Molecular mechanisms of dystonia and spastic paraplegia associated with mutations in ATP5G3

与 ATP5G3 突变相关的肌张力障碍和痉挛性截瘫的分子机制

• 批准号: 10799993
• 负责人: Jesse David Slone
• 金额: $ 40.66万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10799993
• 关键词: Alleles; Amino Acid Substitution; Animal Model; Behavioral; Behavioral Assay; Biochemical; Biological; Biological Models; Cell Culture Techniques; Cells; Characteristics; Clinical Treatment; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; Corpus striatum structure; Corticospinal Tracts; DNA; Defect; Disease; Dominant Genetic Conditions; Dominant-Negative Mutation; Drosophila genus; Dystonia; Electrophysiology (science); Equilibrium; Escherichia coli; Family; Fibroblasts; Focal Dystonias; Functional disorder; Gait abnormality; Genes; Genetic; Genetic Diseases; Heterozygote; Human; Inherited; Insecta; Investigation; Involuntary Movements; Knock-out; Knockout Mice; Leg; Limb structure; Link; Lower Extremity; Mammals; Missense Mutation; Mitochondria; Modeling; Molecular; Molecular Analysis; Movement; Mus; Muscle; Muscle Contraction; Mutant Strains Mice; Mutation; Nature; Neurologic; Neurologic Dysfunctions; Neuronal Plasticity; Orthologous Gene; Osteogenesis Imperfecta; Pathogenicity; Pathologic; Pathology; Pathway interactions; Patients; Pattern; Penetrance; Phenotype; Probability; Production; Proteins; Protons; Publishing; Reporting; Role; Smooth Muscle; Spastic Paraplegia; Synaptic Transmission; System; Testing; Thalamic structure; Variant; age related; autosomal dominant mutation; autosome; base editing; citrate carrier; de novo mutation; effective therapy; experimental study; fly; genetic analysis; in vitro Model; in vivo; insight; limb movement; mouse model; mutant; nervous system disorder; neuromuscular; novel; oligomycin sensitivity-conferring protein; overexpression; spasticity

Dystonia and spastic paraplegia are debilitating neurological conditions with distinct signs and underlying pathophysiology. Dystonia describes a range of conditions characterized by involuntary muscle contraction, while spastic paraplegias are characterized by progressive weakness and stiffness of the leg muscles. Previously, a large family was published displaying an autosomal dominant pattern of progressive, age- dependent spastic paraplegia and incomplete penetrance for generalized and focal dystonia. Genetic analysis showed that both phenotypes were connected to a single novel missense mutation in the gene ATP5G3 (c.318C>G, p.N106K), which encodes subunit c of ATP synthase. Recently, the pathology of this variant was confirmed by the identification of a dystonia patient from a second family who was found to be carrying the same mutation as a de novo mutation. Further experiments demonstrated that ATP production and complex V activity were significantly reduced in patient fibroblasts, consistent with the predicted role of ATP5G3. As additional confirmation, experiments were also performed in Drosophila utilizing overexpression of the equivalent missense mutation, which resulted in a significant disruption in the flies’ locomotor ability and complex V activity. Based on these preliminary results, the central hypothesis of this proposal is that the p.N106K mutation acts in a dominant negative manner to disrupt complex V function, leading to the dystonia and spastic paraplegia phenotype. This hypothesis will be tested using three complementary approaches. First, experiments will be performed to characterize a newly generated mouse model carrying a missense mutation equivalent to the pathogenic human mutation (p.N105K) using behavioral, molecular, and electrophysiological approaches. Second, the biochemical mechanisms of the p.N106K mutation will be elucidated using the E. coli ATP synthase system. Specifically, mutations will generated with the equivalent “humanized” substitutions for suspected autosomal dominant ATP5G3 mutations in the bacterial subunit c protein. The biochemical effects of each of these substitutions will then be explored in the bacterial ATP Synthase, particularly as it relates to assembly of the c-ring, the physical interaction of the F1 the Fo complexes, and/or proton translocation. Finally, effective therapies will be developed for the ATP5G3N106K mutation by using CRISPR-based gene editing to inactivate the dominant p.N106K allele in patient fibroblasts. After optimizing the editing efficiency in cell culture, the feasibility of using CRISPR-based inactivation as an in vivo treatment approach will be evaluated using Atp5g3N105K mutant mice. The results of this experiment will help lay the groundwork for developing CRISPR-based editing as a possible treatment for genetic diseases caused by dominant negative mutation such as osteogenesis imperfecta, which is probably the only option for treatment. This comprehensive characterization of these ATP5G3 mutations will yield valuable insights into dystonia and spastic paraplegia, complex V functionality, and the pathology and treatment of autosomal dominant genetic diseases, particularly those caused by dominant negative mutations.

肌张力障碍和痉挛性截瘫是使人衰弱的神经系统疾病，具有明显的体征和潜在的症状 病理生理学肌张力障碍描述了一系列以不自主肌肉收缩为特征的病症， 而痉挛性截瘫的特征在于腿部肌肉的进行性无力和僵硬。 以前，发表了一个大家族，显示出一种常染色体显性模式，进行性，年龄- 依赖性痉挛性截瘫和全身性和局灶性肌张力障碍的不完全痉挛。遗传分析 结果表明，这两种表型都与ATP 5G 3基因中的一个新的错义突变有关。 （c.318C>G，p.N106K），编码ATP合酶亚基c。最近，这种变异的病理学是 第二个家庭的一名肌张力障碍患者被发现携带同样的基因， 突变为de novo突变。进一步的实验表明，ATP的产生和复合物V的活性 在患者成纤维细胞中显著降低，与ATP 5G 3的预测作用一致。作为附加 为了证实这一点，还在果蝇中进行了实验，利用等同的错义 突变，导致果蝇的运动能力和复合物V活性显著中断。基于 根据这些初步结果，该提议的中心假设是，p.N106K突变在显性遗传中起作用。 负性方式破坏复合体V功能，导致肌张力障碍和痉挛性截瘫表型。这 将使用三种互补的方法对假设进行检验。首先，将进行实验， 表征携带等同于致病人类的错义突变的新生成的小鼠模型 突变（p.N105K）的研究。第二，生化 p.N106K突变的机制将使用E. coli ATP合成酶系统。具体地说， 疑似常染色体显性遗传的等同“人源化”取代将产生突变 细菌C亚基蛋白中的ATP 5G 3突变。每一种替换的生化效应 然后在细菌ATP合酶中进行探索，特别是因为它涉及c环的组装， F1与Fo复合物的相互作用和/或质子移位。最后，将开发有效的治疗方法 对于ATP 5G 3 N106 K突变，通过使用基于CRISPR的基因编辑来消除显性p.N106K等位基因， 患者成纤维细胞。在优化细胞培养中的编辑效率后，使用基于CRISPR的 将使用Atp 5g 3 N105 K突变小鼠评价作为体内治疗方法的灭活。的结果 这项实验将有助于为开发基于CRISPR的编辑奠定基础，作为一种可能的遗传治疗方法。 由显性负突变引起的疾病，如成骨细胞增生症，这可能是唯一的 治疗的选择。这些ATP 5G 3突变的综合表征将产生有价值的见解 肌张力障碍和痉挛性截瘫，复杂V功能，以及常染色体 显性遗传病，特别是由显性负突变引起的疾病。