ER Network Shaping Mechanisms in the Hereditary Spastic Paraplegias

神经遗传学分部细胞生物科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截瘫(HSPs)。这些疾病共同困扰着数以百万计的美国人，多年来还在潜移默化地恶化，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最终是如何导致细胞功能障碍的。 在这个项目中，我们将重点放在HSP上。一个主要的研究主题涉及遗传性痉挛截瘫3A(SPG3A)蛋白atlastin-1的特性和功能分析。2009年，我们在《细胞》杂志上报道，atlastin-1是一个普遍存在的GTP酶家族的成员，它与两个内质网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SPG31蛋白REEP1和SPG4蛋白spastin相互作用，前者是一种内质网形成蛋白，后者是一种微管切断ATPase。2010年，我们在《临床调查杂志》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SPG3A、SPG4和SGP31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。最近发现了许多调节ER形态的其他HSP蛋白，包括CPT1C蛋白(与Kurt Fischbeck博士合作)，这支持了这一点。 在过去的一年里，我们继续开发SPG31(基因敲除)和SPG3A(基因敲除和敲入)的动物模型，以使用体内和体外研究来评估内质网形态变化的程度。我们正在使用高通量电子显微镜(与Mark Terasaki博士合作)和超分辨率共聚焦显微镜来研究神经元轴突内管状ER的变化，以响应这些遗传操作。此外，我们还确定了这些蛋白与热休克蛋白中其他几种突变蛋白的相互作用，扩大了与内质网形成缺陷相关的热休克蛋白病例的数量。此外，我们正在积极地通过生产患者来源的诱导多能干细胞，然后与李学军博士合作，将其分化为遥脑神经元，从而为许多热休克蛋白建立原位模型。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。与Jennifer Lippincott-Schwartz博士、Eric Betzig博士和Harald Hess博士合作，正在使用一些新兴的超分辨率显微镜技术来评估这些和其他细胞中的内质网形态和动力学。最后，我们正在与Niamh O‘Sullivan博士合作建立这些热休克蛋白的飞行模型。 内质网功能的一个关键方面可能与疾病的发病机制有关，即形成脂滴，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，将内质网形态的变化与脂滴生物发生的变化联系起来。作为这些研究的一部分，我们使用CRISPR技术从HeLa和NIH-3T3等细胞系中敲除了所有三种阿特拉斯汀亚型。我们还利用先进的成像技术，如CT扫描和磁共振波谱，以非侵入性的方式研究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Hum Mol Genet上。这些数据被用于规划一项针对三种最常见的常染色体显性遗传性过敏症(SPG4、SPG3A和SPG31)患者的大型临床自然病史试验，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防止这些疾病进展的新疗法。

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) to evaluate the extent of ER morphology changes using both in vivo and ex vivo studies. We are employing both high-throughput electron microscopy (in collaboration with Dr. Mark Terasaki) 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. 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.