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

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的退化。ALS在美国军队中的发病率较高 退伍军人比一般人群，被认为是一个服务连接的条件。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的复杂性。 神经系统，其中包含大量专门的细胞类型。而ALS最初的重点 研究主要集中在运动神经元，非神经元细胞类型也被认为在运动神经元中起着重要作用。 在运动神经元死亡中起关键作用。以前的研究使用了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。然而，关于关键变化的信息， 可能影响ALS大脑中不同类型的细胞仍然有限。一个原因是，影响特定 细胞类型不能从大量脑样本的数据可靠地推断，所述大量脑样本合并了来自所有细胞类型的信号。 大多数ALS病例（约90%）是偶发性（sALS），病因不明，而约10%的病例 被归类为家族性（fALS）。迄今为止，超过50个基因的突变与fALS有关。膨胀 C9 orf 72（C9）基因中六核苷酸重复序列的缺失是ALS最常见的原因， 神经退行性疾病，额颞叶痴呆（FTD），占所有ALS的约11%，占所有ALS的约13%。 FTD病例。我们最近使用尸检的运动和前额叶皮层进行了单核（sn）RNA-seq分析， 来自具有C9突变的ALS和FTD病例以及来自对照的皮质。我们发现了与疾病相关的变化 在许多细胞类型中，包括ALS和FTD的共同作用，以及许多疾病特异性改变。之间 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的基因表达的变化 从C9-ALS病例中，提示可能至少部分地成为ALS基础的特定细胞间通路， 谷氨酸（Glu）兴奋性毒性。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，尚不清楚所观察到的细胞类型依赖性缺陷是否 特异于C9-ALS或也存在于sALS患者中。(2)虽然ALS通常会在3分钟内导致死亡， 在最初症状发作后5年内，大约10%的ALS患者的寿命显著延长（>10年 [2019 - 10 - 15][2019 - 09 - 19][2019 - 09 - 19][2019 - 09][2019 - 10 - 19][2019 - 09]这些的分子基础 差异尚未调查。为解决这些问题，我们提出以下目标： 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 与单细胞分辨率的sALS。假设：我们在C9-ALS患者中发现的转录缺陷 并且，至少部分地解释了ALS相关的Glu兴奋毒性，也存在于军事脑中。 有肌萎缩侧索硬化症的退伍军人。我们将通过在标准持续时间的大脑中进行snRNA-seq来测试这一假设。 sALS病例和对照（Ns=24）来自退伍军人事务部生物储存脑库（VABBB）。 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： 长时程sALS与标准时程sALS相比，既有独特性，又有重叠性 神经毒性通路我们将通过在长期sALS病例中进行snRNA-seq来测试这一假设， VBBB（N=24）和比较标准（Aim 1）和长持续时间sALS之间的转录组。 目的3：验证主要皮质细胞类型中sALS相关缺陷。3a.我们将用我们的小说 核分选方案从四种主要的脑细胞类型（神经元，少突胶质细胞，星形胶质细胞， 和小胶质细胞）。我们将利用这些准备工作来验证 通过qPCR的最显著snRNA-seq发现。3b. RNA-seq研究并没有告知所识别的基因是否 表达的变化转化为蛋白质的变化。在这里，我们将使用免疫染色研究蛋白质 由来自Aims 1 -2的sALS相关基因编码，这些基因将在Aim 3a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.