetic transformation of Drosophila with transposable element vectors[END_REF].

However, one downside of this method was that the insertion site was unpredictable.

Other systems were designed to overcome this issue, like the PhiC31 integrase, which allowed the insertion of transgenes in chosen genomic sites [START_REF] Bischof | An optimized transgenesis system for Drosophila using germ-line-specific φC31 integrases[END_REF]. More recent advances have now made it possible to modify genes even more precisely. That is the case of zinc-finger nucleases [START_REF] Bibikova | Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases[END_REF], transcription activator-like effector (TALEN) [START_REF] Liu | Efficient and specific modifications of the Drosophila genome by means of an easy TALEN strategy[END_REF], and Cas9 nucleases associated to clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) [START_REF] Bassett | Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system[END_REF]. These nucleases can break DNA double-strand at a defined site, allowing either the removal of a portion of the sequence, or the insertion of a new sequence by homologous recombination using a repair template.

Expression systems

The genomic editing tools described in the previous part have been used to generate several expression systems allowing to very easily overexpress or knock-down any Drosophila genes, as well as to express ectopic proteins, in particular useful bioengineered compounds like fluorescent markers, calcium indicators, or pH probes.

Overexpression or ectopic expression are achieved by driving the transcription of the gene of interest sequence, while knock-downs generally rely on the expression of doublestranded RNA interference (RNAi) constructs directed against the gene of interest. Note that Dicer-2 (Dcr-2), a protein that can cleave double-stranded RNAs [START_REF] Bernstein | Role for a bidentate ribonuclease in the initiation step of RNA interference[END_REF], is sometimes co-expressed with RNAis to increase their efficiency.

The most commonly used expression method is the Gal4-UAS binary system, which allows the expression of a transgene in a specific tissue or cell type [START_REF] Brand | Targeted gene expression as a means of altering cell fates and generating dominant phenotypes[END_REF]. To do so, a first parent line contains in its genome the sequence of Gal4, Other expression systems similar to Gal4-UAS have been also developed such as the LexA-LexAop system. It works the same way as Gal4-UAS, with the LexA protein being expressed under the control of a tissue-specific promoter and activating the LexAop sequence, allowing the transcription of the downstream gene of interest. Since both systems are independent from one another, they can be used within the same fly to drive the expression of a first gene in a cell-type in parallel of the expression of a second gene in a different cell type [START_REF] Lai | Genetic mosaic with dual binary transcriptional systems in Drosophila[END_REF].

The Drosophila nervous system 2.3.1. Adult central nervous system

In adult Drosophila, the central nervous system (CNS) is composed of the brain, in the head, and thoracic ganglions forming the ventral nerve cord (VNC), located in the thorax, an equivalent to the spinal cord in mammals. The central brain is flanked by two lobe optics, one on each side, and presents several major structures: the antennal lobes receiving olfactory information, the mushroom bodies involved in learning and memory, and the central complex required for motor control (Figure A18) [START_REF] Wilson | Transformation of olfactory representations in the Drosophila antennal lobe[END_REF][START_REF] Akalal | Roles for Drosophila mushroom body neurons in olfactory learning and memory[END_REF][START_REF] Seelig | Feature detection and orientation tuning in the Drosophila central complex[END_REF].

A model to study the brain

Because of the relatively small size of its brain, yet able to sustain a variety of complex behaviours, and its panoply of available genetic tools, Drosophila is used as a model to study various fundamental neuronal questions. Several neuronal networks have been deciphered, or are still being investigated, in the adult and larval brain, like the ones underlying feeding behaviour [START_REF] Zhou | Mechanosensory circuits coordinate two opposing motor actions in Drosophila feeding[END_REF], courtship behaviour [START_REF] Yamamoto | Genes and circuits of courtship behaviour in Drosophila males[END_REF], or memory and learning [START_REF] Boto | Cellular and circuit mechanisms of olfactory associative learning in Drosophila[END_REF][START_REF] Modi | The Drosophila mushroom body: from architecture to algorithm in a learning circuit[END_REF].

The fly is also a convenient model to study the molecular basis of aging because of its short lifespan and other advantages [START_REF] He | Studying aging in Drosophila[END_REF], as well as several neurodegenerative diseases. Mutations observed in genetic troubles can be re-created by mutating these genes fly homologs, to recapitulate pathological phenotypes. Parkinson disease (PD), a neurodegenerative disease characterized by protein aggregates called the Lewy bodies and a loss of dopaminergic neurons leading to locomotor impairment, can be modelized in flies, for example by mutating the homolog of human PTEN-induced putative kinase 1 (PINK1), a gene associated with early-onset cases. Drosophila PINK1 mutants showed dopaminergic degeneration and locomotor defect reminiscent of the human symptoms [START_REF] Park | Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin[END_REF].

Proteins known to play a role in a pathogenesis can also be ectopically expressed in Drosophila to decipher cellular consequences. This was done in another PD model, where expression of α-Synuclein led to locomotor decline, neuronal degeneration and the formation of cellular inclusions similar to Lewy bodies [START_REF] Feany | A Drosophila model of Parkinson's disease[END_REF][START_REF] Riemensperger | A single dopamine pathway underlies progressive locomotor deficits in a Drosophila model of Parkinson disease[END_REF]. Another approach to study neurotoxic mechanisms consists in exposing flies to certain toxins, notably pesticides such as rotenone or paraquat [START_REF] Coulom | Chronic exposure to rotenone models sporadic Parkinson's disease in Drosophila melanogaster[END_REF][START_REF] Cassar | A dopamine receptor contributes to paraquat-induced neurotoxicity in Drosophila[END_REF].

The Drosophila V-ATPases

Drosophila V-ATPase composition

To our knowledge, there is to date no crystallographic reconstruction of Drosophila V-ATPase. Since the structure is very conserved between organisms as far apart as mammals and yeast, the fly V-ATPase is believed to be no different, with a cytosolic domain V 1 made of subunits A 3 B 3 CDE 3 FG 3 H and membrane-bound domain V0 made of ac 9 c"de. Of note, Drosophila V 0 does not seem to have a c' subunit, being closer in that sense to mammals than yeast V 0 . But unlike mammals, almost all V-ATPase subunits in fly are coded by several genes (Table A2) [START_REF] Dow | The multifunctional Drosophila melanogaster V-ATPase is encoded by a multigene family[END_REF]. Like in mammals or yeast, there is no official f subunit in V 0 as of today. However, the specific protein identified in rat brain V-ATPase, RNAseK [START_REF] Perreira | RNASEK is a V-ATPase-associated factor required for endocytosis and the replication of rhinovirus, influenza A virus, and dengue virus[END_REF], does have a fly homolog.

The two mammalian accessory subunits, ATP6AP1 and ATP6AP2, also present homologs in Drosophila.

Table A2. List of human isoforms for all V-ATPase subunits, and their Drosophila homologs.

V-ATPase domain Subunit Human genes Drosophila genes

V 1 A ATP6V1A Vha68-1 Vha68-2 Vha68-3 V 1 B ATP6V1B1 ATP6V1B2 Vha55 V 1 C ATP6V1C1 ATP6V1C2 Vha44 V 1 D ATP6V1D Vha36-1 Vha36-2 Vha36-3 V 1 E ATP6V1E1 ATP6V1E2 Vha26 V 1 F ATP6V1F Vha14-1 Vha14-2 V 1 G ATP6V1G1 ATP6V1G2 ATP6V1G3 Vha13 V 1 H ATP6V1H VhaSFD V 0 a ATP6V0A1 ATP6V0A2 ATP6VOA3/TCIRG1 ATP6V0A4 Vha100-1 Vha100-2 Vha100-3 Vha100-4
Vha100-5

V 0 c ATP6V0C Vha16-1 Vha16-2 Vha16-3 Vha16-4
Vha16-5

V 0 c" ATP6V0B VhaPPA1-1 VhaPPA1-2 V 0 d ATP6V0D1 ATP6V0D2 VhaAC39-1 VhaAC39-2 V 0 e ATP6V0E1 ATP6V0E2

Drosophila V-ATPase functions

Like in other multicellular organisms, the Drosophila V-ATPase can be found of the plasma membrane of some specific cell types.

The first animal knock-out of a V-ATPase subunit has been described in Drosophila [START_REF] Gausz | Genetic characterization of the 87C region of the third chromosome of Drosophila melanogaster[END_REF]. At that time, the V-ATPase was not even officially named, and the authors had no idea that the observed mutations affected the proton pump.

It was only years later that another study mapped the identified locus to Vha55, the gene coding for subunit B of the V 1 domain (Davies et al., 1996). Mutations of Vha55 produced two phenotypes. The first one was a lethality at various early developmental stages, ranging from embryonic to 1 st instar larva depending on the mutation. The second phenotype was a transparent Malpighian tubule (an equivalent of vertebrate's kidney), unlike the luminal white observed in healthy larvae. V-ATPases are indeed found on the plasma membrane of active principal cells in Malpighian tubules, and participate in the energization of this epithelia and urine acidification [START_REF] Wieczorek | Vacuolar-type proton pumps in insect epithelia[END_REF]. This phenotype is thus believed to result from a failure in acidifying the tubule lumen, preventing the precipitation of uric acid which crystals normally give the luminal white appearance. If this Malpighian phenotype is indeed due to V-ATPase dysfunction, it should also appear when other subunits of the complex are mutated, providing that the right isoform is targeted. Consistent with that, mutations in isoforms of subunits A, D, E, F, G, H, c and e presented a clear luminal tubule, as well as a lethality at early development stages (Allan et al., 2005).

Drosophila V-ATPase is also responsible for the acidification of internal cellular compartments. Mutations or overexpression of V-ATPase subunits have been shown to impair endolysosomal acidification, leading to various developmental defects depending on the affected tissue. Examples include the formation of egg chambers (multicellular assemblies within ovaries, each giving rise to a single egg), eye and wing [START_REF] Yan | The vacuolar proton pump, V-ATPase, is required for notch signaling and endosomal trafficking in Drosophila[END_REF][START_REF] Buechling | Wnt/Frizzled signaling requires dPRR, the Drosophila homolog of the prorenin receptor[END_REF][START_REF] Hermle | Regulation of Frizzled-dependent planar polarity signaling by a V-ATPase subunit[END_REF][START_REF] Mo | Investigation of isoform specific functions of the V-ATPase a subunit during Drosophila wing development[END_REF].

One of the subunits with the highest number of isoforms is the proton port, subunit a of V 0 , with five different genes encoding it. The five isoforms are expressed in different tissues, and two of them, Vha100-3 and Vha100-5, are not necessary for fly development and survival [START_REF] Mo | Investigation of isoform specific functions of the V-ATPase a subunit during Drosophila wing development[END_REF]. The first isoform, Vha100-1, was shown to be only required in neurons (Hiesinger et al., 2005). Its mutation led to defects in the endolysosomal pathway (Williamson et al., 2010), like what has been observed with other subunits in various tissues. This impairment in cellular degradation processes was associated with neurodegeneration, similar to the role imputed to autophagy dysfunction in neurodegenerative diseases [START_REF] Nixon | The role of autophagy in neurodegenerative disease[END_REF]. In addition to these effects, mutation of Vha100-1 affected synaptic transmission by impairing exocytosis of synaptic vesicles.

Thesis Goal

The V-ATPase is a remarkable molecular motor, used in all eukaryotic cells to acidify several organelles as well as the extracellular milieu in multicellular organisms.

The complexity of its structure was overcome by previous discoveries made on other simpler rotary ATPases, and thanks to technological advances, allowing greater resolutions in electron microscopy reconstructions of the complex over the years. If several questions remain unanswered on the detailed interactions between each subunit within the complex, the core mechanism of the proton pump is now well documented.

Because of the multitude of pathways relying on pH-dependent processes, the V-ATPase is at the centre of a complex regulation web. Many parameters need to be integrated into the modulation of the proton pump like which organelle it is located on, or which cell type it is expressed in, in addition to the many cues transcribing the local context, such as the energy status. Several fundamental and conserved regulatory mechanisms have already been uncovered, owing to the contribution of many eukaryotic models, from yeast to mammals. It is now clear that isoform composition plays an important part into subcellular targeting of the V-ATPase, and in cell-type specificity in multicellular organisms. Reversible dissociation of the two domains, V 0 and V 1 , is also used to regulate V-ATPase activity, often in response to contextual cues. Nevertheless, the picture is far from being complete, and a lot remain to be understood about how the general V-ATPase machinery is uniquely adapted to fit every specific need.

The central position of the V-ATPase in many key cellular processes also makes it of special interest in numerous pathologies. Sometimes, pathogenesis involves an up or down-regulation of V-ATPase activity supporting disease progression, like in cancer cells. In other situations, mutations of V-ATPase subunits are directly at the origin of the pathology. In both cases, V-ATPases constitute an attractive therapeutic target. However, this strategy is limited by the secondary effects usually associated with compounds acting on V-ATPases. This problem could be overcome by increasing the specificity of medicines, so that they do not target all V-ATPases, but only the one located in the cell type and on the organelle of interest. To achieve such level of precision, increasing knowledge about V-ATPase specific regulations is necessary.

In neurons, like in other cell types, V-ATPases are involved in the endolysosomal and secretory pathways. In these post-mitotic cells, the proton pump's role in lysosomal acidification is of particular importance, as autophagy dysfunction has been implicated in several neuropathologies. In addition, the V-ATPase plays a critical part in synaptic transmission through the acidification of synaptic vesicles. This implies that V-ATPase must be under specific neuronal regulation to fulfil these functions, but also that V-ATPase activity itself might be a modulator of synaptic transmission.

We have chosen to investigate these questions in the Drosophila melanogaster organism. This model has already greatly contributed to a better understanding of V-ATPase functions in vivo. Its relatively small brain offers the advantage of being simpler than vertebrates models, while still being complex enough to sustain a variety of behaviours. In a previous study focusing on neuronal autophagy, we identified an uncharacterized Drosophila gene whose expression in neurons was required for fly's survival. This gene, that we originally named Lome, was predicted on sequence to belong to the V-ATPase family. Interestingly, several databases suggested that Lome expression was restricted to neurons, making it a potential candidate for specific regulation of neuronal V-ATPase. My PhD project aimed therefore at answering two questions:

-is Lome involved in V-ATPase activity, and if so, specifically in neurons?

-how the regulation of V-ATPase activity can influence synaptic transmission?

We believe that our results, described here, demonstrate that Lome, which we officially renamed VhaAC45Like (VhaAC45L) in regard to its closest homolog VhaAC45, is indeed a new and specific regulator of neuronal V-ATPase in Drosophila.

Because the V-ATPase is highly conserved across species, gaining new insights in Drosophila regulating mechanisms can help to paint a more complete picture of the complex modulations surrounding the proton pump, thus making one more step toward future use of the V-ATPase in therapeutic strategies. We propose to explore this possibility in the second part of the manuscript, in the context of epilepsy.

B. Results

Introduction

Many cellular processes require a specific electrochemical environment for proper functioning, such as post-translational modifications of proteins in the Golgi apparatus, lysosomal degradation, endosomal ligand-receptor dissociation, or hormone concentration (reviewed in Forgac, 2007). Eukaryotic cells use a highly conserved proton pump, called the vacuolar H + -ATPase (V-ATPase), to achieve the adequate level of acidity in different cellular compartments [START_REF] Saroussi | The little we know on the structure and machinery of V-ATPase[END_REF]. This large enzymatic complex must be tightly regulated, as it is essential for it to be localized on the right membrane, and to fit the different pH ranges specific to each organelle and cell type.

In neurons, the V-ATPase plays a crucial role at the synapse, being responsible for acidifying synaptic vesicles and thus providing the driving force for neurotransmitter loading (Moriyama et al., 1992). Recently, neuronal V-ATPase has also gained interest in the context of aging and neurodegenerative diseases, as its dysregulation, and resulting impairment of the autophagy-lysosomal pathway, have been linked to several pathologies such as Alzheimer and Parkinson disease (Colacurcio and Nixon, 2016;Collins and Forgac, 2020). If the core mechanism of the proton pump is now well understood, the regulations conferring the cell-specific functions of neuronal V-ATPase remain largely unknown, considerably limiting its potential use as a therapeutic target.

The V-ATPase complex is composed of a cytoplasmic domain (V 1 ) and a membranebound domain (V 0 ). The V 1 domain contains the catalytic unit responsible for ATP hydrolysis. The energy resulting from this reaction powers a rotational molecular motor spanning from V 1 to V 0 , allowing protons to cross membranes through the port contained in V 0 [START_REF] Vasanthakumar | Structure and Roles of V-type ATPases[END_REF]. The assembly of V 1 to V 0 is necessary for the pump to function, and reversible dissociation of the two domains has been shown to occur as a way to regulate V-ATPase activity (Collins and Forgac, 2020). Though the core mechanism stays the same, one V-ATPase can differ from another by its composition. In vertebrates, as well as in Drosophila, V 0 is made of five subunits (a, c, c'', d and e), while V 1 contains eight subunits (A, B, C, D, E, F, G and H) (Allan et al., 2005). Each subunit can be coded by several genes, and each gene can produce several isoforms, allowing many different possible combinations to form the full V-ATPase complex. These differences of composition can also have regulatory effects on the complex, both on its localization and on its functional properties [START_REF] Vasanthakumar | Structure and Roles of V-type ATPases[END_REF].

The V-ATPase can also be regulated by two accessory subunits, ATP6AP1/Ac45 and ATP6AP2/PRR [START_REF] Jansen | Novel insights into V-ATPase functioning: distinct roles for its accessory subunits ATP6AP1/Ac45 and ATP6AP2/(pro) renin receptor[END_REF]. Both proteins are found in the nervous tissue but are also required in other organs, and their mutation has been linked to cognitive impairments as well as systemic symptoms like immunodeficiency or hepatopathy (Jansen et al., 2016;[START_REF] Cannata Serio | Mutations in ATP6AP2 cause autophagic liver disease in humans[END_REF]. These two subunits interact directly with the V 0 domain and are believed to promote assembly of the membrane and soluble regions of the V-ATPase complex (Abbas et al., 2020). Another potential accessory subunit exists in vertebrates, named ATP6AP1-like (ATP6AP1L), which is homologous to ATP6AP1 and has not yet been studied. Drosophila possesses identified homologs of ATP6AP1/Ac45 and ATP6AP2/PRR, named VhaAC45 and ATP6AP2, respectively.

These proteins also seem to contribute to assembly of the V-ATPase in fly tissues (Schoonderwoert and Martens, 2002-a;Guida et al., 2018).

In this study, we examine the localization and function of another still uncharacterized ATP6AP1/Ac45 homolog in Drosophila, named VhaAC45L, whose characteristic is to be expressed selectively and ubiquitously in neurons. Whereas a complete deficiency of this protein is lethal, we found that partial VhaAC45L knockdown in larval motoneurons impaired synaptic vesicle acidification, leading to reduced quantal size, which is the amplitude of the postsynaptic response to the release of a single synaptic vesicle, and locomotion defects. We also report that VhaAC45L from brain tissue coimmunoprecipitated with V-ATPase subunits, and in particular with ATP6AP2. Overall, our results indicate that VhaAC45L is a novel accessory subunit of the V-ATPase that is specifically involved in the regulation of its activity in neurons.

Results

VhaAC45L is specifically expressed in neurons and addressed to synaptic areas

Here we studied the Drosophila gene CG31030, identified in FlyBase as a paralog of VhaAC45, and so, as another fly ortholog of vertebrate ATP6AP1/AC45 [START_REF] Thurmond | FlyBase 2.0: the next generation[END_REF], that we named therefore VhaAC45-like (VhaAC45L). VhaAC45L and VhaAC45 indeed share 69.9% similarity in amino-acid sequences (Supplementary Figure 1A), and VhaAC45L is also classified in the V-ATPase family group by the InterPro database (accession: Q8IMJ0_DROME) [START_REF] Mitchell | InterPro in 2019: improving coverage, classification and access to protein sequence annotations[END_REF]. According to FlyAtlas (Chintapalli et al., 2007), VhaAC45 is apparently expressed rather ubiquitously in Drosophila tissues, while in contrast VhaAC45L seems to be specifically expressed in the nervous system in both larval and adult flies, making it a possible candidate for the specific regulation of neuronal V-ATPase (Supplementary Figure 1B). To confirm this assumption, we checked by qRT-PCR the repartition of VhaAC45L transcripts in three parts of the adult fly body: the head containing the brain, the thorax containing the ventral nerve cord (VNC), and the abdomen, relatively poor in nervous tissue. Both females and males showed highest expression in the head, minor expression in the thorax and no detectable expression in the abdomen (Figure 1A). The expression of VhaAC45L therefore closely follows the repartition of the nervous system in the fly body.

The single-cell RNA-Seq Scope database [START_REF] Davie | A single-cell transcriptome atlas of the aging Drosophila brain[END_REF] furthermore indicates that, in Drosophila, VhaAC45L is expressed in all neurons, with few or no expression in glial cells. To verify this, we expressed three different VhaAC45L RNAi either with the panneuronal driver elav-Gal4 or the pan-glial driver repo-Gal4 (see Supplementary Table 3 for genotypes of the different Drosophila lines used in this study). The expression of VhaAC45L RNAi1 and VhaAC45L RNAi2 was found to be lethal at embryonic and 1 st larval stages, respectively, while VhaAC45L RNAi3 produced viable adults with a shortened longevity (Supplementary Figure 2), and obvious locomotor impairments. This difference in phenotypes observed with different RNAi constructs could be attributed to a variation in residual levels of the VhaAC45L protein. RT-qPCR experiments showed that the panneuronal expression of VhaAC45L RNAi3 together with the RNAi booster Dicer-2 (Dcr-2)

was sufficient to decrease by more than 80% VhaAC45L transcripts abundance in extracts from the adult heads (Figure 1B). On the other hand, glial expression of this RNAi construct with repo-gal4 had no significant effects on VhaAC45L transcript level (Figure 1B), confirming that this gene is selectively expressed in neurons. It is interesting to note that both VhaAC45L RNAi1 and VhaAC45L RNAi2 also induced lethal phenotypes at various developmental stages when expressed either with a glutamatergic (VGlut-Gal4) or a cholinergic (Cha-Gal4) driver (data not shown), in accordance with the RNA-Seq data of the Scope website suggesting a pan-neuronal expression of this gene.

To validate these observations, we placed the available MIMIC line VhaAC45L MI107 that contains a stop codon inserted in the middle of the gene [START_REF] Nagarkar-Jaiswal | A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila[END_REF] over the deficiency Df(3R)Exel6214 encompassing VhaAC45L [START_REF] Parks | Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome[END_REF]. The resulting mutant, likely to be a null, was found to be embryonic lethal, in agreement with the results obtained with two VhaAC45L RNAi lines. Remarkably, re-expressing the gene selectively in neurons, using a UAS-VhaAC45L construct driven by elav-Gal4, was sufficient to rescue this lethality, producing viable and fertile adults with no obvious behavioral defects (Supplementary Table 1). These results strongly indicate that

VhaAC45L is an essential gene whose expression appears to be specifically required in neurons.

Next, we studied the cellular localization of this protein. We used the CRISPR-Cas9

technique to insert a small V5 epitope tag in frame at the 3' end of its gene, disrupting the stop codon, thus generating the VhaAC45L V5 mutant line (Figure 1C). By immunostaining with a V5-specific antibody, we observed that the general expression pattern of VhaAC45L in adult brain (Figure 1D) and larval CNS (Figure 1E) was widespread and similar to that of the synaptic marker Cadherin-N (CadN), indicative of a predominantly synaptic localization. No specific signal was detected in a control w 1118 line that does not contain the V5-tagged protein (not shown). Some neuronal cell bodies were also marked with the V5 antibody in the VhaAC45L V5 line, both in adult and larva, and the synapse-containing neuropile areas (like the antennal lobes) were not as sharply defined as with the CadN antibody, suggesting that axons could also be immunopositive.

Finally, co-immunostaining of larval body wall muscles with anti-horseradish peroxidase (HRP) antibodies, a marker of Drosophila neurons [START_REF] Jan | Antibodies to horseradish peroxidase as specific neuronal markers in Drosophila and in grasshopper embryos[END_REF], showed precise co-localization with the V5 signal, indicating that VhaAC45L is addressed to synaptic boutons at the larval neuromuscular junction (Figure 1F).

VhaAC45L co-immunoprecipitates with V-ATPase proteins

The VhaAC45L protein is predicted to be part of the InterPro V-ATPase family, but no experimental information is currently available regarding its potential interactors. To determine whether VhaAC45L could interact, directly or indirectly, with subunits of the V-ATPase complex, we carried out co-immunoprecipitation experiments using an anti-V5 antibody on proteins extracted from heads of VhaAC45L V5 mutants and w 1118 control flies, followed by nano LC-MS/MS mass-spectrometry analysis of the precipitated proteins. Three independent experiments were performed to increase reliability, and in total, 410 proteins were identified in all three experiments. Among those, only 12 proteins had at least a two-fold abundance difference with the control in all three experiments (Figure 2A and Supplementary Table 2), one of them being, as expected, the coimmunoprecipitation target VhaAC45L. Remarkably, three of the 11 other proteins were identified as subunits of the V-ATPase complex: Vha100-1, VhaAC39-1 and ATP6AP2

(Figure 2B).

Vha100-1 and VhaAC39-1 code for subunits a and d of the V 0 domain, respectively [START_REF] Vasanthakumar | Structure and Roles of V-type ATPases[END_REF]. The subunit a, coded by five different genes in Drosophila, is the proton port of the pump (Collins and Forgac, 2020). Among the five isoforms, Vha100-1, which co-immunoprecipitated with VhaAC45L in our experiments, has been shown to be specifically required in neurons and present at the synapse (Hiesenger et al., 2005). Subunit d of V 0 is coded by two Drosophila genes: VhaAC39-1 and VhaAC39-2, and only the first co-immunoprecipitated with VhaAC45L. According to FlyAtlas, VhaAC39-1 is expressed in many tissues and enriched in the brain, while VhaAC39-2 seems to be mostly found in testis and salivary glands. For both V 0 subunits, VhaAC45L thus co-precipitated with the likely neuronal isoform. The co-

VhaAC45L knockdown increases the pH of synaptic vesicles

Because VhaAC45L appeared to be mainly localized in synaptic areas (see Figure 1), we chose to look at the physiological effect of its disruption at the Drosophila larval neuromuscular junction, a model that has contributed to the study of many synaptic processes. At synaptic nerve endings, a prominent role of the V-ATPase is to acidify the lumen of synaptic vesicles, the electrochemical gradient generated providing the driving force to load and concentrate the neurotransmitters. Thus, a malfunction of synaptic V-ATPase should induce a decrease of neurotransmitter concentration inside the vesicles, potentially resulting in an altered synaptic transmission.

To test this hypothesis, we co-expressed each of the two strongest VhaAC45L RNAi constructs together with VMAT-pHluorin, a pH-sensitive probe targeted to synaptic vesicles [START_REF] Wu | Loss of vesicular dopamine release precedes tauopathy in degenerative dopaminergic neurons in a Drosophila model expressing human tau[END_REF], in larval motoneurons using the glutamatergic driver OK371-Gal4. Both RNAi1 and RNAi2 induced a lethal phenotype at pupal stage in these conditions. VMAT-pHluorin is an ecliptic pHluorin that is fluorescent at neutral pH, and gets quenched, by protonation, at acidic pH [START_REF] Miesenböck | Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins[END_REF]. Thus, in control condition, VMAT-pHluorin should not be fluorescent in synaptic vesicles, whose pH is at around 5.5, but only when externalized on the presynaptic membrane during exocytosis, and so, in contact with the more neutral synaptic cleft milieu. In the case of an acidification defect of synaptic vesicles, the probe could be fluorescent both in synaptic vesicles, where pH would be abnormally high, and on the presynaptic membrane (Figure 3A, central panel).

In order to evaluate the ratio of the internal fluorescence (from synaptic vesicles) over the external fluorescence (from the presynaptic membrane) at the neuromuscular junction of VhaAC45L knockdown larvae compared to controls, we first quenched the external signal by replacing the physiological milieu by an identical one with pH adjusted to 5.5 (Figure 3A, left panel). This operation resulted in only the internal signal being conserved. In controls, this meant that all signal was abolished, as expected because the synaptic vesicles were normally acidified (Figure 3B, left panel). In contrast and strikingly, a residual signal was still visible in this acidic milieu in both RNAi1 and RNAi2 knockdown larvae (Figure 3B, right panel). Quantification of the ratio of fluorescence area in acidic milieu over neutral milieu showed that about 37% of the total signal remained visible in the RNAi larvae after external quenching (Figure 3C andD).

To verify that the residual signal seen in knockdown larvae was indeed coming from inside vesicles, the opposite strategy was used: instead of quenching the outside signal, we revealed all the internal one by collapsing the pH gradient of synaptic vesicles (Figure 3A, right panel). To do so, we replaced the physiological milieu by an ammonium solution, as previously described [START_REF] Poskanzer | Mobilization and fusion of a non-recycling pool of synaptic vesicles under conditions of endocytic blockade[END_REF]. This solution had the same composition except that 50 mM NaCl were replaced by 50 mM NH 4 Cl. Ammonium and ammonia being in equilibrium (NH 4 + ⇄ NH 3 + H + ), uncharged ammonia crosses membranes and binds to protons leading to an alkalization of vesicle lumen pH. This

VhaAC45L downregulation decreases larval locomotor performance

A reduced pH gradient of synaptic vesicles in larval motoneurons could alter synaptic transmission and, consequently, the larval locomotor behavior. To assess locomotion, we recorded the spontaneous crawling of 3 rd -instar larvae expressing VhaAC45L RNAi1 or RNAi2 in motoneurons with OK371-Gal4 on an agar plate with no food source for 2-min periods. Tracking was then performed using the FIMtrack software [START_REF] Risse | FIM imaging and FIMtrack: two new tools allowing high-throughput and cost effective locomotion analysis[END_REF], allowing measurement of the total distance travelled and of the stride size, defined as the distance crawled during one peristaltic wave of muscle contraction, and duration (Figure 4A). The knockdown larvae obviously moved less than controls on the plate, as confirmed by their trajectory maps (Figure 4B andC), travelling a distance only half as long as controls with both RNAi (Figure 4E andF). This effect was not simply due to a difference in larval size, as the average length and width of recorded larvae were not significantly different (Supplementary Figure 3).

In contrast, the stride size actually showed little or no difference, depending on the RNAi, between knockdown larvae and controls (Figure 4G andH). Instead, we found that the stride duration, which is the time necessary to accomplish one peristaltic wave, was significantly longer in knockdown animals compared to controls for both RNAi (Figure Mean values with 95% confidence intervals are reported on the graphs.

Discussion

In this study, we have investigated the hypothesis that a previously uncharacterized Drosophila protein, called VhaAC45L, is a regulator of the neuronal V-ATPase. At variance with its broadly expressed paralog VhaAC45, we have shown that VhaAC45L is found mainly, if not only, in neurons. We also provide evidence that VhaAC45L interacts with two constitutive subunits and one accessory subunit of the V-ATPase, the constitutive ones being also enriched in neurons, and that it is required to have properly acidified synaptic vesicles. This implies that VhaAC45L is an essential protein for nervous system functioning in Drosophila.

In yeast, all V-ATPase subunits are coded by a single gene, with the exception of the V 0 subunit a. The knock-out of any of the single-gene subunits all present a similar phenotype: the inability to survive in a neutral pH environment [START_REF] Nelson | A journey from mammals to yeast with vacuolar H+-ATPase (V-ATPase)[END_REF]. For subunit a, the same phenotype was only achieved in a double-mutant of both isoforms (Manolson et al., 1994). In multicellular organisms, mutations of V-ATPase subunits, or accessory proteins, also often lead to a lethal phenotype at various developmental stages, whether in mice (Inoue et al., 1999;[START_REF] Sun-Wada | Acidic endomembrane organelles are required for mouse postimplantation development[END_REF]Schoonderwoert et al., 2002b), C. elegans (Lee et al., 2010), or flies (Davies et al., 1996;Allan et al., 2005). Similarly, a VhaAC45L null mutant was found to be embryonic lethal, and, interestingly, this lethality could be rescued to the adult stage by re-expressing VhaAC45L in all neurons,

showing that the protein is specifically required in this cell type for fly survival. The percentage of adult survivors was about three times less than what would be expected in case of a full rescue (Supplementary Table 1) and the missing two-thirds likely died at an early developmental stage as no larval lethality was observed. This could be explained by the fact that VhaAC45L protein re-expression in rescued knock-out flies first required the expression of Gal4 under regulation of the elav promoter, which starts to express rather late in embryos, followed by activation of the UAS sequence upstream the VhaAC45L insert. So, it is possible that part of the mutant embryos did not survive the delay inherent to this ectopic expression process.

We found that VhaAC45L transcripts follows the repartition of the nervous system, being mainly found in Drosophila head. Moreover, pan-neuronal expression of VhaAC45L RNAi3 , the RNAi construct with the weakest effect on fly survival, was sufficient to decrease by more than 80% its level in the head of adult escapers. These results indicate that VhaAC45L expression is mainly neuronal, if not even entirely restricted to neurons. In addition, VhaAC45L cellular localization shows similarity with a synaptic pattern. Cell bodies were also marked, although generally less strongly.

Synaptic protein complexes can be assembled in the cell bodies before being transported in axons, and it is the case for the soluble and membrane-bound domains of synaptic V-ATPase, as was shown in Torpedo [START_REF] Morel | Vacuolar H+-ATPase domains are transported separately in axons and assemble in Torpedo nerve endings[END_REF]. It is difficult to decide whether the signal coming from cell bodies corresponds to a functional site for VhaAC45L or rather to newly produced proteins that have not yet been targeted to synapses. However, the prominent signal in synaptic areas led us to assume that VhaAC45L plays an important role in some synaptic processes.

In presynaptic terminals, the most abundant organelles are synaptic vesicles, which require acidification to provide the driving force for neurotransmitter loading. This acidification is ensured by the V-ATPase pump, which generates an electrochemical gradient by importing protons into the vesicle lumen, thus creating both a membrane potential (ΔΨ, inside positive voltage) and a pH gradient (ΔpH, acidic lumen). Our experiments showed that the knockdown of VhaAC45L at the glutamatergic larval neuromuscular junction increased the internal pH of synaptic vesicles, and so decreased the ΔpH component of the electrochemical gradient generated by the V-ATPase. This result is however by itself insufficient to conclude to a dysfunction of the proton pump, since other players have been shown to influence synaptic vesicle pH gradient downstream V-ATPase activity. For example, cation/H + exchangers, found on synaptic vesicles, can decrease ΔpH while increasing ΔΨ by exchanging cations, like Na + or K + , against luminal H + [START_REF] Takamori | Presynaptic molecular determinants of quantal size[END_REF]. This activity can be upregulated by increased intracellular concentration of these cations [START_REF] Huang | Presynaptic HCN channels regulate vesicular glutamate transport[END_REF]. Glutamate being negatively charged, its transport across vesicular membrane is predominantly driven by ΔΨ (Maycox et al., 1988;[START_REF] Tabb | Glutamate transport into synaptic vesicles. Roles of membrane potential, pH gradient, and intravesicular pH[END_REF]. As a consequence, the increased membrane potential resulting from up-regulation of cation/H+ exchangers actually facilitates glutamate uptake [START_REF] Goh | Presynaptic regulation of quantal size: K+/H+ exchange stimulates vesicular glutamate transport[END_REF][START_REF] Huang | Presynaptic HCN channels regulate vesicular glutamate transport[END_REF]. We measured synaptic quanta size by quantal analysis of mEPSPs of VhaAC45L knockdown larvae and found that it was decreased, contrary to what would be expected in the case of an upregulation of cation/H + exchanger activity. This apparent decrease of vesicular glutamate concentration was corroborated by its phenotypical consequence, namely the locomotion defect exhibited by knockdown larvae. This suggests that the observed diminution of ΔpH is correlated with a diminution of ΔΨ, thus pointing towards a V-ATPase malfunction.

This hypothesis is further supported by the co-immunoprecipitation of VhaAC45L with three V-ATPase subunits, with all of them being associated with the neuronal V-ATPase.

Altogether, these results strongly suggest that VhaAC45L is a neuronal protein necessary to V-ATPase function in synapses. Some V-ATPase subunits isoforms have been shown

to target specific subcellular compartments, like the Torpedo V 0 a1 isoform which is found in synaptic V-ATPase and not in neuronal cell bodies (Morel et al., 2003).

Similarly, VhaAC45L could be specifically involved in the regulation of synaptic V-ATPase, or, alternatively, globally act on all neuronal V-ATPase. Specificity level could even be pushed further, since synaptic vesicles are not the only organelles requiring acidification that are localized in synapses. Thus, a synaptic V-ATPase regulator could be devoted to synaptic vesicles as it could be affecting more generally all synaptic V-ATPase complexes, indifferently of their membrane localization. The answer to such questions could help better understand regulations of synaptic transmission, since synaptic V-ATPase activity is one of the pre-synaptic modulators of quantal response [START_REF] Takamori | Presynaptic molecular determinants of quantal size[END_REF][START_REF] Gowrisankaran | Regulation of synaptic vesicle acidification at the neuronal synapse[END_REF].

While accessory subunits directly interact with V-ATPase domains, other stimulus can indirectly affect activity of the complex, like glucose concentration or serotonin (Sautin et al., 2005;[START_REF] Voss | Stimulusinduced phosphorylation of vacuolar H+-ATPase by protein kinase A[END_REF]. Co-precipitation of VhaAC45L with V-ATPase subunits does not necessarily imply a direct interaction, but suggests a minima an indirect association with the complex, possibly in a non-transient manner. The impairment in V-ATPase activity induced by VhaAC45L downregulation rules out the hypothesis of an inhibitory action of this protein on the proton pump. Nevertheless, the precise requirement of VhaAC45L for activity of the V-ATPase complex still remains unknown to date.

Lethality of the mutant could indicate an essential role of VhaAC45L in V-ATPase function. However, some V-ATPase regulators and accessory subunits have been found

in other signaling pathways, like ATP6AP2 in the renin-angiotensin system [START_REF] Nguyen | The (pro) renin receptor in health and disease[END_REF], so we cannot exclude that lethality could be due to VhaAC45L playing a part in other vital neuronal functions. High signal in axons could also point to a role in targeting the complex to the synapses. The two domains of the V-ATPase, V0 and V1, are believed to be assembled in cellular bodies before being separately transported to synaptic area, V 0 by fast axonal anterograde transport, most likely directly on new synaptic vesicles, and V 1 by slow axonal transport like other cytoplasmic synaptic proteins [START_REF] Morel | Vacuolar H+-ATPase domains are transported separately in axons and assemble in Torpedo nerve endings[END_REF]. VhaAC45L could be involved in the transport of one of the two domains, and effects of VhaAC45L knockdown could then result from a decreased synaptic abundance of the affected V-ATPase domain. However, it is believed that only one copy of the V-ATPase complex is sufficient to properly acidify one synaptic vesicle [START_REF] Takamori | Molecular anatomy of a trafficking organelle[END_REF], a consequence of this being that neurotransmitter loading would be an all-or-none process. Consistent with this, mutation of the Drosophila synaptic V 0 subunit Vha100-1

does not seem to impact quantal size but rather mEPSP frequency, potentially reflecting the presence of an increased number of empty synaptic vesicles (Hiesinger et al., 2005).

Similarly, a reduction in the frequency of spontaneous quantal events with no change in quantal size was observed when the Drosophila vesicular glutamatergic transporter (VGlut) was downregulated, also suggesting that a single copy of VGlut is sufficient for proper acidification of a vesicle [START_REF] Daniels | A single vesicular glutamate transporter is sufficient to fill a synaptic vesicle[END_REF]. The fact that VhaAC45L knockdown changes the quantal size but not significantly mEPSP frequency seems to point toward a role of this accessory protein in V-ATPase efficiency, rather than in a process affecting the abundance of the complex such as synaptic targeting.

In this respect, it is interesting to note that the V-ATPase protein that more consistently co-immunoprecipitated with VhaAC45L in our experiments is the accessory subunit ATP6AP2, the fly homolog of human ATP6AP2/PRR. Together with ATP6AP1/Ac45, ATP6AP2 has been proposed to enable the assembly and disassembly of the catalytic and membrane domains of the V-ATPase in the mammalian brain (Abbas et al., 2020). This suggests that VhaAC45L could play a role in the regulation of these dynamic processes, which could also influence neurotransmitter loading and release in a more continuous way. Our results suggest indeed that modulating V-ATPase activity in presynaptic terminals can finely affect quantal size. Whether such regulation actually occurs in physiological, or pathological, conditions remains to be established. Importantly, human ATP6AP2/PRR is known to be a Parkinsonism candidate gene and its mutations in humans, mice or flies can lead to cognitive impairment, neurodegeneration and epilepsy (Korvatska et al., 2013;Dubos et al. 2015;[START_REF] Ichihara | The (pro) renin receptor in health and disease[END_REF]. It is therefore of major interest to identify a potential new interactor of ATP6AP2 that is specific to the nervous system, as it could help better understand the consequences of V-ATPase dysregulation on synaptic transmission in pathological contexts. Accordingly, it would be very interesting to determine whether human ATP6AP1 and/or ATP6AP1L share a conserved function with Drosophila VhaAC45L in the nervous system. Every new actor identified allows us to paint a more detailed picture of the complex regulations surrounding neuronal V-ATPase specificity, providing new angles for potential therapeutic targets, and a better understanding of fundamental processes such as synaptic transmission.

Materials and methods

Drosophila strains, construction and culture

Flies were raised on standard agar-cornmeal-yeast medium, at 25°C in a 12:12h lightdark cycle. VhaAC45L RNAi strains and mutants were obtained from the Bloomington Drosophila stock center (BDSC) and the Vienna Drosophila Resource Center (VDRC).

Detailed genotypes and references of these lines are provided in Supplementary Table 3.

To construct the UAS-VhaAC45L strain, the VhaAC45L cDNA was PCR amplified from the clone RH09162 obtained from the Drosophila Genomics Resource Center, Indiana University, USA, using the following primers with added restriction sites: P1-EcoRI (forward) 5'-CCATCCGAATTCAAAATGCAGCTGATTCTCGT and P2-XhoI (reverse) 5'-TGGCTGCTCGAGATCTATTGGGTTATGAGAGA. The 1160 bp PCR fragment was inserted into pUAST [START_REF] Brand | Targeted gene expression as a means of altering cell fates and generating dominant phenotypes[END_REF], verified by sequencing (GATC Biotech) and sent to BestGene, Chino Hills, CA, USA, for P-element transformation by random insertion in w 1118 background. A 2d-chromosome insertion of UAS-VhaAC45L that yielded strong expression of the transgene was used thereafter.

Reverse transcription-coupled qPCR

Total RNAs were extracted from 20 heads (or 15 thoraces or 15 abdomens) of 8-day-old flies using the QIAzol Lysis reagent (Qiagen). The Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, ref. K1671) was used with oligo(dT)20 primers to synthesize the cDNAs. Relative quantitative PCR assays were carried out using a

LightCycler 480 and the SYBR Green I Master mix (Roche LifeScience), with Act5C as internal control for normalization of mRNA levels. All reactions were performed in triplicate. The specificity of amplification products was assessed by melting curve analyses. The following forward and reverse primers, were used: for VhaAC45L, 5'-GGCTTCGTTGTAGGCCAACAGA and 5'-CACCAGGTATCCCAAGTTCCAGA; for Act5C, 5'-CGTCGACCATGAAGATCAAG and 5'-TTGGAGATCCACATCTGCTG.

CRISPR/Ca9 gene tagging

The sequence of a V5 tag was inserted in frame after the coding sequence of the VhaAC45L gene, using a homology-directed repair CRISPR-Cas9 method (see Figure 1C). The following guide RNA sequence: 5'-TTCACCGTACAGGAGTAAGG-3' was cloned into the BbsI site of pCFD3: U6:3-gRNA plasmid [START_REF] Port | Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila[END_REF] (kind gift of Hervé Tricoire, Université de Paris, Paris, France). This plasmid was then injected, at a concentration of 500 ng/µL, with the following single-stranded oligodeoxynucleotide (ssODN) donor repair template: 5'-GTTCGCGCAGCAAACAGTTGACCTTCACCGTACAGGAGTACGCAGGTAAGC CTATCCCTAACCCTCTCCTCGGTCTAGATTCTACGTAAGGAGGTCATAAGTC TCTGATGAACCAATAGATCTCGGGC-3' (synthetized by Integrated DNA Technology, Leuven, Belgium), also at a concentration of 500 ng/µL (the sequence underlined corresponds to the in-frame V5 tag), into nos-cas9 embryos (genotype y 1 , P(nos-cas9, w+), M(3xP3-RFP.attP)ZH-2A, w*) [START_REF] Port | Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila[END_REF]). An alanine was added before the V5 tag (dashed underline) to prevent the creation of a potential tyrosine phosphorylation site. Embryo injections were performed by BestGene (Chino Hills, CA, USA). Single F 0 flies were crossed over the TM6C(Sb) balancer to establish stable lines.

DNA was then extracted from three flies of each of these independent lines, and V5 insertion events were detected by dot blot using a mouse anti-V5 tag monoclonal antibody (Thermo Fisher Scientific,. Positive strains were outcrossed in a w 1118 background and their genomic DNA was sequenced to check for proper in-frame V5 integration in VhaAC45L. One of these equivalent VhaAC45L V5 mutant line was selected for further studies.

Immunohistochemistry

Adult brains of 8-day-old females, or 3 rd -instar larva CNS, were dissected in Drosophila For immunostaining of the larval muscles and neuromuscular junctions, the Alexa Fluor 488 Tyramide SuperBoost Kit (Thermo Fisher, ref. B40912) was used to increase the V5 signal that was otherwise faint in this tissue. The working protocol was as recommended by the manufacturer, with anti-V5 diluted to 1:100, then followed by classical immunostaining, as described above, to co-stain for the nerve terminals with an anti-HRP antibody (Jackson ImmunoResearch,1:200).

Longevity assay

About 110 virgin females from each genotype were collected on their hatching day, and placed in clean bottles, with no more than 25 flies per bottle. Flies were transferred in new clean bottles, and survivors were counted every two days for 60 days.

Co-immunoprecipitation

About 200 heads from 8 day-old VhaAC45L V5 and w 1118 flies were lysed using glass beads in 500 µL of ice-cold lysis buffer: 50 mM Tris-HCl pH 7. -11), that had been previously washed as recommended by the manufacturer. Samples were incubated with gentle agitation at 4°C for 4 hours. The supernatants were removed using a magnetic rack, and beads were washed three times with 500 µM of ice-cold lysis buffer before being resuspended in 10 µL of milliQ water.

Mass spectrometry

The MS/MS analysis was performed at the Proteomics facility of the Institut Jacques Only proteins identified in at least one group in two independent experiments were kept in the analysis. Missing values were set to the minimum abundance of the experiment. A more detailed description of the LC-MS/MS procedure is provided in the Supplementary Information.

VMAT-pHluorin experiments

Third-instar larvae expressing VMAT-pHluorin with or without VhaAC45L RNAi in motor neurons using the OK371-Gal4 driver, were dissected to expose the body wall muscles in Ca 2+ -free HL3 saline solution (70 mM NaCl, 5 mM KCl, 70 mM MgCl 2 , 10 mM NaHCO 3 , 115 mM sucrose, 5 mM trehalose, 5 mM HEPES, pH 7.6). Two other solutions were used: an acidic Ca 2+ -free HL3 saline (pH 5.5) and a neutral HL3-NH 4 Cl saline (pH 7.6), in which 50 mM NaCl was replaced by 50 mM NH 4 Cl. After dissection, larvae fillets were allowed to settle in Ca 2+ -free HL3 saline for 10 min before being scanned a first time directly in a drop of the solution using a Nikon A1R confocal microscope. Ca 2+ -free HL3 saline was then replaced either by the acidic Ca 2+ -free HL3 saline for low pH-induced quenching experiments, or by the HL3-NH 4 Cl saline for pH gradient collapse experiments. In both cases, larval fillets were rinsed three times with the modified solution, and incubated for 3 min, before being scanned a second time, in a drop of modified solution. Quantification was done on Z-projections (set to maximal intensity) of confocal stacks. Using the Fiji software, a fixed threshold was applied to all images to get rid of the background and select only synaptic areas. Percentage of area over the threshold was used as a measure of the signal intensity and ratios of the values obtained in modified solutions over standard HL3, i.e. the second scan over the first scan, were calculated for quantifications.

Larval locomotion assays

Larval locomotion was assessed on an in-house made version of the FIMtable system [START_REF] Risse | FIM imaging and FIMtrack: two new tools allowing high-throughput and cost effective locomotion analysis[END_REF]. Third-instar larvae were collected and briefly rinsed in water to remove traces of food, before being gently placed on the recording table precoated with a thin layer of 1.2% agar gel. Only four larvae of the same genotype were recorded simultaneously, to avoid collisions between animals, for a period of 2 min with a Basler ace acA2040-25gm camera at 12.5 frames/s. Larvae that burrowed themselves into the agar plate or escaped the arena before the end of recording were excluded from the results.

Tracking was done using the FIMtrack software, as described in [START_REF] Risse | FIM imaging and FIMtrack: two new tools allowing high-throughput and cost effective locomotion analysis[END_REF]. The number of peristaltic waves was computed from the variations in larval area. More precisely, the curves of area variation were first smoothed with a Savitzky-Golay filter to get rid of unwanted noise, then the number of waves was defined as the number of peaks on the curve (which was automatically computed by a custom-made Python script). Stride size was then calculated as the distance travelled by the larva divided by the number of peristaltic waves, while stride duration was defined as the recording time (i.e. 120 seconds) divided by the number of peristaltic waves.

Electrophysiological recording and quantal analysis

Third-instar larvae expressing VhaAC45L RNAi in motor neurons using the OK371-Gal4 driver, and appropriate controls, were dissected to expose the body wall muscles, and the brain removed, in Ca 2+ -free HL3 saline solution (70 mM NaCl, 5 mM KCl, 70 mM MgCl 2 , 10 mM NaHCO 3 , 115 mM sucrose, 5 mM trehalose, 5 mM HEPES, pH 7.6) (Stewart et al., 1994;Cattaert and Birman, 2001). Spontaneous miniature EPSPs (mEPSPs) were recorded in the presence of tetrodotoxin (TTX) 10 -6 M, so that no spike could occur, from the ventral longitudinal abdominal muscle 6 in segment A3. Quantal analysis was performed following the theoretical background described in [START_REF] Kuno | Quantum aspects of central and ganglionic synaptic transmission in vertebrates[END_REF] and [START_REF] Castellucci | A quantal analysis of the synaptic depression underlying habituation of the gill-withdrawal reflex in Aplysia[END_REF]. Distribution histograms of mEPSP size were built from each muscle fiber recording with a 0.01 mV bin size. These histograms provided an estimate of the mean size of a unitary EPSP, since the peaks represent integer multiples of the unitary size. A theoretical distribution was then computed by convolving a binomial distribution, accounting for quantal content (number of quanta released), and a Gaussian distribution, allowing for variations in size of individual quanta. A detailed description of the mathematical treatment is provided in the Supplementary Information.

Statistical Analysis

Statistical analysis was performed with the GraphPad Prism 6 software. 1 12 proteins were identified with at least twice higher abundance in VhaAC45L V5 compared to the w 1118 control in three independent co-immunoprecipitations with anti-V5 antibodies, followed by mass-spectrometry analysis experiments. Among these proteins, three are known to be constitutive or accessory subunits of the V-ATPase complex. They are indicated in the first ochrehighlighted lines of the

Quantal analysis

The probability for an EPSP to contain " quanta followed a binomial distribution:

P " = ∁ & ' ×) ' ×(1 -)) (&.') (1)
in which ) is the average probability of release, and / is the total number of releasable quanta. In the frequency distribution of EPSP amplitude, successive peaks represent increasing numbers of quanta.

In order to predict how these events are distributed between different bins in a histogram, it is necessary to allow for variations in quantal size. To do this, the largest peaks of the histogram were fitted to a Gaussian curve scaled in width to have a variance proportional to quantal size, and scaled in height so that its area corresponded to the predicted number of events [START_REF] Del Castillo | Quantal components of the end-plate potential[END_REF]. From the mean (0) and standard deviation (1) of the Gaussian curve, the content (2) of each bin (3) is given by:

2 3 = 1 251 6 -1 2 ( 3-0 1 ) 2 (2)
The standard deviation (σ) of each peak depends on the number of quanta it contains. It was calculated as follows:

σ = "× σ 8 (3)
with " the number of quanta in the peak, and σ 8 the standard deviation of a single quanta.

The amplitude of Gaussian distribution for each peak is scaled to the probability 9(") for that peak (see [START_REF]References[END_REF]).

The complete theoretical distribution, allowing for variance and peak overlap was then obtained by pooling the Gaussians for all peaks. In this way, the theoretical distribution could be superimposed on the histogram for direct comparison with the data.

Other results

2.1. Fertility study of VhaAC45L knocked-down flies

Methods

Here, we detail specific protocols used in this Results section.

Fertility test

Flies were raised on standard agar-cornmeal-yeast medium at 25°C in a 12:12 h light-dark cycle. Virgins females from the genotypes of interest were selected, as well as virgin wild-type Canton S males. Egg-laying plates coated with a nutritive layer were prepared using the following recipe: 150 mL distilled water, 50 mL grapefruit juice, 4.5 g agar, 5 g sucrose, 0.3 g of methyl 4-hydroxybenzoate (methylparaben). Just before the test, a small patch of yeast paste was added in the centre of the plate.

When the selected virgins were two-day old after eclosion, they were transferred in bottle with food medium, without anaesthesia. Each bottle contained a single virgin female, either of the tested genotype or of one of the controls, and two wild-type males.

Flies were incubated for three days at 25°C to allow mating. They were then transferred to empty bottles, again without anaesthesia, that were placed on an egg-laying plate for 24 h. Plates were then removed and the number of eggs laid was counted (Figure B1).

Plates were then incubated at 25°C for 24 h before being checked again to verify if eggs had hatched, in order to include only the fertilized ones in the results.

Results on fertility study in VhaAC45L knocked-down context

In the first part of the Results section, the RNAi constructs that were mainly used were VhaAC45L RNAi1 and VhaAC45L RNAi2 , because of their strong effects. When expressed with a pan-neuronal driver, these two RNAis induced lethality at early developmental stage. In contrast, VhaAC45L RNAi3 produced viable adult flies, mostly females, when expressed in all neurons (see Supplementary Table 3 for description of the three RNAis used). These flies were very unhealthy, as explained previously, as they had obvious locomotion defects and a reduced longevity. In addition to that, we noticed that they produced almost no progeny, and that, accordingly, they laid very few eggs. This low fertility phenotype was observed both for knockdown (KD) females and KD males, when crossed with wild-type flies.

This could be explained in two ways: either all females were laying a dramatically decreased amount of eggs, or some females displayed regular oviposition while others laid no eggs. To distinguish between these two scenarios, we performed individual egglaying experiments. Among the controls, 100% of the control females laid eggs during the test, against only 33% of the KD females (Figure B4.A). The number of fertilized eggs laid by fertile females was in average slightly lower for egg-laying KD flies than for controls, but not significantly (Figure B4.B). These two results led us to the conclusion that about two thirds of KD females did not lay fertilized eggs, while the remaining third had a close to normal egg-laying behaviour.

B6.A). This difference was not due to a lack of wild-type male interest towards KD females, as male courtship behaviour occurred in every case. Moreover, the delay to successful courtship was also increased in mated KD females compared to controls (Figure B6.B).

A similar experiment was performed on KD males. Note that pan-neuronal expression with the elav-Gal4 driver, located on the X chromosome, is stronger in males than females due to dosage compensation of the X-chromosome [START_REF] Jonson | Systematic Aβ analysis in Drosophila reveals high toxicity for the 1-42[END_REF]. KD males are even weaker than KD females, and have greater difficulty to move. During courtship, males need to be very active, to be able to follow and turn rapidly around the females while displaying their various behaviours, like singing. KD males were not capable of such level of activity, and 0% of them successfully mated (Figure B6.C).

Although they started vibrating their wings when females were in close proximity, this never lasted more than a second as females would not stay still and KD males were too weak to follow them. The lack of mating of KD males is therefore likely to be caused by their general weakness.

In conclusion, the mating rate was found to be strongly reduced in females and males with partial VhaAC45L KD, but apparently for different reasons. This could explain the low number of eggs laid by the KD females, although a partial defect in oviposition could also contribute to this last phenotype. Further investigation would be necessary to understand in which neurons and through which cellular mechanisms VhaAC45L KD affect courtship and egg-laying behaviour.

as a result of another medical condition like stroke, a brain tumour, head trauma or brain infection [START_REF] Bhalla | Etiologies of epilepsy: a comprehensive review[END_REF]. Genetic also seems to play a part in most epilepsy disorders, either directly or indirectly. In some very rare familial cases, mutations of a single gene have been identified as the cause of seizures occurrence. These genes include voltage-gated ion channels, GABA receptors and neuronal nicotinic acetylcholine receptors [START_REF] Scheffer | The genetics of human epilepsy[END_REF].

Anti-epileptic drugs (AEDs) are the standard treatment for epilepsy, but despite the large number of AEDs able to prevent of suppress seizures, 35% of patients still experienced uncontrolled seizures [START_REF] Abou-Khalil | Antiepileptic drugs: advantages and disadvantages[END_REF]. Moreover, prolonged drug administration can lead to pharmaco-resistance of patients, forcing a temporary break in the treatment or a change of the prescribed drug. Other therapeutic strategies, like seizure foci resection or brain stimulation, are very invasive, thus limiting the number of the potential beneficiaries. The need for novel and improved treatments calls for the finding of new therapeutic prospects. In this context, vesicular neurotransmitter transporters have been proposed as a therapeutic target, since affecting their activity could help contain hyperexcitability of epileptic brains by decreasing neurotransmitter loading [START_REF] Van Liefferinge | Are vesicular neurotransmitter transporters potential treatment targets for temporal lobe epilepsy?[END_REF]. This led us to question whether V-ATPase genes could also represent an interesting potential target for epilepsy. Mutations of several V-ATPase subunits in humans can result in neurodevelopmental defects and epilepsy, indicating that there is indeed a link between V-ATPase function and this pathological condition [START_REF] Gupta | A splice site mutation in ATP6AP2 causes X-linked intellectual disability, epilepsy, and parkinsonism[END_REF][START_REF] Fassio | De novo mutations of the ATP6V1A gene cause developmental encephalopathy with epilepsy[END_REF][START_REF] Mucha | A new microdeletion syndrome involving TBC1D24, ATP6V0C, and PDPK1 causes epilepsy, microcephaly, and developmental delay[END_REF]. We have shown in the first part of Results that VhaAC45L knockdown in Drosophila glutamatergic motoneurons resulted in a decrease in quantal size, which suggests that acting on V-ATPase activity in excitatory neurons could potentially decrease hyperexcitability in flies. Genetic constructions required to test this hypothesis are still in progress, so we do not have any results on this topic yet. However, we used this opportunity to develop a new behavioural paradigm, that will be described in this chapter, to assess seizure phenotypes in Drosophila.

Induction of epilepsy in flies by photosensitivity 2.2.2.1. Mechanical and electroconvulsive shock protocols

Development of new treatments can only be achieved by better understanding the mechanisms of the pathology, mainly through the help of animal models. There are many types of epilepsy, which differ in their causes as well as their symptomatic manifestations.

As such, there cannot be one animal model recapitulating the whole range of epileptic disorders. Instead, various animal models can help understand precise questions about specific aspects of epilepsy. They can be used both to investigate molecular and cellular mechanisms of the pathogenesis, and screen for possible treatments. The many models that have helped to better understand epilepsy disorders over the years include baboon, chicken, cat, dog, Mongolian gerbil, zebrafish, Drosophila and C. elegans [START_REF] Avoli | Feline generalized penicillin epilepsy[END_REF][START_REF] Bertorelli | The Mongolian gerbil in experimental epilepsy[END_REF][START_REF] Menini | The photosensitive epilepsy of the baboon: a model of generalized reflex epilepsy[END_REF][START_REF] Batini | An avian model of genetic reflex epilepsy[END_REF][START_REF] Lohi | Expanded repeat in canine epilepsy[END_REF][START_REF] Baraban | Emerging epilepsy models: insights from mice, flies, worms and fish[END_REF].

Drosophila has been used as a model for reflex epilepsy, i.e. epilepsy where seizures are consistently induced by identified triggers such as visual, auditory or tactile stimulation. In flies, several mutants are known for their seizure-like phenotype in response to a mechanical stimulus. These mutants are designated as bang sensitive (BS).

To induce seizures in BS mutants, flies can simply be introduced in an empty tube that is vortexed at high speed for 10 s (Figure B7). Following this drastic stimulation, BS mutants experience a seizure-like behaviour characterized by intense abnormal uncoordinated contraction, wing-flapping and leg-shaking. This only lasts for a short period (about 2 s), and is then followed by temporary paralysis (20 to 300 s, depending on the mutation), ending by a second short seizure period called "recovery seizure" (about 2 s), before flies start behaving normally again [START_REF] Song | From bench to drug: human seizure modeling using Drosophila[END_REF]). This episode is followed by a refractory period, that can last between 5 min to over an hour, during which flies are immune to attempts at eliciting new seizures [START_REF] Ganetzky | Indirect suppression involving behavioral mutants with altered nerve excitability in Drosophila melanogaster[END_REF].

Wild-type flies are insensitive to this protocol, and do not display any seizure-like behaviour in response to vortex mixing.

Seizure-like activity can also be elicited by a brain electroconvulsive shock (ECS) in every Drosophila strains, wild-type as well as mutants. High frequency (HF) electrical impulse provokes intense erratic HF firing of the recorded neurons for a few seconds, followed by a period of synaptic failure. This seizure-like activity seems to propagate through out the entire brain as every examined neuron displayed the same abnormal firing. This protocol also induces a refractory period during which ECS are not able anymore to elicit abnormal activity [START_REF] Kuebler | Modifications of seizure susceptibility in Drosophila[END_REF]. In this protocol, the difference between control strains and BS mutants resides in the voltage threshold necessary to induce seizure activity. Wild-type flies need a stimulation of about 30 V to generate HF abnormal firing, while BS mutants have a much lower threshold, usually below 10 V.

Unlike ECS, the vortex protocol offers the advantage of physiological relevance, as seizures are provoked by external triggers, which happens also in the human pathology.

In contrast, it does not allow electrophysiological recordings or imaging of the brain during abnormal activity, limiting investigation of the cellular processes governing the phenotype. On the other hand, such examination is possible with ECS, but this protocol remains a non-physiological way to elicit seizures, thus potentially differing in its mechanisms from more natural triggers. In addition, shaking the flies with a vortex allows little flexibility over the stimulus parameters. The rpm of the vortex can be adjusted and the time of mixing increased or decreased, but the stimulus being very chaotic, there is no control over what individual flies experience during these few violent seconds. For these reasons, we decided to try another approach to elicit seizures in BS mutants. Several publications mentioned an unpublished communication from Prof. Ronald Hoy at Cornell University, USA, indicating that BS mutants can also be sensitive to stroboscopic light [START_REF] Song | From bench to drug: human seizure modeling using Drosophila[END_REF]). Since we were not able to obtain any protocols about this or any further information from Pr. Hoy, we decided to try it by ourselves anyway.

activity. The enzyme ethanolamine kinase is necessary for the synthesis of phosphatidylethanolamine, a major constituent of phospholipid membranes. Sensitivity of the eas mutant to mechanical shock is believed to be due to a defect in phospholipid composition, altering excitability [START_REF] Pavlidis | The Drosophila easily shocked gene: a mutation in a phospholipid synthetic pathway causes seizure, neuronal failure, and paralysis[END_REF].

slamdance (sda), where the gene coding for Aminopeptidase N is mutated. The mechanism underlying seizure sensitivity is unclear, but the mutation is believed to downregulate sda expression [START_REF] Zhang | The Drosophila slamdance gene: a mutation in an aminopeptidase can cause seizure, paralysis and neuronal failure[END_REF].

Note that eas and sda mutants are actually double mutants since they are on the commonly used w 1118 background. The white gene (w) encodes an ATP-binding cassette (ABC) transporter, carrying the precursors of red and brown pigments into the eye pigment cells, giving it its red colour [START_REF] Mackenzie | Mutations in the white gene of Drosophila melanogaster affecting ABC transporters that determine eye colouration[END_REF]. Flies carrying the w 1118 mutation have white eyes, and are believed to be easily blinded by bright light [START_REF] Cosens | A switch phenomenon in the compound eye of the white-eyed mutant of Drosophila melanogaster[END_REF], which could be a problem in our case since our protocol relies on light stimulation. The bss mutant is in wild-type background, and as such has red eyes.

The experimental set-up is composed of (Figure B8): -a plate made of three arenas, each containing 20 flies and a small ration of food (Figure B9), cut in Acrylic glass (courtesy of the ESPCI mechanical workshop).

triggers' properties responsible for seizures, as well as on the the cellular processes that lead to these dramatic reactions in the brain.

Discussion

V-ATPase regulations

Many cellular processes are pH-dependent and rely on V-ATPases, which implies that the proton pump needs to be tightly controlled. Several regulatory mechanisms are therefore available in cells to influence different parameters of the V-ATPase. These parameters can be categorized into two groups: the ones affecting the complex spatial localization and the ones changing its activity or efficiency. Indeed, the V-ATPase is present in different cellular compartments, as well as in the plasma membrane of specific cells in multicellular organisms, so it has to be spatially controlled. Different organelles require different levels of acidification, and these levels are not fixed in time but can vary depending on the context. The V-ATPase activity should therefore also be adaptable (Figure C1).

How are these parameters regulated? Here again, V-ATPase modulators can be segregated into two groups: the ones intrinsic to the complex, and the external ones.

Intrinsic properties include the structure, composition and core mechanism of the complex. Altogether they define the basal activity of the proton pump. Intrinsic properties can also influence the spatial localization of the complex, for example isoform composition was shown to be be involved in V-ATPase targeting. External regulations, like for example the energetic status of the cell, can increase or decrease the basal activity of the proton pump, for example through reversible assembly and disassembly of the two domains of the complex. Can these external regulators also change the original spatial localization defined by the intrinsic properties? We do not know of any physiological examples of such regulation, but in yeast with no Vph1p, overexpression of Stv1p (the remaining isoform of this subunit normally targeted to the Golgi apparatus), leads to the apparition of Stv1p-containing V-ATPases also on vacuoles (Kawasaki-Nishi et al., 2001b). This shows that originally programmed targeting can be modified when needed, suggesting that external regulators might in some cases alter V-ATPase spatial localization.

However, it seems logical that impairing synaptic vesicles acidification would only be viable until a certain degree, and that too much alkalination of the vesicles would lead to a shut down of synaptic transmission and eventually death. Is this impairment of acidification enough to conclude that VhaAC45L acts on V-ATPase activity rather than localization? Surely not, since an incorrect assembly or mistargeting of the complex would also result in a defect in acidification.

V-ATPases are quite large molecular complexes, with a diameter of about 10 nm.

As a comparison, the diameter of synaptic vesicles is usually around 40 nm. If we also take into account the fact that V-ATPases are just one of the many components necessary for synaptic vesicle structure and function (Figure C2), it is not surprising that each vesicle was estimated to present only one or two V-ATPase complex on its surface [START_REF] Takamori | Molecular anatomy of a trafficking organelle[END_REF]. A consequence of this is that a reduction in V-ATPase quantity would generate a subset of synaptic vesicles devoid of proton pump. These vesicles would be unable to uptake neurotransmitters and thus remain empty, but should still be able to undergo exocytosis [START_REF] Zhou | Effects of reduced vesicular filling on synaptic transmission in rat hippocampal neurones[END_REF]. In any case, whether these empty vesicles are released or not, the effect on postsynaptic receptors would be the same, i.e. none, which should result in a decrease of the postsynaptic spontaneous events frequency. In our situation, knock-down of VhaAC45L did not significantly affect events frequency, but rather decreased the quantal size. These results suggest that the quantity of V-ATPase is unlikely to be altered by VhaAC45L knock-down. Instead, it looks like vesicles contained less neurotransmitters. This could happen if V-ATPase activity was impaired, for example through weakest or shortest association of the two domains, resulting in a limiting acidification of vesicles which could decrease the level neurotransmitter uptake.

In conclusion, our results could point towards a role of VhaAC45L in the regulation of V-ATPase activity rather than on its spatial localization, although we have no direct evidence of that. Another interesting question is whether VhaAC45L is an intrinsic or external regulators of the complex?

A question of gear size

In the Introduction (part 1.1.3), we described the V-ATPase as a nanoscale rotary motor, but in reality each of its domain is already a rotary motor by itself, functioning on different fuels. V 1 is powered by ATP hydrolysis and drives clockwise rotation, while V 0 is powered by the proton gradient and drives counter-clockwise direction [START_REF] Stewart | Rotary ATPases: models, machine elements and technical specifications[END_REF]. The actual direction of the full assembled complex rotation is decided by the relative strength of these two opposing motors: if V 1 is stronger then the complex will work as a proton pump consuming ATP, but if V 0 is stronger then the complex will produce ATP using the proton gradient. Except that the V-ATPase is believed to only work as a proton pump and not the other way around like the F-ATPase and the A-ATPase [START_REF] Wilkens | Rotary molecular motors[END_REF]. So what in its design prevents the rotation from changing direction?

The relative strength of V 0 and V 1 depends on several parameters. To better understand the powers at play, it is simpler to visualize the complex as two gears stacked on top of each other, connected by a central shaft (Figure C3). Each gear is trying to force the other one to turn in its own rotational direction. ATP molecules and protons are "workers" pushing the gears, each in an opposite direction. If the workers of one gear overpower the others, then they impose the rotation direction. V-, F-, and A-ATPases all have the same number of "ATP workers": they all need three ATP to complete a 360° revolution [START_REF] Wilkens | Rotary molecular motors[END_REF]. The number of "proton workers" is a more debated question.

This number corresponds to the maximum amount of protons being transported for one complete revolution of the complex. It is dictated by the number of c subunit integrated in the c-ring of the membrane-bound domain. It is usually considered that each V-ATPase has 9 c subunits and one c" subunit, thus totalizing 10 protons potentially transported per revolution (Collins and Forgac, 2020). F-ATPases with variable numbers of c subunits, ranging from 10 to 15, have been found [START_REF] Stock | Molecular architecture of the rotary motor in ATP synthase[END_REF][START_REF] Pogoryelov | The oligomeric state of c rings from cyanobacterial F-ATP synthases varies from 13 to 15[END_REF]. An

ATPase able to transport 15 protons per revolution would have a powerful membranebound domain (highest fuel displacement). So a reasonable proton gradient would be able to drive ATP synthesis against ATP hydrolysis. Thus the V-ATPase might owe its unidirectional rotation to an inferior number of c subunits (Figure C3).

V 1 while reinforcing V 0 . This is exactly what was done to prove that the V-ATPase could also function as an ATP synthase [START_REF] Hirata | Regulation and reversibility of vacuolar H+-ATPase[END_REF]. However, creating such extreme conditions was barely sufficient to reverse the rotation as the efficiency of the complex to produce ATP was still very low. This confirms that the V 1 motor is far stronger than V 0 and so it is very unlikely that the V-ATPase rotation will ever be reversed in vivo.

It is important to better understand what exact intrinsic properties of the V-ATPase underlie its unidirectional rotation, and what subunits or regulators are necessary to maintain it, as pathological V-ATPase mutations might act by altering the balance between the two competing motors.

Accessory but necessary

In this work, we provided evidence that VhaAC45L is required for V-ATPasemediated acidification of synaptic vesicles. Could we determine whether this regulation is internal or external to the complex? In other terms, does VhaAC45L participate to the structure and the intrinsic properties of synaptic vesicle V-ATPase or indirectly modulate its activity?

Most subunits composing the complex are now well described. However, as mentioned in the Introduction (part 1.1.2), another potential subunit of V 0 was recently discovered: the f subunit (Mazhab-Jafari et al., 2016;Abbas et al., 2020). Because V 0 is membrane embedded, its purification is more delicate than for the cytosolic domain V 1 , making analysis of its composition more difficult. The f subunit was identified in yeast and in rat brain, two very different models suggesting that it could actually be present in all V-ATPases, and that its presence might have eluded previous studies because of technical difficulties. It is not the first subunit in this case, as the V 0 e subunit discovery also came along after the description of the other core subunits, probably for technical issues as well [START_REF] Sambade | The yeast vacuolar proton-translocating ATPase contains a subunit homologous to the Manduca sexta and bovine e subunits that is essential for function[END_REF]. Interestingly, the two already known accessory subunits, ATP6AP1/AC45 and ATP6AP2/PRR, also associate with the V 0 domain [START_REF] Feng | Cytoplasmic terminus of vacuolar type proton pump accessory subunit Ac45 is required for proper interaction with V0 domain subunits and efficient osteoclastic bone resorption[END_REF]Abbas et al., 2020). Originally, these proteins were designated as "accessory" not because they are dispensable for V-ATPase functioning (their knock-outs are generally lethal [START_REF] Sun-Wada | Acidic endomembrane organelles are required for mouse postimplantation development[END_REF][START_REF] Zima | Prorenin Receptor Homologue VHA-20 is Critical for Intestinal pH Regulation, Ion and Water Management and Larval Development in C. elegans[END_REF]), but because they were not identified on every purified complexes. One could wonder if this could not simply be due to technical limitations as well, and whether the distinction between "accessory" and other core subunits is really justified. Since the two accessory proteins seem to associate in a non-transient way to the proton pump (Abbas et al., 2020), we could argue that they most likely take part in its core mechanism and so could be considered as intrinsic regulators. Could VhaAC45L be a late discovered subunit essential for the V-ATPase core mechanism?

Here we showed that VhaAC45L co-immunoprecipitated with several V-ATPase subunits. Among the three independent experiments performed, 3 subunits consistently came out with at least a two-fold difference in abundance between the sample of interest and the negative control: VhaAC39-1 (subunit d of V 0 ), Vha100-1 (subunit a of V 0 ) and ATP6AP2 (accessory subunit associated with V 0 ). These results suggest a clear bias of VhaAC45L interaction toward the membrane-bound domain. Note that ATP6AP2 was consistently detected solely in the sample of interest and never in the negative control throughout all these experiments, making it the strongest probable interactor of VhaAC45L. Other V-ATPase subunits co-precipitated with VhaAC45L only in one or two of the three experiments, including other V 0 subunits (subunits e and c), as well as a few V 1 subunits. Interestingly, two V 0 subunits never came out in any of the three experiments: the c" subunit and the accessory protein VhaAC45/ATP6AP1, which is the closest homolog of VhaAC45L. This suggests that VhaAC45L could replace VhaAC45 within the V-ATPase complex in neurons. However, because VhaAC45/ATP6AP1 is expressed ubiquitously in Drosophila, and in particular in neurons according to the Scope database [START_REF] Davie | A single-cell transcriptome atlas of the aging Drosophila brain[END_REF], this replacement could only concern part of the neuronal V-ATPase complexes, likely including those that are targeted to synaptic vesicles.

The precise function of AC45/ATP6AP1 in multicellular organism is still unclear, but the protein is suspected to be the functional homolog of the yeast assembly factor Voa1p (Jansen et al., 2016). ATP6AP1 and ATP6AP2 have been shown to interact with each other, and the complex they form could operate in V-ATPase assembly [START_REF] Rujano | Mutations in the X-linked ATP6AP2 cause a glycosylation disorder with autophagic defects[END_REF]Abbas et al., 2020). In addition, expression of Drosophila ATP6AP2 in the double knock-out yeast mutant of the two assembly factors Vma21 and Voa1 can rescue the Vma -phenotype, and this rescue was even improved with a co-expression of ATP6AP1 (Guida et al., 2018). The interaction of VhaAC45L and ATP6AP2 suggested by co-immunoprecipitation experiments, and the apparent lack of coimmunoprecipitation of VhaAC45L with VhaAC45, could be an argument in favour of knock-out of Vma21 and Voa1 in yeast led to a decrease in V 0 level, believed to be due to its lack of assembly in the ER. However, knock-out of Voa1 alone did not alter V-ATPase quantity but only decreased its activity [START_REF] Ryan | Voa1p functions in V-ATPase assembly in the yeast endoplasmic reticulum[END_REF]. In our experiments, electrophysiological recordings of VhaAC45L knockdown larvae did not suggest a difference in V-ATPase quantity, since event frequency was not significantly changed, but rather a change in activity level, as suggested by the decrease in quantal size. This could be comparable to what was observed in Voa1 single knock-out. The complete absence of VhaAC45L, or ATP6AP2 as mentioned in the Introduction, is lethal in Drosophila, but it would be interesting to look at the double knockdown of these proteins in neurons to determine whether the quantity of V 0 would be affected in this situation, like what is observed in the double knock-out mutant in yeast.

Regulated or regulating?

The difference between being a target of regulation or being a regulator is mostly a question of referential, since pathways are made of multiple cascades of regulation. If

A regulates B, which in turn regulates C, then from the perspective of A, B is a target of regulation, but from the point of view of C, B is a regulator. In this sense, the V-ATPase is regulated as much as it regulates since when its activity is modulated, the whole pathway depending on it is consequently affected.

Our results indicated that decreasing V-ATPase activity in synaptic vesicles led to a decrease in quantal size, raising the question of whether V-ATPases could regulate synaptic transmission (Figure C5). Indeed, quantal size, which refers to the postsynaptic response to the release of a single vesicle, is a key determinant of synaptic transmission.

Its variations are usually attributed to postsynaptic alterations, such as changes in receptors density or sensitivity. More recently, a presynaptic modulation of quantal size has also been shown to be possible. For example, overexpression of vesicular neurotransmitter transporters like VGlut1 or VMAT2 increased neurotransmitter uptake, resulting in increased quantal size [START_REF] Pothos | Synaptic vesicle transporter expression regulates vesicle phenotype and quantal size[END_REF][START_REF] Wilson | Presynaptic regulation of quantal size by the vesicular glutamate transporter VGLUT1[END_REF]. However, these genetic modifications do not inform on the potential physiological occurrence of such regulations. At the Drosophila neuromuscular junction, separate glutamatergic motoneurons were shown to have different synaptic vesicle volumes associated with different quantal sizes [START_REF] Karunanithi | Quantal size and variation determined by vesicle size in normal and mutant Drosophila glutamatergic synapses[END_REF]. Expression of the vesicular glutamate transporter VGlut1 was found to be endogenously regulated during development, and by levels of activity [START_REF] Wilson | Presynaptic regulation of quantal size by the vesicular glutamate transporter VGLUT1[END_REF]. These studies suggest that quantal size modulation through regulation of neurotransmitter loading could take place physiologically.

Since V-ATPase controls neurotransmitter transporters through synaptic vesicle acidification, it could also influence quantal size (Figure C4). V-ATPase activity is sensitive to several factors, one of them being ATP concentration: high level of ATP increases V-ATPase activity, while limited ATP decreases it. In isolated synaptic vesicles, low ATP concentration was shown to decrease glutamate uptake [START_REF] Naito | Characterization of glutamate uptake into synaptic vesicles[END_REF], suggesting that modulating V-ATPase activity can result in variation of the level of neurotransmitter loading. This result is consistent with our own observation of a decreased quantal size associated with increased synaptic vesicle pH. So V-ATPase down-regulation can diminish synaptic transmission, but is the opposite true: can V-ATPase up-regulation increase quantal size?

Overexpression of vesicular transporters increases neurotransmitter uptake, which makes them a limiting factor in this process. Studies showing this result did not look at V-ATPase activity in overexpression context. But if we consider that it is unchanged compared to controls, as we do not have any argument to believe otherwise, this would mean that the proton pump can easily power more vesicular transporters if needed. This suggests that unlike the number of transporters, the V-ATPase is not a limiting factor for neurotransmitter uptake in normal conditions. Moreover, since neurotransmitter transporters are acting downstream of V-ATPase action, the effect of an increase in proton pump activity would always be restricted by the limiting rate of vesicular transporters. So increasing acidification of synaptic vesicles would probably not be sufficient to upregulate quantal size, except in case of V-ATPase deficiency.

result in epileptic-like phenotypes. Interestingly, ATP6AP2 and ATP6V0C are also listed as variants related to epilepsy by the database EpilepsyGene [START_REF] Ran | EpilepsyGene: a genetic resource for genes and mutations related to epilepsy[END_REF]. ATP6V0C

is associated with Severe myoclonic epilepsy of infants (SMEI or Dravet syndrome), a drug-resistant form of epilepsy manifesting within the first year of life. Epilepsy can also develop as a co-morbidity of other disorders, for example 40% of patients with autism also experience seizures [START_REF] Tuchman | Convulsing toward the pathophysiology of autism[END_REF]. ATP6AP2 is one of the genes of interest shared between epilepsy and autism.

Seizures are due to a hyperexcitability leading to uncontrolled activity throughout the whole brain (generalized seizures) or a restricted area (focal seizures). Imbalance between excitatory and inhibitory are believed to be the cause of these deleterious bursts of activity. Since V-ATPase is essential to synaptic transmission, it is conceivable that dysregulation of its activity could lead to the development of abnormal neuronal networks facilitating seizures occurrence. The implication of mutations of several of its genes in epileptic-like behaviours seems to support this theory. So could V-ATPase also be a potential therapeutic target against seizures? Right at this moment, probably not. As seen previously in the manuscript, altering V-ATPase activity in higher eukaryotes is a delicate enterprise because of its involvement in a wild type of pathways in all cell types.

Preventing seizures through V-ATPase would require to specifically act on neuronal V-ATPase, or even more precisely on synaptic V-ATPase, and in the right subtype of neurons. Indeed, reducing acidification of synaptic vesicles in inhibitory neurons could aggravate seizures rather than prevent it, by weakening inhibitory transmission. Our knowledge of what makes the specificity of neuronal V-ATPase is probable not yet deep enough to allow the development of such therapeutic strategy. Future work could fill these gaps and enable V-ATPase to be an interesting target in epilepsy, as well as in other neurologic disorders where its dysregulation has been implicated.

We recently attempted to investigate the role of neuronal V-ATPase in the context of epilepsy in the Drosophila model. Seizures can be triggered by various environmental stimuli. This type of seizures is called reflex epilepsy. Several Drosophila mutants have been found to display seizure-like phenotype when subjected to mechanical shocks.

Because a large proportion of the fly brain is dedicated to visual processing, we thought that light might also trigger such phenotypes in this model. A new protocol using a visual trigger could help decipher the mechanisms underlying the generation of abnormal activity by sensory input. Our results indicated that the Drosophila mutant bss is indeed sensitive to stroboscopic light, a stimulus that can also trigger seizures in epileptic patients [START_REF] Harding | Photic-and pattern-induced seizures: expert consensus of the Epilepsy Foundation of America Working Group[END_REF]. In these patients, light flashing at a frequency between 15 and 20 Hz usually elicit seizures [START_REF] Da Silva | Photosensitivity and epilepsy: current concepts and perspectives-a narrative review[END_REF]. In humans, visual temporal resolution, i.e. the capacity to distinguish two consecutive stimuli, depends on the intensity of the light: low-light levels give a resolution of 15 Hz, while high-light levels give a maximum resolution close to 60 Hz [START_REF] Kalloniatis | Temporal resolution. Webvision: The Organization of the Retina and Visual System[END_REF]. A 20-Hz signal is therefore already relatively fast for the human eye. In our setup, the maximum effect was obtained for the light flashing at the maximal speed: 25 Hz. The Drosophila eye, despite having a poorer spatial resolution than the human eye, is believed to have a much higher temporal resolution, up to 200-300 Hz [START_REF] Borst | Visual motion detection in Drosophila[END_REF]. So 25 Hz might not be such a high speed for the fly eye, and it would be interesting to test whether higher frequencies, closer to 100 Hz, would also elicit seizures, and if this is the case, if parameters such as onset time would differ compared to 25 Hz.

Mutation of the human gene homolog of para (the fly gene responsible for the bss mutant phenotype) is associated with SMEI, a childhood form of epilepsy particularly pharmacoresistant. Up to 40% of children with SMEI are photosensitive [START_REF] Mickler | Occlusive Patch Therapy for Reduction of Seizures in Dravet Syndrome[END_REF]. The new photosensitivity protocol proposed here could help to better understand the underlying mechanisms of this mutation, and potentially screen for compounds able to prevent seizures triggered by light.

General conclusion

The V-ATPase is an impressive molecular motor capable of pumping protons across membranes. It is used in all eukaryotic cells to acidify several types of organelles, and even the extracellular medium in multicellular organisms. This function of acidification makes the V-ATPase a key player in endocytic and secretory pathways, so the complex has been a subject of intense interest. Its structure has been thoroughly described and its core mechanism is now well understood. The V-ATPase shares many similarities with other rotary ATPases, but it also presents clear differences probably underlying its specialization as a proton pump rather than an ATP synthase. Some questions still remain unanswered, especially concerning the detailed structure of the membrane-bound domain, but there is no doubt that continuous progress of technical tools, especially the increasing resolution of crystallographic reconstruction, will soon help uncover the last mysteries hidden in the pump.

Because of the number of cellular processes depending on its activity, V-ATPases have been implicated in a variety of pathologies. Dysregulation of the proton pump participate to metastasis in cancer, and mutations of different subunits isoforms can lead to various diseases. Among them, several have neurological symptoms. This naturally raises the question of the potential use of the V-ATPase as a therapeutic target. However, for such strategy to be successful, understanding the general mechanism of the pump is not sufficient. It is necessary to describe the complex web of regulations governing V-ATPase activity and what makes the specificity of each proton pump.

Our project has been part of this effort, and focused on the neuronal V-ATPase of Drosophila. We showed that Lome, a previously uncharacterized protein, predicted from its sequence to be part of the V-ATPase protein family, is specifically expressed in the nervous system and only required in neurons for fly survival. We provided evidence that this protein closely interacts with some V-ATPase subunits, including ATP6AP2.

Moreover, Lome proved to be necessary for acidification of synaptic vesicles at the neuromuscular junction, suggesting that it is required for proper V-ATPase activity in this organelle and for synaptic transmission. We finally renamed this protein VhaAC45L, in reference to its closest Drosophila homolog, VhaAC45. Further investigation will be needed to understand the precise function of this new V-ATPase regulator specific to neurons. We believe that its identification and the initial characterization carried out in this study will bring a significant element to the incomplete picture of the neuronal V-ATPase in Drosophila and other species.

1. 1 . 2 .

 12 V-ATPase regulations ................................................................................ 1.2. Intrinsic properties ................................................................................... 1.2.1. A question of gear size ................................................................................................. 1.2.2. Accessory but necessary .............................................................................................. 1.3. Regulated or regulating? .......................................................................... 1.4. When things go wrong… ........................................................................... General conclusion ..................................................................................... D. References ................................................................................... E. Annexes .......................................................................................

Figure A1 :

 A1 Figure A1: Number of publications about V-APase in Pubmed.

Figure A2 :

 A2 Figure A2: Structure of the V-ATPase.

Figure A3 :

 A3 Figure A3:The family of the rotary ATPases.

Figure A4 :

 A4 Figure A4: Representations of the V-ATPase, divided in its two domains or according to stator and rotor parts.

Figure A5 :

 A5 Figure A5: Mechanism of proton translocation by the V-ATPase.

Figure A6 :

 A6 Figure A6: Summary of V-ATPase function in intracellular compartments.

Figure A7 :

 A7 Figure A7: Examples of plasma membrane V-ATPase functions.

Figure A8 :

 A8 Figure A8: Reversible assembly of the two domains of the V-ATPase.

Figure A9 :

 A9 Figure A9: Assembly of the yeast V 0 domain in the ER.

Figure A10 :

 A10 Figure A10: Position of ATP6AP1 and ATP6AP2 on the V-ATPase.

Figure A11 :

 A11 Figure A11: V-ATPase in pathologies.

Figure A12 :

 A12 Figure A12: Neurotransmitter uptake by different vesicular transporters.

Figure A13 :

 A13 Figure A13: V-ATPase reversible assembly during synaptic vesicle exocytosis cycle.

Figure A14 :

 A14 Figure A14: Life cycle of the Drosophila.

Figure A15 :

 A15 Figure A15: Analogy between Drosophila and human internal organs.

Figure A16 :

 A16 Figure A16: Tissue-specific expression of a target gene with the Gal4-UAS system.

Figure A17 :

 A17 Figure A17: Temporal control of tissue restricted expression by Gal4.

Figure A18 :

 A18 Figure A18: Drosophila adult CNS.

Figure A19 :

 A19 Figure A19: Neuron morphology in mammals and Drosophila.

Figure A20 :

 A20 Figure A20: Drosophila larval CNS.

Figure A21 :

 A21 Figure A21: Drosophila larval NMJ.

Figure A22 :

 A22 Figure A22: Differential expression of different isoforms of Drosophila V-ATPase subunits.

Figure 1 :

 1 Figure 1: VhaAC45L is expressed in neurons and addressed to synaptic areas.

Figure 2 .

 2 Figure 2. VhaAC45L co-immunoprecipitates with V-ATPase subunits.

Figure 3 .

 3 Figure 3. VhaAC45L knockdown larvae have a synaptic vesicle acidification defect.

Figure 4 .

 4 Figure 4. VhaAC45L downregulation in motoneurons decreases larval locomotor performance.

Figure B10 :

 B10 Figure B10: Effects of stroboscopic lamp parameters on fly seizure.

Figure B11 :

 B11 Figure B11:The photosensitivity protocol induced a short refractory period.

Figure B12 :Figure C1 :

 B12C1 Figure B12: Reciprocal influence of photosensitivity and mechanical tests.

Figure C2 :

 C2 Figure C2: Model of a synaptic vesicle surface proteins.

Figure C3 :

 C3 Figure C3: Schematic representation of the two rotary motor composing an ATPase.

Figure C4 :

 C4 Figure C4: Consequence of VhaAC45L knockdown on quantal size.

Figure A3 .

 A3 Figure A3. Structure of the three types of rotary ATPases. Equivalent subunits are represented in the same colour within each complex. (From Song et al., 2013)

  the expression patterns of almost all Drosophila genes are available. The FlyAtlas project(Chintapalli et al., 2007) compiles mRNA enrichment (compared to the whole fly) in different tissues, measured by Affymetrix. According to this database, different isoforms of a same V-ATPase subunit present typical patterns of expression (Figure A22.A, B), showing tissue specificity like in mammals, while monogenic subunits are ubiquitously expressed, all displaying similar tissue repartition (Figure A22.C).

  blue) in place of the original stop codon (in green) in the VhaAC45L gene using the CRISPR-Cas9 technology. (D, E) Anti-V5 immunostaining in the VhaAC45L V5 strain revealed that VhaAC45L is mainly addressed to synaptic areas in the adult brain (D) and larval CNS (E), as indicated by its apparent colocalization with the presynaptic marker Cadherin-N (CadN). OL: optic lobe, AL: antennal lobe, SO: suboesophageal ganglion. (F) A V5-immunopositive signal was also detected at the neuromuscular junction of VhaAC45L V5 larvae, and found to co-localize with anti-horseradish peroxidase (HRP) immunostaining that labels neuronal membranes, confirming the synaptic localization of VhaAC45L. Scale bars: 100 µm in D and E, and 10 µm in F.

  increase of vesicular pH is maintained during NH 4 Cl exposure. In control condition, this gradient collapse should reveal the VMAT-pHluorin probe present in synaptic vesicles and thus highly increase the fluorescent signal. On the contrary, in the case of an acidification defect, the signal should remain fairly stable since synaptic vesicles are already fluorescent. Results were consistent with this hypothesis: while the fluorescence of controls increased about 3 folds in the pH collapsing ammonium solution, the signal in VhaAC45L knockdown larvae hardly rose by 10 to 20% depending on the RNAi construct (Figure 3E, F and G). Taken together, these results indicate that VhaAC45L knockdown significantly decreases protons concentration in synaptic vesicles of motoneuron terminals. the neutral HL3 solution should lead to a collapse of the pH gradients due to the free diffusion of NH3 in membranes, so that fluorescence will be emitted both by the presynaptic membrane and synaptic vesicles both in control and mutant conditions. (B) Representative pictures showing the effect of perfusing an acidic HL3 solution on VMAT-pHluorin fluorescence in control and VhaAC45L knockdown larvae. (C, D) Quantification of the ratio of the fluorescence level at pH 5.5 over the original signal at pH 7.6. Whereas the low pH extinguished fluorescence in control flies, about 37% of the signal persisted after quenching in VhaAC45L knockdown larvae using two different RNAi constructs. (E) Representative pictures showing the effect of collapsing the synaptic vesicle pH by perfusing HL3-NH 4 Cl in control and VhaAC45L knockdown larvae. (F, G) Quantification of the ratio of the signal in HL3-NH 4 Cl over the original signal in HL3 showed that fluorescence increased about 3-fold in controls while it only rose by 10 to 20% depending on the RNAi in VhaAC45L knockdown larvae. Results of four independent experiments, with 3-5 larvae analyzed per genotype in each experiment. Paired Student's t-test, *p < 0.05 **p < 0.01. Mean values with 95% confidence intervals are reported on the graphs. Scale bar: 15 µm in B and E.

  as the distance crawled during one peristaltic wave of muscle contraction, while stride duration is the time necessary for the completion of one peristaltic wave. (B-D) Locomotor trails of individual larvae recorded over a period of 2 min. Larvae expressing VhaAC45L RNAi in motoneurons show reduced spontaneous movements (C) compared to the driver and UAS controls (B and D, respectively). Scale bar: 25 mm. (E, F) Quantification of the travelled distances confirmed that knocking down VhaAC45L in motoneurons with two different RNAi induced significant locomotor defects. (G-J) Stride size (G, H) appears to be much less affected than stride duration (I, J) in the knocked-down larvae. Result of five independent experiments, with 3-4 larvae analyzed per genotype in each experiment. Repeated-measures one-way ANOVA with Dunnett's post-test for multiple comparisons, **p < 0.01, ***p < 0,001. Mean values with 95% confidence intervals are reported on the graphs.Quantal size is reduced in VhaAC45L knockdown larvaeThe locomotor deficit of VhaAC45L knockdown larvae could originate from an impairment of synaptic transmission at the neuromuscular junction or a defect in the central control of motor behavior, or both. To determine if neurotransmission was affected, we carried out electrophysiological recordings to measure the quantal size, which is the postsynaptic response to the release of one synaptic vesicle, at the larval neuromuscular junction. We expressed VhaAC45L RNAi1 or RNAi2 in motoneurons with the OK371-Gal4 driver, and recorded spontaneous miniature excitatory postsynaptic potentials (mEPSPs) intracellularly from ventral longitudinal abdominal muscle 6 of segment A3 (Figure5A). These muscles are innervated by synaptic boutons that are clearly marked by OK371-Gal4, as shown by the co-localization of membrane-associated GFP and the postsynaptic marker Discs large (Dlg) in OK371>mCD8::GFP flies (Figure5B). Representative amplitude distributions of mEPSPs in a control and RNAi1 larvae are shown in Figure5C. Quantal analysis of recorded events confirmed that both RNAi1 and RNAi2 knockdown larvae have a significantly reduced quantal size compared to controls (Figure5D), suggesting a decrease in glutamate vesicular uptake, and potentially linking vesicle acidification defect and locomotor impairment. The V-ATPase dysfunction in synapses of VhaAC45L knockdown larvae apparently led to incomplete loading of vesicles, thus decreasing the amount of neurotransmitter released per unit of time during a peristaltic wave, and therefore slowing down larval locomotion. Although the mean frequency of mEPSPs of both RNAi1 and RNAi2 larvae appeared lower than controls, were recorded intracellularly from the ventral longitudinal abdominal muscle 6 in segment A3. (B) Expression of membrane-associated mCD8::GFP with the glutamatergic driver OK371-Gal4 strongly labels the presynaptic nerve endings at the neuromuscular junction of muscles 6-7 in segment A3. The scaffolding protein Discs large (Dlg) was used as a postsynaptic marker. Scale bar: 20 µm. (C) Representative distributions of spontaneous mEPSPs recorded in a control larva (top panel, in grey) and a VhaAC45L RNAi knockdown larva (bottom panel, in orange). The dotted lines represent actually recorded amplitudes and the plain lines the computed theoretical distributions. Genotypes and quantal size (q) are indicated on each graph. (D) Quantal analysis of recorded events showed that knockdown larvae have a significantly reduced synaptic quanta size compared to controls, both with VhaAC45L RNAi1 (top panel, in orange) or RNAi2 (bottom panel, in blue). (E) Although both RNAi1 (left panel) and RNAi2 (right panel) larvae had a lower mean EPSP frequency than controls, this difference was not statistically significant. One-way ANOVA with Dunnett's post-test for multiple comparisons, *p < 0.05, **p < 0.01, ***p < 0.001.

  Ringer's solution or hemolymph-like saline solution (HL3) (70 mM NaCl, 5 mM KCl, 1.5 mM CaCl 2 , 70 mM MgCl 2 , 10 mM NaHCO 3 , 115 mM sucrose, 5 mM trehalose, 5 mM HEPES, with pH adjusted to 7.6), respectively, and fixed in 4% paraformaldehyde (Thermo Fischer Scientific) for one hour. After three 20-min washes in PBS plus 0.5% Triton X-100 (PBT), brains were blocked in PBT + 2% bovine serum albumin for two hours. They were then incubated in primary antibodies diluted in blocking solution for 24 h at 4°C. The primary antibodies used were: mouse monoclonal anti-V5 (Thermo Fisher Scientific, ref. R960-25, 1:200) and rat anti-CadN (DSHB, ref.DN-Ex #8, 1:20). Brains were then washed three times, for 20 min each, in PBT before being incubated in secondary antibodies for 2 hours. Secondary antibodies used were: Alexa Fluor 488 antimouse and Alexa Fluor 555 anti-rat (Fisher Scientific, ref.A11029 and ref. A21434 respectively), all diluted at 1:1000. After two 20-min washes in PBT followed by two 20min washes in PBS, brains were mounted in Prolong Gold Antifade Mountant (Thermo Fisher Scientific, ref. P36930). Imaged were acquired on a Nikon A1R confocal microscope.

  Monod (Université de Paris, Paris, France). Briefly, proteins were directly digested on the beads by trypsin (Promega, Madison, WI, USA) overnight at 37°C in a 25-mM NH 4 HCO 3 buffer (0.2 µg trypsin in 20 µL). The resulting peptides were desalted on a ZipTip µ-C18 Pipette Tips (Pierce Biotechnology, Rockford, IL, USA). Eluates were analyzed using either an Orbitrap Fusion or an Orbitrap Q-Exactive Plus, coupled respectively to a Nano-LC Proxeon 1200 or a Nano-LC Proxeon 1000, both equipped with an easy spray ion source (Thermo Fisher Scientific, Waltham, MA, USA). Raw data were processed on Proteome Discoverer 2.4 with the mascot node (Mascot version 2.5.1), with a maximum of 2 missed cleavages authorized, against the Swissprot/TrEMBL protein database (https://www.uniprot.org/news/2017/06/07/release) 12 for Drosophila melanogaster. The following post-translational modifications were included as variable:Acetyl (Protein N-term), Oxidation (M), Phosphorylation (STY). Peptide identifications were validated with a 1% FDR (false discovery rate) threshold calculated with the Percolator algorithm. Label-free quantification was done in TOP 3 abundance calculation mode with pairwise ratio-based calculation and t-test (background based) hypothesis test.

-

  an infrared camera, connected to the computer (Basler Ace acA2040-25mg, with an infrared filter: Schneider Kreuznach Infrared Pass Filter IF 093) -an infrared light -a stroboscopic RGB (Red Green Blue) lamp (Ignition Co6 Flood IP65), controlled by a DMX console (AONCO Contrôleur DM X 512 192).

Table 2 .

 2 Expected percentage of adult progeny flies of each genotype in case of full rescue. Actual percentage obtained in the experiments. 24 rescued adults were recovered out of a total of 373 progeny flies. List of the genes encoding proteins that co-immunoprecipitated with VhaAC45L in Drosophila head extracts.

	The paired

2 3

Table 3 .

 3 Table and circled in blue in Fig. 2. The grey line indicates the coimmunoprecipitation target VhaAC45L. Drosophila strains used in this study.

	Results

Rescue of embryonic lethality was assayed by crossing elav-Gal4; VhaAC45L MI107 /TM6B(Tb)

Acknowledgements

Acknowledgements

We thank Dr Philippe Marin for helpful advice about the quantitative analysis of mass spectrometry experiments, Drs Hervé Tricoire and Elodie Martin for helpful suggestion on the CRISPR/Cas9 mutagenesis, Pr David Krantz for providing the UAS-VMAT-pHluorin strain, Dr Hervé Tricoire for the pCFD3: U6:3-gRNA plasmid, and Dr. Céline Petitgas and Manon Dobrigna for participating in preliminary experiments during their student internships. This work was supported by a grant from the Fondation de France (Subvention n° 0086407) to SB and recurrent fundings to his lab from the ESPCI Paris and CNRS. AD, ARI and JS were recipient of PhD fellowships from PSL University and Labex MemoLife, the Doctoral School 3C and the China Scholarship Council, respectively.

List of Tables

A. Introduction

Table A1: Human isoforms for all V-ATPase subunits and their yeast homologs.

Table A2:

Human isoforms for all V-ATPase subunits and their Drosophila homologs.

B. Results

Supplementary Table

Competing interests

No competing interest to declare.

A novel and specific regulator of neuronal V-ATPase in

Drosophila

Amina Dulac, Abdul-Raouf Issa, Jun Sun, Giorgio Matassi, Baya Chérif-Zahar,

Daniel Cattaert and Serge Birman

Supplementary information

Supplementary figures

Supplementary tables

Supplementary methods

Supplementary tables

Supplementary Table 1. Pan-neuronal expression of VhaAC45L rescues the embryonic lethality of VhaAC45L-deficient flies up to the adult stage.

Genotype of the progeny flies 1 Expected percentage

Supplementary methods

Detailed LC-MS/MS procedure

Proteins on beads were digested overnight at 37°C with trypsin (Promega, Madison, WI, USA) in 25 mM NH 4 HCO 3 buffer (0.2 µg trypsin in 20 µL). The resulting peptides were desalted using ZipTip µ-C18 Pipette Tips (Pierce Biotechnology, Rockford, IL, USA).

Samples were analyzed using either an Orbitrap Fusion or an Orbitrap Q-Exactive Plus, coupled respectively to a Nano-LC Proxeon 1200 or a Nano-LC Proxeon 1000, both equipped with an easy spray ion source (Thermo Fisher Scientific, Waltham, MA, USA).

On the Orbitrap Fusion instrument, peptides were loaded with an online preconcentration method and separated by chromatography using a Pepmap-RSLC C18 column (0. Sci, 314(1165), 1-340. Whitton, B., Okamoto, H., Packham, G., & Crabb, S. J. (2018). Vacuolar ATPase as a potential therapeutic target and mediator of treatment resistance in cancer. Cancer medicine, 7(8), 3800-3811.

E. Annexes