Cleavage of the Vesicular GABA Transporter under Excitotoxic Conditions Is Followed by Accumulation of the Truncated Transporter in Nonsynaptic Sites
Cleavage of the Vesicular GABA Transporter under Excitotoxic Conditions Is Followed by Accumulation of the Truncated Transporter in Nonsynaptic Sites
复制标题
DOI：
10.1523/jneurosci.3541-10.2011
复制
发表时间：
2011-03-23
影响因子：
5.3
通讯作者：
Duarte, Carlos B.
中科院分区：
文献类型：
--
作者：
Gomes, Joao R.;Lobo, Andrea C.;Duarte, Carlos B.
GABA是CNS中主要的抑制性神经递质，GABA能神经传递的变化影响神经元网络的整体活性。GABA进入突触囊泡的摄取是由囊泡GABA转运蛋白（VGAT）介导的，转运蛋白表达的变化直接调节神经递质的释放。在这项工作中，我们研究了缺血和兴奋性毒性条件下VGAT蛋白水平的变化，这可能会影响死亡过程。我们发现，VGAT被切割的钙蛋白酶兴奋性毒性刺激海马神经元与谷氨酸，产生一个稳定的截短的裂解产物（tVGAT）。小鼠短暂性大脑中动脉闭塞（脑缺血模型）和海马内注射红藻氨酸后也观察到VGAT切割，但在过表达钙蛋白酶抑制剂钙蛋白酶抑制剂的转基因小鼠中没有观察到任何影响。用重组钙蛋白酶孵育分离的皮层突触囊泡也诱导VGAT的裂解和稳定的tVGAT的形成。使用靶向VGAT不同区域的抗体的免疫印迹实验和N-末端测序分析表明，钙蛋白酶在N-末端区域的氨基酸52和60处切割转运蛋白。表达GFP融合蛋白与全长VGAT或tVGAT的GABA能纹状体神经元的免疫细胞化学显示，转运蛋白的切割诱导突触传递的损失，导致蛋白质沿神经突沿着均匀分布。我们的研究结果表明，兴奋性毒性下调全长VGAT，伴随着tVGAT的产生，这可能会影响GABA能神经传递，并可能影响缺血期间的细胞死亡。
GABA is the major inhibitory neurotransmitter in the CNS and changes in GABAergic neurotransmission affect the overall activity of neuronal networks. The uptake of GABA into synaptic vesicles is mediated by the vesicular GABA transporter (VGAT), and changes in the expression of the transporter directly regulate neurotransmitter release. In this work we investigated the changes in VGAT protein levels during ischemia and in excitotoxic conditions, which may affect the demise process. We found that VGAT is cleaved by calpains following excitotoxic stimulation of hippocampal neurons with glutamate, giving rise to a stable truncated cleavage product (tVGAT). VGAT cleavage was also observed after transient middle cerebral artery occlusion in mice, a cerebral ischemia model, and following intrahippocampal injection of kainate, but no effect was observed in transgenic mice overexpressing calpastatin, a calpain inhibitor. Incubation of isolated cerebrocortical synaptic vesicles with recombinant calpain also induced the cleavage of VGAT and formation of stable tVGAT. Immunoblot experiments using antibodies targeting different regions of VGAT and N-terminal sequencing analysis showed that calpain cleaves the transporter in the N-terminal region, at amino acids 52 and 60. Immunocytochemistry of GABAergic striatal neurons expressing GFP fusion proteins with the full-length VGAT or tVGAT showed that cleavage of the transporter induces a loss of synaptic delivery, leading to a homogeneous distribution of the protein along neurites. Our results show that excitotoxicity downregulates full-length VGAT, with a concomitant generation of tVGAT, which is likely to affect GABAergic neurotransmission and may influence cell death during ischemia.