Investigating the pathogenesis of CoQ10 deficiencies

描述（由申请人提供）：辅酶Q10 （CoQ10）是一种小亲脂分子，由苯醌环和疏水类异戊二烯尾部组成，几乎存在于所有细胞膜中。在线粒体呼吸链中，辅酶q10对于电子从复合体I和复合体II到复合体III的传输至关重要。它也是抗氧化剂、膜稳定剂和细胞凋亡调节剂。人类辅酶q10缺乏症与四种临床表型相关。所有形式的辅酶q10缺乏症患者在口服补充后都有所改善，因此认识到这种可治疗的遗传疾病是很重要的。在过去的十年中，候选人和她的导师收集了84名患者（71个家庭）的生物样本，这些患者在肌肉和/或成纤维细胞中记录了辅酶q10缺乏，或根据临床表现以及对辅酶q10补充的反应怀疑辅酶q10缺乏。共有54名患者（48个家庭）在肌肉、成纤维细胞或两者中均存在辅酶q10缺乏症。2006年，研究小组报告了CoQ10生物合成基因COQ2的首次突变，COQ2编码4-对羟基苯甲酸酯：聚戊烯基转移酶；PDSS2编码十烯二磷酸合成酶的亚基2。此外，在一个有四个人患有小脑性共济失调和辅酶q10缺乏症的家庭中，他们发现了APTX基因的致病突变，该基因编码一种参与单链断裂修复的蛋白质。因此，这些研究表明辅酶q10缺乏症可能是原发性的，也可能是继发性的。不足为奇的是，CoQ10缺乏会导致呼吸链活性缺陷（复合物I+III和II+III的活性降低）。呼吸链缺陷、ROS产生和细胞凋亡在辅酶q10缺乏症发病机制中的相对重要性尚不清楚。研究小组研究了COQ2和PDSS2突变的培养皮肤成纤维细胞中CoQ10严重缺乏对生物能量学、氧化应激和抗氧化防御的影响。CoQ10生物合成途径的前两个步骤中的缺陷会产生不同的生化改变。与对照细胞相比，PDSS2突变型成纤维细胞的辅酶q10含量为12%，ATP合成明显减少，但没有表现出活性氧（ROS）产生增加、氧化应激迹象或抗氧化防御标记物增加。相比之下，COQ2突变型成纤维细胞含有30% CoQ10， ATP合成部分缺陷，ROS生成和脂质和蛋白质氧化显著增加。为了更好地了解辅酶q10缺乏的发病机制，研究小组描述了不同程度的辅酶q10缺乏对具有不同辅酶q10生物合成遗传缺陷的细胞中ROS产生和线粒体生物能量学的影响。他们证实了之前的发现，并进一步观察到辅酶q10水平与活性氧产生之间的相关性遵循抛物线曲线；10-15%残留CoQ10和60-70%残留CoQ10与ROS生成无关，而30-50%残留CoQ10与ROS生成的最大增加相关。此外，活性氧的增加似乎与最初的超极化，随后的去极化和细胞死亡有关。在CoQ10缺乏患者的成纤维细胞中使用CoQ10和其他抗氧化剂治疗的初步结果证实了这些数据。

DESCRIPTION (Provided by Applicant): Coenzyme Q10 (CoQ10) is a small lipophilic molecule composed of a benzoquinone ring and a hydrophobic isoprenoid tail which is present in virtually all cell membranes. In the mitochondrial respiratory chain, CoQ10 is vital for the transport of electrons from complex I and complex II to complex III. It is also an antioxidant, membrane stabilizer, and modulator of apoptosis. Human CoQ10-deficiency has been associated with four clinical phenotypes. Patients with all forms of CoQ10-deficiency have improved with oral supplementation, therefore recognition of this treatable genetic condition is important. In the last decade, the candidate and her mentors have collected biological samples from 84 patients (71 families) with documented CoQ10 deficiency in muscle and/or fibroblasts, or suspected CoQ10 deficiency based on the clinical manifestations as well as the response to CoQ10 supplementation. A total of 54 patients (48 families) have documented CoQ10 deficiency in muscle, fibroblasts, or both. In 2006, the investigative team reported the first mutations in CoQ10 biosynthetic genes, COQ2, which encodes 4-para-hydroxybenzoate: polyprenyl transferase; and PDSS2, which encodes subunit 2 of decaprenyl diphosphate synthase. In addition, in a family with four individuals with cerebellar ataxia and CoQ10 deficiency, they identified a pathogenic mutation in the APTX gene, which encodes a protein involved in single-strand break repair. Thus, these studies have revealed that CoQ10 deficiency can be primary or secondary. Not surprisingly, CoQ10 deficiency causes defects of respiratory chain activities (reduced activities of complexes I+III and II+III). The relative importance of respiratory chain defects, ROS production, and apoptosis in the pathogenesis of CoQ10-deficiency is unknown. The investigative team studied the consequences of severe CoQ10 deficiency on bioenergetics, oxidative stress, and antioxidant defenses in cultured skin fibroblasts harboring COQ2 and PDSS2 mutations. Defects in the first two committed steps of the CoQ10 biosynthetic pathway produce different biochemical alterations. PDSS2 mutant fibroblasts have 12% CoQ10 relative to control cells and markedly reduced ATP synthesis, but do not show increased reactive oxygen species (ROS) production, signs of oxidative stress, or increased antioxidant defense markers. In contrast, COQ2 mutant fibroblasts have 30% CoQ10 with partial defect in ATP synthesis, and significantly increased ROS production and oxidation of lipids and proteins. To better understand the pathogenesis of CoQ10 deficiency, the investigative team has characterized the effects of varying severity of CoQ10 deficiency on ROS production and mitochondrial bioenergetics in cells harboring different genetic defects of CoQ10 biosynthesis. They confirmed their previous findings and further observed that the correlation between level of CoQ10 and ROS production follows a parabolic curve; 10-15% residual CoQ10 and 60-70% are not associated with significant ROS production, whereas 30-50% residual CoQ10 is associated with the maximum increases in ROS production. Moreover, increase in reactive oxygen species appears to be associated with initial hyperpolarization followed by depolarization and cell death. These data are corroborated by preliminary results of treatment with CoQ10 and other antioxidants in fibroblasts from the CoQ10 deficient patients. To better understand the pathogenesis of human CoQ10 deficiency the candidate proposes the following three specific aims: Aim 1: To identify novel genetic causes of CoQ10 deficiency. Aim 2: To understand the mitochondrial bioenergetics and oxidative stress consequences of different degrees of CoQ10 deficiency in the same genetic background, she will modulate COQ2 and PDSS2 expression using RNA interference (RNAi). Aim 3: To test ROS scavenging as a potential therapeutic strategy, she will overexpress the enzyme superoxide manganese dismutase (MnSOD) in COQ2 mutant fibroblasts and will assess level of ROS, oxidative stress, and apoptosis. NARRATIVE: Defects of mitochondria cause diverse human diseases. A subtype of mitochondrial disease is caused by deficiency of coenzyme Q10 (CoQ10), an essential component of the mitochondria involved in energy production. Patients with CoQ10 deficiency often improve dramatically with CoQ10 supplementation. The candidate will study patients with this disease and she will attempt to understand why the mutations cause CoQ10 deficiency. Knowing the cause of CoQ10 deficiency will likely enhance our scientific knowledge of CoQ10 biosynthesis, and will provide molecular tests for accurate genetic counseling, prenatal diagnosis, and more rapid initiation of the therapy.