Biology of Tauopathies Studied with HSV Amplicons

描述（由申请人提供）：一种用于研究基因组复杂性的新型研究工具由感染性细菌人工染色体（iBAC）代表。iBAC系统基于将整个基因组基因座包装到HSV扩增子中，然后感染性转移到细胞中。通过显示三个大基因组位点（>100 Kb）到细胞中的递送、选择性剪接和表达，已经完成了有效性的初步证明。微管相关蛋白Tau参与了多种神经退行性痴呆，这是一种没有有效治疗的公共卫生问题。最近，tau基因的突变与额颞叶痴呆和17号染色体相关的帕金森综合征（FTDP-17）有关。在与神经变性相关的HI/HI遗传单倍型的背景下，在该基因的外显子10之后的一个内含子中发现了几个突变。Tau蛋白的调控经历了相对复杂的选择性剪接，在成人脑中产生六种亚型。同种型的区别特征是存在三个与四个串联重复微管结合结构域。三个与四个重复同种型的比率通常为1，但在内含子突变的tau中，该比率由于外显子10剪接被破坏而改变。在遗传群体研究中，tau内含子突变与改变的同种型比例和神经退行性表型相关。相关的是，即使在常见的散发性痴呆（如阿尔茨海默病）中，Tau同种型比率的细微改变也被假定与神经变性有关（Goedert等人，2000年）。因此，需要生物学研究和模型来解释改变的Tau亚型比例和神经毒性之间的因果关系。在该提议中，野生型tau基因组基因座、其内含子剪接突变体将各自在与痴呆相关的单倍型或不相关的单倍型的背景下被改造成iBAC。野生型和异常tau剪接的模式然后将在每种构建体的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（目的1）。内含子tau突变的作用将通过研究表达每种iBAC-tau的神经元中的Tau磷酸化和微管结合的变化来进一步测定（Aim 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的神经元的活力和功能（Aim 3）。iBAC将为进一步分析Tau相关发病机制中内含子突变的生物学意义以及高通量发现可能影响与Tau突变体相关的神经变性的药物提供极好的工具。

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.