Zeng Chunmei, Lei Danqing, Lu Yuling, Huang Qi, Wu Ying, Yang Shengyu, Wu Yuan
Department of Neurology, The First Affiliated Hospital, Guangxi Medical University, #6 Shuangyong Road, Nanning, 530021, Guangxi, China.
Experimental Center of Life Sciences Institutes, Guangxi Medical University, #22 Shuangyong Road, Nanning, 530021, Guangxi, China.
Arch Biochem Biophys. 2023 Jan 15;734:109499. doi: 10.1016/j.abb.2022.109499. Epub 2022 Dec 29.
Parvalbumin-expressing neurons are a type of inhibitory intermediate neuron that play an important role in terminating seizures. The aim of the present study was to use lentiviral construction and packaging technology to overexpress and silence the parvalbumin gene in pheochromocytoma (PC12) cells, and to evaluate how parvalbumin influences the metabolic pathway involving glutamate and γ-aminobutyric acid (GABA). In this work, Immunofluorescence staining was used to verify the differentiation of PC12 cells into neurons after adding nerve growth factor (NGF). Western blotting and real-time quantitative polymerase chain reaction (qRT-PCR) were used to confirm lentivirus-mediated knockdown or overexpression of parvalbumin. Expression of parvalbumin, the 65-kDa GAD isoform (GAD65), and the 67-kDa GAD isoform (GAD67) in neuronal cells was examined at the mRNA and protein levels using qRT-PCR, western blotting and immunofluorescence staining, while intracellular glutamate and GABA levels were determined by high performance liquid chromatography (HPLC). We demonstrate that the expression of parvalbumin is associated with GAD65 and GAD67. Interestingly, overexpression of parvalbumin up-regulated GAD65 and GAD67, increased GABA concentration, and decreased glutamate concentration. Silencing of parvalbumin led to the opposite effects. Altogether, parvalbumin affected the expression of GAD65 and GAD67, thereby influencing the metabolic pathway involving glutamate and GABA.
表达小白蛋白的神经元是一种抑制性中间神经元，在癫痫发作终止中起重要作用。本研究的目的是利用慢病毒构建和包装技术在嗜铬细胞瘤（PC12）细胞中过表达和沉默小白蛋白基因，并评估小白蛋白如何影响涉及谷氨酸和γ-氨基丁酸（GABA）的代谢途径。在这项工作中，免疫荧光染色用于验证添加神经生长因子（NGF）后PC12细胞向神经元的分化。蛋白质印迹法和实时定量聚合酶链反应（qRT-PCR）用于确认慢病毒介导的小白蛋白敲低或过表达。使用qRT-PCR、蛋白质印迹法和免疫荧光染色在mRNA和蛋白质水平检测神经元细胞中小白蛋白、65-kDa谷氨酸脱羧酶同工型（GAD65）和67-kDa谷氨酸脱羧酶同工型（GAD67）的表达，同时通过高效液相色谱法（HPLC）测定细胞内谷氨酸和GABA水平。我们证明小白蛋白的表达与GAD65和GAD67相关。有趣的是，小白蛋白的过表达上调了GAD65和GAD67，增加了GABA浓度，并降低了谷氨酸浓度。小白蛋白的沉默导致相反的效果。总之，小白蛋白影响GAD65和GAD67的表达，从而影响涉及谷氨酸和GABA的代谢途径。
