Elucidating the etiology of SPAST-based Hereditary Spastic Paraplegia

项目摘要 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 进行性虚弱、下肢痉挛和步态缺陷。该病主要表现为成人- 皮质脊髓束（CST）的发病退变。SPAST，也称为SPG 4，编码痉挛素， 一种切断微管的酶SPAST是HSP中最常见的基因突变。到 迄今为止，由功能性痉挛素水平降低引起的单倍不足一直是普遍的机制， 对HSP-SPG 4的解释。然而，单倍不足不能解释为什么没有发育性的 HSP患者的异常以及为什么轴突变性主要局限于CST。此外，SPG 4 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PI实验室中，专门设计用于测试突变痉挛蛋白的功能获得毒性 是CST消退和HSP样运动缺陷的原因。这一建议的核心假设是， 突变型spastin蛋白的毒性是HSP-SPG 4的原因，而功能性spastin蛋白的减少是HSP-SPG 4的原因。 水平不引起HSP，但使轴突更容易受到疾病特异性打击。机制假说 将通过转基因小鼠模型进行研究（包括在PI实验室建立的新小鼠， SPAST基因敲除小鼠，以及通过两者杂交产生的小鼠），以及前脑 来源于同基因人诱导多能干细胞（hiPSC）的神经元培养物 线将对小鼠进行猫步步态分析和CST解剖学评估，以比较和评估小鼠的运动能力。 将由突变体spastins的毒性引起的表型与由功能降低的spastins引起的表型进行对比， 痉挛水平。将检验这一假设，即两个小鼠品系杂交将导致更极端的HSP- 相似的表型。累积突变的剂量依赖性细胞毒性 将评估痉挛蛋白，这是该疾病的功能获得机制的关键预测。降低 在受损轴突中观察到的微管乙酰化被认为是由较高的组蛋白去乙酰化酶6引起的 （HDAC 6）活性引起的突变spastins，并假定是主要原因的枯死退化， CST。将探索更大HDAC 6活性的潜在机制解释。还原微管 微管断裂减少（由于功能性痉挛减少）导致的活动性被认为是主要的 这是轴突对突变spastins更脆弱的原因。当代分子生物学，活细胞 将使用成像、解剖和行为方法来检验这些假设。成功解决 这些问题将为治疗HSP-SPG 4患者带来更好的前景， 基于微管的机制可能是常见的热休克蛋白引起的其他基因的突变。

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.