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

描述（由申请方提供）：拟定研究的目的是深入了解GM1-神经节苷脂沉积症CNS发病机制的分子机制，GM1-神经节苷脂沉积症是一种由2-半乳糖苷酶（2-gal）缺乏引起的灾难性神经退行性溶酶体贮积病（LSD）。在这种疾病中，GM1-神经节苷脂（GM1）的大量和进行性积累，特别是在中枢神经系统中被认为在疾病的发病机制中起着重要作用，尽管所涉及的分子途径仍然在很大程度上未被探索。神经节苷脂是含有唾液酸的鞘糖脂（GSL），在神经系统中特别丰富.作为细胞内Ca2+通量的关键调节剂，它们涉及Ca2+信号传导下游的细胞过程。因此，其化学成分和浓度的变化可以改变正常的细胞功能并导致细胞死亡。我们成功地产生了与人类疾病非常相似的2-gal-/-小鼠模型，使我们能够对儿童这种疾病的神经变性特征的分子基础进行全面分析。我们已经证明，在这些小鼠中，过多的细胞内浓度的GM1诱导细胞凋亡的内质网（ER）的Ca2+存储和激活的未折叠蛋白反应（UPR）的耗尽。鉴于ER和线粒体之间的空间相互作用和Ca2+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Ca2+稳态来诱导ER应激和ESTA介导的凋亡。本建议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水平增加对调节细胞内Ca2+浓度的ER膜通道和泵的潜在影响。2)我们将专注于GM1的作用，对特定的膜组件的ESTA相关的膜或MAMs，ER和线粒体膜，调节这些细胞器之间的Ca2+通量的动力学之间的并列的网站。3)我们将研究ER Ca2+耗竭对线粒体的下游影响。考虑到Bcl-2蛋白家族在ER和Bcl-2介导的细胞凋亡中的作用，以及它们对这些细胞器中Ca2+水平的反应，4）我们将探索这些蛋白在GM1-神经节苷脂沉积症特征性神经退行性事件中的推定的额外参与。 公共卫生相关性：GM1-神经节苷脂沉积症是一种影响婴儿和儿童的灾难性神经退行性疾病，目前尚无治疗方法。我们能够充分了解这种疾病中导致大脑细胞死亡的事件。我们相信，这些研究将能够确定新的发病机制，并可能揭示由GM1控制的基本生物学过程。这些知识对于设计治疗GM1-神经节苷脂沉积症儿童以及可能患有其他GSL储存疾病的儿童的新疗法至关重要。

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.