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，其特征在于脑和脊髓中的运动神经元变性，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TDP-43蛋白的泛素化和过度磷酸化包涵体。TDP-43的类似内含物是另一种神经退行性疾病的标志，额颞叶变性（FTLD-TDP），这是中晚期痴呆的主要原因。了解ALS和FTLD-TDP中控制TDP-43病理学的机制对于设计神经保护策略至关重要。TDP-43被激酶CDC 7直接磷酸化，并且这种磷酸化驱动TDP-43神经毒性和由此产生的神经变性。该提案描述了探索促进TDP-43靶向CDC 7激酶活性的细胞和分子变化的实验，重点是开发用于治疗TDP-43蛋白病（如ALS和FTLD-TDP）的治疗干预措施。 疾病特异性CDC 7的变化可能促进TDP-43磷酸化。事实上，在TDP-43磷酸化的哺乳动物细胞模型中，观察到增加的CDC 7蛋白水平和CDC 7的翻译后修饰与病理性TDP-43磷酸化平行。将在小鼠原代神经元培养物、哺乳动物细胞培养物和转基因C.使用分子生物学、生物化学和遗传学技术，建立了显示TDP-43磷酸化的线虫模型。表征CDC 7中的疾病相关变化将进一步了解ALS和FTLD-TDP中TDP-43病理学的原因。 为了鉴定控制神经元中CDC 7激酶活化的途径，已经调查了CDC 7激酶调节剂、途径基因和辅因子对TDP-43磷酸化的影响。DNA损伤反应（DDR）途径通过该筛选被鉴定为TDP-43磷酸化的关键调节剂。DDR通路的组分将使用小鼠原代神经元C.线虫和哺乳动物细胞模型的TDP-43蛋白质病。确定调节TDP-43靶向CDC 7激酶活性的基因和基因途径将提供对疾病病理生物学的深入了解，并可能确定新的干预治疗靶点。 CDC 7是用于抑制在ALS和FTLD-TDP中观察到的病理性TDP-43磷酸化的有希望的治疗靶标。然而，在体内具有适当药代动力学的特异性针对CDC 7的小分子抑制剂尚未商购获得。因此，将组装结构上类似于CDC 7抑制剂的小分子的靶向文库。将调查这些小分子对CDC 7激酶活性的特异性抑制，并在C. elegans和哺乳动物细胞培养模型中用于保护免受TDP-43驱动的神经变性。这是开发ALS和FTLD-TDP可行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.