Molecular Neurobiology ( IF 4.6 ) Pub Date : 2022-08-25 , DOI: 10.1007/s12035-022-03003-1 Farhana Naznin 1, 2 , T M Zaved Waise 1 , Paul Fernyhough 1, 2
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Impairments in mitochondrial physiology play a role in the progression of multiple neurodegenerative conditions, including peripheral neuropathy in diabetes. Blockade of muscarinic acetylcholine type 1 receptor (M1R) with specific/selective antagonists prevented mitochondrial dysfunction and reversed nerve degeneration in in vitro and in vivo models of peripheral neuropathy. Specifically, in type 1 and type 2 models of diabetes, inhibition of M1R using pirenzepine or muscarinic toxin 7 (MT7) induced AMP-activated protein kinase (AMPK) activity in dorsal root ganglia (DRG) and prevented sensory abnormalities and distal nerve fiber loss. The human neuroblastoma SH-SY5Y cell line has been extensively used as an in vitro model system to study mechanisms of neurodegeneration in DRG neurons and other neuronal sub-types. Here, we tested the hypothesis that pirenzepine or MT7 enhance AMPK activity and via this pathway augment mitochondrial function in SH-SY5Y cells. M1R expression was confirmed by utilizing a fluorescent dye, ATTO590-labeled MT7, that exhibits great specificity for this receptor. M1R antagonist treatment in SH-SY5Y culture increased AMPK phosphorylation and mitochondrial protein expression (OXPHOS). Mitochondrial membrane potential (MMP) was augmented in pirenzepine and MT7 treated cultured SH-SY5Y cells and DRG neurons. Compound C or AMPK-specific siRNA suppressed pirenzepine or MT7-induced elevation of OXPHOS expression and MMP. Moreover, muscarinic antagonists induced hyperpolarization by activating the M-current and, thus, suppressed neuronal excitability. These results reveal that negative regulation of this M1R-dependent pathway could represent a potential therapeutic target to elevate AMPK activity, enhance mitochondrial function, suppress neuropathic pain, and enhance nerve repair in peripheral neuropathy.
中文翻译:
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毒蕈碱乙酰胆碱 1 型受体的拮抗作用通过人神经母细胞瘤 SH-SY5Y 细胞和原代神经元中的 AMPK 增强线粒体膜电位和呼吸链成分的表达
线粒体生理学受损在多种神经退行性疾病的进展中起作用,包括糖尿病中的周围神经病变。用特异性/选择性拮抗剂阻断毒蕈碱乙酰胆碱 1 型受体 (M 1 R) 可防止线粒体功能障碍和逆转周围神经病变体外和体内模型中的神经变性。具体来说,在 1 型和 2 型糖尿病模型中,抑制 M 1R 使用哌仑西平或毒蕈碱毒素 7 (MT7) 在背根神经节 (DRG) 中诱导 AMP 活化蛋白激酶 (AMPK) 活性,并防止感觉异常和远端神经纤维丢失。人神经母细胞瘤 SH-SY5Y 细胞系已被广泛用作体外模型系统来研究 DRG 神经元和其他神经元亚型的神经变性机制。在这里,我们检验了哌仑西平或 MT7 增强 AMPK 活性并通过该途径增强 SH-SY5Y 细胞线粒体功能的假设。M 1 R 的表达通过使用一种荧光染料 ATTO590 标记的 MT7 得到证实,该染料对该受体表现出极大的特异性。米1SH-SY5Y 培养物中的 R 拮抗剂处理增加了 AMPK 磷酸化和线粒体蛋白表达 (OXPHOS)。在哌仑西平和 MT7 处理的培养的 SH-SY5Y 细胞和 DRG 神经元中线粒体膜电位 (MMP) 增加。化合物 C 或 AMPK 特异性 siRNA 抑制哌仑西平或 MT7 诱导的 OXPHOS 表达和 MMP 升高。此外,毒蕈碱拮抗剂通过激活 M 电流诱导超极化,从而抑制神经元兴奋性。这些结果表明,这种 M 1 R 依赖性途径的负调节可能代表了提高 AMPK 活性、增强线粒体功能、抑制神经性疼痛和增强周围神经病变中神经修复的潜在治疗靶点。