Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.
Neurobiol Dis. 2011 Dec;44(3):292-303. doi: 10.1016/j.nbd.2011.07.010. Epub 2011 Jul 26.
Glutamate is loaded into synaptic vesicles by vesicular glutamate transporters (VGLUTs), and alterations in the transporters expression directly regulate neurotransmitter release. We investigated changes in VGLUT1 and VGLUT2 protein levels after ischemic and excitotoxic insults. The results show that VGLUT2 is cleaved by calpains after excitotoxic stimulation of hippocampal neurons with glutamate, whereas VGLUT1 is downregulated to a lower extent. VGLUT2 was also cleaved by calpains after oxygen/glucose deprivation (OGD), and downregulated after middle cerebral artery occlusion (MCAO) and intrahippocampal injection of kainate. In contrast, VGLUT1 was not affected after OGD. Incubation of isolated synaptic vesicles with recombinant calpain also induced VGLUT2 cleavage, with a little effect observed for VGLUT1. N-terminal sequencing analysis showed that calpain cleaves VGLUT2 in the C-terminus, at Asn(534) and Lys(542). The truncated GFP-VGLUT2 forms were found to a great extent in non-synaptic regions along neurites, when compared to GFP-VGLUT2. These findings show that excitotoxic and ischemic insults downregulate VGLUT2, which is likely to affect glutamatergic transmission and cell death, especially in the neonatal period when the transporter is expressed at higher levels.
谷氨酸通过囊泡谷氨酸转运体（VGLUTs）被装载到突触小泡中，而转运体表达的改变直接调节神经递质的释放。我们研究了缺血和兴奋毒性刺激后 VGLUT1 和 VGLUT2 蛋白水平的变化。结果表明，谷氨酸刺激海马神经元后，钙蛋白酶可使 VGLUT2 被切割，而 VGLUT1 的下调程度较低。钙蛋白酶还可切割氧/葡萄糖剥夺（OGD）后的 VGLUT2，以及大脑中动脉闭塞（MCAO）和海马内注射海人藻酸后的 VGLUT2。相比之下，OGD 后 VGLUT1 不受影响。用重组钙蛋白酶孵育分离的突触小泡也可诱导 VGLUT2 切割，而对 VGLUT1 的影响较小。N 端测序分析表明，钙蛋白酶在 C 端切割 VGLUT2，在 Asn（534）和 Lys（542）处切割。与 GFP-VGLUT2 相比，发现 GFP-VGLUT2 形成的截断形式在神经突的非突触区域中大量存在。这些发现表明，兴奋毒性和缺血性损伤可下调 VGLUT2，这可能会影响谷氨酸能传递和细胞死亡，尤其是在转运体表达水平较高的新生儿期。
