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，与 神经炎。尽管越来越多的证据支持这样一种观点，即神经炎症可能在 在肌萎缩侧索硬化症发病机制中的作用，抗炎治疗必须不提供或最小限度地改变疾病 对肌萎缩侧索硬化症的影响。在此，我们假设，由于免疫细胞表现出表型的异质性，有效地 ALS的免疫反应调节疗法需要靶向神经炎症的特定成分 而不是广泛地抑制其信号。这项研究的基本原理是，一旦基因组签名 ALS的中枢神经系统(CNS)和外周神经系统(PNS)中的免疫细胞 已知的、有意义的生物标志物可以被识别出来，创新的治疗策略也可以被设计出来。因此， 提出了以下三个目标。为了定义中枢神经系统内免疫细胞反应的异质性， 在目标1中，我们将使用新鲜提取的免疫细胞进行单细胞RNA测序(ScRNASeq) ALS患者的脊髓(ALS易感区)和海马区(ALS抵抗区) 广泛使用和验证的ALS转基因(TG)突变体SOD1(Mutod1)小鼠模型，来自前 出现终末期瘫痪的症状。然后，我们将以计算方式利用该大规模多变量数据集 构建完整的与MN退变相关的中枢神经系统免疫细胞应答信号。自电机 轴突变性是ALS病理的一个重要特征，发生在中枢神经系统之外，在AIM 2中，我们将 利用scRNASeq对周围神经侵袭适应性和先天免疫细胞进行表型分析 使用与AIM 1相同的分析管道，在ALS患者和Tg musod1小鼠的坐骨神经中进行研究。 然后，我们将通过计算构建一个整合了CNS和PNS免疫细胞的双室模型 产生肌萎缩侧索硬化症的神经炎性特征的信息。最后，由于脊髓的不同区域 在肌萎缩侧索硬化症中，我们将使用在AIM 1和2中产生的整合的神经炎性信号 ：(I)询问ALS患者和TG现有的全面空间转录组数据库 用免疫荧光和共聚焦显微镜(II)对不同的脊髓进行定位 相应组织中的免疫细胞亚群。成功完成拟议的调查将 在三叉神经节和中枢神经系统建立ALS免疫细胞表型的异质性 神经退行性变。这些发现将产生重要的积极影响，因为它们将提供机会 用于ALS和ALS的新的致病假说、生物标志物的识别和治疗干预 相关的障碍。

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.