Department of Pharmacy, Unit of Pharmacology and Toxicology and Center of Excellence for Biomedical Research, University of Genoa, 16148 Genoa, Italy.
Department of Neuroscience and Brain Technologies, Fondazione Istituto Italiano di Tecnologia, 16163 Genoa, Italy; Department of Experimental Medicine, University of Genoa, 16132 Genoa, Italy.
Neurobiol Dis. 2015 Feb;74:314-24. doi: 10.1016/j.nbd.2014.12.004. Epub 2014 Dec 10.
The impact of synaptic vesicle endo-exocytosis on the trafficking of nerve terminal heterotransporters was studied by monitoring membrane expression and function of the GABA transporter-1 (GAT-1) and of type-1/2 glycine (Gly) transporters (GlyT-1/2) at spinal cord glutamatergic synaptic boutons. Experiments were performed by inducing exocytosis in wild-type (WT) mice, in amphiphysin-I knockout (Amph-I KO) mice, which show impaired endocytosis, or in mice expressing high copy number of mutant human SOD1 with a Gly93Ala substitution (SOD1(G93A)), a model of human amyotrophic lateral sclerosis showing constitutively excessive Glu exocytosis. Exposure of spinal cord synaptosomes from WT mice to a 35mM KCl pulse increased the expression of GAT-1 at glutamatergic synaptosomal membranes and enhanced the GAT-1 heterotransporter-induced [(3)H]d-aspartate ([(3)H]d-Asp) release. Similar results were obtained in the case of GlyT-1/2 heterotransporters. Preventing depolarization-induced exocytosis normalized the excessive GAT-1 and GlyT-1/2 heterotransporter-induced [(3)H]d-Asp release in WT mice. Impaired endocytosis in Amph-I KO mice increased GAT-1 membrane expression and [(3)H]GABA uptake in spinal cord synaptosomes. Also the GAT-1 heterotransporter-evoked release of [(3)H]d-Asp was augmented in Amph-I KO mice. The constitutively excessive Glu exocytosis in SOD1(G93A) mice resulted in augmented GAT-1 expression at glutamatergic synaptosomal membranes and GAT-1 or GlyT-1/2 heterotransporter-mediated [(3)H]d-Asp release. Thus, endo-exocytosis regulates the trafficking of GAT-1 and GlyT-1/2 heterotransporters sited at spinal cord glutamatergic nerve terminals. As a consequence, it can be hypothesized that the excessive GAT-1 and GlyT-1/2 heterotransporter-mediated Glu release, in the spinal cord of SOD1(G93A) mice, is due to the heterotransporter over-expression at the nerve terminal membrane, promoted by the excessive Glu exocytosis.
通过监测 GABA 转运体-1（GAT-1）和 1/2 型甘氨酸（Gly）转运体（GlyT-1/2）在脊髓谷氨酸能突触末梢的膜表达和功能，研究了突触小泡内吞-外排对神经末梢异源转运体运输的影响。在野生型（WT）小鼠、内吞作用受损的 amphiphysin-I 敲除（Amph-I KO）小鼠或表达高拷贝数突变型人 SOD1（G93A）的小鼠中诱导外排，进行了实验，G93A），一种表现出持续过度 Glu 外排的人类肌萎缩侧索硬化症模型。WT 小鼠脊髓突触体暴露于 35mM KCl 脉冲增加了谷氨酸能突触小体膜上 GAT-1 的表达，并增强了 GAT-1 异源转运体诱导的[(3)H]d-天冬氨酸([(3)H]d-Asp)释放。GlyT-1/2 异源转运体也得到了类似的结果。防止去极化诱导的外排使 WT 小鼠过度的 GAT-1 和 GlyT-1/2 异源转运体诱导的[(3)H]d-Asp 释放正常化。Amph-I KO 小鼠的内吞作用受损增加了脊髓突触体中的 GAT-1 膜表达和[(3)H]GABA 摄取。GlyT-1/2 异源转运体诱导的[(3)H]d-Asp 释放也在 Amph-I KO 小鼠中增加。SOD1(G93A)小鼠中持续过度的 Glu 外排导致谷氨酸能突触小体膜上 GAT-1 表达增加，以及 GAT-1 或 GlyT-1/2 异源转运体介导的[(3)H]d-Asp 释放增加。因此，内吞-外排调节了位于脊髓谷氨酸能神经末梢的 GAT-1 和 GlyT-1/2 异源转运体的运输。因此，可以假设 SOD1(G93A)小鼠脊髓中过度的 GAT-1 和 GlyT-1/2 异源转运体介导的 Glu 释放是由于神经末梢膜上异源转运体的过度表达，这是由过度的 Glu 外排所促进的。
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