Defining immune cell heterogeneity in human ALS and mouse model of the disease

肌萎缩侧索硬化症（ALS）是一种致命的运动神经元（MN）疾病，其与以下特征相关： 神经炎症尽管越来越多的证据支持神经炎症可能在神经系统疾病中发挥积极作用的观点， 由于抗炎疗法在ALS发病机制中的作用，抗炎疗法必须不提供或提供最小程度的疾病修饰作用， 对ALS的影响在此，我们假设，由于免疫细胞表现出表型异质性，因此有效的免疫抑制剂可能是免疫细胞。 ALS的免疫应答调节疗法需要靶向神经炎症的特定成分 而不是广泛抑制其信号传导。这项研究的基本原理是，一旦基因组特征 ALS的中枢神经系统（CNS）和外周神经系统（PNS）中的免疫细胞的数量减少。 可以鉴定已知的、有意义的生物标志物，并且可以设计创新的治疗策略。因此，在本发明中， 提出了以下三个目标。为了确定CNS内免疫细胞应答的异质性， 在AIM 1中，我们将使用来自以下组织的新鲜提取的免疫细胞进行单细胞RNA测序（scRNASeq）： 脊髓（ALS易感区）和海马（ALS抵抗区）的ALS患者以及 广泛使用和验证的ALS转基因（Tg）突变体SOD 1（mutSOD 1）小鼠模型， 会导致晚期瘫痪然后，我们将利用这个大规模的多变量数据集计算 构建与MN变性相关的整合CNS免疫细胞应答特征。因为马达 轴突变性是ALS病理学的一个重要特征，发生在CNS之外，在AIM 2中，我们将 通过scRNASeq进行外周神经浸润适应性和先天性免疫细胞的表型分析 在ALS患者和Tg mutSOD 1小鼠的坐骨神经中，使用与AIM 1中相同的分析管道。 然后，我们将通过计算构建一个整合CNS和PNS免疫细胞的双房室模型 信息来产生ALS的神经炎症特征。最后，由于脊髓的分区 在ALS中不均匀地退化，我们将使用AIM 1和2中产生的整合的神经炎症特征， （i）询问来自ALS患者和Tg两者的现有的、全面的空间转录组数据库 mutSOD 1小鼠，并通过免疫荧光和共聚焦显微镜（ii）定位不同的脊髓 免疫细胞亚群在各自的组织。成功完成拟议的调查将 在PNS和CNS中建立ALS中免疫细胞表型的异质性， 神经变性这些研究结果将产生重要的积极影响，因为它们将提供机会 用于新的致病假说，用于鉴定生物标志物和用于ALS的治疗干预， 相关疾病。

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron (MN) disease that is associated with features of neuroinflammation. Although mounting evidence supports the notion that neuroinflammation may play an active role in ALS pathogenesis, anti-inflammatory therapies have to provide either no or minimal disease-modifying effect in ALS. Herein, we hypothesize that since immune cells exhibit a phenotypic heterogeneity, effective immune response-modifying therapy for ALS requires the targeting of specific components of neuroinflammation rather than broadly inhibiting its signaling. The rationale for this research is that, once the genomic signatures of immune cells in the central nervous system (CNS) and the peripheral nervous system (PNS) of ALS are known, meaningful biomarkers can be identified and innovative therapeutic strategies can be devised. Thus, the following three aims are proposed. To define the heterogeneity of the immune cell response within the CNS, in AIM 1, we will perform single-cell RNA-sequencing (scRNASeq) using freshly extracted immune cells from spinal cord (ALS susceptible region) and hippocampus (ALS resistant region) of patients with ALS as well as of the extensively used and validated transgenic (Tg) mutant SOD1 (mutSOD1) mouse model of ALS, from pre- symptomatic to end-stage paralysis. We will then utilize this large-scale multivariable dataset computationally to construct an integrated CNS immune cell response signature associated with MN degeneration. Since motor axon degeneration is a critical feature of ALS pathology and takes place outside of the CNS, in AIM 2, we will perform a phenotypical analysis of peripheral nerve infiltrating adaptive and innate immune cells by scRNASeq in sciatic nerves from both ALS patients and Tg mutSOD1 mice using the same analytic pipeline as in AIM 1. We will then computationally construct a bi-compartmental model that integrates the CNS and PNS immune cell information to generate a neuroinflammatory signature of ALS. Lastly, since subregions of the spinal cord degenerate unevenly in ALS, we will use the integrated, neuroinflammatory signatures generates in AIM 1 & 2 to: (i) interrogate an existing, comprehensive spatial transcriptome database from both ALS patients and Tg mutSOD1 mice, and by immunofluorescence and confocal microscopy (ii) localize the different spinal cord immune cell subpopulations in the respective tissue. Successful completion of the proposed investigations will establish heterogeneity of the immune cell phenotype in ALS in both the PNS and CNS in response to neurodegeneration. These findings will have an important positive impact in that they will provide opportunities for novel pathogenic hypothesis, for identification of biomarkers and for therapeutic interventions in ALS and related disorders.