| The release of neurotransmitter from synaptic vesicle is an important step for neuronal communication. SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins comprise the core machinery of synaptic exocytosis. Moreover, many other proteins play important regulating roles in this machinery. Munc-18 has been known to operate as the chief commander of the exocytotic SNARE team, controlling every step in the exocytotic pathway in central nervous system. In 2000, an article first reported the identification of high-titer antibodies against Munc-18 in the serum and cerebrospinal fluid from a patient with Rasmussen's Encephalitis (RE) which is a disease of childhood characterized by progressive destruction of cerebral hemisphere and manifests as an intractable seizure disorder. In 2008, Hirotomo Saitsu, et al. found a missense mutation in the gene encoding protein of Munc-18 in a girl with EIEE (Early infantile epileptic encephalopathy with suppression-burst). These studies suggest that Munc-18 plays an important role in the central nervous system, and the dysfunction of this protein may induce occurrence of diseases of central nervous system. Previous studies mainly focused their attention on how to regulate neurotransmitter releasing process by Munc-18 in cytoplasm. However, Vandana M. Sharma et al. reported the nuclear localization of Munc-18 in neurons in 2005. They also demonstrated the binding of Munc-18-1 to double stranded DNA, suggesting Munc-18 may be involved in the regulation of gene expression. So far, little is known about the relationship between disorders of nervous system and the distribution of Munc-18 in nucleus. On the basis of the studies mentioned above, we therefore investigated the effect of neuronal excitation on the redistribution of Munc-18 in neuronal nucleus based on kainic-acid (KA)-induced epileptic model and primary cultured neurons treated by glutamate. The results are summarized as follow:1. First we have confirmed the presence of Munc-18 in cytoplasm and nucleus in neurons from rat hippocampus by immunohistochemistry and immunoelectron microscopy. The nucleus of neuron and glia from rat hippocampus were isolated by differential centrifugation, and analyzed by Western blot which results showed that Munc-18 mainly distributed in the neuronal nucleus, meanwhile the very weak signal of Munc-18 was detected in the fraction of glia nucleus. 2. Two epileptic animal models were used in this study, which included the KunMing mice peritoneally injected with Kainic acid (KA) (10mg/kg) and SD rats intrahippocampusly injected with KA (1μl,1μg/μl). After appearing epileptic seizures, the animals were sacrificed, and the brain slices of these rats were analyzed by immunohistochemical staining for Munc-18. We found that the immunopositive cells were decreased in CA3 and Dentate Gyrus of hippocampus, meanwhile we observed that immunopositive signal significantly decreased in the neuronal nucleus. Western blot analysis showed that, compared with control group, Munc-18 expression did not significantly change in hippocampus of epileptic animals induced by KA, but the distribution of Munc-18 in nucleus fraction decreased as well as in cytoplasm fraction of hippocampus neurons of rats. Meanwhile, Western blot analysis also showed SNAP-25 expression in hippocampus of epileptic animals induced by KA increased compared with control group.3. After 50μM glutamate acid treatment for 3 hours, immunoflorescent staining showed that distribution of Munc-18 in neurites of primary cultured neurons increased and in nucleus of neurons decreased. Western blot analysis showed that, compared with control group, no significant changes of Munc-18 expression in primary cultured neurons received 50μM glutamate treatment for 3 hr were observed, but the distribution of Munc-18 in nucleus of neurons received glutamate treatment obviously decreased and in cytoplasm of primary cultured neurons increased. Meanwhile, Western blot analysis showed SNAP-25 and Tra-2βexpressions in primary cultured neurons received 50μM glutamate treatment increased compared with control group.4. Analysis of amino acid sequences of Munc-18 showed there are two clusters of amino acid residues at# 20-31 and# 457-467 that are characteristic of bipartite nuclear localization signal (NLS) sequence, and a cluster of amino acids at residue # 405-414 representing an potential nuclear export signal (NES), suggesting its ability to be imported and exported out of the nucleus. The recombinant eukaryotic expression vector pDsRed-N1-munc-18-FL (full length) and pDsRed-N1-munc-18-N (carboxyl-terminus deleted which contains one NLS sequence) was constructed by respectively cloning full length and carboxyl-terminus deleted munc-18 cDNA into the vector pDsRed-N1, and subsequently transfected in SH-SY5Y cells. At 24 hr after transfection, we observed that full length and carboxyl-terminus deleted Munc-18 which fused a sequence of red fluorescent protein both could be located in the nucleus of SH-SY5Y cell. It suggests that the two nuclear localization signal sequences both involved in the process of Munc-18 entering into the nucleus.Above all, our results showed that, when neuron was excited, the distribution of Munc-18 in nucleus of neuron decreased and SNAP-25 and Tra-2βexpressions in neuron increased. It suggests that the distribution variation of Munc-18 in nucleus of neuron is regulated by the state of neuronal activity. Whether the increasing of SNAP-25 and Tra-2βexpression in neuron is directly related to the decreasing of Munc-18 in the nucleus of neuron remains to be further studied... |
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