ER Network Shaping Mechanisms in the Hereditary Spastic Paraplegias

遗传性痉挛性截瘫的 ER 网络塑造机制

• 批准号: 9563114
• 负责人: Craig Blackstone
• 金额: $ 205.9万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9563114
• 关键词: ATP phosphohydrolase; American; Amyotrophic Lateral Sclerosis; Animal Model; Area; Axon; Biogenesis; CRISPR/Cas technology; Cell Line; Cells; Cellular biology; Charcot-Marie-Tooth Disease; Clinical; Collaborations; Confocal Microscopy; Cytoskeleton; Data; Defect; Disease; Dystonia; Electron Microscopy; Endoplasmic Reticulum; Family; Fatty acid glycerol esters; Functional disorder; Genes; Genetic; Genets; Goals; Guanosine Triphosphate Phosphohydrolases; Hela Cells; Hereditary Spastic Paraplegia; Human; Imaging Techniques; In Situ; Inherited; Investigation; Journals; Knock-in; Knock-out; Laboratories; Lead; Length; Lipids; Magnetic Resonance Spectroscopy; Maps; Membrane; Microscopy; Microtubules; Mitochondrial Diseases; Modeling; Molecular; Molecular Biology; Molecular Genetics; Morphology; Motor Neurons; Mutant Strains Mice; Mutate; Mutation; Natural History; Neurodegenerative Disorders; Neurons; Pathogenesis; Pathway interactions; Patients; Process; Production; Protein Isoforms; Proteins; Publishing; Recruitment Activity; Reporting; Research; Resolution; Science; Shapes; Spastic Paraplegia; Spastic Paraplegia, Hereditary, Autosomal Dominant; Stem cells; Techniques; Tissues; Tubular formation; United States National Institutes of Health; Work; X-Ray Computed Tomography; axonopathy; clinical investigation; clinically relevant; fly; functional group; gene product; genetic manipulation; hereditary neuropathy; in vivo; induced pluripotent stem cell; insight; member; mouse model; nervous system disorder; neurogenetics; novel; prevent; response; spastin; structural biology; trafficking

Research in the Cell Biology Section, Neurogenetics Branch focuses on the molecular mechanisms underlying a number of neurodegenerative disorders, including mitochondrial disorders, dystonia, and the hereditary spastic paraplegias (HSPs). These disorders, which together afflict millions of Americans, worsen insidiously over a number of years, and treatment options are limited for many of them. Our laboratory is investigating inherited forms of these disorders, using molecular and cell biology approaches to study how mutations in disease genes ultimately result in cellular dysfunction. In this project, we are focusing on the HSPs. One major research theme involves the characterization and functional analysis of the hereditary spastic paraplegia type 3A (SPG3A) protein, atlastin-1. In 2009, we reported in the journal Cell that atlastin-1 is a member of a ubiquitous family of GTPases that interact with two families of ER shaping proteins to generate the tubular endoplasmic reticulum (ER) network. Interestingly, atlastin-1 interacts with the SPG31 protein REEP1, which is an ER shaping protein, as well as the SPG4 protein spastin, a microtubule-severing ATPase. In 2010, we published a study in the Journal of Clinical Investigation demonstrating that these three proteins interact with one another to organize the tubular ER network in conjunction with the microtubule cytoskeleton. Since SPG3A, SPG4, and SGP31 account for well over 50% of all HSP cases, we suggest ER network defects as the predominant neuropathologic mechanism for the HSPs. This is supported by the recent identification of numerous other HSP proteins that regulate ER morphology, including CPT1C protein (in collaboration with Dr. Kurt Fischbeck). Over the past year, we have continued to develop animal models for SPG31 (knock out) and SPG3A (knockout and knock in), as well as double mutant mice, to evaluate the extent of ER morphology changes using both in vivo and ex vivo studies. We are employing both high-throughput FIB-SEM electron microscopy and super-resolution confocal microscopy to examine the changes in tubular ER within neuronal axons in response to these genetic manipulations. In addition, we have identified interactions of these proteins with several other proteins mutated in the HSPs, expanding the number of HSP cases related to defects in ER network formation. Furthermore, we are actively generating in situ models for many of the HSPs through the production of patient-derived, induced pluripotent stem cells that are then differentiated into telecephalic neurons, in collaboration with Dr. Xue-Jun Li. A number of these studies were published in 2014 in the journals Stem Cells and Human Molecular Genetics. ER morphology and dynamics in these and other cells are being evaluated using a number of emerging super-resolution microscopy techniques in collaboration with Drs. Jennifer Lippincott-Schwartz, Eric Betzig, and Harald Hess; soime of this work was published in Science in 2016. Finally, we are working with Dr. Niamh O'Sullivan on fly models of these HSPs. A key aspect of ER function possibly related to disease pathogenesis is the formation of lipid droplets, and this is an area of emphasis for our cellular and organismal studies. We are in the process of completing several studies tying changes in ER morphology to alterations in lipid droplet biogenesis. As part of these studies, we have used CRISPR technologies to knock out all three atlastin isoforms from cell lines such as HeLa and NIH-3T3. We have also utilized advanced imaging techniques such as CT scans and MR spectroscopy to study changes in fat tissue in HSP mouse models non-invasively; one such study was published in Hum Mol Genet in 2016. These data were used for the planning of a large clinical natural history trial in patients with the three most common forms of autosomal dominant HSP (SPG4, SPG3A, and SPG31), which has just begun recruiting. Taken together, we expect that our studies will advance our understanding of the molecular pathogenesis of the HSPs. Such an understanding at the molecular and cellular levels will hopefully lead to novel treatments to prevent the progression of these disorders.

神经遗传学分支细胞生物学部分的研究重点是一些神经退行性疾病的分子机制，包括线粒体疾病，肌张力障碍和遗传性痉挛性截瘫（HSP）。 这些疾病共同折磨着数百万美国人，在数年内不知不觉地恶化，其中许多人的治疗选择有限。 我们的实验室正在研究这些疾病的遗传形式，使用分子和细胞生物学方法来研究疾病基因的突变如何最终导致细胞功能障碍。 在这个项目中，我们专注于HSPs。一个主要的研究主题涉及遗传性痉挛性截瘫3A型（SPG 3A）蛋白质atlastin-1的表征和功能分析。 2009年，我们在Cell杂志上报道，atlastin-1是一个普遍存在的GTP酶家族的成员，它与两个ER成形蛋白家族相互作用，产生管状内质网（ER）网络。 有趣的是，atlastin-1与SPG 31蛋白REEP 1相互作用，这是一种ER成形蛋白，以及SPG 4蛋白spastin，一种微管切断ATP酶。 2010年，我们在临床研究杂志上发表了一项研究，证明这三种蛋白质相互作用，与微管细胞骨架一起组织管状ER网络。 由于SPG 3A、SPG 4和SGP 31占所有HSP病例的50%以上，我们认为ER网络缺陷是HSP的主要神经病理机制。 这得到了最近鉴定的许多其他调节ER形态的HSP蛋白的支持，包括CPT 1C蛋白（与Kurt Fischbeck博士合作）。 在过去的一年中，我们继续开发SPG 31（敲除）和SPG 3A（敲除和敲入）以及双突变小鼠的动物模型，以使用体内和体外研究评估ER形态学变化的程度。 我们采用高通量的FIB-SEM电子显微镜和超分辨率共聚焦显微镜来检查这些遗传操作在神经元轴突内的管状ER的变化。 此外，我们还确定了这些蛋白质与HSP中突变的其他几种蛋白质的相互作用，扩大了与ER网络形成缺陷相关的HSP病例数量。 此外，我们正在与李学军博士合作，通过生产患者来源的诱导多能干细胞，然后分化为远头神经元，积极为许多HSP生成原位模型。其中一些研究于2014年发表在《干细胞和人类分子遗传学》杂志上。 这些细胞和其他细胞中的ER形态和动力学正在与Jennifer Lippincott-Schwartz，Eric Betzig和Harald Hess博士合作，使用许多新兴的超分辨率显微镜技术进行评估;这项工作的成果于2016年发表在Science上。最后，我们正在与Niamh O 'Sullivan博士合作研究这些HSP的飞行模型。 可能与疾病发病机制相关的ER功能的一个关键方面是脂滴的形成，这是我们细胞和生物体研究的重点领域。 我们正在完成几项研究，将ER形态的变化与脂滴生物发生的改变联系起来。 作为这些研究的一部分，我们使用CRISPR技术从HeLa和NIH-3 T3等细胞系中敲除所有三种atlastin亚型。 我们还利用先进的成像技术，如CT扫描和MR光谱，以非侵入性方式研究HSP小鼠模型中脂肪组织的变化;其中一项研究于2016年发表在《Genet》杂志上。 这些数据被用于规划一项大型临床自然史试验，该试验在三种最常见的常染色体显性HSP（SPG 4、SPG 3A和SPG 31）患者中进行，该试验刚刚开始招募。 综上所述，我们希望我们的研究将促进我们对热休克蛋白分子发病机制的理解。 在分子和细胞水平上的这种理解将有望导致新的治疗方法，以防止这些疾病的进展。