Mechanisms Underlying Axonopathy in Hereditary Spastic Paraplegia

项目摘要 皮质脊髓束内的轴突变性导致几种神经系统疾病，包括 遗传性痉挛性截瘫（HSP）是一组临床和遗传异质性步态， 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 瘫痪利用平行动物（大鼠）和基于诱导多能干细胞（iPSC）的模型，我们的目标是 为了更好地理解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， 治疗疾病的方法。使用CRISPR介导的基因组编辑，我们在生理上开发了 相关模型，概括了HSP患者表现出的表型。具体来说，CRISPR- 表达SPG4（spastin）和SPG57（TFG）的病理变体的改良大鼠表现出早发性 后肢痉挛和共济失调，其迅速发展为后肢瘫痪。其他老鼠模型，包括那些 携带先前在患者中鉴定的SPG80（UBAP 1）的截短物，表现出晚发型疾病表型， 使我们能够在多种独特的环境中检查疾病进展。我们现在有一个前所未有 有机会确定所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，以测试的假设， 特定因子的运输，包括先前参与神经退行性疾病的神经丝蛋白 疾病，有助于HSP中受损的神经元功能。我们还将确定神经丝的运输 基于肌电图研究，组织病理学， 对啮齿类动物运动进行了全面的步态和运动学分析，如痉挛和肌无力。 此外，我们将确定HSP突变影响神经元兴奋性的机制， 同样使用活细胞成像方法，而且还使用体外生物化学和遗传研究。总的来说，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。 为今后开发药物筛选方法奠定基础。

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.