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

• 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
• 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

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.

肌萎缩性侧索硬化症（ALS）是一种毁灭性的人类神经退行性疾病，是 表现为上和下运动神经元的变性。 ALS在美国军方中发生了更高的事件 退伍军人比普通人群被认为是与服务相关的条件。了解 ALS的生物基础仍然是一个主要挑战，这主要是由于人类中心的复杂性 神经系统，其中包含大量专门细胞类型。而ALS的原始重点 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 在运动神经元死亡中的关键作用。先前的研究使用了整体基因表达的全基因组分析 脑组织评估与ALS相关的转录变化。但是，有关关键更改的信息 可能影响ALS大脑中的不同细胞类型仍然有限。原因之一是影响特定的变化 细胞类型不能从将所有细胞类型的信号混合的大脑样品的数据中可靠地推断出来。 大多数ALS病例（〜90％）偶尔出现（SALS），病因未知，约有10％的病例发生 被归类为家族性（伪造）。迄今为止，超过50个基因的突变与伪造有关。扩张 C9orf72（C9）基因中的六核苷酸重复是ALS的最常见原因和另一种原因 神经退行性疾病，额颞痴呆（FTD），占所有ALS的11％，占所有ALS的13％ FTD案例。我们最近使用尸体式电动机和前额叶进行了单核（SN）RNA-seq分析 来自ALS和FTD病例的皮质，具有C9突变以及对照组。我们确定了与疾病有关的变化 在许多细胞类型中，包括ALS和FTD中的共享效果，以及许多特定疾病的改变。之中 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中基因表达的变化 从C9-ALS病例提出了特定的细胞间途径，至少部分可能是ALS- 相关的谷氨酸（GLU）兴奋性。我们的申请旨在解决以下问题：（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定义是 针对C9-AL的特异性或在患有SAL的患者中也存在。 （2）尽管ALS通常会导致死亡3 初始症状发作后的5年，大约10％的ALS患者寿命明显更长（> 10年 症状发作后；此后将“长持续时间ALS”命名为[10]。这些的分子基础 差异尚未被调查。为了解决这些问题，我们提出以下目的： 目标1：研究美国退伍军人大脑中特定的细胞类型转录失调 与单细胞分辨率中的盐。假设：转录定义了我们在C9-ALS患者中发现的 至少部分解释了与ALS相关的GLU兴奋性毒性，也存在于军事大脑中 萨尔斯的退伍军人。我们将通过在标准持续时间的大脑中进行SNRNA-SEQ来检验该假设 退伍军人事务部生物卫生脑库（VABBB）的萨尔案件和对照（NS = 24）。 目的2：阐明长持续时间SALS表型的细胞类型特异性基础。假设： 与标准持续时间萨尔相比，长持续时间萨尔斯的特征是独特和重叠 神经毒性途径。我们将通过在长时间的案例中进行snRNA-seq来检验该假设 VBBB（n = 24），并比较标准（AIM1）和持续时间sals之间的转录组。 目的3：验证与SALS相关的主要皮质细胞类型中的定义。 3a。我们将采用我们的小说 核排序方案可将核与四种主要脑细胞类型分离（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中使用的样品的姐妹等分试样。我们将使用这些准备工作来验证 QPCR最重要的SnRNA-seq发现。 3b。 RNA-seq研究未告知已鉴定的基因 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AIMS1-2与SALS相关基因编码，这些基因将在AIM3A中得到验证。