Cell-type-specific molecular pathology of ALS in U.S. military Veterans

美国退伍军人 ALS 的细胞类型特异性分子病理学

基本信息

批准号：
10254543
负责人：
STELLA DRACHEVA
金额：
--
依托单位：
JAMES J PETERS VA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-10-01 至 2025-09-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10254543
关键词：
ALS pathology ALS patients Accounting Address Affect Aliquot Amyotrophic Lateral Sclerosis Anatomy Astrocytes Autopsy Biological Brain C9ORF72 Cell Nucleus Cells Cessation of life Communities Data Disease Endothelial Cells Environmental Exposure Etiology Fluorescence Frontotemporal Dementia Gene Expression Gene Expression Profiling General Population Genes Genetic Predisposition to Disease Genetic Transcription Glutamates Human Incidence Individual Large-Scale Sequencing Link Masks Microglia Molecular Motor Cortex Motor Neurons Mutation Names Neuraxis Neurodegenerative Disorders Neuroglia Neurons Nuclear Oligodendroglia Pathology Pathway interactions Patients Phenotype Play Prefrontal Cortex Preparation Proteins Protocols documentation Reporting Research Resolution Rest Role Sampling Services Severities Signal Transduction Sister Small Nuclear RNA Sorting - Cell Movement Specimen Symptoms Testing Translating United States Department of Veterans Affairs Veterans base biobank brain cell brain tissue case control cell type excitatory neuron excitotoxicity familial amyotrophic lateral sclerosis frontotemporal lobar dementia-amyotrophic lateral sclerosis genome-wide analysis illness length insight military veteran molecular pathology neuron loss neurotoxic novel sporadic amyotrophic lateral sclerosis transcriptome transcriptome sequencing

项目摘要

Amyotrophic lateral sclerosis (ALS) is a devastating human neurodegenerative disorder that is manifested in the degeneration of upper and lower motor neurons. ALS has higher incidence in U.S. military Veterans than in the general population and is considered a service-connected condition. Understanding the biological basis of ALS remains a major challenge, which is largely due to the complexity of the human central nervous system, which contain vast numbers of specialized cell types. Whereas the original focus of ALS research was concentrated on motor neurons, the non-neuronal cell types have also been suggested to play a crucial role in motor neuron death. Previous studies used genome-wide analysis of gene expression in bulk brain tissues to assess transcriptional changes associated with ALS. However, information on key changes that could affect different cell types in ALS brain remains limited. One reason is that changes affecting a particular cell type cannot be reliably inferred from data on bulk brain specimens that conflate signals from all cell types. The majority of ALS cases (~90%) occur sporadically (sALS) with unknown etiology, while ~10% of cases are classified as familial (fALS). To date, mutations in more than 50 genes have been linked to fALS. Expansion of the hexanucleotide repeat in C9orf72 (C9) gene is the most common cause of ALS and another neurodegenerative disorder, frontotemporal dementia (FTD), accounting for ~ 11% of all ALS and ~13% of all FTD cases. We recently performed single nucleus (sn)RNA-seq analysis using autopsied motor and prefrontal cortices from ALS and FTD cases with a C9 mutation and from controls. We identified disease-related changes in many cell types, including shared effects in ALS and FTD, and numerous disease-specific alterations. Among other findings, we detected changes in gene expression in endothelial cells, astrocytes, and excitatory neurons from C9-ALS cases that suggest a specific intercellular pathway that might, at least in part, underlie an ALS- associated glutamate (Glu) excitotoxicity. Our application aims to address the following issues: (1) Our snRNA- seq studies were limited to C9 cases; therefore, it is not known if the observed cell-type-dependent deficits are specific for C9-ALS or are also present in patients with sALS. (2) Although ALS typically leads to death within 3 to 5 years after initial symptom onset, approximately 10% of patients with ALS live significantly longer (>10 years after symptom onset; hereafter named “long duration ALS”) [10]. The molecular underpinnings of these differences have not been investigated. To address these issues, we propose the following Aims: Aim 1: To study cell-type-specific transcriptional dysregulation in the brains of U.S. military Veterans with sALS in single cell resolution. Hypothesis: Transcriptional deficits that we identified in C9-ALS patients and which, at least in part, explain the ALS-associated Glu excitotoxicity, are also present in the brain of military Veterans with sALS. We will test this hypothesis by performing snRNA-seq in the brains of standard duration sALS cases and controls (Ns=24) from the Department of Veterans Affairs Biorepository Brain Bank (VABBB). Aim 2: To elucidate cell-type-specific underpinnings of the long duration sALS phenotype. Hypothesis: Compared with standard duration sALS, long duration sALS is characterized by both unique and overlapping neurotoxic pathways. We will test this hypothesis by performing snRNA-seq in long duration sALS cases from VBBB (N=24) and comparing transcriptomes between standard (Aim1) and long duration sALS. Aim 3: To validate sALS-associated deficits in major cortical cell types. 3a. We will employ our novel nuclear sorting protocol to isolate nuclei from four major brain cell types (neurons, oligodendrocytes, astrocytes, and microglia) from the sister aliquots of samples used in Aims 1-2. We will use these preparations to validate the most significant snRNA-seq findings by qPCR. 3b. RNA-seq studies do not inform if the identified gene expression changes translate into changes in proteins. Here we will use immunostaining to investigate proteins encoded by sALS-associated genes from Aims1-2 that will have been validated in Aim3a.

肌萎缩侧索硬化症（ALS）是一种毁灭性的人类神经退行性疾病，表现为上、下运动神经元退化，ALS在美军中发病率较高。退伍军人比一般人群多，被认为是与服役有关的条件。 ALS 的生物学基础仍然是一个重大挑战，这很大程度上是由于人类中枢神经系统的复杂性神经系统，包含大量特殊的细胞类型。研究集中在运动神经元上，非神经元细胞类型也被建议发挥作用先前的研究使用了批量基因表达的全基因组分析。脑组织来评估与 ALS 相关的转录变化。对 ALS 大脑中不同细胞类型的影响仍然有限，原因之一是影响特定细胞类型的变化。无法从混合了所有细胞类型信号的大量脑样本数据中可靠地推断出细胞类型。大多数 ALS 病例 (~90%) 是散发性的 (sALS)，病因不明，而~10% 的病例迄今为止，已有超过 50 个基因的突变与 fALS 相关。 C9orf72 (C9) 基因中六核苷酸重复的缺失是 ALS 的最常见原因，也是 ALS 的另一个常见原因。神经退行性疾病、额颞叶痴呆 (FTD)，约占所有 ALS 的 11% 和约 13% 我们最近使用尸检运动和前额叶进行了单核 (sn)RNA-seq 分析。我们从具有 C9 突变的 ALS 和 FTD 病例以及对照的皮质中发现了与疾病相关的变化。在许多细胞类型中，包括 ALS 和 FTD 的共同影响，以及许多疾病特异性改变。其他发现，我们检测到内皮细胞、星形胶质细胞和兴奋性神经元基因表达的变化来自 C9-ALS 病例，表明特定的细胞间途径可能（至少部分）是 ALS-的基础我们的应用旨在解决以下问题：（1）我们的 snRNA- seq 研究仅限于 C9 病例；因此，尚不清楚观察到的细胞类型依赖性缺陷是否存在 C9-ALS 特异性或也存在于 sALS 患者中 (2) 尽管 ALS 通常会在 3 天内导致死亡。初始症状出现后 5 年内，大约 10% 的 ALS 患者寿命显着延长（>10 年）症状出现后；以下称为“长期 ALS”）[10]。为了解决这些问题，我们提出以下目标：目标 1：研究美国退伍军人大脑中细胞类型特异性转录失调假设：我们在 C9-ALS 患者中发现的转录缺陷。至少部分解释了 ALS 相关的谷氨酸兴奋性毒性，这种毒性也存在于军人的大脑中我们将通过在标准持续时间的大脑中执行 snRNA-seq 来测试患有 sALS 的退伍军人。来自退伍军人事务部生物储存脑库 (VABBB) 的 sALS 病例和对照 (Ns=24)。目标 2：阐明长持续时间 sALS 表型的细胞类型特异性基础。与标准持续时间 sALS 相比，长持续时间 sALS 具有独特性和重叠性的特点我们将通过在长期 sALS 病例中进行 snRNA-seq 来检验这一假设。 VBBB (N=24) 并比较标准 (Aim1) 和长持续时间 sALS 之间的转录组。目标 3：验证主要皮质细胞类型中与 sALS 相关的缺陷。核分选方案，用于从四种主要脑细胞类型（神经元、少突胶质细胞、星形胶质细胞、和小胶质细胞）来自目标 1-2 中使用的姐妹等分样品，我们将使用这些制剂来验证。 qPCR 3b 最重要的 snRNA-seq 结果并不能说明所识别的基因。表达变化转化为蛋白质的变化。在这里，我们将使用免疫染色来研究蛋白质。由来自 Aims1-2 的 sALS 相关基因编码，该基因将在 Aim3a 中得到验证。