Investigation of modifiers of TDP-43 neurotoxicity in ALS models

描述(由申请人提供)： 肌萎缩侧索硬化症(ALS)是一种严重的进行性神经退行性疾病，其特征是大脑和脊髓运动神经元变性，导致神经源性肌肉萎缩、瘫痪和死亡。近95%的ALS病例的病理特征是神经元和神经胶质细胞中泛素化和过度磷酸化的TDP-43蛋白包涵体。类似的TDP-43包涵体是另一种神经退行性疾病-额颞叶变性(FTLD-TDP)的特征，这是中老年痴呆的主要原因。了解肌萎缩侧索硬化症和FTLD-TDP中控制TDP-43病理的机制对于设计神经保护策略至关重要。TDP-43被CDC7直接磷酸化，这种磷酸化导致TDP-43的神经毒性和最终的神经退行性变。这项建议描述了探索促进TDP-43靶向CDC7激酶活性的细胞和分子变化的实验，重点是开发治疗TDP-43蛋白病的干预措施，如ALS和FTLD-TDP。CDC7的疾病特异性变化可能促进TDP-43的磷酸化。事实上，在TDP-43磷酸化的哺乳动物细胞模型中，CDC7蛋白水平的增加和CDC7的翻译后修饰与TDP-43的病理磷酸化是平行的。将利用分子生物学、生物化学和遗传学技术，在小鼠原代神经元培养、哺乳动物细胞培养和表现出TDP-43磷酸化的转基因线虫模型中评估CDC7蛋白水平、定位、翻译后修饰和基因表达。表征CDC7的疾病相关变化将进一步加深我们对ALS和FTLD-TDP中TDP-43病理原因的理解。为了确定控制神经元中CDC7K激活的通路(S)，我们考察了CDC7K调节因子、通路基因和辅助因子对TDP-43磷酸化的影响。DNA损伤反应(DDR)通路是TDP-43磷酸化的关键调节因子。DDR途径的组成部分将使用小鼠原代神经元、线虫和TDP-43蛋白病的哺乳动物细胞模型进行详细评估。调控TDP-43靶向CDC7激酶活性的基因和基因通路的确定将有助于深入了解疾病的病理生物学，并有可能确定新的干预治疗靶点。CDC7是抑制ALS和FTLD-TDP病理性TDP-43磷酸化的有前景的治疗靶点。然而，针对CDC7的小分子抑制剂在体内具有适当的药代动力学，目前还没有商业化应用。因此，将组装一个结构类似于CDC7抑制剂的靶向小分子文库。这些小分子将被研究CDC7激酶活性的特异性抑制，并在线虫和哺乳动物细胞培养模型中进行体内测试，以防止TDP-43引起的神经退化。这是为ALS和FTLD-TDP开发可行的治疗方法的关键一步。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a severe progressive neurodegenerative disease characterized by degeneration of motor neurons in the brain and spinal cord, resulting in neurogenic muscle wasting, paralysis, and death. Nearly 95% of ALS cases have pathology featuring ubiquitinated and hyperphosphorylated inclusions of the TDP-43 protein in neurons and glial cells. Similar inclusions of TDP-43 are hallmarks of another neurodegenerative disease, frontotemporal lobar degeneration (FTLD-TDP), which is a major cause of mid- to late-life dementia. An understanding of the mechanisms controlling TDP-43 pathology in ALS and FTLD-TDP is critical to the design of neuroprotective strategies. TDP-43 is directly phosphorylated by the kinase CDC7, and this phosphorylation drives TDP-43 neurotoxicity and resultant neurodegeneration. This proposal describes experiments exploring the cellular and molecular changes that promote TDP-43 targeted CDC7 kinase activity, with a focus on the development of therapeutic interventions for the treatment of TDP-43 proteinopathies such as ALS and FTLD-TDP. Disease specific changes in CDC7 may promote TDP-43 phosphorylation. In fact, increased CDC7 protein levels and post-translational modifications of CDC7 are observed in parallel with pathological TDP-43 phosphorylation in a mammalian cell model of TDP-43 phosphorylation. CDC7 protein levels, localization, post-translational modifications, and gene expression will be evaluated in mouse primary neuron culture, mammalian cell culture, and transgenic C. elegans models exhibiting TDP-43 phosphorylation using molecular biology, biochemistry, and genetics techniques. Characterizing disease-relevant changes in CDC7 will further our understanding of the causes of TDP-43 pathology in ALS and FTLD-TDP. To identify pathway(s) controlling CDC7 kinase activation in neurons, CDC7 kinase regulators, pathway genes, and co-factors have been surveyed for effects on TDP-43 phosphorylation. The DNA damage response (DDR) pathway was identified by this screen as a key regulator of TDP-43 phosphorylation. Components of the DDR pathway will be evaluated in detail using mouse primary neurons, C. elegans and mammalian cell models of TDP-43 proteinopathy. The determination of the genes and gene pathways regulating TDP-43 targeted CDC7 kinase activity will provide insight into disease pathobiology, and potentially identify novel therapeutic targets for intervention. CDC7 is a promising therapeutic target for th inhibition of pathological TDP-43 phosphorylation that is observed in ALS and FTLD-TDP. However, small molecule inhibitors specific for CDC7 with appropriate pharmacokinetics in vivo are not commercially available. Therefore, a targeted library of small molecules structurally similar to CDC7 inhibitors will be assembled. These small molecules will be surveyed for specific inhibition of CDC7 kinase activity, and tested in vivo in C. elegans and in mammalian cell culture models for protection against TDP-43 driven neurodegeneration. This is a critical step towards developing viable treatments for ALS and FTLD-TDP.