Elucidating the etiology of SPAST-based Hereditary Spastic Paraplegia

项目总结 遗传性痉挛截瘫是一种遗传性神经退行性疾病，患者患有 进行性虚弱、肢体痉挛和步态缺陷。本病主要表现为成人-- 起病的皮质脊髓束退行性变(CST)。Spast，也称为SPG4，编码spastin， 这是一种切断微管的酶。到目前为止，spast是HSP中最常见的突变基因。至 目前，功能性痉挛蛋白水平降低导致的单倍体功能不全一直是普遍存在的机制。 对HSP-SPG4的解释。然而，单倍性不足不能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。一种新的小鼠模型 在实验室里，PI已经被专门设计来测试突变的痉挛蛋白的功能获得毒性 作为CST死背和热休克蛋白样运动缺陷的原因。这项提议的中心假设是 突变的spastin蛋白的毒性特性是HSP-SPG4的原因，而功能性痉挛减少 水平不会导致HSP，但会使轴突更容易受到疾病特异性打击。机械论假说 将通过转基因小鼠模型进行研究(包括在PI的实验室建立的一只新小鼠， Spast基因敲除小鼠，以及两者杂交产生的小鼠)，以及前脑 同基因人诱导多能干细胞来源的谷氨酸能神经元培养 台词。将对小鼠进行走秀步态分析和CST解剖学评估，以比较和 比较突变型Spastins的毒性和功能减退引起的表型 痉挛蛋白水平。这一假设将得到检验，即跨越两个小鼠品系将导致更极端的HSP- 与亲本中的任何一个所显示的表型相似。累积突变的细胞毒作用呈剂量依赖性 将对spastin蛋白质进行评估，该蛋白质是该病功能获得机制的关键预测。减少 在受累轴突中观察到的微管乙酰化可能是由较高的组蛋白脱乙酰酶6引起的。 (HDAC6)由突变型spastins引起的活性，被认为是死背退化的主要原因 CSTS.将探索HDAC6活性更大的潜在机制解释。缩小的微管 微管切断减少(由于功能减弱的痉挛)导致的活动能力被认为是主要的 这是因为轴突对突变型脊椎蛋白的脆弱性更大。当代分子生物学、活细胞 成像、解剖学和行为学方法将用来检验这些假说。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 基于微管的机制可能在由其他基因突变引起的热休克蛋白中普遍存在。

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.