Microglia are the brain resident immune cells that maintain brain homeostasis by responding to cytokines and eliminating substances by phagocytosis. Microglial activation and phagocytotic clearance of aggregated amyloid ß (Aß) and tau aggregates limits Alzheimer’s disease (AD) pathology. There is an urgent need to elucidate the mechanisms that promote these actions and to develop novel approaches to control AD pathology. We recently discovered that Sulf2, an extracellular heparan sulfate (HS) sulfatase, selectively suppresses microglial response to interleukin 4, a cytokine that restricts microglia activation. Furthermore, our preliminary result showed that Sulf2 facilitated microglial phagocytotic clearance of Aß deposits ex vivo. The aims of the proposed project are: i) to clarify the molecular mechanism underlying Sulf2 effects on these microglia functions and ii) to investigate whether HS remodeling by Sulf2 could reduce Aß load and tau aggregates in AD brain in vivo by facilitating sustained and controlled microglial phagocytosis activity over time. The HS compositions and structure of HS S-domains present in Sulf2-transduced microglial cell line and primary mouse microglia; in the brains of microglia-specific Sulf2 transgenic/J20 hAPP-Tg and P301S tau-Tg AD model mice; in AD patient brains, will be determined by state-of-the-art methods, including mass spectrometry-based methods and a protein nanopore oligosaccharide sequencing technique. RNA-seq transcriptome analysis will be also performed for these microglial cells and AD model mouse brains. We will evaluate AD pathogenesis in vivo in these Sulf2-transgenic AD model mice. The partners’ expertise in glycoscience, neurology, and structural biology strengthens the accomplishment of the proposed project. As a whole, our ultimate goal is to develop a novel strategy for AD treatment with the idea of contributing to better health and well-being as well as social and economic welfare.

.Skeletal dysplasias with multiple dislocations (SDM) are severe disorders characterized by dislocations of large joints, scoliosis and pre and postnatal growth retardation. More than 10 recessive disorders have been described so far and the majority of them have been linked to pathogenic variants in genes encoding glycosyltransferases (“linkeropathies”), sulfotransferases, epimerases or sulfate transporters, all requested for the biosynthesis of the heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycan (GAG) chains attached to HS and/or CS proteoglycan (HSPG and CSPG) core protein. These findings support the recognition of a new group of inborn errors leading to defects in GAG biosynthesis. This process is tightly regulated in the Golgi, especially through ion homeostasis. In our cohort of patients with SDM, pathogenic variants have been also identified in genes encoding proteins with no known functions directly related to proteoglycan synthesis, such as calcium activated nucleotidase-1 (CANT-1), that were associated with defective proteoglycans synthesis. More recently, we identified homozygous mutations in SLC10A7, which encodes a member of the SLC family of uncharacterized transporters. Furthermore, we developed a Slc10a7-deficient mouse model that mimics the human bone phenotype. Preliminary results, interestingly, demonstrated a strong specific decrease in HS level in both patient fibroblasts and Slc10a7-/- mouse cartilages and a congenital disorder of glycosylation type II (CDG II) profile, i.e. resulting in the presence of truncated and abnormal N-glycan structures, in SLC10A7 deficient patients. The link of SLC10A7 with divalent ion homeostasis is not clear but suggested by yeast studies showing that SLC10A7 orthologs could act as negative regulator of cytosolic calcium homeostasis. This proposal is built on the hypothesis that mutations identified in genes encoding transporters or ion binding proteins, such as SLC10A7 or CANT-1 lead to impaired Golgi ion homeostasis then resulting in Golgi glycosylation and specific GAG synthesis defects. As such, it is ambitious to hypothesize that the restoration of Golgi ion homeostasis will lead to a normalization of the GAG biosynthesis process, and will open the field of novel therapies for SDM patients. In order to avoid a scattering of our efforts, we will strategically focus our work on SLC10A7, for which both patient fibroblasts and Slc10a7-/- mouse model are already available. The ambition is to develop a framework that will then serve as a model to study the functions of the other identified transporters. We will combine the synergistic complementary expertise of three teams) in skeletal dysplasia and ossification process 2) in Golgi glycosylation and Golgi homeostasis and 3) in GAG biosynthesis. This team complementarity will be essential in pursuing our three main objectives: WP1: Elucidation of the roles of SLC10A7 in Golgi glycosylation and ion homeostasis (Team 2) The contribution of SLC10A7 in Ca2+/ Mn2+ homeostasis will be fully studied. WP 2: Analysis of SLC10A7 deficiency specific consequences on endochondral and membranous ossification using Slc10a7-/- mouse model (Teams 1 and 3). Ion supplementation efficiency will be tested to reverse the GAG synthesis and glycosylation defects in Slc10a7-/- mice WP 3: Golgi glycosylation, toward new therapeutics (Teams 1, 2 and 3). It will include i) a structural analysis of glycoconjugates and a characterization of GAG synthesis defects in patient and mouse samples, ii) whole exome analysis on samples from SDM patients with still unknown molecular bases. The established framework will be applied to any relevant new gene.

Osteoarthritis (OA) is the most common joint disease, characterized by gradual loss of articular cartilage due to abnormal extracellular matrix (ECM) and changes in chondrocyte morphology and metabolism, associated to sub-endochondral bone remodeling and local synovitis. The burden of this disease has been gradually gaining importance in the last few decades with the aging of the population and the obesity epidemic. Beyond the huge healthcare costs for treatment of OA affecting 70 million individuals in Europe, there is no treatment that can repair the cartilage and stop the progress of OA. Existing therapies, based on Hyaluronic acid and Chondroitin sulfate injections, are symptomatic and pursue only pain alleviation with no effect on slowing disease progression and on restoring cartilage and chondrocytes functions. In parallel, new therapeutic strategies are currently based on stem cells, but these fragile cells are injected in an inflammatory microenvironment detrimental to their survival and clinical efficacy. Therefore, a more suitable middle is highly mandatory. GAGOSOME project is born from the observation that OA is closely related to a loss of proteoglycans (PGs), one of the largest component of the ECM. These PGs are not only structural components, but regulators of cell functions also since they interact with growth factors, cytokines, proteinases, adhesion receptors and extracellular matrix components through their sulfated glycosaminoglycan (GAGs) chains. As a consequence, these polysaccharides are new important classes of molecular targets in the fields of biochemistry, pathology and pharmacology. However, due to the natural extractive source of PGs and GAGs and their inherent complexity in terms of relative molecular mass, charge density, sulfation patterns, the relationship with functions are difficult to elucidate and their therapeutic and commercial use is complicated according to their poorly defined structures. Use of well-defined biomimetic structures are therefore the valuable alternatives for therapeutic strategies. Attemps have relied on the idea that a limited number of sulfate groups on a smaller oligosaccharide scaffold may overcome the difficulties of working with GAGs or PGs. Examples of these include GAG related polysaccharides of non-mammal origin that have shown to stimulate healing of tissues. And, very recently, glycopolymers with oligomers of GAGs as side chains revealed fascinating ability to recapitulate biological features of natural PGs. Even for these short GAGs, their syntheses still pose significant challenges. These shortcomings can be remedied, through the GAG-LIKE project, by designing readily accessible GAG oligosaccharide mimetics with size-homogeneous and enabling controlled sulfation pattern. Additionally, as for native PGs, multi-presentation of GAG mimetics on a polymeric backbone will be achieved. Up to date, these approaches have never been investigated, especially in order to promote the articular cartilage homeostasis. The GAG-LIKE project aims to develop PGs-like biopolymers made of architecturally defined grafted polyesters having sulfonates functional groups or simplified sulfated GAG mimetics. These glycomimetics will be assessed for their abilities to potentiate functional properties of mesenchymal stem cells and chondrocytes with applications in cartilage repair. The panel of biomimetic structures would greatly facilitated the fundamental explorations into the importance of macromolecular structure (e.g., sulfation pattern, density, distance, molecular weight and multivalency) and relationships with physicochemical properties in solution and with biological activities. Forces aligned within this consortium will combine crucial expertise in chemo-enzymatic modification of oligosaccharides (CERMAV-Grenoble), in the preparation of functional polyesters (ICMPE-Paris Est), and in the study of GAGs on the mesenchymal stem cells properties (CRRET-Paris Est & IBS-Grenoble).

We have performed an exome sequencing of a family of patients with an aggregation of very severe forms of ankylosing spondylitis. We identified a rare missense variant of the BbSp gene carried by all family members with a severe form of the disease. The corresponding amino acid is highly conserved across mammals, attesting its functional importance. This gene is involved in bone metabolism and we wish to confirm its causal role in the ossifications of AS by i) exome sequencing of 200 patients with a severe form of SA ii) phenotypic study in CT and histology of an animal mouse model carrying the same mutation of the murine BbSp gene; we will look for ossifications of the spine and entheses iii) the use of a cell culture model of chondrocytes and osteoblasts from these mice to understand the functional consequences of this mutation on their maturation and cellular functions.

Regenerative therapy based on the use of mesenchymal stem cells (MSC) is a promising approach for the treatment of stroke. The beneficial effects of MSC appears to be mainly related to the secretion of cellular factors and/or extracellular vesicles (EV). Heparan sulfates present on the surface of producing and recipient cells as well as on EV could play a critical role in the EV-mediated communication. The MAESTROVE project will explore, through the use of innovative approaches and models developed by 4 partners, the ability of combining human MSC-derived EV with a HS mimetic (HSm) agent, i.e. OTR4132, to enhance EV-mediated tissue regeneration and functional recovery following stroke, thus opening a new and rapid perspective for a development more easily industrialized for the treatment of stroke. The research hypothesis of the project lies on the fact that OTR4132 will create/restore a favourable tissue environment in which MSC-derived EV will be satisfactorily trapped and thus exert their beneficial effects in an optimal manner in the damaged brain tissue after stroke. Moreover, in vitro MSC priming with OTR4132 could improve EV biogenesis, cargo composition and regenerative properties. Combining HSm-based matrix therapy and MSC-derived EV therapy has never been studied. Another originality of this project is to test a priming strategy of MSC with this HSm-based matrix therapy to improve EV cargo constitution. This project will also evaluate for the first time a high-yield and scalable turbulence EV production approach for post-ischemic stroke treatment. This therapeutic strategy will be tested in relevant animal models of stroke towards the clinic, including the integration of the main comorbidity factor, i.e. the pre-existence of chronic arterial hypertension and an original non-human primate model. Overall, this project aims to provide an improved EV-based therapy that could represent a new clinically cell-free feasible paradigm for stroke. MAESTROVE gathers 4 partners with complementary expertise, most of them collaborating for a long time together with publications, patents and joint funding including 3 academic laboratories (Partner 1: ISTCT unit, Partner 3: Gly-CRRET unit and Partner 4: MSC unit) and 1 SME (Partner 2: OTR3). This project is subdivided into 5 Work Packages with WP1 for project management; WP2 for EV production by turbulence and characterization; WP3 for in vitro potency studies of EV (derived from MSC primed or not with OTR4132) in association or not with OTR4132 on neuronal, glial and endothelial cells survival submitted to an ischemic-like stress; WP4 for effects of combined therapies (OTR4132/EV) in different stroke models in the rat and marmoset; WP5 for effects of combined therapies (OTR4132/EV) on endogenous GAG modifications induced by stroke and blood markers identification of treatment efficacy. The MAESTROVE project proposes to develop a new therapy concept with the combination of OTR4132 and EV produced by human MSC. The success of the project is strongly supported by the proven effectiveness of OTR4132 and combined OTR4132/MSC in stroke tissues. Partner 2 has exclusive and worldwide rights for the engineering of matrix agents (RGTA®, ReGeneraTing Agents including OTR4132) and its applications in nervous system pathologies. Besides, the development of GMP EV production by turbulence from human MSC is on ingoing for a future clinical via a bioproduction start-up (EverZom) exploiting the patent from Partner 4.