Defective Mitophagy in XPA via PARP1 activation and NAD+/SIRT1-depletion: Implications for neurodegeneration (mouse). Mus musculus

Mitochondrial dysfunction is a common feature in neurodegeneration and aging. We identify mitochondrial dysfunction in xeroderma pigmentosum group A (XPA), a nucleotide excision DNA repair disorder with severe neurodegeneration, in silico and in vivo. XPA deficient cells show defective mitophagy with excessive cleavage of PINK1 and increased mitochondrial membrane potential. The mitochondrial abnormalities appear to be caused by decreased activation of the NAD+-SIRT1-PGC-1α axis triggered by hyperactivation of the DNA damage sensor PARP1. This phenotype is rescued by PARP1 inhibition or by supplementation with NAD+ precursors that also rescue the lifespan defect in xpa-1 nematodes. Importantly, this pathogenesis appears common to ataxia-telangiectasia and Cockayne syndrome, two other DNA repair disorders with neurodegeneration, but absent in XPC, a DNA repair disorder without neurodegeneration. Our findings reveal a novel nuclear-mitochondrial cross-talk that is critical for the maintenance of mitochondrial health. Overall design: Mice carrying WT, or CX (Csa-/-/Xpa-/-) alleles in a C57BL/6 background were maintained under standard laboratory conditions and allowed free access to water and control casein pelleted diet (Research Diets D12450B). At 3 months of age, 3 replicates of each of the CX and WT mice were given subcutaneous interscapular injections of 500 mg of Nicotinamide riboside/kg body weight/day or the equivalent volume of saline for 14 consecutive days at 4:00 pm. On day 15, the mice were sacrificed and half of the cerebellum was harvested for purification of mitochondria, with the left half snap-frozen, homogenized, and aliquoted for RNA isolation. Total RNA extraction was done using a TRIzol Plus RNA purification kit as per manufacturer’s protocol. Quality and quantity of the total RNA was tested using the Agilent 2100 Bio-Analyzer and RNA 6000 nano kits. The RNA was labeled using the standard Illumina protocol and hybed overnight to Mouse Ref-8 Illumina arrays. The arrays were scanned using the Beadstation 500 X from Illumina.

线粒体功能障碍是神经退行性变与衰老的共同特征。本研究通过计算机模拟与体内实验，在着色性干皮病A型（XPA）——一种伴随严重神经退行性变的核苷酸切除修复类DNA修复疾病——中鉴定出线粒体功能障碍现象。XPA缺陷细胞表现出线粒体自噬功能缺陷，伴随PINK1过度剪切及线粒体膜电位升高。上述线粒体异常由DNA损伤传感器PARP1（多聚ADP核糖聚合酶1）过度激活所触发的NAD+-SIRT1-PGC-1α信号轴激活不足所导致。
该表型可通过PARP1抑制或补充NAD+前体得以挽救，后者同时可修复xpa-1线虫的寿命缺陷。值得注意的是，这一发病机制同样存在于另外两种伴随神经退行性变的DNA修复疾病——共济失调毛细血管扩张症与科凯恩综合征，但在不伴随神经退行性变的DNA修复疾病XPC（着色性干皮病C型）中未观察到此现象。本研究揭示了一条全新的核-线粒体交互通路，其对维持线粒体健康至关重要。
整体实验设计：在C57BL/6遗传背景下，携带野生型（WT）或CX（Csa-/-/Xpa-/-）等位基因的小鼠在标准实验室饲养条件中饲养，自由饮水并喂食标准酪蛋白颗粒饲料（Research Diets D12450B）。小鼠3月龄时，CX组与WT组各取3只生物学重复，于每日下午4:00进行肩胛间皮下注射：500mg/kg体重的烟酰胺核糖，或等量生理盐水，连续给药14天。第15天处死小鼠，取一半小脑组织用于线粒体纯化，另一半小脑组织经快速冷冻、匀浆后分装，用于RNA提取。总RNA提取采用TRIzol Plus RNA purification kit，严格按照制造商说明书操作。总RNA的质量与浓度通过Agilent 2100生物分析仪及RNA 6000纳米试剂盒进行检测。RNA按照标准Illumina实验方案进行标记，随后与小鼠Ref-8 Illumina芯片进行过夜杂交。芯片扫描采用Illumina Beadstation 500 X平台。