Mechanisms Underlying Axonopathy in Hereditary Spastic Paraplegia

项目摘要 皮质脊髓束内的轴突变性会导致几种神经系统疾病，包括 遗传性痉挛截瘫(HSPs)是一组临床和遗传上不同的步态 以平衡不良、痉挛和进行性肌肉无力为特征的疾病，最终可能导致 处于瘫痪状态。利用平行动物(大鼠)和诱导多能干细胞(IPSC)模型，我们的目标是 为了更好地了解导致神经退行性变的病理机制， 导致HSP的基因突变，长期目标是利用这些模型作为平台来识别新的 对抗疾病的治疗学。使用CRISPR介导的基因组编辑，我们已经在生理上开发了 相关模型概括了患有过敏性紫杉醇患者的表型。具体而言，CRISPR- 表达SPG4(Spastin)和SPG57(TFG)病理变体的修饰大鼠早期发病 后肢痉挛和共济失调，迅速发展为后肢瘫痪。其他大鼠模型，包括 携带先前在患者中发现的SPG80(UBAP1)的截断，表现出较晚发病的疾病表型， 使我们能够在多个独特的背景下检查疾病的发展。我们现在有一个前所未有的 有机会确定观察到的轴突病变的机制基础。特别是，我们计划使用高- 分辨率、活细胞共聚焦成像和电子断层扫描来验证这一假设 特定因子的运输，包括以前与神经退行性变有关的神经丝蛋白 疾病，导致过敏性紫杉醇神经元功能受损。我们还将确定神经丝交易如何 观察到的缺陷与疾病的发病有关，基于肌电研究，组织病理学， 以及随着痉挛和肌肉无力而产生的啮齿动物运动的全面步态和运动学分析。 此外，我们将确定HSP背后的突变影响神经元兴奋性的机制， 再次使用活细胞成像方法，还进行了体外生物化学和遗传学研究。总而言之，这 这项工作将有助于揭示在患者中观察到的导致神经元功能障碍的几种机制 为今后药物筛选方法的发展奠定了基础。

Project Summary Axonal degeneration within the corticospinal tract leads to several neurological diseases, including hereditary spastic paraplegias (HSPs), which are a clinically and genetically heterogeneous group of gait disorders characterized by poor balance, spasticity, and progressive muscle weakness that can ultimately result in paralysis. Leveraging parallel animal (rat) and induced pluripotent stem cell (iPSC)-based models, our goal is to develop a better understanding of the pathomechanisms that underlie neurodegeneration resulting from mutations in genes that cause HSP, with a longer term goal of using these models as platforms to identify new therapeutics to combat disease. Using CRISPR-mediated genome editing, we have developed physiologically relevant models that recapitulate phenotypes exhibited by patients suffering from HSP. Specifically, CRISPR- modified rats expressing pathological variants of SPG4 (spastin) and SPG57 (TFG) demonstrate early onset hind limb spasticity and ataxia, which rapidly progresses to hind limb paralysis. Other rat models, including those harboring a truncation of SPG80 (UBAP1) identified previously in patients, exhibit later onset disease phenotypes, enabling us to examine disease progression in multiple, unique contexts. We now have an unprecedented opportunity to determine the mechanistic basis of the axonopathies observed. In particular, we plan to use high- resolution, live cell confocal imaging and electron tomography to test the hypothesis that changes in the trafficking of specific factors, including neurofilament proteins implicated previously in neurodegenerative disease, contribute to impaired neuronal function in HSP. We will also determine how neurofilament trafficking defects observed relate to disease onset based on a combination of electromyography studies, histopathology, and comprehensive gait and kinematic analysis of rodent movement as spasticity and muscle weakness ensues. Furthermore, we will determine mechanisms by which mutations that underlie HSP impact neuronal excitability, again using live cell imaging approaches, but also in vitro biochemistry and genetic studies. Collectively, this work will help to uncover several of the mechanisms that contribute to neuronal dysfunction observed in patients with HSP and lay the foundation for the future development of drug screening approaches.