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肌肉不自主收缩为特征的病症， 而痉挛性截瘫的特点是腿部肌肉进行性无力和僵硬。 此前，发表的一个大家族显示出渐进的、年龄相关的常染色体显性模式。 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外显率。遗传分析 显示这两种表型都与 ATP5G3 基因中的一个新错义突变有关 (c.318C>G, p.N106K)，编码 ATP 合酶的 c 亚基。最近，该变异的病理学是 经第二个家庭的一名肌张力障碍患者的鉴定证实，该患者被发现携带同样的肌张力障碍 突变为从头突变。进一步的实验表明 ATP 产生和复合 V 活性 患者成纤维细胞中的 ATP5G3 显着减少，与 ATP5G3 的预测作用一致。作为附加 确认后，还在果蝇中进行了利用等效错义过表达的实验 突变，导致果蝇的运动能力和复合 V 活性显着破坏。基于 根据这些初步结果，该提案的中心假设是 p.N106K 突变以显性作用 负面方式破坏复合体 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来检验假设。首先，将进行实验 描述了新生成的小鼠模型，其携带与致病性人类相当的错义突变 使用行为、分子和电生理学方法进行突变（p.N105K）。二、生化 p.N106K 突变的机制将使用大肠杆菌 ATP 合酶系统来阐明。具体来说， 突变将通过对疑似常染色体显性遗传的等效“人源化”替换而产生 细菌 c 亚基蛋白中的 ATP5G3 突变。这些替代的生化效应将 然后在细菌 ATP 合酶中进行探索，特别是因为它与 C 环的组装有关，即物理 F1 与 Fo 复合物的相互作用，和/或质子易位。最终，将开发出有效的治疗方法 通过使用基于 CRISPR 的基因编辑来灭活 ATP5G3N106K 突变中的显性 p.N106K 等位基因 患者的成纤维细胞。优化细胞培养中的编辑效率后，使用基于 CRISPR 的可行性 将使用 Atp5g3N105K 突变小鼠评估失活作为体内治疗方法。这样做的结果 实验将有助于为开发基于 CRISPR 的编辑作为遗传性疾病的可能治疗方法奠定基础 由显性失活突变引起的疾病，例如成骨不全症，这可能是唯一的 治疗的选择。这些 ATP5G3 突变的全面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 功能以及常染色体病理学和治疗 显性遗传疾病，特别是由显性失活突变引起的疾病。