Biology of Tauopathies Studied with HSV Amplicons

用 HSV 扩增子研究 Tau蛋白病的生物学

• 批准号: 6921193
• 负责人: E. Antonio Chiocca
• 金额: $ 29.68万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-19 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6921193
• 关键词: Alzheimer' s disease; Herpesviridae; artificial chromosomes; cerebral degeneration; gene mutation; genetic mapping; laboratory mouse; microtubule associated protein; neurotoxicology; phosphorylation; protein structure function; tau proteins

DESCRIPTION (provided by applicant): A novel investigative tool for studying genome complexity is represented by the infectious bacterial artificial chromosome (iBAC). The iBAC system is based on packaging an entire genomic locus into an HSV amplicon, followed by infectious transfer into cells. Preliminary demonstration of validity has been accomplished by showing delivery, alternative splicing and expression of three large genomic loci (>100 Kb) into cells. The microtubule-associated protein Tau is involved in a variety of neurodegenerative dementias, a public health problem without effective therapy. Recently, mutations of the tau gene have been linked to frontotemporal dementias and Parkinsonism associated with chromosome 17 (FTDP-17). Several mutations are found in one of the introns that follow exon 10 of the gene, in the context of the HI/HI genetic haplotype associated with neurodegeneration. Tau regulation undergoes relatively complex alternative splicing to produce six isoforms in adult brain. A distinguishing characteristic of the isoforms is the presence of a three vs. a four tandem repeat microtubule binding domain. The ratio of three vs. four repeats isoforms is normally 1, but in intronically-mutated tau this ratio becomes altered because of disrupted exon 10 splicing. In genetic population studies, tau intronic mutations are associated with altered isoform ratios and neurodegenerative phenotypes. Of relevance, even in common sporadic dementias (such as Alzheimer's disease) subtle alterations in Tau isoform ratios have been postulated to be linked to neurodegeneration (Goedert et al., 2000). Biologic studies and models are thus needed to decipher cause-effect relationships between altered Tau isoform ratios and neurotoxicity. In this proposal, the wild-type tau genomic locus, its intronic splice mutants will each be retrofitted into iBACs in the context of haplotypes linked to dementia or haplotypes that are not linked. The pattern of wild-type and aberrant tau splicing will then be recapitulated in neurons after iBAC transfer of each construct and correlated with effects on Tau function and neurotoxicity (Aim 1). The effect of intronic tau mutations will be further assayed by investigating changes in Tau phosphorylation and microtubule binding, in neurons expressing each iBAC-tau (Aim 2). Finally, we will determine if abnormal amyloid will affect the viability and function of neurons that express each iBAC-tau (Aim 3). The iBAC should provide an excellent tool for further analyses of the biological significance of intronic mutations in Tau-associated pathogenesis and for high-throughput discovery of drugs that may affect the neurodegeneration associated with Tau mutants.

描述(申请人提供)：传染性细菌人工染色体(IBAC)是研究基因组复杂性的一种新的研究工具。IBAC系统的基础是将整个基因组位点包装成HSV扩增子，然后感染性地转移到细胞中。通过展示三个大的基因组座位(&gt；100kb)的递送、选择性剪接和表达，初步证明了其有效性。微管相关蛋白Tau与多种神经退行性痴呆有关，这是一个公共卫生问题，没有有效的治疗方法。最近，tau基因的突变被认为与额颞性痴呆和17号染色体相关的帕金森症(FTDP-17)有关。在该基因第10外显子之后的一个内含子中发现了几个突变，与神经退行性变相关的HI/HI遗传单倍型。Tau调控在成人大脑中经历了相对复杂的选择性剪接，产生了六种异构体。这些异构体的一个显著特征是存在三个或四个串联重复的微管结合域。三对四重复异构体的比例通常为1，但在内在突变的tau中，由于外显子10剪接中断，这一比例发生了变化。在遗传群体研究中，tau内含子突变与异构体比率改变和神经退行性变表型相关。相关的是，即使在常见的散发性痴呆(如阿尔茨海默病)中，Tau异构体比率的细微变化也被假定与神经退化有关(Goedert等人，2000年)。因此，需要生物学研究和模型来破译改变的Tau异构体比率和神经毒性之间的因果关系。在这项建议中，野生型tau基因组座位及其内含子剪接突变体将分别在与痴呆症相关的单倍型或未连锁的单倍型的背景下改造成iBacs。野生型和异常tau剪接模式将在每个构建的IBAC转移后在神经元中重现，并与对Tau功能和神经毒性的影响相关(目标1)。将通过研究表达每个IBAC-tau的神经元中tau磷酸化和微管结合的变化来进一步分析tau内含子突变的影响(目标2)。最后，我们将确定异常淀粉样蛋白是否会影响表达每个IBAC-tau的神经元的活性和功能(目标3)。IBAC将为进一步分析内含子突变在Tau相关发病机制中的生物学意义以及高通量发现可能影响与Tau突变相关的神经变性的药物提供一个极好的工具。