人类 ALS 大脑和脊髓运动神经元中 DNA 损伤不断积累，并启动了相应的反应，而 SOD1 突变的 iPSC 衍生运动神经元中可激活 DNA 修复。
DNA damage accumulates and responses are engaged in human ALS brain and spinal motor neurons and DNA repair is activatable in iPSC-derived motor neurons with SOD1 mutations.
机构信息
Department of Pathology, Johns Hopkins University School of Medicine, 558 Ross Building, 720 Rutland Avenue, Baltimore, MD, 21205-2196, USA.
Division of Neuropathology, the Pathobiology Graduate Training Program, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
出版信息
Acta Neuropathol Commun. 2020 Jan 31;8(1):7. doi: 10.1186/s40478-019-0874-4.
DNA damage is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, relationships between DNA damage accumulation, DNA damage response (DDR), and upper and lower motor neuron vulnerability in human ALS are unclear; furthermore, it is unknown whether epigenetic silencing of DNA repair pathways contributes to ALS pathogenesis. We tested the hypotheses that DNA damage accumulates in ALS motor neurons along with diminished DDR, and that DNA repair genes undergo hypermethylation. Human postmortem CNS tissue was obtained from ALS cases (N = 34) and age-matched controls without neurologic disease (N = 15). Compared to age-matched controls, abasic sites accumulated in genomic DNA of ALS motor cortex and laser capture microdissection-acquired spinal motor neurons but not in motor neuron mitochondrial DNA. By immunohistochemistry, DNA damage accumulated significantly in upper and lower motor neurons in ALS cases as single-stranded DNA and 8-hydroxy-deoxyguanosine (OHdG) compared to age-matched controls. Significant DDR was engaged in ALS motor neurons as evidenced by accumulation of c-Abl, nuclear BRCA1, and ATM activation. DNA damage and DDR were present in motor neurons at pre-attritional stages and throughout the somatodendritic attritional stages of neurodegeneration. Motor neurons with DNA damage were also positive for activated p53 and cleaved caspase-3. Gene-specific promoter DNA methylation pyrosequencing identified the DNA repair genes Ogg1, Apex1, Pnkp and Aptx as hypomethylated in ALS. In human induced-pluripotent stem cell (iPSC)-derived motor neurons with familial ALS SOD1 mutations, DNA repair capacity was similar to isogenic control motor neurons. Our results show that vulnerable neurons in human ALS accumulate DNA damage, and contrary to our hypothesis, strongly activate and mobilize response effectors and DNA repair genes. This DDR in ALS motor neurons involves recruitment of c-Abl and BRCA1 to the nucleus in vivo, and repair of DNA double-strand breaks in human ALS motor neurons with SOD1 mutations in cell culture.
DNA 损伤与肌萎缩侧索硬化症（ALS）的发病机制有关。然而，在人类 ALS 中，DNA 损伤的积累、DNA 损伤反应（DDR）以及上下运动神经元的易损性之间的关系尚不清楚；此外，尚不清楚表观遗传沉默的 DNA 修复途径是否有助于 ALS 的发病机制。我们检验了以下假设：在 ALS 运动神经元中，DNA 损伤会累积，同时 DDR 减弱，并且 DNA 修复基因发生超甲基化。我们从 ALS 病例（N=34）和无神经疾病的年龄匹配对照（N=15）的人死后中枢神经系统组织中获得了人类死后 CNS 组织。与年龄匹配的对照相比，ALS 运动皮层和激光捕获微切割获得的脊髓运动神经元的基因组 DNA 中积累了碱基缺失，而不是运动神经元线粒体 DNA。通过免疫组化，与年龄匹配的对照相比，ALS 病例中的单链 DNA 和 8-羟基脱氧鸟苷（OHdG）在上、下运动神经元中积累了大量的 DNA 损伤。在 ALS 运动神经元中，DDR 明显被激活，证据是 c-Abl、核 BRCA1 和 ATM 的激活积累。在运动神经元的萎缩前阶段和整个神经退行性变的体树突萎缩阶段，都存在 DNA 损伤和 DDR。有 DNA 损伤的运动神经元也有激活的 p53 和切割的 caspase-3 阳性。基因特异性启动子 DNA 甲基化焦磷酸测序确定 DNA 修复基因 Ogg1、Apex1、Pnkp 和 Aptx 在 ALS 中呈低甲基化。在具有家族性 ALS SOD1 突变的人诱导多能干细胞（iPSC）衍生的运动神经元中，DNA 修复能力与同基因对照运动神经元相似。我们的研究结果表明，在人类 ALS 中，易损神经元会积累 DNA 损伤，与我们的假设相反，强烈激活并动员反应效应器和 DNA 修复基因。这种在 ALS 运动神经元中的 DDR 涉及到 c-Abl 和 BRCA1 在体内向核内募集，以及在具有 SOD1 突变的人 ALS 运动神经元中修复 DNA 双链断裂，这些都在细胞培养中得到证实。
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