Zacharie Taoufiq

Current proteomic studies clarified canonical synaptic proteins that are common to many types of synapses. However, proteins of diversified functions in a subset of synapses are largely hidden because of their low abundance or structural similarities to abundant proteins. To overcome this limitation, we have developed an "ultra-definition" (UD) subcellular proteomic workflow. Using purified synaptic vesicle (SV) fraction from rat brain, we identified 1,466 proteins, three times more than reported previously. This refined proteome includes all canonical SV proteins, as well as numerous proteins of low abundance, many of which were hitherto undetected. Comparison of UD quantifications between SV and synaptosomal fractions has enabled us to distinguish SV-resident proteins from potential SV-visitor proteins. We found 134 SV residents, of which 86 are present in an average copy number per SV of less than one, including vesicular transporters of nonubiquitous neurotransmitters in the brain. We provide a fully annotated resource of all categorized SV-resident and potential SV-visitor proteins, which can be utilized to drive novel functional studies, as we characterized here Aak1 as a regulator of synaptic transmission. Moreover, proteins in the SV fraction are associated with more than 200 distinct brain diseases. Remarkably, a majority of these proteins was found in the low-abundance proteome range, highlighting its pathological significance. Our deep SV proteome will provide a fundamental resource for a variety of future investigations on the function of synapses in health and disease.

Cerebral malaria, one of the most serious complications of Plasmodium falciparum infection, is characterized by the sequestration of parasitized red blood cells (PRBCs) in cerebral microvascular beds. The precise mechanisms involved in the onset of neuropathology remain unknown, but parasite sequestration in the brain, metabolic disturbances, and host immune responses all play a role. Sequestration of PRBCs is mediated by different endothelial cell surface receptors, mainly ICAM-1 and CD36. In vitro studies demonstrated that PRBC adhesion to endothelial cells induces over-expression of various adhesion molecules including ICAM-1, expression of iNOS, oxidative stress and finally apoptosis in endothelial cells. In vivo studies, in humans and in mice models of cerebral malaria brought striking evidence of the implication of brain infiltrating cytotoxic effector CD8T lymphocytes in the development of murine cerebral malaria pathogenesis. These cells probably act by direct cytotoxicity against endothelial cells. Cytotoxicity and apoptosis potentially lead blood-brain-barrier disruption and could contribute to the development of cerebral malaria. We propose a key role for endothelial cells in the pathogenesis of cerebral malaria, both by suicide / apoptosis, and / or by murder / cytotoxicity.

-Synuclein is a presynaptic protein the function of which has yet to be identified, but its neuronal content increases in patients of synucleinopathies including Parkinson's disease. Chronic overexpression of -synuclein reportedly expresses various phenotypes of synaptic dysfunction, but the primary target of its toxicity has not been determined. To investigate this, we acutely loaded human recombinant -synuclein or its pathological mutants in their monomeric forms into the calyces of Held presynaptic terminals in slices from auditorily mature and immature rats of either sex. Membrane capacitance measurements revealed significant and specific inhibitory effects of WT monomeric -synuclein on vesicle endocytosis throughout development. However, the -synuclein A53T mutant affected vesicle endocytosis only at immature calyces, whereastheA30Pmutanthadnoeffectthroughout.TheendocyticimpairmentbyWT-synucleinwasrescuedbyintraterminalcoloadingofthe microtubule (MT) polymerization blocker nocodazole. Furthermore, it was reversibly rescued by presynaptically loaded photostatin-1, a photoswitcheable inhibitor of MT polymerization, in a light-wavelength-dependent manner. In contrast, endocytic inhibition by the A53T mutant at immature calyces was not rescued by nocodazole. Functionally, presynaptically loaded WT -synuclein had no effect on basal synaptic transmission evoked at a low frequency, but significantly attenuated exocytosis and impaired the fidelity of neurotransmission during prolonged high-frequency stimulation. We conclude that monomeric WT -synuclein primarily inhibits vesicle endocytosis via MT overassembly, thereby impairing high-frequency neurotransmission.

Acute clinical manifestations of falciparum malaria, such as multiorgan failure and cerebral malaria, occur unpredictably and lead to coma and death within hours if left untreated. Despite the emergency administration of effective antimalarial drugs, 15%-20% of patients die. Other therapeutic approaches are therefore urgently needed. There is increasing evidence that endothelial changes play a key role in the pathogenesis of severe malaria. We therefore used coculture models to study interactions between infected erythrocytes and endothelium. We found that adhesion of Plasmodium falciparum to endothelial cells in vitro activated the Rho kinase signaling pathway, which is strongly involved in various vascular diseases. When added concomitantly with parasites, the Rho kinase inhibitor fasudil (HA-1077), a drug already in clinical use, decreased both NF-kappaB activation and endothelial cell apoptosis. Fasudil also helped to restore endothelial barrier integrity after P. falciparum adhesion. Rho kinase inhibition thus appears to be a promising adjunctive therapeutic approach to the management of severe human malaria.