Cholesterol reduction impairs exocytosis of synaptic vesicles.

Cholesterol and sphingolipids are abundant in neuronal membranes, where they help the organisation of the membrane microdomains involved in major roles such as axonal and dendritic growth, and synapse and spine stability. The aim of this study was to analyse their roles in presynaptic physiology. We first confirmed the presence of proteins of the exocytic machinery (SNARES and Ca(v)2.1 channels) in the lipid microdomains of cultured neurons, and then incubated the neurons with fumonisin B (an inhibitor of sphingolipid synthesis), or with mevastatin or zaragozic acid (two compounds that affect the synthesis of cholesterol by inhibiting HMG-CoA reductase or squalene synthase). The results demonstrate that fumonisin B and zaragozic acid efficiently decrease sphingolipid and cholesterol levels without greatly affecting the viability of neurons or the expression of synaptic proteins. Electron microscopy showed that the morphology and number of synaptic vesicles in the presynaptic boutons of cholesterol-depleted neurons were similar to those observed in control neurons. Zaragozic acid (but not fumonisin B) treatment impaired synaptic vesicle uptake of the lipophilic dye FM1-43 and an antibody directed against the luminal epitope of synaptotagmin-1, effects that depended on the reduction in cholesterol because they were reversed by cholesterol reloading. The time-lapse confocal imaging of neurons transfected with ecliptic SynaptopHluorin showed that cholesterol depletion affects the post-depolarisation increase in fluorescence intensity. Taken together, these findings show that reduced cholesterol levels impair synaptic vesicle exocytosis in cultured neurons.

[Recent advance in research for myasthenia gravis, in relation to various antibodies affecting synaptic structure and function].

Autoantibodies impair acetylcholine receptor (AChR) in myasthenia gravis (MG) and P/Q-type voltage-gated calcium channel (VGCC) in Lambert-Eaton myasthenic syndrome (LEMS). (1) Some of MG and LEMS patients are "seronegative" for respective antibodies or modified by antibodies that recognize other proteins than AChR and VGCC such as MuSK, AChR allosteric site, membrane Na+ channel and ryanodine receptor-1 (RyR1) in MG, and synaptotagmin-1 in LEMS. (2) Autoimmune responses affect the proteins participating in the mechanisms to compensate for synaptic disorders on the basis of presynaptic Ca2+ homeostasis provided by VGCC and non-VGCC (receptor-operated TRPCs): they act as enhancers of Ca(2+) -mediated ACh release via phospholipase C signaling pathways including M1-type presynaptic muscarinic AChR, neurotrophin receptor (TrkB), and fast-mode of synaptic vesicle recycling. (3) The pathophysiology contributive to contractile fatigue in MG includes RyR1 and also TRPC3. The TRPC3 also forms a complex with STIM1 and Orail to make up for Ca2+ after sarcoplasmic Ca2+ release. The prevalent detection of anti-TRPC3 antibodies in MG with thymoma could affect muscle contractile machineries in addition to anti-RyR1-induced affection. (4) When one faces "seronegative" MG, one should be cautious to conformation-specific antibodies and also congenital myasthenic syndromes.

JunB is required for IgE-mediated degranulation and cytokine release of mast cells.

Mast cells are effector cells of IgE-mediated immune responses frequently found at the vicinity of blood vessels, the margins of diverse tumors and at sites of potential infection and inflammation. Upon IgE-mediated stimulation, mast cells produce and secrete a broad spectrum of cytokines and other inflammatory mediators. Recent work identified JunB, a member of the AP-1 transcription factor family, as critical regulator of basal and induced expression of inflammatory mediators in fibroblasts and T cells. To study the impact of JunB on mast cell biology, we analyzed JunB-deficient mast cells. Mast cells lacking JunB display a normal in vivo maturation, and JunB-deficient bone marrow cells in vitro differentiated to mast cells show no alterations in proliferation or apoptosis. But these cells exhibit impaired IgE-mediated degranulation most likely due to diminished expression of SWAP-70, Synaptotagmin-1, and VAMP-8, and due to impaired influx of extracellular calcium. Moreover, JunB-deficient bone marrow mast cells display an altered cytokine expression profile in response to IgE stimulation. In line with these findings, the contribution of JunB-deficient mast cells to angiogenesis, as analyzed in an in vitro tube formation assay on matrigel, is severely impaired due to limiting amounts of synthesized and secreted vascular endothelial growth factor. Thus, JunB is a critical regulator of intrinsic mast cell functions including cross-talk with endothelial cells.

An antibody to synaptotagmin I facilitates synaptic transmission.

Proper functioning of the nervous system requires precise control of neurotransmitter release. Synaptotagmin, a synaptic vesicle protein, is crucial for the temporal control of neurotransmitter release. The mechanism of synaptotagmin function is still under debate. To investigate the mechanism by which synaptotagmin controls neurotransmitter release, we injected an antibody of rat synaptotagmin I into a crayfish motor axon. We found that the antibody enhanced synaptic transmission at crayfish neuromuscular junctions by increasing the amplitude of the evoked synaptic response. This effect was antibody-dose dependent. The antibody also reduced the rise time of the synaptic potentials. These effects were accompanied by a reduction in the Hill coefficient for Ca(2+)-dependence of synaptic transmission. Our findings support the hypothesis that synaptotagmin inhibits neurotransmitter release in the absence of Ca(2+).

[Noninvasive detection of experimental vulnerable atherosclerotic plaques with 99Tc m labeled C2A domain of synaptotagmin I].

OBJECTIVE: (99)Tc(m) labeled C2A domain of synaptotagmin I ((99)Tc(m)-Syt I-C2A) is used for noninvasive detection of vulnerable atherosclerotic plaque. METHODS: Recombinant C2A domain of synaptotagmin I, overexpressed in E. Coli, was thiolated with 2-iminothiolane (2-IT) and labeled with (99)Tc(m). Atherosclerotic plaques were produced in 5 rabbits by deendothelialization of the abdominal aorta and the rabbits were fed with cholesterol diet for 3 months. Three rabbits not manipulated served as normal controls. All animals were injected with (99)Tc(m)-Syt I-C2A and underwent in vivo imaging thereafter. Aortas were then explanted for ex vivo imaging and histological characterization. RESULTS: In deendothelialized animals, intense radio-uptake in abdominal aorta, showed by gamma camera at 2 h after injection, was visualized and T/B was 3.25 +/- 0.51 by ROI measurement, quantitative uptake ratio of abdominal aortas with atherosclerotic lesions to thoracic aortas was 8.39 +/- 1.74 in ex vivo imaging. The mean uptake in specimens of abdominal aortas with lesions was 12.6-fold higher than in control abdominal aortas, and 10.2-fold higher than in thoracic aortas of deendothelialized animals by gamma-counter. CONCLUSION: (99)Tc(m)-Syt I-C2A has a high affinity for vulnerable atherosclerotic plaque and is a suitable a gent for the noninvasive detection of vulnerable atherosclerotic plaque.