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

基本信息

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
项目类别：
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

项目摘要

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 是一种快速进展且致命的运动神经系统疾病。表现为肌肉颤动并在临床神经生理学测试中捕获1-3，是一种早期的影响 10% 家族性 ALS (fALS) 患者的病理特征，这些患者携带显性突变超过 25 个 ALS 基因之一，并且 90% 的明显散发性 ALS (sALS) 患者没有基因受影响的家庭成员4。然而，我们不知道过度兴奋是否会导致运动神经。小鼠、无脊椎动物和人类诱导多能干细胞（iPSC）模型均已显示出退化。异常的运动神经兴奋性5-8 然而，过度兴奋和异常之间的机械联系。运动神经元变性仍不清楚6,9,10。在本提案中，我们将使用来自 fALS 患者的 iPSC 来衍生运动神经元并测试之间的联系我们将确定多种 fALS 突变是否会增加。运动神经元兴奋性，过度兴奋是否先于神经突回缩和细胞应激，以及是否过度兴奋会导致神经突回缩、细胞应激和运动神经元死亡。检查运动神经元神经突之前、期间和之后多个 fALS 系的转录组特征我们预计，通过基因本体论鉴定，离子通道转录物将出现在神经突之前。然后我们将确定是否有潜在的离子通道转录物。直接使用贴片电生理学的变化负责增加兴奋性。 RNA 测序数据集的交集，我们将确定 RNA-seq 签名是否来自 iPSC 运动神经元与散发性 ALS 患者脊髓组织重叠。由于我们在高收益方面的经验，拟议的项目在三年内是可行的运动神经元分化方案、基因编辑、高内涵成像技术、电生理学和拟议的工作将确定多个因素之间是否存在因果关系。过度兴奋和运动神经元变性之间的 fALS 遗传亚组该项目结果将。对试图使患者运动神经元兴奋性正常化的临床试验有直接影响。神经突回缩之前、期间和之后多个 fALS 系的转录组特征将使我们能够确定运动神经元神经变性的共同特征，这些特征也可能适用于 sALS，因此最后，我们对患者组织的接触使我们能够验证具体的发现。我们预计这与兴奋性和转录组特征有关，以确定其临床相关性。该提案将确定过度兴奋是否是由 fALS 突变引起并有助于 ALS 中的神经退行性变直接影响正在进行的临床试验和治疗开发。