Faculty Bibliography

Despite intense research efforts, the physiological function and molecular environment of the amyloid precursor protein has remained enigmatic. Here we describe the application of time-controlled transcardiac perfusion cross-linking, a method for the in vivo mapping of protein interactions in intact tissue, to study the interactome of the amyloid precursor protein (APP). To gain insights into the specificity of reported protein interactions the study was extended to the mammalian amyloid precursor-like proteins (APLP1 and APLP2). To rule out sampling bias as an explanation for differences in the individual datasets, a small scale quantitative iTRAQ (isobaric tags for relative and absolute quantitation)-based comparison of APP, APLP1, and APLP2 interactomes was carried out. An interactome map was derived that confirmed eight previously reported interactions of APP and revealed the identity of more than 30 additional proteins that reside in spatial proximity to APP in the brain. Subsequent validation studies confirmed a physiological interaction between APP and leucine-rich repeat and Ig domain-containing protein 1, demonstrated a strong influence of Ig domain-containing protein 1 on the proteolytic processing of APP, and consolidated similarities in the biology of APP and p75

RNA amplification is a series of molecular manipulations designed to amplify genetic signals from small quantities of starting materials (including single cells and homogeneous populations of individual cell types) for microarray analysis and other downstream genetic methodologies. A novel methodology named terminal continuation (TC) RNA amplification has been developed in this laboratory to amplify RNA from minute amounts of starting material. Briefly, an RNA synthesis promoter is attached to the 3' and/or 5' region of cDNA utilizing the TC mechanism. The orientation of amplified RNAs is 'antisense' or a novel 'sense' orientation. TC RNA amplification is utilized for many downstream applications, including gene expression profiling, microarray analysis, and cDNA library/subtraction library construction. Input sources of RNA can originate from a myriad of in vivo and in vitro tissue sources. Moreover, a variety of fixations can be employed, and tissues can be processed for histochemistry or immunocytochemistry prior to microdissection for TC RNA amplification, allowing for tremendous cell type and tissue specificity of downstream genetic applications