Parkinson’s disease (PD) is a major progressive neurodegenerative disorder affecting several regions of the brain, especially the substantia nigra (SN). Although symptomatic treatments are available, there are no protective or curative treatments and diagnosis is established only when degeneration is well advanced. There is thus a major medical need to develop disease modifying treatment and biomarkers of early stage PD. In this regard, genetics has revealed a-synuclein, a protein enriched in Lewy bodies of PD brains, and LRRK2, a complex signalling protein, as major disease factors involved in both familial and sporadic PD. Recent data suggest that a-synuclein, localized in presynaptic nerve terminals, regulates the function of SNAREs, the core machinery mediating intracellular membrane fusion and traffic. VAMP7 is a v-SNARE related to synaptic VAMP2 and is enriched in neuronal cells particularly in 1) somatodendritic compartment of SN neurons where dopaminergic (DA) neurons affected in PD are located and 2) nerve terminals where neuronal activity is regulated by DA released by dendrites of SN neurons and 3) the striatum where DA neurons project. Growing evidence also shows a role for LRRK2 and its close homolog LRRK1 in autophagy, and inhibition of LRRK2 induces protective autophagy. Recently VAMP7 was shown to interact with LRRK1 and a-synuclein to bind to VAMP2 and block synaptic SNARE-dependent vesicle fusion. These observations led us to the working hypothesis that VAMP7-dependent organelle biogenesis and exocytosis in brain may be regulated by LRRK2 and a-synuclein and that this mechanism may be defective in PD. The important physiopathological implication would be that early PD symptoms may result from defective vesicular homeostasis mediated by VAMP7. Modulating VAMP7-dependent secretion may thus be a new promising target for treating early-diagnosed PD patients. Our main objective is therefore to characterize the structure/function of the biochemical connections between PD-related genes LRRK2 and a-synuclein in VAMP7-dependent autophagy and secretion particularly in DA neurons in culture and in vivo. Our strategy is based on the strong synergy between our teams combining biochemistry, biophysics, cell biology, electrophysiology and mouse genetics in 3 workpackages. WP-1: we will biochemically characterize the physical and functional interaction between VAMP7 and LRRK1, LRRK2 and a-synuclein in vitro using binding assays and artificial membrane based assays. We will focus on potential phosphorylation dependent activity in the VAMP7:LRRK complexes. WP-2: we will determine regulation by VAMP7, LRRK2 and a-synuclein of autophagosome and neuromelanin granule biogenesis, and exocytosis in PC12 cells and DA neurons. We will set up cell models for VAMP7, LRRK2, a-synuclein using CRISPR and viral vector technology and determine the role of these proteins in autophagosome biogenesis. We will characterize the biogenesis of neuromelanin granules, the secretome, and track the dynamics of VAMP7 exocytosis. We will test the effect of autophagy inducers and LRRK2 inhibitors. WP-3: we aim to determine secretory defects in DA neurons of VAMP7, LRRK2 and a-synuclein mutants’ brains. We will use mesencephalic brain slices of VAMP7, LRRK2 and a-synuclein KO mice to characterize the secretome and the activity of GABA neurons which sense the release of dendritic DA. We will test the models for the expression of PD related proteins of the endosomal/lysosomal system. Finally, we will assess the therapeutic potential of the pharmacological induction of autophagy in the AAV-A-syn PD rodent model. This project will lead to a better understanding of the role of key PD proteins in the regulation of vesicular trafficking. It will enable to identify specific therapeutic targets and potential biomarkers of PD related to vesicular physiology.

Congenital defects afflict more than 3% of births. These developmental diseases are a major public healthcare burden and today the molecular bases of the tissue-tissue interactions that ensure correct tissue morphogenesis in humans remain largely unknown. This is also the case for three-dimensional cellular interactions in humans, which specify the development of neurons in the peripheral nervous system and the growth of their axons. We have recently been able to combine whole-mount immunostaining and 3D imaging of solvent-cleared organs with light-sheet fluorescence microscopy, to study the 3D organization of several organs and systems in transparent human embryos and fetuses. The aim of this project is to form a consortium whose research will improve current knowledge of the development of the peripheral nervous system, particularly neuromuscular, in humans during the first trimester of gestation. This will allow us to establish a unique interactive database for neuroscientists and clinicians. We will focus on the cephalic region, the limbs and some internal organs, the development of which is poorly understood, and will improve 3D imaging methods for whole organs. Currently available scaling immunohistochemical detection to large tissue volumes has limitations due to restricted multiplexing capability of antibody labels. Moreover, human specimens in good condition are rare, especially pathological cases. Therefore, optimizing their exploitation is a priority and this aim seeks improving 3D-labeling methods. We will develop novel immunohistochemistry (IHC) multiplexed strategy to visualize up 8-10 antigens within the same sample. We will also further seek to develop whole-body IHC using single-chain variable fragments of conventional antibodies, which could outperform conventional antibodies when it comes to tissue penetration. This approach will thus improve immunostaining of very large samples, such as fetal organs and/or whole fetuses at gestational weeks 10-15 (GW10-GW15). In parallel, we will use multiplexed single-molecule in situ hybridization methods (MSM-FISH) combined with IHC on both tissue sections and intact human embryos, to dissect human fetal tissues in more detail in terms of their cellular composition and high-resolution architecture. Our second objective is to revisit through 3D imaging the embryonic development of peripheral, sensory, motor and autonomic human nerves using a large battery of markers (largely already validated) specific to different populations. We will astudy the expression pattern of the main axonal guidance molecules and their receptors in the peripheral nervous system. Another main objective is to decipher the phenotype of human motor neurons (MNs), according to their topographical distribution. MN phenotyping relies upon knowledge accumulated in mice, and the genetic fate map of MN pools and subtype is largely unknown in human embryos. We will characterize with a large panel of markers the topographic distribution of MNs in human embryos and fetuses. We will delineate the period of developmental cell death of human MNs. Last ,we will map the development of motor nerves in correlation with their target muscles. Finally, we will study the development of autonomic innervation (vegetative system) of the pancreas, as there is evidence to suggest that abnormal development of this innervation may promote diabetes. We will compare the pancreatic innervation of embryos from lean or obese women. Theiscombination of novel strategies and approaches, focused on human embryos, will enable us to address important questions in the field of human embryogenesis and ultimately to better understand the etiology of certain neurodevelopmental and muscular diseases. The two partners, Dr Chédotal (Paris) and Dr Giacobini (Lille) are recognized experts in their field and have all the required technical know-how to perform the planned experiments, as demonstrated by their collaborative track-record.

The concept that astrocytes are actively involved in neural development, synapse formation and synaptic activity in the healthy brain has emerged during the last 15 years. In the diseased brain, such as in Alzheimer’s Disease (AD), we are just starting to probe whether dysregulated astrocytes significantly contribute to the pathology. Neurofibrillary tangles, made of hyperphosphorylated and aggregated Tau proteins, are an essential pathological neuronal feature of the AD brain. In the latter, progression of neuronal « Tau pathology », scored as the Braak stages, from entorhinal cortex to hippocampus and lastly neocortex, is associated with progression of clinical symptoms supporting a pivotal contribution of neuronal Tau pathology to cognitive deficits. Mechanisms for Tau pathology-induced cognitive deficits in AD remain poorly understood and most studies focus on neuron-autonomous dysregulations. Conversely, relationships between neuronal Tau pathology and astrocytes remain largely unknown. Our preliminary observations raise the hypothesis that neuronal Tau pathology induces astrocyte dysfunctions, possibly through a Tau transfer from neurons to astrocytes. In addition, we recently identified adenosine A2A receptors (A2ARs), G-protein-coupled receptors whose endogenous ligand is adenosine, as a mediator of these interactions between neuronal Tau pathology and astrocytes. In this context, our overall objective is to uncover the impact of neuronal Tau pathology towards astrocyte function and establish astroglial A2ARs as druggable regulators of the development of Tau pathology and associated memory deficits. We will explore this link using several mouse models combined with innovative viral methods and functional tools to specifically address several questions in FACS-sorted acstrocytes, regarding astrocyte morphology and activity, as well as modifications of astrocytic transcriptome and epigenome. Specifically, we will 1) evaluate whether and how neuronal Tau pathology affects astrocyte function including chromatin structure and gene profiles and 2) establish astrocyte A2A receptors as a druggable switch of Tau-induced astrocyte dysfunction. To achieve our goals, we have assembled the unique ADORASTrAU consortium which brings together three research teams sharing a common interest towards AD pathophysiology, with highly complementary, non-overlapping and non-interchangeable scientific expertise. Our project will provide original insights in the mechanisms by which Tau pathology impacts the function of astrocytes and determine to which extent adenosine A2A receptors could be a molecular switch of Tau-induced cognitive dysfunctions.

Amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD) are two fatal adult-onset neurodegenerative diseases with a lifetime risk of 1/1000. Both ALS and FTD patients share a very poor prognosis and limited treatment options. ALS and FTD share common genetic risk factors, and co-occur in families and/or in individual patients. A subset of ALS cases (10-20%) is inherited, and referred to as familial ALS or FTD. Three mutually exclusive neuropathological inclusions are found in ALS/FTD patients: cytoplasmic inclusions of TDP-43 (TDP-43 proteinopathy), FUS (FUSopathy) or TAU proteins (so-called TAUopathy). TDP-43 and FUS are RNA-binding proteins involved in multiple steps of RNA metabolism, from transcription to alternative splicing and transport. TAU protein is a cytoskeletal protein that is critical for the stability of microtubule structure and functions in neurons. Further strengthening the role of these three proteins in ALS/FTD, mutations in TDP-43 and FUS genes have been identified in fALS and mutations in TAU are associated with a significant proportion of familial FTD. In all, pathology and genetics converge to ascribe a predominant pathogenic role for TDP-43, TAU and FUS in the development of ALS/FTD. Defining the mechanistic relationships between TDP-43, FUS and TAU is the subject of the current application. Mechanistic relationships between TAU and FUS have been described, and our consortium has available both mouse models and zebrafish models of Fus and Tau mediated diseases. Thus, we will focus our work on the TAU/FUS epistatic interaction and our major objective is to evaluate the contribution of FUS to TAUopathies, and of TAU to FUSopathies. In parallel, we will explore possible interactions between TAU and TDP-43 mostly in zebrafish models. Likewise, we will also extend our analysis to other RNA-binding proteins such as ATXN2, TAF15 and EWSR1 in zebrafish models. We propose to answer to three complementary yet independent scientific questions: Aim 1) Does cytoplasmic mislocalization of FUS alter TAU metabolism and trigger FTD? Aim 2) Are TAUopathies associated with altered function or localisation of FUS or TDP-43? Aim 3) Does TAU contribute to neurodegeneration due to mutations in FUS, TDP-43 and in other RNA-binding proteins? The originality of our project lies in different aspects. First, we have been using gene targeting, rather than classical transgenesis for generating our mouse models. We think this will likely lead to very relevant insights into disease mechanisms. Second, our experimental approach is multi-organismal, from zebrafish and mouse models to human patients. Third, our project is seeking to delineate a coherent pathogenic pathway for currently unrelated FTD causes. Overall, we believe that this proposal has the potential to impact durably our understanding of ALS and FTLD pathogenesis by using unique animal models to identify cell type specific alterations, validate these alterations in the human tissue which together will represent novel and valuable therapeutic targets.

Alzheimer’s disease (AD) is the most prevalent cause of dementia in elderly aged of 65 and over. Yet, there are no curative drugs and current treatments remain symptomatic. AD is a multifactorial slow and progressive dementing disease that combines two pathophysiological mechanisms: the amyloid pathology and the Tau pathology. The first one results from extraneuronal aggregation of amyloid-beta (Aß) peptides that derive from cleavages of a large transmembrane precursor named amyloid protein precursor (APP). The second one corresponds to intraneuronal accumulation and aggregation of abnormally modified microtubule-associated tau proteins, to form the so-called neurofibrillary tangles. So far, most efforts have been focused on either part of the pathology, especially the amyloid pathology. However, current clinical trials against amyloid pathology failed to show an improvement of the cognitive status of patients in clinical phase III. Protein mis-folding, protein aggregation and prion-like diffusion of protein pathogens are mechanisms common to several neurodegenerative diseases including AD. Protein homeostasis and degradation systems such as the proteasome, autophagy, endosome/lysosome and endoplasmic reticulum associated degradation (ERAD) are central cellular mechanisms for the clearance of misfolded proteinaceous aggregates. At the intersection of autophagy, ubiquitin-proteasome system and ERAD, is VCP (Valosin Containing Protein), also referred to as p97, TER94 or CDC48. VCP belongs to the family AAA-ATPase (ATPase with multiple cellular activities) which is essential for several cellular processes including the removal of misfolded proteins. Therefore, VCP constitutes a potential therapeutic target for the treatment of neurodegenerative diseases. In our collaborative project targeting AD pathophysiology between chemists and neurobiologists of Inserm UMR-S1172 (formerly, UMR 837 and EA4481 GRIOTT) and AlzProtect company, several compounds named MSBD (Melnyk et al. WO 2006 051489) have been developed and one compound (compound 29 in Melnyk et al. 2015) is currently in clinical phase I. To our knowledge, these compounds are the first to act on both APP metabolism (increase in APP-CTFs and sAPPa, decrease in Aß) and the Tau pathology by improving the cognitive deficits in transgenic mouse models of both amyloid and Tau pathologies. The investigation of the MSBD molecular target led to the identification of VCP. Our preliminary data suggest that MSBD repress the production of Aß and promote Tau proteolysis, both in vitro and in vivo, through a VCP-dependent mechanism. However, the precise mechanism remains to be unraveled. Therefore, one of the main questions we have to address is how MSBD modify the activity of this AAA-ATPase and consequently modulate both the amyloid and tau pathology. The present research proposal aims at addressing this question and further validate VCP as a therapeutic target for the treatment of neurodegenerative diseases and develop more potent families of VCP modulators as potential drugs against AD.