RNA干扰endophilin A2抑制大鼠海马神经元突触囊泡内吞

RNA interference of endophilin A2 inhibits synaptic vesicle endocytosis in rat hippocampal neurons

目的:探讨内吞蛋白(endophilin)A2对大鼠海马神经元突触囊泡内吞的影响。方法:设计干扰endophilin A2的小分子干扰片段,通过Western blot的方法筛选出针对endophilin A2有效的特异性干扰片段;用免疫荧光的方法验证干扰片段对神经元内源性endophilin A2的干扰效果;利用双全细胞膜片钳的方法,记录不同刺激方案下的兴奋性突触后电流(excitatory postsynaptic currents,EPSCs)的情况,以明确endophilin A2对突触囊泡循环的影响。结果:(1)免疫荧光结果显示endophilin A2的干扰片段能显著减少海马神经元内源性endophilin A2的表达(P<0.05);(2)双全细胞膜片钳结果显示,0.1 Hz的低频刺激下,干扰endophilin A2表达的神经元EPSCs大小与阴性对照组没有明显差异;而无论是单串高频刺激下还是多串高频刺激下,干扰endophilin A2表达的神经元标准化EPSCs的下降程度都明显增强(P<0.05)。结论:(1)成功筛选出特异性干扰endophilin A2的有效干扰片段;(2)干扰endophilin A2的表达不影响海马神经元突触囊泡胞吐;(3)干扰endophilin A2的表达可抑制大鼠海马神经元突触囊泡内吞。

AIM： To investigate the effect of endophilin A2 on synaptic vesicle endocytosis in rat hippocampal neurons. METHODS： The siRNAs for endophilin A2 isoform were designed,and the efficacy and specificity of these siRNAs were determined by Western blot. Immunostaining was performed to verify the interference efficiency of the siRNA for endogenous endophilin A2 in the hippocampal neurons. Excitatory postsynaptic currents（ EPSCs） were recorded on cultured hippocampal neurons using dual whole-cell recordings by evoking the transfected presynaptic neurons in various stimulation patterns. RESULTS： The result of immunostaining in cultured hippocampal neurons demonstrated that the siRNA of endophilin A2 inhibited the expression of endogenous endophilin A2 significantly（ P〈0. 05）. The results of dual wholecell recordings showed that no significant difference in the EPSCs amplitude evoked between endophilin A2 knockdown neurons and control was observed when stimulated at low frequency（ 0. 1 Hz）. The amplitude of normalized EPSCs evoked in endophilin A2 knockdown neurons decreased significantly for both single-train and multiple-train stimulations（ P〈0. 05）.CONCLUSION： An effective siRNA of endophilin A2 was screened out successfully. Knockdown of endophilin A2 isoform does not affect synaptic vesicle exocytosis,but inhibits synaptic vesicle endocytosis in rat hippocampal neurons.