GM1 at the ER-mitochondrion microdomains regulates Ca2+ signaling and apoptosis

描述(申请人提供)：拟议研究的目标是深入了解GM1-神经节苷脂沉积症(GM1)中枢神经系统发病的分子机制，这是一种由2-半乳糖苷酶(2-GAL)缺乏引起的灾难性神经退行性溶酶体储存疾病(LSD)。在这种疾病中，大量和进行性的GM1-神经节苷脂(GM1)积聚，特别是在中枢神经系统，被认为在疾病的发病机制中发挥了重要作用，尽管涉及的分子途径在很大程度上仍未被探索。神经节苷脂是一种含唾液酸的神经节苷脂(GSLS)，在神经系统中特别丰富。作为细胞内钙离子流量的关键调节因子，它们参与了细胞内钙信号的下游过程。因此，它们的化学成分和浓度的变化会改变正常的细胞功能，导致细胞死亡。我们成功地建立了一个与人类疾病非常相似的2-半/小鼠模型，使我们能够启动对儿童这种疾病所特有的神经退行性变的分子基础的全面分析。我们已经证明，在这些小鼠中，过高的细胞内GM1浓度通过耗尽内质网(ER)钙离子储存和激活未折叠蛋白反应(UPR)来诱导细胞凋亡。鉴于内质网和线粒体之间的空间相互作用以及钙离子在传递这些隔室发出的凋亡信号中的作用，我们现在计划研究GM1是否可以通过影响这些细胞器中的钙稳态来诱导内质网应激和线粒体介导的细胞凋亡。这项提案的两个目标中提出的研究旨在阐明以下几点。1)我们将研究GM1水平升高对内质网膜通道和调节细胞内钙离子浓度的泵的潜在影响(S)。2)我们将重点研究GM1在线粒体相关膜(MAM)特定膜成分上的作用，线粒体相关膜是内质网和线粒体膜并置的位置，调节这些细胞器之间的钙离子流动动力学。3)研究内质网钙离子耗竭对线粒体下游的影响。考虑到Bcl2蛋白家族在内质网和线粒体介导的细胞凋亡中的作用，以及它们对这些细胞器中钙离子水平的反应，4)我们将探索这些蛋白在神经节苷脂GM1特有的神经退行性事件中可能的额外参与。 公共卫生相关性：GM1神经节苷脂沉积症是一种灾难性的神经退行性疾病，影响婴儿和儿童，目前尚无治疗方法。我们有能力充分了解这种疾病中导致大脑细胞死亡的事件。我们相信，这些研究将能够识别新的发病机制，并可能揭示GM1控制的基本生物学过程。这些知识对于设计新的治疗GM1神经节苷脂沉积症和可能患有其他GSL储存性疾病的儿童是必不可少的。

DESCRIPTION (provided by applicant): The goal of the proposed research is to achieve in-depth understanding of the molecular mechanisms underlying the CNS pathogenesis in GM1-gangliosidosis, a catastrophic neurodegenerative lysosomal storage disease (LSD) caused by deficiency of 2-galactosidase (2-gal). In this disease massive and progressive accumulation of GM1-ganglioside (GM1) particularly in the CNS is thought to play a major role in disease pathogenesis, albeit the molecular pathways involved remain largely unexplored. Gangliosides are sialic acid- containing glycosphingolipids (GSLs) particularly abundant in the nervous system. As key modulators of intracellular Ca2+ flux, they have been implicated in cellular processes downstream of Ca2+ signaling. Changes in their chemical composition and concentration can therefore alter normal cell function and lead to cell death. Our successful generation of a 2-gal-/- mouse model that closely resembles the human disease has enabled us to initiate a comprehensive analysis of the molecular bases of neurodegeneration characteristic of this disorder in children. We have demonstrated that in these mice excessive intracellular concentration of GM1 induces apoptosis by depletion of endoplasmic reticulum (ER) Ca2+ stores and activation of an unfolded protein response (UPR). Given the spatial interplay between the ER and the mitochondria and the role of Ca2+ in conveying apoptotic signals emanating from these compartments, we now plan to investigate whether GM1 can induce both ER stress- and mitochondria-mediated apoptosis by influencing Ca2+ homeostasis in these organelles. The studies put forward in the two Aims of this proposal are designed to elucidate the following points. 1) We will examine the potential effect(s) of increased levels of GM1 on ER membrane channels and pump that regulate intracellular Ca2+ concentration. 2) We will focus on the role of GM1 on specific membrane components of the mitochondria-associated membranes or MAMs, the sites of juxtaposition between ER and mitochondrial membranes that regulate the kinetics of Ca2+ flux between these organelles. 3) We will study the downstream effects of ER Ca2+ depletion on the mitochondria. Considering the role of the Bcl-2 family of proteins in both ER- and mitochondria-mediated apoptosis, and their response to Ca2+ levels in these organelles, 4) we will explore a putative, additional involvement of these proteins in the neurodegenerative events characteristic of GM1-gangliosidosis. PUBLIC HEALTH RELEVANCE: GM1-gangliosidosis is a catastrophic neurodegenerative disease that affects infants and children for which there is no treatment. We are in the position to gain full understanding of the events in this disease that cause cell death in the brain. We are confident that these studies will enable the identification of novel mechanisms of pathogenesis and may reveal basic biological processes controlled by GM1. This knowledge is essential for designing new therapies for children with GM1-gangliosidosis and possibly those with other GSL storage diseases.