THE ROLE OF CD2AP IN APP PROCESSING

Abstract

Genome-wide association studies (GWAS) have identified >20 risk loci for late-onset Alzheimer's disease (LOAD). Several of these risk genes are involved in endocytosis, a process believed to be disrupted during the early stages of LOAD. The CD2-associated protein (CD2AP) gene is strongly associated with LOAD risk (p=5.2x10-11) and plays a role in receptor-mediated endocytosis. We hypothesised that CD2AP contributes to amyloid precursor protein (APP) metabolism and subsequent amyloid –beta (Aβ) generation through its regulation of clathrin-mediated endocytosis. To test this, we initially characterised the effects of CD2AP knockdown on APP metabolism and Aβ production in a human neuronal cell line. A recent study found that another GWAS-identified risk gene PICALM, regulates trafficking of Aβ across the endothelial cells which form the blood brain barrier (BBB). We hypothesise that CD2AP acts in a similar way to PICALM and that knocking down CD2AP diminishes Aβ trafficking across the BBB, decreasing the amount of Aβ cleared from the brain. To investigate this, we are characterising the effects of CD2AP knockdown in an in vitro model of the BBB. Endogenous CD2AP expression was knocked down in H4 and hMEC/D3 cells using siRNA. Knockdown was quantified by western blot and Image J analysis. Levels of APP, Aβ, and APP metabolites were quantified using ELISAs. The rate of receptor-mediated endocytosis was measured by a transferrin uptake assay. Co-localisation of CD2AP with APP and endosomal markers was assessed using immunocytochemistry. Transcytosis in hMEC/D3 cells will be assessed using Transwell assays. We observed a significant 30.27±0.07 % increase in Aβ40 expression (p=0.0046). Expression levels of APP and APP processing metabolites was not significantly altered. Results from our experiments in progress will be made available at AAIC 2016. Knockdown of CD2AP expression resulted in a significant increase in extracellular amyloid-B (Aβ) levels, which did not appear to be directly related to changes in APP processing. This study is ongoing and novel data will be presented at AAIC 2016. Overall, our study provides valuable insights into how CD2AP contributes to LOAD susceptibility. Effect of CD2AP siRNA treatment of H4 cells on extracellular Aβ40 protein levels. Results are expressed as a percentage of oligofectamine only treated cells. Four biological replicates were analysed for each assay. Statistical significance of CD2AP siRNA vs. control siRNA treatment was determined by two-tailed student's t-test., **p = 0.0046; *= 0.0223 Error bars = SEM.