The Alzheimer's related disease vascular dementia is due to an age-dependent accumulation of small blood vessel ruptures in the brain. It affects almost a third of people over age 70. There is no treatment that reverses the dementia that reflects the damage to the brain caused by broken vessels. A better understanding of the molecular mechanisms underlying age associated cerebral blood vessel weakness is the most likely path to improved diagnostics and dementia prevention. The goal of this supplement is develop fly strains expressing variants of ApoE and mutations involved in the inherited blood vessel weakness Hereditary Hemorrhagic Telangiectasia (HHT) that will be employed to test two hypotheses for the causes of Alzheimer's disease and vascular dementia. The hypotheses are: 1) that germline mutations generating HHT are recapitulated over time by somatic mutations leading to age associated brain blood vessel weakness and 2) that HHT mutations synergyze with APOE4 to accelerate blood vessel ruptures. This supplement is within the scope of the parent award since we are funded to create a resource for the Drosophila 4th chromosome. This resource includes new fly strains with loss of function mutations for each gene on the 4th, new fly strains expressing each gene on the 4th and new fly strains expressing the two closest human homologs for each 4th chromosome gene. In this supplement we will create new fly strains expressing human genes with APOE4 and HHT mutations and employ them in a pilot project. The connection between the parent and supplement is that 4 of the 5 genes in the pilot are on the 4th chromosome. We have already created many of the necessary strains and thus expect to complete the pilot in one year. First we will create loss of function mutations in 5 fly genes (Apolpp and four genes with homologs implicated in HHT). These mutations are engineered to drive transgene expression in the mutant gene's native expression pattern. To set a baseline, expression of the fly gene corresponding to the mutant is employed to rescue the mutant phenotype. Rescue experiments will focus on defects in the fly blood brain barrier to mimic an APOE4 mutant phenotype and as an analog to the human vasculature for HHT mutations. We then humanize the mutant flies by overexpressing the cognate human homolog to evaluate the degree of rescue. The hypothesis is then tested by expressing ApoE variants and human homologs with HHT mutations individually and in combinations. Combinatorial experiments allow assessment of enhancing and suppressing interactions. Phenotypic differences between males and females as well as age-dependent increases in penetrance will be noted. The strains we generate can be employed to study other ApoE and HHT related diseases in humanized flies by the community. Our results will likely stimulate efforts to replicate our findings in vertebrate models of aging, dementia and Alzheimer's disease. Vertebrate model experiments could provide the nuanced understanding of the molecular mechanisms underlying vascular dementia necessary for identifying predictive diagnostics and perhaps preventive therapeutics.

PUBLIC PURPOSE We propose a supplemental project that will advance our understanding of vascular dementia. We will test an innovative hypothesis in a pilot project and create new resources for scientists. We will utilize the fruit fly model system to test the hypothesis that mutations causing an inherited form of blood vessel weakness are also the cause of age associated blood vessel weakness in the brain (the basis of vascular dementia). Our studies will shed light on basic biological mechanisms of brain blood vessel breakdown and suggest new preventive therapeutics.