Formichi Patrizia, Radi Elena, Giorgi Eleonora, Gallus Gian Nicola, Brunetti Jlenia, Battisti Carla, Rufa Alessandra, Dotti Maria Teresa, Franceschini Rossella, Bracci Luisa, Federico Antonio
Department of Medicine, Surgery and Neurosciences, University of Siena, V.le Bracci, 2, 53100 Siena, Italy.
Department of Medical Biotechnologies, University of Siena, V. A. Moro, 2, 53100 Siena, Italy.
J Neurol Sci. 2015 Apr 15;351(1-2):99-108. doi: 10.1016/j.jns.2015.02.047. Epub 2015 Mar 6.
Autosomal dominant optic atrophy (ADOA) is a hereditary optic neuropathy characterized by bilateral symmetrical visual loss, decrease in retinal ganglion cells and a loss of myelin within the optic nerve. ADOA is associated to mutations in Optic atrophy 1 gene (OPA1), which encodes a mitochondrial protein involved in cristae remodeling, maintenance of mitochondrial membrane integrity, mitochondrial fusion and apoptosis regulation. We thus evaluated the rate of apoptosis and the expression levels of OPA1 isoforms in ADOA and control cells. Peripheral blood lymphocytes from eight patients with OPA1 mutation and age matched controls were cultivated both in basal conditions or with 2-deoxy-D-ribose, a reducing sugar that induces apoptosis through oxidative stress. Apoptosis was analyzed by flow cytometry, phosphatidylserine translocation, mitochondrial membrane depolarization and caspase 3 activation. We also analyzed the expression levels of OPA1 isoforms in ADOA and control cells cultured with and without 2-deoxy-D-ribose. We showed an increased percentage of apoptotic cells in ADOA patients compared to controls, both in basal culture conditions and after 2-deoxy-D-ribose treatment. This suggested a great susceptibility of ADOA cells to oxidative stress and a strong correlation between OPA1 protein dysfunctions and morphological-functional alterations to mitochondria. Moreover OPA1 protein expression was significantly decreased in lymphocytes from the ADOA patients after 2-deoxy-D-ribose treatment, implying a great sensitivity of the mutated protein to free radical damage. Concluding, we could confirm that oxidative stress-induced apoptosis may play a key role in the pathophysiological process bringing to retinal ganglion cells degeneration in ADOA.
常染色体显性遗传性视神经萎缩（ADOA）是一种遗传性视神经病变，其特征为双侧对称性视力丧失、视网膜神经节细胞减少以及视神经髓鞘缺失。ADOA与视神经萎缩1基因（OPA1）突变有关，该基因编码一种参与嵴重塑、维持线粒体膜完整性、线粒体融合及凋亡调控的线粒体蛋白。因此，我们评估了ADOA细胞和对照细胞中的凋亡率以及OPA1亚型的表达水平。采集了8例OPA1突变患者及年龄匹配的对照者的外周血淋巴细胞，分别在基础条件下或与2-脱氧-D-核糖共同培养，2-脱氧-D-核糖是一种还原糖，可通过氧化应激诱导细胞凋亡。通过流式细胞术、磷脂酰丝氨酸易位、线粒体膜去极化和半胱天冬酶3激活来分析细胞凋亡情况。我们还分析了在添加和不添加2-脱氧-D-核糖的情况下培养的ADOA细胞和对照细胞中OPA1亚型的表达水平。结果显示，与对照者相比，无论是在基础培养条件下还是在2-脱氧-D-核糖处理后，ADOA患者的凋亡细胞百分比均增加。这表明ADOA细胞对氧化应激高度敏感，且OPA1蛋白功能障碍与线粒体形态功能改变之间存在密切关联。此外，2-脱氧-D-核糖处理后，ADOA患者淋巴细胞中的OPA1蛋白表达显著降低，这意味着突变蛋白对自由基损伤高度敏感。总之，我们可以确认氧化应激诱导的细胞凋亡可能在导致ADOA患者视网膜神经节细胞变性的病理生理过程中起关键作用。
