Center for Neuroscience and Cell Biology, Department of Life Sciences, University of Coimbra, Coimbra, Portugal.
PLoS One. 2010 Apr 12;5(4):e10139. doi: 10.1371/journal.pone.0010139.
Glutamic acid decarboxylase is responsible for synthesizing GABA, the major inhibitory neurotransmitter, and exists in two isoforms--GAD65 and GAD67. The enzyme is cleaved under excitotoxic conditions, but the mechanisms involved and the functional consequences are not fully elucidated. We found that excitotoxic stimulation of cultured hippocampal neurons with glutamate leads to a time-dependent cleavage of GAD65 and GAD67 in the N-terminal region of the proteins, and decrease the corresponding mRNAs. The cleavage of GAD67 was sensitive to the proteasome inhibitors MG132, YU102 and lactacystin, and was also abrogated by the E1 ubiquitin ligase inhibitor UBEI-41. In contrast, MG132 and UBEI-41 were the only inhibitors tested that showed an effect on GAD65 cleavage. Excitotoxic stimulation with glutamate also increased the amount of GAD captured in experiments where ubiquitinated proteins and their binding partners were isolated. However, no evidences were found for direct GADs ubiquitination in cultured hippocampal neurons, and recombinant GAD65 was not cleaved by purified 20S or 26S proteasome preparations. Since calpains, a group of calcium activated proteases, play a key role in GAD65/67 cleavage under excitotoxic conditions the results suggest that GADs are cleaved after ubiquitination and degradation of an unknown binding partner by the proteasome. The characteristic punctate distribution of GAD65 along neurites of differentiated cultured hippocampal neurons was significantly reduced after excitotoxic injury, and the total GAD activity measured in extracts from the cerebellum or cerebral cortex at 24h postmortem (when there is a partial cleavage of GADs) was also decreased. The results show a role of the UPS in the cleavage of GAD65/67 and point out the deregulation of GADs under excitotoxic conditions, which is likely to affect GABAergic neurotransmission. This is the first time that the UPS has been implicated in the events triggered during excitotoxicity and the first molecular target of the UPS affected in this cell death process.
谷氨酸脱羧酶负责合成 GABA，即主要的抑制性神经递质，存在两种同工酶形式——GAD65 和 GAD67。该酶在兴奋毒性条件下被切割，但涉及的机制和功能后果尚未完全阐明。我们发现，谷氨酸对培养的海马神经元的兴奋毒性刺激导致蛋白质 N 端区域的 GAD65 和 GAD67 发生时间依赖性切割，并降低相应的 mRNA。GAD67 的切割对蛋白酶体抑制剂 MG132、YU102 和 lactacystin 敏感，并且 E1 泛素连接酶抑制剂 UBEI-41 也能阻断其切割。相比之下，MG132 和 UBEI-41 是唯一对 GAD65 切割有影响的测试抑制剂。用谷氨酸进行兴奋毒性刺激也增加了在分离泛素化蛋白及其结合伴侣的实验中捕获的 GAD 的量。然而，在培养的海马神经元中没有发现直接 GADs 泛素化的证据，并且重组 GAD65 未被纯化的 20S 或 26S 蛋白酶体制剂切割。由于钙激活蛋白酶 calpains 在兴奋毒性条件下 GAD65/67 切割中起关键作用，结果表明 GADs 在未知结合伴侣的泛素化和蛋白酶体降解后被切割。兴奋毒性损伤后，分化培养的海马神经元中 GAD65 沿着神经突的特征性点状分布明显减少，死后 24 小时（此时 GADs 部分切割）从小脑或大脑皮质提取物中测量的总 GAD 活性也降低。结果表明 UPS 在 GAD65/67 的切割中起作用，并指出兴奋毒性条件下 GADs 的失调，这可能影响 GABA 能神经传递。这是 UPS 首次被牵连到兴奋毒性过程中触发的事件中，也是 UPS 首次成为这种细胞死亡过程中受影响的分子靶点。
