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. [unreadable] [unreadable]