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

肌萎缩侧索硬化症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的变性。肌萎缩侧索硬化症在美国军方的发病率较高 退伍军人人数高于一般人群，被认为是与服役有关的疾病。了解 ALS的生物学基础仍然是一个重大挑战，这在很大程度上是由于人类中枢 神经系统，其中包含大量的特化细胞类型。鉴于肌萎缩侧索硬化最初的焦点 研究集中在运动神经元上，非神经元细胞类型也被认为起到了 在运动神经元死亡中起着至关重要的作用。以前的研究大量使用全基因组的基因表达分析。 以评估与肌萎缩侧索硬化症相关的转录变化。然而，有关关键变化的信息 对ALS大脑中不同类型细胞的影响仍然有限。一个原因是，影响到一个特定的 从大量大脑样本的数据中无法可靠地推断出细胞类型，这些样本融合了所有细胞类型的信号。 大多数ALS病例(约90%)是散发性的(SAL)，病因不明，而约10%的病例 被归类为家族性(FAL)。到目前为止，已有50多个基因的突变与FALS有关。扩展 C9orf72(C9)基因的六核苷酸重复是ALS最常见的原因，另一个原因是 神经退行性疾病，额颞叶痴呆(FTD)，占所有ALS的约11%和约13% FTD病例。我们最近使用尸检的运动和前额叶进行了单核(Sn)rna-seq分析。 来自具有C9突变的ALS和FTD病例的皮质和来自对照的皮质。我们发现了与疾病相关的变化 在许多细胞类型中，包括在ALS和FTD中的共同影响，以及许多疾病特异性改变。其中 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病例中发现了一种特殊的细胞间通路，可能至少部分地构成了ALS- 谷氨酸(Glu)兴奋性毒性。我们的应用程序旨在解决以下问题：(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缺陷是否 C9-ALS特异或也存在于SALS患者中。(2)尽管肌萎缩侧索硬化症通常会在3天内导致死亡 在最初症状出现后的5年内，大约10%的ALS患者的存活时间显著延长(&>10年 症状出现后；此后称为“长期ALS”)[10]。它们的分子基础 分歧尚未得到调查。为了解决这些问题，我们提出了以下目标： 目的1：研究美国退伍军人大脑中细胞类型特异性转录失调 其中SALS为单单元分辨率。假设：我们在C9-ALS患者中发现的转录缺陷 这至少部分解释了肌萎缩侧索硬化症相关的谷氨酸兴奋性毒性，也存在于军人的大脑中 有SALS的退伍军人。我们将通过在标准持续时间的大脑中执行SnRNA-seq来验证这一假设 SALS病例和对照(NS=24)，来自退伍军人事务部生物信息库脑库(VABBB)。 目的2：阐明长程SALS表型的细胞类型特异性基础。假设： 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 神经毒性通路。我们将通过在长期SALS病例中执行SNRNA-SEQ来检验这一假设 VBBB(N=24)，并比较标准(Aim1)和长时程SALS的转录本。 目的3：验证主要皮质细胞类型中SALS相关的缺陷。3A.我们将利用我们的小说 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AIMS 1-2中使用的样品的姐妹等分。我们将利用这些准备工作来验证 定量聚合酶链式反应最显著的SnRNA-seq发现。3B.Rna-seq研究不会告知已识别的基因 表达的变化会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，将在Aim3a中得到验证。

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.