Expression Patterns And Function Of Synaptic Vesicle Protein 2A In Astrocytes Of Hippocampus In Temporal Lobe Epilepsy

BackgroundTemporal lobe epilepsy(TLE) is the most common form of medically intractable epilepsy in adults, but its mechanism is largely unknown. Reactive astrogliosis in epileptogenic zone, one of the most important pathologic changes in temporal lobe epilepsy, may participate in the process of epileptogenesis. Thus, more research focused on the astrocytic function may shed new light on the treatment of epilepsy.Astrocytes release gliotransmitters to regulate neural activities synchronously. Like the way of neural quantal release, exocytosis mediated by synaptic vesicles is a vital path for gliotransmission, which requires a series of secretory proteins interacted. Synaptic vesicle protein 2A(SV2A), which makes a role in exocytosis and calcium homeostasis of synaptic vesicle constituents, has been proved to be an positive molecular in synaptic transmission, as well as the binding site of novel antiepileptic drug levetiracetam. Proepileptic phenotype of SV2A-deficient mice in previous studies suggested SV2 A may lead to increase seizure vulnerability and accelerated epileptogenesis. Preliminary experiment suggested astrocytes expressed SV2 A under pathologic conditions. So we led to the hypothesis that, SV2 A expression in activated astrocyte may promote excitability of local neurocircut and finally contribute to epileptogenicity. ObjectiveTo observe the spatiotemporal expression of SV2 A in hippocampus from TLE patients and rat lithium-pilocarpine model, then define astrocyte-specific expression and subcellular distribution of SV2 A in chronic phase and finally investigate the role of SV2 A in excitatory mechanism of activated astrocytes in vitro. MethodsIn the first part, we observe SV2 A expression in hippocampus and temporal lobe resected from patients with TLE-HS as well as its distribution in subfield of hippocampus in TLE model 24 hours, 1week, 4weeks and 8weeks after SE by Immunohistochemical(IHC) method. Then we analyzed SV2 A total m RNA and protein level quantitatively in hippocampus corresponding to different phases above. In the second part, we examined astrocytic SV2 A expression both in rat and patients during progression of epilepsy by immunofluorescence, especially its subcellular localization 4weeks after SE by immune electron microscopy. Finally, we treated cultured astrocytes with LPS to mimic astrocytes in sclerotic hippocampus to detect SV2 A protein and distribution after 24 hours and 48 hours. On the other side, SV2 A protein change was also detected when astrocytes were given Ca2+ mobilization inducer ATP alone or combined with LPS after 12 hours and 24 hours. Down regulation of astrocytic SV2 A expression with si RNA, we observe Ca2+ sensor Syt-4 m RNA expression and its tendency under the same condition that elevated SV2 A expression. Results(1) IHC showed that SV2 A decreased in hippocampus and temporal lobe resected from patients with TLE-HS as well as in stratum lucidum and hilus in pilocarpine model 1week and 4weeks after SE(P<0.05).Hippocampal SV2 A m RNA and protein level coincidently decreased 24 hours, 1week and 4weeks after SE(P<0.05).(2) Positive expression rate of astrocytic SV2 A showed significant increase in sclerotic hippocamps. SV2 A seldom expressed in astrocytes of saline control and in the acute phase after SE, but progressively increased in latent and chronic period. Detected with immune electron microscopy, positive SV2 A puncta was in a pattern around astrocytic somata and process in epileptic rats but was absent in control.(3) SV2 A expression increased when cultured astrocytes activated with LPS after 24 hours and 48hours(P<0.05) accompanied by wide distribution in cytoplasm and process. ATP alone didn’t affect SV2 A protein level but showed elevation with addition of LPS after 24 hours. Syt-4 m RNA expression was decreased when SV2 A was down regulated 48 hours; meanwhile Syt-4 m RNA level showed increased tendency when interfered with LPS or combined with ATP after 6, 12 and 24 hours, which also elevated SV2 A expression. ConclusionReduced expression of SV2 A presented in specimens of temporal lobe and sclerotic hippocampus, similarly as a region-specifically decrease in TLE model showed,which can be partly due to neuronal degeneration and damage of synaptic connections. Abnormal astrocytic and neural distribution of SV2 A in hippocampus during chronic phase indicated a possible contribution of SV2 A to the epileptogenicity of TLE. Ca2+ mobilization inducer ATP could elevate SV2 A expression on activated astrocytes in vitro; SV2 A influenced Syt-4 expression, a key molecular involved in calcium-dependent glutamate release in astrocyte.Thus, functional alterations of SV2 A on astrocytes may regulate Syt-4 to enhanced glutamate release and participate in synaptic reorganization during epileptogenisis.