GM1 as apoptotic signal in neurodegenerative GM1-gangliosidosis

描述（由申请人提供）：拟议研究的目标是深入了解神经退行性GM1-神经节脂质病（GM1）中中枢神经系统发病机制的分子机制。这种溶酶体贮积病（LSD）是由溶酶体2-半乳糖苷酶（2-gal）缺乏引起的，导致溶酶体降解和储存GM1-神经节苷脂（GM1）受损。神经节苷脂是细胞膜的基本成分，而GM1是脊椎动物大脑中的主要神经节苷脂。GM1的积累有助于疾病的发病机制，但所涉及的分子途径在很大程度上仍未被探索。我们成功培育了2- gal-/-小鼠，这是一种与人类GM1非常相似的动物模型，它促进了在儿童身上很难或不可能进行的研究。在最后一个给予期，我们开始了对2-gal-/-小鼠模型中神经元细胞死亡的全面分析。这些研究的发现有助于深入了解GM1在正常细胞代谢中的作用，并使我们能够为本提案制定以下2个具体目标。在目的1中，我们将阐明在2-gal-/-小鼠中，在内质网膜水平上GM1积累导致Ca2+释放的病理后果，进而激活UPR和神经元细胞死亡。我们将确定GM1是否直接影响er特异性Ca2+泵和控制细胞内Ca2+水平的通道的生化特性。我们将采用复杂的体外和体内试验来验证GM1与这些内质网膜蛋白的潜在物理相互作用。在Aim 2中，我们提出研究在2-gal-/-小鼠细胞内Ca2+稳态破坏介导的神经元凋亡过程中，内质网和线粒体之间是否发生串扰。这些研究是基于这样的假设，即从内质网过量释放Ca2+会引起细胞质中Ca2+的不平衡，这反过来可能影响其他细胞器，特别是线粒体。线粒体形态和功能的分析将平行于细胞内Ca2+运输的直接测量。我们还将检测GM1是否直接影响线粒体膜的通透性，通过检测这种神经节苷脂是否被纳入线粒体膜，从而扰乱线粒体完整性。考虑到Bcl-2蛋白家族在内质网和线粒体介导的细胞凋亡中的关键作用，以及它们与内质网和线粒体膜组分的相互作用，我们还将确定GM1是否可能通过激活这些凋亡因子而作用于内质网Ca2+释放的上游。相关性：GM1是一种影响婴儿和儿童的灾难性神经退行性疾病。我们能够充分了解GM1中导致大脑细胞死亡的事件。提出的研究也可能揭示由GM1中积累的分子控制的基本生物过程。这一知识对于为患有GM1的儿童以及可能患有其他lsd的儿童设计新的治疗方法至关重要。

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 neurodegenerative GM1-gangliosidosis (GM1). This lysosomal storage disease (LSD) is caused by deficiency of lysosomal 2-galactosidase (2-gal) that results in impaired lysosomal degradation and storage of GM1-ganglioside (GM1). Gangliosides are basic components of cell membranes, and GM1 is the primary ganglioside in the vertebrate brain. The accumulation of GM1 contributes to disease pathogenesis, but the molecular pathways involved remain largely unexplored. Our successful generation of 2- gal-/- mice, an animal model that closely resembles GM1 in humans, has facilitated studies that would be difficult or impossible to undertake in children. During the last granting period, we initiated a comprehensive analysis of neuronal cell death in the 2-gal-/- mouse model. Findings from those studies have contributed insight about the role of GM1 in normal cell metabolism and enabled us to develop the following 2 Specific Aims for this proposal. In Aim 1, we will elucidate the pathologic consequences of GM1 accumulation at the level of the ER membrane that leads to Ca2+release and, in turn, activation of the UPR and neuronal cell death in 2-gal-/- mice. We will determine whether GM1 directly influences the biochemical properties of the ER-specific Ca2+ pump and channels that control intracellular Ca2+ levels. We will apply sophisticated in vitro and in vivo assays to verify a potential physical interaction of GM1 with these ER membrane proteins. In Aim 2 we propose to investigate whether a cross-talk between the ER and the mitochondria occurs during the neuronal apoptosis mediated by the disruption of intracellular Ca2+ homeostasis in the 2-gal-/- mice. These studies are based on the hypothesis that excessive release of Ca2+ from the ER provokes Ca2+ imbalance in the cytosol, which, in turn, may impact on other organelles, in particular the mitochondria. Analyses of mitochondrial morphology and function will be paralleled by direct measurement of intracellular Ca2+ trafficking. We will also examine whether GM1 directly affects mitochondrial membrane permeability by testing if this ganglioside is incorporated in the mitochondrial membranes and, hence, perturbs mitochondrial integrity. Considering the pivotal role assigned to the Bcl-2 family of proteins in both ER- and mitochondria-mediated apoptosis, and their interplay with both ER and mitochondrial membrane components we will also determine whether GM1 may act upstream of ER Ca2+ release by activating these apoptogenic factors. RELEVANCE: GM1 is a catastrophic neurodegenerative disease that affects infants and children. We are in the position to gain full understanding of the events in GM1 that cause cell death in the brain. The proposed studies may also reveal basic biological processes controlled by the molecules that are accumulated in GM1. This knowledge is essential for designing new therapies for children with GM1 and possibly those with other LSDs.