Nerve terminal damage by beta-bungarotoxin: its clinical significance.
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We report here original data on the biological basis of prolonged neuromuscular paralysis caused by the toxic phospholipase A2 beta-bungarotoxin. Electron microscopy and immunocytochemical labeling with anti-synaptophysin and anti-neurofilament have been used to show that the early onset of paralysis is associated with the depletion of synaptic vesicles from the motor nerve terminals of skeletal muscle and that this is followed by the destruction of the motor nerve terminal and the degeneration of the cytoskeleton of the intramuscular axons. The postjunctional architecture of the junctions were unaffected and the binding of fluorescein-isothiocyanate-conjugated alpha-bungarotoxin to acetylcholine receptor was not apparently affected by exposure to beta-bungarotoxin. The re-innervation of the muscle fiber was associated by extensive pre- and post-terminal sprouting at 3 to 5 days but was stable by 7 days. Extensive collateral innervation of adjacent muscle fibers was a significant feature of the re-innervated neuromuscular junctions. These findings suggest that the prolonged and severe paralysis seen in victims of envenoming bites by kraits (elapid snakes of the genus Bungarus) and other related snakes of the family Elapidae is caused by the depletion of synaptic vesicles from motor nerve terminals and the degeneration of the motor nerve terminal and intramuscular axons. (+info)
A neomorphic syntaxin mutation blocks volatile-anesthetic action in Caenorhabditis elegans.
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The molecular mechanisms underlying general anesthesia are unknown. For volatile general anesthetics (VAs), indirect evidence for both lipid and protein targets has been found. However, no in vivo data have implicated clearly any particular lipid or protein in the control of sensitivity to clinical concentrations of VAs. Genetics provides one approach toward identifying these mechanisms, but genes strongly regulating sensitivity to clinical concentrations of VAs have not been identified. By screening existing mutants of the nematode Caenorhabditis elegans, we found that a mutation in the neuronal syntaxin gene dominantly conferred resistance to the VAs isoflurane and halothane. By contrast, other mutations in syntaxin and in the syntaxin-binding proteins synaptobrevin and SNAP-25 produced VA hypersensitivity. The syntaxin allelic variation was striking, particularly for isoflurane, where a 33-fold range of sensitivities was seen. Both the resistant and hypersensitive mutations decrease synaptic transmission; thus, the indirect effect of reducing neurotransmission does not explain the VA resistance. As assessed by pharmacological criteria, halothane and isoflurane themselves reduced cholinergic transmission, and the presynaptic anesthetic effect was blocked by the resistant syntaxin mutation. A single gene mutation conferring high-level resistance to VAs is inconsistent with nonspecific membrane-perturbation theories of anesthesia. The genetic and pharmacological data suggest that the resistant syntaxin mutant directly blocks VA binding to or efficacy against presynaptic targets that mediate anesthetic behavioral effects. Syntaxin and syntaxin-binding proteins are candidate anesthetic targets. (+info)
The synaptophysin-synaptobrevin complex: a hallmark of synaptic vesicle maturation.
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Exocytosis of synaptic vesicles requires the formation of a fusion complex consisting of the synaptic vesicle protein synaptobrevin (vesicle-associated membrane protein, or VAMP) and the plasma membrane proteins syntaxin and soluble synaptosomal-associated protein of 25 kDa (or SNAP 25). In search of mechanisms that regulate the assembly of the fusion complex, it was found that synaptobrevin also binds to the vesicle protein synaptophysin and that synaptophysin-bound synaptobrevin cannot enter the fusion complex. Using a combination of immunoprecipitation, cross-linking, and in vitro interaction experiments, we report here that the synaptophysin-synaptobrevin complex is upregulated during neuronal development. In embryonic rat brain, the complex is not detectable, although synaptophysin and synaptobrevin are expressed and are localized to the same nerve terminals and to the same pool of vesicles. In contrast, the ability of synaptobrevin to participate in the fusion complex is detectable as early as embryonic day 14. The binding of synaptoporin, a closely related homolog of synaptophysin, to synaptobrevin changes in a similar manner during development. Recombinant synaptobrevin binds to synaptophysin derived from adult brain extracts but not to that derived from embryonic brain extracts. Furthermore, the soluble cytosol fraction of adult, but not of embryonic, synaptosomes contains a protein that induces synaptophysin-synaptobrevin complex formation in embryonic vesicle fractions. We conclude that complex formation is regulated during development and is mediated by a posttranslational modification of synaptophysin. Furthermore, we propose that the synaptophysin-synaptobrevin complex is not essential for exocytosis but rather provides a reserve pool of synaptobrevin for exocytosis that can be readily recruited during periods of high synaptic activity. (+info)
A nerve growth factor mimetic TrkA antagonist causes withdrawal of cortical cholinergic boutons in the adult rat.
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Cholinergic neurons respond to the administration of nerve growth factor (NGF) in vivo with a prominent and selective increase of choline acetyl transferase activity. This suggests the possible involvement of endogenous NGF, acting through its receptor TrkA, in the maintenance of central nervous system cholinergic synapses in the adult rat brain. To test this hypothesis, a small peptide, C(92-96), that blocks NGF-TrkA interactions was delivered stereotactically into the rat cortex over a 2-week period, and its effect and potency were compared with those of an anti-NGF monoclonal antibody (mAb NGF30). Two presynaptic antigenic sites were studied by immunoreactivity, and the number of presynaptic sites was counted by using an image analysis system. Synaptophysin was used as a marker for overall cortical synapses, and the vesicular acetylcholine transporter was used as a marker for cortical cholinergic presynaptic sites. No significant variations in the number of synaptophysin-immunoreactive sites were observed. However, both mAb NGF30 and the TrkA antagonist C(92-96) provoked a significant decrease in the number and size of vesicular acetylcholine transporter-IR sites, with the losses being more marked in the C(92-96) treated rats. These observations support the notion that endogenously produced NGF acting through TrkA receptors is involved in the maintenance of the cholinergic phenotype in the normal, adult rat brain and supports the idea that NGF normally plays a role in the continual remodeling of neural circuits during adulthood. The development of neurotrophin mimetics with antagonistic and eventually agonist action may contribute to therapeutic strategies for central nervous system degeneration and trauma. (+info)
A case of synchronous double primary lung cancer with neuroendocrine features.
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We report a case of unique double primary lung cancers with neuroendocrine features in a 63-year-old male smoker. The mass in the left lower lobe (LLL) was a small cell/large cell carcinoma with spindle cell sarcomatous areas and organoid structure. The mass in the left upper lobe (LUL) was a tubular adenocarcinoma with neuroendocrine features including organoid nests showing occasional rosette formation, nuclear palisading in the periphery of the nests and positive immunoreaction for CD56, chromogranin A and synaptophysin. The difference in histological structures between the two masses led us to diagnose double primary lung cancer. The combination of small cell lung carcinoma and spindle cell carcinoma is very uncommon. The relationship between LLL and LUL tumors remains unclear. Multiple lung cancers with neuroendocrine features have only rarely been reported in the literature. The patient in our case died of widespread cancer 2 years and 4 months after the surgery without adjuvant chemotherapy, a longer postoperative survival time than in cases of ordinary extensive small cell lung cancer. Multiple lung cancers with neuroendocrine features are extremely rare and similar cases have not been reported in the literature. Neuroendocrine differentiation has attracted widespread attention and, therefore, examining neuroendocrine features in lung cancers is important. (+info)
Expression of human apolipoprotein E3 or E4 in the brains of Apoe-/- mice: isoform-specific effects on neurodegeneration.
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Apolipoprotein (apo) E isoforms are key determinants of susceptibility to Alzheimer's disease. The apoE4 isoform is the major known genetic risk factor for this disease and is also associated with poor outcome after acute head trauma or stroke. To test the hypothesis that apoE3, but not apoE4, protects against age-related and excitotoxin-induced neurodegeneration, we analyzed apoE knockout (Apoe-/-) mice expressing similar levels of human apoE3 or apoE4 in the brain under control of the neuron-specific enolase promoter. Neuronal apoE expression was widespread in the brains of these mice. Kainic acid-challenged wild-type or Apoe-/- mice had a significant loss of synaptophysin-positive presynaptic terminals and microtubule-associated protein 2-positive neuronal dendrites in the neocortex and hippocampus, and a disruption of neurofilament-positive axons in the hippocampus. Expression of apoE3, but not of apoE4, protected against this excitotoxin-induced neuronal damage. ApoE3, but not apoE4, also protected against the age-dependent neurodegeneration seen in Apoe-/- mice. These differences in the effects of apoE isoforms on neuronal integrity may relate to the increased risk of Alzheimer's disease and to the poor outcome after head trauma and stroke associated with apoE4 in humans. (+info)
Impairments in high-frequency transmission, synaptic vesicle docking, and synaptic protein distribution in the hippocampus of BDNF knockout mice.
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Brain-derived neurotrophic factor (BDNF) promotes long-term potentiation (LTP) at hippocampal CA1 synapses by a presynaptic enhancement of synaptic transmission during high-frequency stimulation (HFS). Here we have investigated the mechanisms of BDNF action using two lines of BDNF knockout mice. Among other presynaptic impairments, the mutant mice exhibited more pronounced synaptic fatigue at CA1 synapses during high-frequency stimulation, compared with wild-type animals. Quantitative analysis of CA1 synapses revealed a significant reduction in the number of vesicles docked at presynaptic active zones in the mutant mice. Synaptosomes prepared from the mutant hippocampus exhibited a marked decrease in the levels of synaptophysin as well as synaptobrevin [vesicle-associated membrane protein (VAMP-2)], a protein known to be involved in vesicle docking and fusion. Treatment of the mutant slices with BDNF reversed the electrophysiological and biochemical deficits in the hippocampal synapses. Taken together, these results suggest a novel role for BDNF in the mobilization and/or docking of synaptic vesicles to presynaptic active zones. (+info)
Immunolocalization of mitsugumin29 in developing skeletal muscle and effects of the protein expressed in amphibian embryonic cells.
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The temporal appearance and subcellular distribution of mitsugumin29 (MG29), a 29-kDa transmembrane protein isolated from the triad junction in skeletal muscle, were examined by immunohistochemistry during the development of rabbit skeletal muscle. MG29 appeared in the sarcoplasmic reticulum (SR) in muscle cells at fetal day 15 before the onset of transverse tubule (T tubule) formation. In muscle cells at fetal day 27, in which T tubule and triad formation is ongoing, both SR and triad were labeled for MG29. In muscle cells at newborn 1 day, the labeling of the SR had become weak and the triads were well developed and clearly labeled for MG29. Specific and clear labeling for MG29 was restricted to the triads in adult skeletal muscle cells. When MG29 was expressed in amphibian embryonic cells by injection of the cRNA, a large quantity of tubular smooth-surfaced endoplasmic reticulum (sER) was formed in the cytoplasm. The tubular sER was 20-40 nm in diameter and appeared straight or reticular in shape. The tubular sER was formed by the fusion of coated vesicles [budded off from the rough-surfaced endoplasmic reticulum (rER)] and vacuoles of rER origin. The present results suggest that MG29 may play important roles both in the formation of the SR and the construction of the triads during the early development of skeletal muscle cells. (+info)