Elucidating the etiology of SPAST-based Hereditary Spastic Paraplegia

阐明基于 SPAST 的遗传性痉挛性截瘫的病因

• 批准号: 10117879
• 负责人: Liang Qiang
• 金额: $ 37.8万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-15 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10117879
• 关键词: Accounting; Acetylation; Adult; Age; Alleles; Anatomy; Animals; Axon; Back; Behavioral; Biological; Cell Line; Corticospinal Tracts; Cultured Cells; Data; Deacetylase; Defect; Development; Disease; Dose; Enzymes; Etiology; Experimental Models; Gait; Gene Mutation; Genes; Glutamates; HDAC6 gene; Hereditary Spastic Paraplegia; Heritability; Human; Induced pluripotent stem cell derived neurons; Knock-out; Knockout Mice; Knowledge; Laboratories; Lead; Lower Extremity; Microtubules; Minor; Molecular; Morphology; Mus; Mutate; Mutation; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; Parents; Pathogenicity; Patients; Pharmaceutical Preparations; Phenotype; Phosphorylation; Predisposition; Property; Prosencephalon; Protein Isoforms; Proteins; Reporting; Resolution; Role; Squid; Stress; Swelling; Symptoms; Synapses; Testing; Toxic effect; Transgenic Mice; Tubulin; axonal degeneration; base; behavior test; cytotoxicity; design; effective therapy; fly; gain of function; gait examination; induced pluripotent stem cell; insight; live cell imaging; loss of function; motor deficit; mouse model; mutant; novel; response; spasticity; spastin

PROJECT SUMMARY Hereditary Spastic Paraplegia (HSP) is a heritable neurodegenerative disorder in which patients suffer from progressive weakness, spasticity of lower limbs and gait deficiencies. The disease mainly manifests as adult- onset die-back degeneration of the corticospinal tracts (CSTs). SPAST, also called SPG4, encodes spastin, which is an enzyme that severs microtubules. By far, SPAST is the most common gene mutated in HSP. To date, haploinsufficiency resulting from reduced functional spastin levels has been the prevalent mechanistic explanation for HSP-SPG4. However, haploinsufficiency fails to explain why there are no developmental abnormalities in HSP patients and why axonal degeneration is mostly confined to the CSTs. In addition, SPG4 knockout (KO) mice display only very mild motor deficits, with no reports of CST die-back. A new mouse model in the laboratory of the PI has been designed specifically to test gain-of-function toxicity of mutant spastin proteins as the cause of CST die-back and HSP-like motor deficits. The central hypothesis of this proposal is that the toxic properties of mutant spastin proteins are the cause of HSP-SPG4, whereas reduced functional spastin levels do not cause HSP but render axons more vulnerable to the disease-specific hit. Mechanistic hypotheses will be investigated via transgenic mouse models (including a new mouse established in the PI’s laboratory, the SPAST knockout mouse, and the mouse that is generated by crossing the two), as well as forebrain glutamatergic neuronal cultures derived from isogenic human induced pluripotent stem cell (hiPSC) lines. Catwalk gait analyses and CST anatomical assessments on the mice will be conducted to compare and contrast the phenotypes resulting from toxicity of mutant spastins with those resulting from reduced functional spastin levels. The hypothesis will be tested that crossing the two mouse lines will result in a more extreme HSP- like phenotype than displayed by either of the parent lines. Dose dependent cytotoxicity of accumulated mutated spastin proteins, a key prediction of a gain-of-function mechanism for the disease, will be evaluated. Decreased microtubule acetylation observed in the afflicted axons is posited to result from higher histone deacetylase 6 (HDAC6) activity elicited by mutant spastins and is posited to be the main cause of the die-back degeneration of CSTs. Potential mechanistic explanations for the greater HDAC6 activity will be explored. Reduced microtubule mobility resulting from reduced microtubule severing (due to less functional spastin) is posited to be the main cause of the greater vulnerability of the axon to the mutant spastins. Contemporary molecular biological, live-cell imaging, anatomical and behavioral approaches will be used to test these hypotheses. Successful resolution of these issues will lead to better prospects for treating patients with HSP-SPG4, and also provide insights into microtubule-based mechanisms that may be common across HSPs caused by mutations of other genes.

项目概要 遗传性痉挛性截瘫（HSP）是一种遗传性神经退行性疾病，患者患有 成人时主要表现为进行性无力、下肢痉挛和步态缺陷。 皮质脊髓束（CST）的发病性死亡变性，也称为 SPG4，编码 spastin， SPAST 是一种切断微管的酶，是 HSP 中最常见的突变基因。 迄今为止，由于功能性 spastin 水平降低而导致的单倍体不足已成为普遍的机制 然而，单倍体不足无法解释为什么没有发育。 HSP 患者的异常以及为什么轴突变性主要局限于 CST。此外，SPG4。 基因敲除 (KO) 小鼠仅表现出非常轻微的运动缺陷，没有 CST 死亡的报道。 在 PI 实验室中专门设计用于测试突变型 spastin 蛋白的功能获得毒性 作为 CST 枯萎和 HSP 样运动缺陷的原因 该提议的中心假设是 突变型 spastin 蛋白的毒性是 HSP-SPG4 的原因，而功能性 spastin 减少 水平不会引起 HSP，但会使轴突更容易受到疾病特异性的打击。 将通过转基因小鼠模型（包括 PI 实验室建立的新小鼠， SPAST基因敲除小鼠，以及两者交叉产生的小鼠），以及前脑 来自同基因人类诱导多能干细胞 (hiPSC) 的谷氨酸能神经培养物 对小鼠进行猫步步态分析和 CST 解剖学评估，以进行比较和分析。 将突变型 spastin 的毒性引起的表型与功能降低引起的表型进行对比 假设将测试交叉两个小鼠品系将导致更极端的 HSP-。 与任一亲本系所显示的相似表型，累积突变的剂量依赖性细胞毒性。 spastin 蛋白是疾病功能获得机制的一个关键预测，将被评估减少。 在受影响的轴突中观察到的微管乙酰化被认为是由较高的组蛋白脱乙酰酶 6 引起的 (HDAC6) 活性由突变型 spastin 引起，并被认为是引起枯萎退化的主要原因。 将探讨 HDAC6 活性降低的潜在机制。 微管切断减少（由于功能性 spastin 减少）导致的流动性被认为是主要的 轴突更容易受到当代分子生物学活细胞突变的影响。 成像、解剖和行为方法将用于测试这些假设的成功解决。 这些问题将为治疗 HSP-SPG4 患者带来更好的前景，并为以下方面提供见解： 基于微管的机制可能在其他基因突变引起的 HSP 中很常见。