Characterization of an FAD pathogenic PS1 mutation with complete loss of activity

完全丧失活性的 FAD 致病性 PS1 突变的表征

• 批准号: 8366242
• 负责人: Hannah Louise Brautigam
• 金额: $ 4.22万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2014-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8366242
• 关键词: Active Sites; Affect; Alternative Splicing; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Animal Model; Animals; Antibodies; Aspartic Endopeptidases; Behavioral; Binding; Biological Assay; Cell Culture Techniques; Cessation of life; Cleaved cell; Co-Immunoprecipitations; Complex; Confocal Microscopy; DNA; Data; Deletion Mutation; Deposition; Disease; Embryo; Enzyme-Linked Immunosorbent Assay; Enzymes; Epidemic; Exhibits; Exons; Family; Family member; Genes; Genetic; Goals; Hippocampus (Brain); Human; In Vitro; Inflammation; Intervention; Knock-out; Knockout Mice; Laboratories; Lead; Membrane Proteins; Memory impairment; Methods; Minor; Missense Mutation; Modeling; Molecular; Molecular Conformation; Motor; Mus; Mutation; Neocortex; Neurites; Neurofibrillary Tangles; Neuronal Dysfunction; Neurons; Pathology; Pattern; Peptides; Phenotype; Proteins; Proteolysis; Senile Plaques; Structure; Synapses; Techniques; Technology; Testing; Therapeutic Intervention; United States; Western Blotting; Work; amyloid pathology; amyloid structure; base; conditioned fear; dimer; extracellular; familial Alzheimer disease; hyperphosphorylated tau; in vivo; loss of function mutation; motor deficit; mouse model; mutant; neuropathology; novel; presenilin-1; protein function; secretase; tau Proteins

DESCRIPTION (provided by applicant): The neuropathological hallmarks of Alzheimer's disease (AD) consist of extracellular amyloid plaques, amyloid angiopathy, neurofibrillary tangles, inflammation, dystrophic neurites, and neuronal death. Rare, autosomal dominant familial forms of AD (FAD) have provided us with most of what is currently known of the molecular basis of the disease. Most FAD cases result in mutations in a polytopic membrane protein, presenilin 1 (PS1), that contains the active site for the 3-secretase enzyme, and which, in turn, determines the carboxyl terminus of the amyloid beta peptide (A2). Most A2 peptides end at residue 40, but a minor quantity ends at residue 42 and are highly prone to aggregation. The amyloid hypothesis of AD suggests that this increase in A2-42/40 results in a cascade that ultimately leads to hyperphosphorylated tau, synaptic dysfunction, and neuronal death. When new FAD families are identified, routinely, the PS1 gene is sequenced, and if a PS1 mutation is the cause, then either missense or deletion mutations will be found in the DNA of affected but not unaffected family members. Such was the case of the Tasmanian (Tas-1) family. Kwok et al. (2003) described the pathology and the genetics of the Tas-1 family; the pathogenic mutation in Tas-1 is a missense mutation, L271V, which disrupts alternative splicing of the PS1 gene, and results in a gene that lacks exon 8 (PS1 8). The goal of the current project is to characterize this novel FAD pathogenic PS1 mutation. Based on the work of Kwok et al. (2003), the overall objectives and methods of the project will be to determine if PS1 8 is catalytically inactive. Preliminary data showed that crossing the PS1 8 mutation with a PS1 (-/-) knock-out (KO) mouse did not rescue the KO phenotype seen in PS1 (-/-) KO animals, as they are embryonic lethal. In the current proposal, behavioral deficits and levels of A2-42, A2-42/40, and A2 oligomers will be determined in PS1 8 mice crossed with a mouse model harboring a mutation in APP. Also, histological analyses will be utilized and APP cleavage will be determined in these mouse models. Finally, further to evaluate whether PS1 8 is a loss of function mutation and to distinguish between the older dimer model and the more recent 1:1:1:1 model of the 3-secretase complex, cell culture, co-immunoprecipitation, confocal microscopy, and alpha screen techniques will be employed to evaluate whether PS1 8 is interacting with wt PS1 resulting in 3-secretase's altered substrate cleavage. Resolving the function of this protein in vivo and in vitro will allow us to further understand the mechanisms underlying FAD and possibly elucidate PS1 as a putative target of therapeutic intervention for AD.

描述（由申请人提供）：阿尔茨海默氏病的神经病理学标志（AD）由细胞外淀粉样pla，淀粉样蛋白血管病，神经纤维纤维缠结，炎症，营养不良神经产生和神经元死亡组成。罕见的，常染色体主导的AD（FAD）为我们提供了目前已知的疾病分子基础的大部分内容。大多数FAD病例都会导致在多重膜蛋白（PS1）中发生突变，其中包含3-分泌酶的活性位点，并依次确定淀粉样蛋白β肽的羧基末端（A2）。大多数A2肽在残基40结束，但在残基42处少量结束，并且很容易聚集。 AD的淀粉样假说表明，A2-42/40的增加导致级联反应最终导致高磷酸化的TAU，突触功能障碍和神经元死亡。通常，定期确定新的时尚家族，将对PS1基因进行测序，如果PS1突变是原因，则在受影响但未受到影响的家庭成员的DNA中发现错义或缺失突变。塔斯马尼亚州（Tas-1）家族就是这种情况。 Kwok等。 （2003年）描述了Tas-1家族的病理和遗传学； TAS-1中的致病突变是一种错义突变L271V，它破坏了PS1基因的替代剪接，并导致缺乏外显子8（PS1 8）的基因。当前项目的目的是表征这种新型的FAD致病PS1突变。根据Kwok等人的工作。 （2003年），项目的总体目标和方法将是确定PS1 8是否催化无效。初步数据表明，将PS1 8突变与PS1（ - / - ）敲除（KO）小鼠跨越胚胎杀伤力，并未挽救PS1（ - / - ）KO动物中看到的KO表型。在当前的建议中，将在A2-8小鼠中确定A2-42，A2-42/40和A2低聚物的行为缺陷和水平的水平，并与APP中具有突变的小鼠模型交叉的PS1 8小鼠。同样，将利用组织学分析，并将在这些小鼠模型中确定应用裂解。 Finally, further to evaluate whether PS1 8 is a loss of function mutation and to distinguish between the older dimer model and the more recent 1:1:1:1 model of the 3-secretase complex, cell culture, co-immunoprecipitation, confocal microscopy, and alpha screen techniques will be employed to evaluate whether PS1 8 is interacting with wt PS1 resulting in 3-secretase's altered substrate cleavage.解决该蛋白质在体内和体外的功能将使我们能够进一步理解FAD的基础机制，并可能阐明PS1作为AD治疗干预的推定靶标。