Damiano Maria, Gautier Clément A, Bulteau Anne-Laure, Ferrando-Miguel Rosa, Gouarne Caroline, Paoli Marc Giraudon, Pruss Rebecca, Auchère Françoise, L'Hermitte-Stead Caroline, Bouillaud Frédéric, Brice Alexis, Corti Olga, Lombès Anne
Inserm, U 975, CRICM, Hôpital de la Pitié-Salpêtrière, Paris, France; UPMC Université Paris 06, UMR_S975, Paris, France; CNRS, UMR 7225, Paris, France.
Inserm U 1016, Institut Cochin, Paris, France; CNRS UMR 8104, Paris, France; Université Paris 05, UMR_S1016, Paris, France.
PLoS One. 2014 Jun 24;9(6):e99898. doi: 10.1371/journal.pone.0099898. eCollection 2014.
Loss of Parkin, encoded by PARK2 gene, is a major cause of autosomal recessive Parkinson's disease. In Drosophila and mammalian cell models Parkin has been shown in to play a role in various processes essential to maintenance of mitochondrial quality, including mitochondrial dynamics, biogenesis and degradation. However, the relevance of altered mitochondrial quality control mechanisms to neuronal survival in vivo is still under debate. We addressed this issue in the brain of PARK2-/- mice using an integrated mitochondrial evaluation, including analysis of respiration by polarography or by fluorescence, respiratory complexes activity by spectrophotometric assays, mitochondrial membrane potential by rhodamine 123 fluorescence, mitochondrial DNA content by real time PCR, and oxidative stress by total glutathione measurement, proteasome activity, SOD2 expression and proteins oxidative damage. Respiration rates were lowered in PARK2-/- brain with high resolution but not standard respirometry. This defect was specific to the striatum, where it was prominent in neurons but less severe in astrocytes. It was present in primary embryonic cells and did not worsen in vivo from 9 to 24 months of age. It was not associated with any respiratory complex defect, including complex I. Mitochondrial inner membrane potential in PARK2-/- mice was similar to that of wild-type mice but showed increased sensitivity to uncoupling with ageing in striatum. The presence of oxidative stress was suggested in the striatum by increased mitochondrial glutathione content and oxidative adducts but normal proteasome activity showed efficient compensation. SOD2 expression was increased only in the striatum of PARK2-/- mice at 24 months of age. Altogether our results show a tissue-specific mitochondrial defect, present early in life of PARK2-/- mice, mildly affecting respiration, without prominent impact on mitochondrial membrane potential, whose underlying mechanisms remain to be elucidated, as complex I defect and prominent oxidative damage were ruled out.
由PARK2基因编码的帕金蛋白缺失是常染色体隐性帕金森病的主要病因。在果蝇和哺乳动物细胞模型中，已证实帕金蛋白在维持线粒体质量所必需的各种过程中发挥作用，包括线粒体动力学、生物合成和降解。然而，线粒体质量控制机制改变与体内神经元存活之间的相关性仍存在争议。我们使用综合线粒体评估方法，包括通过极谱法或荧光法分析呼吸作用、通过分光光度法测定呼吸复合体活性、通过罗丹明123荧光测定线粒体膜电位、通过实时PCR测定线粒体DNA含量以及通过总谷胱甘肽测量、蛋白酶体活性、超氧化物歧化酶2（SOD2）表达和蛋白质氧化损伤来评估氧化应激，从而在PARK2基因敲除小鼠的大脑中解决了这个问题。通过高分辨率呼吸测定法发现PARK2基因敲除小鼠大脑中的呼吸速率降低，但标准呼吸测定法未发现此现象。这种缺陷是纹状体特有的，在神经元中较为突出，而在星形胶质细胞中则不太严重。它存在于原代胚胎细胞中，并且在9至24个月龄的体内并未恶化。它与任何呼吸复合体缺陷无关，包括复合体I。PARK2基因敲除小鼠的线粒体内膜电位与野生型小鼠相似，但纹状体中随着年龄增长对解偶联的敏感性增加。纹状体中通过增加的线粒体谷胱甘肽含量和氧化加合物提示存在氧化应激，但正常的蛋白酶体活性显示出有效的补偿作用。仅在24月龄的PARK2基因敲除小鼠纹状体中SOD2表达增加。总之，我们的结果显示了一种组织特异性的线粒体缺陷，在PARK2基因敲除小鼠生命早期就已存在，并轻度影响呼吸作用，对线粒体膜电位没有显著影响，其潜在机制仍有待阐明，因为复合体I缺陷和明显的氧化损伤已被排除。
