DataSheet1_Restoration of Sarco/Endoplasmic Reticulum Ca2+-ATPase Activity Functions as a Pivotal Therapeutic Target of Anti-Glutamate-Induced Excitotoxicity to Attenuate Endoplasmic Reticulum Ca2+ Depletion.docx

Glutamate-induced excitotoxicity is a pathological basis of many acute/chronic neurodegenerative diseases. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2b) is a membrane-embedded P-type ATPase pump that manages the translocation of calcium ions (Ca2+) from cytosol into the lumen of the endoplasmic reticulum (ER) calcium stores. It participates in a wide range of biological functions in the central nervous system (CNS). However, the role of SERCA2b in glutamate-induced excitotoxicity and its mechanism must be elucidated. Herein, we demonstrate that SERCA2b mutants exacerbate the excitotoxicity of hypo-glutamate stimulation on HT22 cells. In this study, SERCA2b mutants accelerated Ca2+ depletion through loss-of-function (reduced pumping capacity) or gain-of-function (acquired leakage), resulting in ER stress. In addition, the occurrence of ER Ca2+ depletion increased mitochondria-associated membrane formation, which led to mitochondrial Ca2+ overload and dysfunction. Moreover, the enhancement of SERCA2b pumping capacity or inhibition of Ca2+ leakage attenuated Ca2+ depletion and impeded excitotoxicity in response to hypo-glutamate stimulation. In conclusion, SERCA2b mutants exacerbate ER Ca2+-depletion-mediated excitotoxicity in glutamate-sensitive HT22 cells. The mechanism of disruption is mainly related to the heterogeneity of SERCA2b mutation sites. Stabilization of SRECA2b function is a critical therapeutic approach against glutamate-induced excitotoxicity. These data will expand understanding of organelle regulatory networks and facilitate the discovery and creation of drugs against excitatory/inhibitory imbalance in the CNS.

谷氨酸诱导的兴奋性毒性是许多急性/慢性神经退行性疾病病理学的基础。肌/内质网Ca2+-ATP酶（SERCA2b）是一种膜嵌入的P型ATP酶泵，负责将钙离子（Ca2+）从细胞质转运至内质网（ER）的钙储存腔室。它在中枢神经系统（CNS）中参与广泛的生物学功能。然而，SERCA2b在谷氨酸诱导的兴奋性毒性中的作用及其机制尚需阐明。本研究中，我们发现SERCA2b突变体加剧了低谷氨酸刺激对HT22细胞的兴奋性毒性。在本研究中，SERCA2b突变体通过功能丧失（泵送能力降低）或功能获得（获得泄漏）加速了钙的耗竭，导致内质网应激。此外，内质网钙的耗竭发生增加了线粒体相关膜的形成，进而导致线粒体钙超载和功能障碍。此外，增强SERCA2b的泵送能力或抑制钙泄漏减轻了钙的耗竭，并阻碍了低谷氨酸刺激引起的兴奋性毒性。总之，SERCA2b突变体加剧了谷氨酸敏感的HT22细胞中由内质网Ca2+-耗竭介导的兴奋性毒性。这种破坏的机制主要与SERCA2b突变位点的异质性相关。SRECA2b功能的稳定化是针对谷氨酸诱导的兴奋性毒性的关键治疗策略。这些数据将拓展对细胞器调控网络的了解，并促进中枢神经系统兴奋/抑制失衡相关药物的研究与开发。