Defining the molecular connections between hyperexcitability and neurodegeneration in ALS

定义 ALS 中过度兴奋性和神经退行性变之间的分子联系

• 批准号: 10044093
• 负责人: Aaron Harry Held
• 金额: $ 6.76万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10044093
• 关键词: ALS patients; Accounting; Affect; Amyotrophic Lateral Sclerosis; Autopsy; Axon; Categories; Cell Culture Techniques; Cell Death; Cell Line; Cellular Stress; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Computational Technique; Computer Analysis; Cytoskeletal Proteins; Data Set; Disease; Disease model; Electrophysiology (science); Engineering; Familial Amyotrophic Lateral Sclerosis; Family; Genes; Genetic; Genetic Transcription; Genotype; Goals; Human; Image; Imaging technology; Immunohistochemistry; Inherited; Institution; Invertebrates; Ion Channel; Link; Measures; Modeling; Molecular; Motor Neurons; Mus; Muscle fasciculation; Mutation; Nerve Degeneration; Nervous system structure; Neural Conduction; Neurites; Neurons; Ontology; Pathologic; Pathology; Patients; Phenotype; Prevalence; Protocols documentation; RNA; RNA Binding; Reporting; Research; Role; Scientist; Series; Spinal; Spinal Cord; Subgroup; Techniques; Testing; Therapeutic; Time; Tissues; Transcript; Work; clinically relevant; designer receptors exclusively activated by designer drugs; differential expression; disease phenotype; excitotoxicity; experience; induced pluripotent stem cell; motor control; motor disorder; motor neuron degeneration; mouse model; neuron loss; neurophysiology; new therapeutic target; proteostasis; stem cell model; stem cells; superoxide dismutase 1; temporal measurement; therapeutic development; transcriptome sequencing; transcriptomics; treatment trial

ALS is a rapidly progressive and fatal disease of the motor nervous system. Motor neuron hyperexcitability, manifested as muscle fasciculations and captured in clinical neurophysiological tests1–3, is an early pathological feature that affects both the 10% of familial ALS (fALS) patients who harbor dominant mutations in one of over 25 ALS genes, and the 90% of apparently sporadic ALS (sALS) patients who do not have an affected family member4. However, we do not know whether hyperexcitability causes motor neuron degeneration. Mouse, invertebrate, and human induced pluripotent stem cell (iPSC) models have all shown abnormal motor neuron excitability5–8. However, the mechanistic connection between hyperexcitability and motor neuron degeneration remains unclear6,9,10. In this proposal, we will use iPSCs from fALS patients to derive motor neurons and test the link between hyperexcitability and motor neuron degeneration. We will determine whether multiple fALS mutations increase motor neuron excitability, whether hyperexcitability precedes neurite retraction and cellular stress, and whether hyperexcitability can cause neurite retraction, cellular stress, and motor neuron death. Furthermore, we will examine transcriptomic signatures of multiple fALS lines before, during, and after motor neuron neurite retraction. We anticipate that ion channel transcripts, as identified by gene ontology, will precede neurite retraction in support of an excitotoxicity model. We will then determine whether potential ion channel transcript changes are directly responsible for increased excitability using patch electrophysiology. Finally, using the intersection of RNA-sequencing data sets, we will determine whether RNA-seq signatures from iPSC derived motor neurons overlap with sporadic ALS patient spinal cord tissue. The proposed project is feasible within the three-year time frame because of our experience with high-yield motor neuron differentiation protocols, gene editing, high-content imaging technology, electrophysiology, and computational analyses. The proposed work will determine whether there is a causative link within multiple fALS genetic subgroups between hyperexcitability, and motor neuron degeneration. The project results will have a direct impact on clinical trials attempting to normalize motor neuron excitability in patients. Furthermore, the transcriptomic signatures of multiple fALS lines before, during, and after neurite retraction will allow us to identify common features of motor neuron neurodegeneration that may also apply to sALS, and thus have larger therapeutic implications. Finally, our access to patient tissue allows us to validate specific findings related to excitability and transcriptomic signatures to determine their clinical relevance. We anticipate that this proposal will determine whether hyperexcitability is caused by fALS mutations and contributes to neurodegeneration in ALS, directly impacting ongoing clinical trials and therapeutic development.

ALS是运动神经系统的快速进行性和致命疾病。运动神经元过度兴奋性， 表现为肌肉束缚，并在临床神经生理测试中捕获1-3是早期 病理特征会影响10％的家庭ALS（FALS）患者，这些患者含有主导突变 超过25个ALS基因之一，没有90％的零星ALS（SALS）患者 受影响的家庭成员4。但是，我们不知道过度兴奋性是否引起运动神经元 退化。小鼠，无脊椎动物和人类诱导的多能干细胞（IPSC）模型均显示 运动神经元异常兴奋性5-8。但是，过度兴奋性和 运动神经元变性仍然不明确6,9,10。 在此提案中，我们将使用FALS患者的IPSC来得出运动神经元，并测试 过度兴奋性和运动神经元变性。我们将确定多个伪造突变是否增加 令人兴奋的运动神经元，过度兴奋性是否先于神经元缩回和细胞应激，以及是否是否 过度刺激性会导致神经落缩回，细胞应激和运动神经元死亡。此外，我们会的 检查运动神经元神经元之前，期间和之后的多个伪装线的转录组特征 撤回。我们预计基因本体论确定的离子通道转录本将在神经落之前 支持兴奋毒性模型的缩回。然后，我们将确定潜在的离子通道转录本是否 变化直接导致使用斑块电生理学增加兴奋。最后，使用 RNA序列数据集的交点，我们将确定来自IPSC的RNA-Seq签名是否得出 运动神经元与零星ALS患者脊髓组织重叠。 由于我们在高收益方面的经验，该提议的项目在三年的时间内是可行的 运动神经元分化方案，基因编辑，高内核成像技术，电生理学和 计算分析。拟议的工作将确定多个内部是否存在严重的联系 在过度兴奋性和运动神经元变性之间伪造遗传亚组。项目结果将 对试图使患者令人兴奋的运动神经元标准化的临床试验有直接影响。此外， 在神经蛋白回收之前，之中和之后，多个伪装线的转录组特征将使我们得以 确定可能也适用于SALS的运动神经元神经退行性的共同特征，因此 更大的治疗意义。最后，我们获得患者组织的机会使我们能够验证特定的发现 与令人兴奋性和转录组特征有关，以确定其临床相关性。我们预料到了 提案将确定过度兴奋性是否是由伪造突变引起的，并有助于 ALS中的神经变性，直接影响正在进行的临床试验和治疗发育。