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: 10319132
• 负责人: Liang Qiang
• 金额: $ 37.88万
• 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/10319132
• 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.

项目摘要 遗传性痉挛性截瘫（HSP）是一种可遗传的神经退行性疾病，患者患有患者 下肢和步态缺陷的渐进无力，痉挛。该疾病主要表现为成人 皮质脊髓区（CSTS）的发作模具退化。点点峰，也称为SPG4，编码Spastin， 这是几个微管的酶。到目前为止，痉挛是HSP中最常见的基因。到 日期，由于功能痉挛水平降低而导致的单倍不足，这是普遍的机械 HSP-SPG4的说明。但是，单倍不足效率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。另外，SPG4 敲除（KO）小鼠仅显示非常温和的运动定义，没有CST倒入的报道。新的鼠标模型 在PI的实验室中，已专门设计用于测试突变型痉挛蛋白的功能获得毒性 作为CST倒退和类似HSP的电动机的原因，定义了。该提议的核心假设是 突变型痉挛蛋白的毒性特性是HSP-SPG4的原因，而功能性痉挛降低 水平不会引起HSP，而是使轴突更容易受到特定疾病的打击。机械假设 将通过转基因小鼠模型进行研究（包括在PI实验室中建立的新小鼠， 用敲除鼠标和通过越过两者产生的鼠标以及前脑生成的鼠标 源自同源性人类诱导多能干细胞（HIPSC）的谷氨酸能神经元培养物（HIPSC） 线。时装秀步态分析和CST对小鼠的解剖学评估将进行比较和 对比突变体氨纶毒性引起的表型与功能降低引起的表型 翼丁级。该假设将测试，即越过两种小鼠线将导致更极端的HSP- 像表型一样，比任何一个父行显示的表型。累积突变的剂量依赖性细胞毒性 将评估Spastin蛋白是该疾病功能获得机制的关键预测。减少 在受苦的轴突中观察到的微管乙酰化分配为由较高组蛋白脱乙酰基酶6 （HDAC6）突变翼蛋白引起的活性，并被定位为死亡的主要原因 CSTS。将探讨更大的HDAC6活性的潜在机械解释。微管还原 降低微管切断（由于功能性痉挛较少）而导致的迁移率定位为主要 轴突对突变型痉挛的脆弱性更大的原因。现代分子生物学，活细胞 成像，解剖学和行为方法将用于检验这些假设。成功解决 这些问题将为治疗HSP-SPG4患者的前景更好，并提供有关 基于微管的机制，这些机制可能是由其他基因突变引起的HSP中常见的。