The Effect And Potential Mechanism Of TUBB3 Involved In Epilepsy

Part one: The expression of TUBB3 in temporal lobe epileptic foci in patients and experimental animalsObjective: Here we investigated the expression of TUBB3 in temporal lobe epileptic foci in intractable epilepsy patients and experimental animals in order to explore the probable relationship between TUBB3 expression and temporal lobe epilepsy.Method:1. Human brain tissues from 30 patients undergoing operation for intractable TLE were randomly selected from brain tissue bank. Meanwhile, 10 patients tissue with no history of epilepsy who undergoing surgery because of head trauma were also obtained from the brain tissue bank used as controls.2. Hippocampus and cortex between spontaneously group and non-spontaneously group in lithium chloride- pilocarpine induced epileptic male SD rat weighing 220±20 g were obtained 2 month after SE(n=8).3. Hippocampus and cortex of rats with fully kindled and without fully kindled in PTZ kindling model were obtained(n=8).4. All the tissues above were examined by Western-blot and Immunofluorescent staining for investigation TUBB3 expression between epileptic groups and control groups.Result:Western blot: The expression of TUBB3 was significantly higher in temporal neocortex of TLE patients than that in controls(★P<0.05). In the cortex and hippocampus of two chronic epilepsy rat models: TUBB3 expression in spontaneous seizures group was significantly increased than in non-spontaneous seizures group(★★★P < 0.001, # p < 0.05) in pilocarpine model when spontaneously seizure demonstrated; TUBB3 expression in rats with fully kindled was significantly increased than rats without fully kindled(★P<0.05, ##p<0.01).To explore the function of TUBB3 in epilepsy, we observed that TUBB3 was colocalized with GAD67, a marker for the production of the inhibitory neurotransmitter GABA, and associated with inhibitory postsynaptic clusters of gephyrin in sections of human cortex. However, TUBB3 was not colocalized with astrocytic marker glial fibrillary acidic protein(GFAP). We observed the pre-determined outcomes both in the hippocampus and cortex of epileptic rat brain.Conclusion: TUBB3 was showed increased in TLE patient and rats in two chronic epilepsy rat models. TUBB3 was enriched in these neurons and colocalized with GAD67 and associated with inhibitory postsynaptic clusters of gephyrin in sections of brain tissue. These indicated that TUBB3 may play an important role in the development of epilepsy.Part two: The effect of TUBB3 on chronic seizure recurrence and seizure precipitation in two epilepsy modelsObjective: To further study whether TUBB3 play a role on epilepsy, we investigated the behavioral changes of epileptic rats performed by adeno-associated viral(AAV) mediated knockdown and overexpression of TUBB3 in the hippocampus.Method:1. We constructed the AAV vectors encoding a short hairpin RNA(sh RNA) directed against TUBB3, as well as TUBB3 overexpression vectors. Control vectors expressed GFP under the control of the same promoter.2. To confirm that the AAV vectors were efficient, Western-blot and Immunofluorescent staining were performed to detect hippocampal AAV-TUBB3-sh and AAV-TUBB3 in rats at 3 and/or 9 weeks after injection.3. Behavioral assays. We chose different pilocarpine and PTZ doses to test specific hypotheses to decrease the overall number of animals required and to avoid ceiling and floor effects. For example, to test the hypothesis that the TUBB3 overexpression rats have more severe seizures than controls, we used a PTZ dose(33 mg/kg, injected every other day) that would fully kindled in a small percentage of the control rats. The opposite was true for the experiments with the TUBB3 knockdown rats; to efficiently test the hypothesis that the rats knocked down TUBB3 have less severe seizures, we used a PTZ dose(40 mg per kg) that fully kindled in a large percentage of the control rats. The video recording was analyzed by two independent observers who were blinded to the group allocation.Result: 1. AAV bearing GFP was localized in the hippocampus 3 and 9 weeks after the AAV injection. The expression of TUBB3 was significantly reduced 3 and 9 weeks in all rats treated with AAV-TUBB3-sh compared with the control and AAV-GFP groups, TUBB3 overexpression group was significantly increased compared with the control and AAV-GFP groups(★★P<0.01).2. In PTZ-induced kindling model, downregulation of TUBB3 effectively delayed the development of PTZ-induced kindling in rats. This was demonstrated by a significant decrease in the seizure severity scores in all assessed periods in AAV-TUBB3-sh group compared with that in the AAV-GFP group. TUBB3 overexpression had opposite effect(★P<0.05, ★★P<0.01).3. In chloride-pilocarpine induced epilepsy model, downregulation of TUBB3 reduced the number and severity of SRS from week 3 to 6 after SE in the AAV-TUBB3-sh group, compared with that in the AAV-GFP group. TUBB3 overexpression had opposite effect(★P<0.05, ★★P<0.01, ★★★P<0.001).Conclusion: 1. The expression of endogenous TUBB3 in the hippocampus was successfully suppressed/increased in vivo by AAV-TUBB3-sh/ AAV-TUBB3, and the efficiency get to a stable level at week 3 till week 9.2. Downregulation of TUBB3 effectively delayed the development of PTZ-induced kindling in rats. TUBB3 overexpression had opposite effect.3. Downregulation of TUBB3 reduces the number and severity of SRS in chloride-pilocarpine induced epilepsy model. TUBB3 overexpression had opposite effect.Part three: The potential mechanism of TUBB3 involved in epilepsyObjective: To further explore probable underlying mechanisms of TUBB3 involved in epilepsy, we investigated the synaptic inhibition of epileptic rats performed by adeno-associated viral mediated knockdown and overexpression of TUBB3 in the hippocampus.Method: 1. Three epileptic groups with hippocampal injection of AAV-GFPAAV-TUBB3-sh RNA, and AAV-TUBB3, respectivly.2. Whole cell patch-clamp recordings of CA1 pyramidal neurons in brain slices were performed, and the miniature inhibitory postsynaptic currents(m IPSCs) and paired-pulse ratio(PPR) were recorded.3. Western blot analysis was used for investigate of the total or the surface / total ratio of GABA-A recepters expression in the epileptic rats brain.4. Immunoprecipitation was used for analysis the relationship between TUBB3 with GABARAP and GABA-A receptors in the epileptic rats.Result:1. We found that m IPSC amplitude was significantly increased in AAV-TUBB3-sh group(★P<0.05). TUBB3 overexpression had opposite effect(★P<0.05). However, the frequency was not statistically different between two groups. Consistent with the significant changed m IPSC amplitude, this effect was probably due to a alteration in postsynaptic function as there were no significant differences between AAV-GFP and AAV-TUBB3-sh group in PPR, which is indicative of presynaptic release probability at inhibitory synapses(P>0.05).2. Western blot analysis showed a significant increase in the surface / total ratio of GABA-AR β2/3 subunits in AAV-TUBB3-sh group compared with the AAV-GFP group(★P<0.05), whereas that of Glu R2/3 was unchanged. TUBB3 overexpression had opposite effect.3. Immunoprecipitation showed TUBB3 coimmunoprecipitates with GABARAP and GABA-A receptors in epileptic rats brain lysate.Conclusion: Downregulation of TUBB3 could enhance GABA-mediated m IPSC in amplitude. TUBB3 overexpression had opposite effect. Thus, TUBB3 could regulate postsynaptic GABA-A receptor mediated synaptic transmission in epileptic condition.