The increasing capacity of DNA sequencing provides an unprecedented opportunity LY294002 for such large-scale studies using patient samples, and our fl-Htt brain interactome may provide
converging information on candidates that may have both a genetic and proteomic link to Htt. Thus, our study lends strong support to a systems biology strategy of vertically integrating large genetic, genomic, and interactome data sets (Geschwind and Konopka, 2009) derived from HD models of different organismal complexity to unravel the conserved mechanism related to Htt biology and HD pathogenesis. Our study supports the view that the majority of Htt-interacting proteins are relatively stable across brain tissue and age (Figures 2A and 2B), while a portion of the Htt interactome is quite dynamic. The latter group of proteins, particularly those that consistently complex with mHtt in a brain-regional-specific or age-specific
manner (Figures 2C–2D), could be interesting candidates to study for their contribution to selective neuronal vulnerability and age-dependent pathogenesis in HD. Our studies also raised the intriguing possibility that age-dependent changes in the normal brain proteome (e.g., Sirt2) may alter Htt interactions, which could in turn contribute to the presently unexplained role of aging in disease pathogenesis (Maxwell et al., 2011). A major advance Dolutegravir supplier in this study is the use of a systems biology approach to construct in vivo protein interaction networks exclusively using first proteomic interactome data sets generated from complex tissue. We applied, for the first time, WGCNA to analyze all the unique peptide count information for an entire group of Htt complexed proteins in our spatiotemporal AP-MS data set. WGCNA provides an unbiased systems-level organization of gene expression modules in both normal and diseased brains (Voineagu
et al., 2011) and has been demonstrated to be among the most powerful methods for global network construction (Allen et al., 2012). Several lines of evidence support the validity and value of WGCNA analyses of our in vivo Htt interactome data set. We were able to show that the pairwise correlation measure leads to a meaningful ranking of Htt-related proteins with respect to the external annotated knowledge of HD-related proteins (Huntington’s Disease Signaling in IPA; Figure 3C). WGCNA identified six significant Htt-correlated modules with distinct tissue- or age-specific overrepresentation and significant enrichment of distinct biological function previously implicated in Htt biology (Figure 6), effectively providing an in silico dissection of the molecular processes related to fl-Htt biology. Several experimental factors were instrumental to the construction of WGCNA networks based on our AP-MS data set. First, the relative level of bait protein (fl-Htt) brought down by IP is markedly, but reproducibly, variable across all samples.