P4-300: Gene expression profiling to identify microvascular changes in Alzheimer's disease mouse models

Dense-core amyloid-β (Aβ) plaques and congophilic amyloid angiopathy are pathological hallmarks of Alzheimer's disease (AD). However, it is not yet clear how these deposits initially aggregate, i.e. whether aggregation is spontaneous or is mediated by specific interactions of Aβ with other brain proteins. We recently showed that dense-core plaques in Tg2576 and PSAPP mouse models and in Flemish APP A692G AD patients are centered on vessel walls and this is also supported by similar recent observations on AD and Down's syndrome patients. Even more importantly, considerable microvascular damage and blood-brain barrier abnormalities were also identified in both amyloid-associated and non-amyloidogenic vessels in the AD mouse models and in AD patients. Objectives: To identify seeding factors responsible for the vascular entrapment of Aβ and to elucidate changes occurring in blood vessels even prior to amyloid deposition by a mixed transcriptomic and proteomic approach. Tg2576, TgN, BRI-Aβ42, and BRI-Aβ40 AD mouse models and littermate controls of different ages are utilized to isolate vascular early dense-core plaques and non-amyloidotic vessels by laser microdissection (PALM MicroBeam, Zeiss) for transcriptional profiling by Agilent microarrays. Frontal neocortical and entire hippocampal tissue of Tg2576 mice and littermate controls are also laser-microdissected as these are the first regions where Aβ is being deposited in these mice. In addition, 2D-DIGE and MALDI-TOF MS/MS analyses will also be performed on this tissue. For optimal transcript preservation, a short twenty-minutes staining protocol has been optimized to visualize early Aβ deposits and/or vessels in brains of transgenic mice and littermate controls. Frozen brain sections were fixed with ice-cold 70% ethanol and fluorescently stained with Thioflavin-S and collagen IV. High-quality total RNA suitable for microarray studies as measured with Experion HighSens chip was extracted from the laser-microdissected tissue. Currently, microarray analyses are being done to elucidate changes in the gene expression profile. Our data suggest that extraction of high-quality RNA from small amounts of laser-microdissected tissue is possible. The strategy discussed here will be important to elucidate the molecular mechanisms of plaque formation and the vascular changes that occur in blood vessels prior to Aβ deposition in Alzheimer Disease.