Synaptotagmin-11 regulates immune functions of microglia in vivo.

Membrane trafficking pathways mediate key microglial activities such as cell migration, cytokine secretion, and phagocytosis. However, the underlying molecular mechanism remains poorly understood. Previously, we found that synaptotagmin-11 (Syt11), a non-Ca2+ -binding Syt associated with Parkinson's disease (PD) and schizophrenia, inhibits cytokine release and phagocytosis in primary microglia. Here we reported the in vivo function of Syt11 in microglial immune responses using an inducible microglia-specific Syt11-conditional-knockout (cKO) mouse strain. Syt11-cKO resulted in activation of microglia and elevated mRNA levels of IL-6, TNF-α, IL-1ß, and iNOS in various brain regions under both resting state and LPS-induced acute inflammation state in adult mice. In a PD mouse model generated by microinjection of preformed α-synuclein fibrils into the striatum, a reduced number of microglia migrated toward the injection sites and an enhanced phagocytosis of α-synuclein fibrils by microglia were found in Syt11-cKO mice. To understand the molecular mechanism of Syt11 function, we identified its direct binding proteins vps10p-tail-interactor-1a (vti1a) and vti1b. The linker domain of Syt11 interacted with both proteins and a peptide derived from it competitively inhibited the interaction of Syt11 with vti1a/vti1b in vitro and in cells. Importantly, application of this peptide induced more cytokine secretion in wild-type microglia upon LPS treatment, phenocopying defects in Syt11 knockdown cells. Altogether, we propose that Syt11 inhibits microglial activation in vivo and regulates cytokine secretion through interactions with vti1a and vti1b.

Synaptotagmin-11 Inhibits Synaptic Vesicle Endocytosis via Endophilin A1.

Synaptic vesicle (SV) endocytosis is a critical and well-regulated process for the maintenance of neurotransmission. We previously reported that synaptotagmin-11 (Syt11), an essential non-Ca2+-binding Syt associated with brain diseases, inhibits neuronal endocytosis (Wang et al., 2016). Here, we found that Syt11 deficiency caused accelerated SV endocytosis and vesicle recycling under sustained stimulation and led to the abnormal membrane partition of synaptic proteins in mouse hippocampal boutons of either sex. Furthermore, our study revealed that Syt11 has direct but Ca2+-independent binding with endophilin A1 (EndoA1), a membrane curvature sensor and endocytic protein recruiter, with high affinity. EndoA1-knockdown significantly reversed Syt11-KO phenotype, identifying EndoA1 as a main inhibitory target of Syt11 during SV endocytosis. The N-terminus of EndoA1 and the C2B domain of Syt11 were responsible for this interaction. A peptide (amino acids 314-336) derived from the Syt11 C2B efficiently blocked Syt11-EndoA1 binding both in vitro and in vivo Application of this peptide inhibited SV endocytosis in WT hippocampal neurons but not in EndoA1-knockdown neurons. Moreover, intracellular application of this peptide in mouse calyx of Held terminals of either sex effectively hampered both fast and slow SV endocytosis at physiological temperature. We thus propose that Syt11 ensures the precision of protein retrieval during SV endocytosis by inhibiting EndoA1 function at neuronal terminals.SIGNIFICANCE STATEMENT Endocytosis is a key stage of synaptic vesicle (SV) recycling. SV endocytosis retrieves vesicular membrane and protein components precisely to support sustained neurotransmission. However, the molecular mechanisms underlying the regulation of SV endocytosis remain elusive. Here, we reported that Syt11-KO accelerated SV endocytosis and impaired membrane partition of synaptic proteins. EndoA1 was identified as a main inhibitory target of Syt11 during SV endocytosis. Our study reveals a novel inhibitory mechanism of SV endocytosis in preventing hyperactivation of endocytosis, potentially safeguarding the recycling of synaptic proteins during sustained neurotransmission.

Dissection of complement and Fc-receptor-mediated pathomechanisms of autoantibodies to myelin oligodendrocyte glycoprotein.

Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) have recently been established to define a new disease entity, MOG-antibody-associated disease (MOGAD), which is clinically overlapping with multiple sclerosis. MOG-specific antibodies (Abs) from patients are pathogenic, but the precise effector mechanisms are currently still unknown and no therapy is approved for MOGAD. Here, we determined the contributions of complement and Fc-receptor (FcR)-mediated effects in the pathogenicity of MOG-Abs. Starting from a recombinant anti-MOG (mAb) with human IgG1 Fc, we established MOG-specific mutant mAbs with differential FcR and C1q binding. We then applied selected mutants of this MOG-mAb in two animal models of experimental autoimmune encephalomyelitis. First, we found MOG-mAb-induced demyelination was mediated by both complement and FcRs about equally. Second, we found that MOG-Abs enhanced activation of cognate MOG-specific T cells in the central nervous system (CNS), which was dependent on FcR-, but not C1q-binding. The identification of complement-dependent and -independent pathomechanisms of MOG-Abs has implications for therapeutic strategies in MOGAD.

Pathogenic Mutations Differentially Regulate Cell-to-Cell Transmission of α-Synuclein.

Recent studies suggest that the cell-to-cell spread of pathological α-synuclein (α-syn) plays important roles in the development of Parkinson's disease (PD). PD patients who carry α-syn gene mutations often have an earlier onset and more severe clinical symptoms and pathology than sporadic PD cases who carry the wild-type (WT) α-syn gene. However, the molecular mechanism by which α-syn gene mutations promote PD remains unclear. Here, we hypothesized that pathogenic mutations facilitate the intercellular transfer and cytotoxicity of α-syn, favoring an early disease onset and faster progression. We investigated the effects of eight known pathogenic mutations in human α-syn (A18T, A29S, A30P, E46K, H50Q, G51D, A53E, and A53T) on its pathological transmission in terms of secretion, aggregation, intracellular level, cytotoxicity, seeding, and induction of neuroinflammation in SH-SY5Y neuroblastoma cells, cultured rat neurons, and microglia, and the rat substantia nigra pars compacta. We found that 2 of the 8 mutations (H50Q and A53T) significantly increased α-syn secretion while 6 mutations (A18T, A29S, A30P, G51D, A53E, and E46K) tended to enhance it. In vitroα-syn aggregation experiments showed that H50Q promoted while G51D delayed aggregation most strongly. Interestingly, 3 mutations (E46K, H50Q, and G51D) greatly increased the intracellular α-syn level when cultured cells were treated with preformed α-syn fibrils (PFFs) compared with the WT, while the other 5 had no effect. We also demonstrated that H50Q, G51D, and A53T PFFs, but not E46K PFFs, efficiently seeded in vivo and acutely induced neuroinflammation in rat substantia nigra pars compacta. Our data indicate that pathogenic mutations augment the prion-like spread of α-syn at different steps and blockade of this pathogenic propagation may serve as a promising therapeutic intervention for PD.

Synaptotagmin-11 regulates the functions of caveolae and responds to mechanical stimuli in astrocytes.

Caveolae play crucial roles in intracellular membrane trafficking and mechanosensation. In this study, we report that synaptotagmin-11 (Syt11), a synaptotagmin isoform associated with Parkinson's disease and schizophrenia, regulates both caveolae-mediated endocytosis and the caveolar response to mechanical stimuli in astrocytes. Syt11-knockout (KO) accelerated caveolae-mediated endocytosis. Interestingly, the caveolar structures on the cell surface were markedly fewer in the absence of Syt11. Caveolar disassembly in response to hypoosmotic stimuli and astrocyte swelling were both impaired in Syt11-KO astrocytes. Live imaging revealed that Syt11 left caveolar structures before cavin1 during hypoosmotic stress and returned earlier than cavin1 after isoosmotic recovery. Chronic hypoosmotic stress led to proteasome-mediated Syt11 degradation. In addition, Syt11-KO increased the turnover of cavin1 and EH domain-containing protein 2 (EHD2), accompanied by compromised membrane integrity, suggesting a mechanoprotective role of Syt11. Direct interactions between Syt11 and cavin1 and EHD2, but not caveolin-1, are found. Altogether, we propose that Syt11 stabilizes caveolar structures on the cell surface of astrocytes and regulates caveolar functions under physiological and pathological conditions through cavin1 and EHD2.

Phagocytosis Assay for α-Synuclein Fibril Uptake by Mouse Primary Microglia.

Microglia are professional phagocytes in the brain and deficiency in their phagocytic activity plays an important role in Parkinson's disease. This protocol mainly describes the phagocytosis assay for uptake of α-synuclein preformed fibrils, a pathologic form of α-synuclein, by primary microglia.

Synaptotagmin-11 inhibits cytokine secretion and phagocytosis in microglia.

Cytokine secretion and phagocytosis are key functions of activated microglia. However, the molecular mechanisms underlying their regulation in microglia remain largely unknown. Here, we report that synaptotagmin-11 (Syt11), a non-Ca2+ -binding Syt implicated in Parkinson disease and schizophrenia, inhibits cytokine secretion and phagocytosis in microglia. We found Syt11 expression in microglia in brain slices and primary microglia. Interestingly, Syt11-knockdown (KD) increased cytokine secretion and NO release in primary microglia both in the absence and presence of lipopolysaccharide. NF-κB was activated in untreated KD microglia together with enhanced synthesis of IL-6, TNF-α, IL-1ß, and iNOS. When the release capacity was assessed by the ratio of extracellular to intracellular levels, only the IL-6 and TNF-α secretion capacity was increased in Syt11-KD cells, suggesting that Syt11 specifically regulates conventional secretion. Consistently, Syt11 localized to the trans-Golgi network and recycling endosomes. In addition, Syt11 was recruited to phagosomes and its deficiency enhanced microglial phagocytosis. All the KD phenotypes were rescued by expression of an shRNA-resistant Syt11, while overexpression of Syt11 suppressed cytokine secretion and phagocytosis. Importantly, Syt11 also inhibited microglial phagocytosis of α-synuclein fibrils, supporting its association with Parkinson disease. Taken together, we propose that Syt11 suppresses microglial activation under both physiological and pathological conditions through the inhibition of cytokine secretion and phagocytosis.