Person:

Tanzi, Rudolph

A major rate-limiting step for Aβ generation and deposition in Alzheimer’s disease brains is BACE1-mediated cleavage (β-cleavage) of the amyloid precursor protein (APP). We previously reported that APP undergoes palmitoylation at two cysteine residues (Cys186 and Cys187) in the E1-ectodomain. 8–10% of total APP is palmitoylated in vitro and in vivo. Palmitoylated APP (palAPP) shows greater preference for β-cleavage than total APP in detergent resistant lipid rafts. Protein palmitoylation is known to promote protein dimerization. Since dimerization of APP at its E1-ectodomain results in elevated BACE1-mediated cleavage of APP, we have now investigated whether palmitoylation of APP affects its dimerization and whether this leads to elevated β-cleavage of the protein. Here we report that over 90% of palAPP is dimerized while only ~20% of total APP forms dimers. PalAPP-dimers are predominantly cis-oriented while total APP dimerizes in both cis- and trans-orientation. PalAPP forms dimers 4.5-times more efficiently than total APP. Overexpression of the palmitoylating enzymes DHHC7 and DHHC21 that increase palAPP levels and Aβ release, also increased APP dimerization in cells. Conversely, inhibition of APP palmitoylation by pharmacological inhibitors reduced APP-dimerization in coimmunoprecipitation and FLIM/FRET assays. Finally, in vitro BACE1-activity assays demonstrate that palmitoylation-dependent dimerization of APP promotes β-cleavage of APP in lipid-rich detergent resistant cell membranes (DRMs), when compared to total APP. Most importantly, generation of sAPPβ-sAPPβ dimers is dependent on APP-palmitoylation while total sAPPβ generation is not. Since BACE1 shows preference for palAPP dimers over total APP, palAPP dimers may serve as novel targets for effective β-cleavage inhibitors of APP as opposed to BACE1 inhibitors.

A goal of genomics is to understand the relationships between biological processes. Pathways contribute to functional interplay within biological processes through complex but poorly understood interactions. However, limited functional references for global pathway relationships exist. Pathways from databases such as KEGG and Reactome provide discrete annotations of biological processes. Their relationships are currently either inferred from gene set enrichment within specific experiments, or by simple overlap, linking pathway annotations that have genes in common. Here, we provide a unifying interpretation of functional interaction between pathways by systematically quantifying coexpression between 1,330 canonical pathways from the Molecular Signatures Database (MSigDB) to establish the Pathway Coexpression Network (PCxN). We estimated the correlation between canonical pathways valid in a broad context using a curated collection of 3,207 microarrays from 72 normal human tissues. PCxN accounts for shared genes between annotations to estimate significant correlations between pathways with related functions rather than with similar annotations. We demonstrate that PCxN provides novel insight into mechanisms of complex diseases using an Alzheimer’s Disease (AD) case study. PCxN retrieved pathways significantly correlated with an expert curated AD gene list. These pathways have known associations with AD and were significantly enriched for genes independently associated with AD. As a further step, we show how PCxN complements the results of gene set enrichment methods by revealing relationships between enriched pathways, and by identifying additional highly correlated pathways. PCxN revealed that correlated pathways from an AD expression profiling study include functional clusters involved in cell adhesion and oxidative stress. PCxN provides expanded connections to pathways from the extracellular matrix. PCxN provides a powerful new framework for interrogation of global pathway relationships. Comprehensive exploration of PCxN can be performed at http://pcxn.org/.

The heterogeneous precipitates of Aβ that accumulate in the brain cortex in Alzheimer's disease possess varying degrees of resistance to resolubilization. We previously found that Aβ1–40 is rapidly precipitated in vitro by physiological concentrations of zinc, a neurochemical that is highly abundant in brain compartments where Aβ is most likely to precipitate. We now present evidence that the zinc-induced precipitation of Aβ is mediated by a peptide dimer and favored by conditions that promote α-helical and diminish β-sheet conformations. The manner in which the synthetic peptide is solubilized was critical to its behaviorin vitro. Zinc-induced Aβ aggregation was dependent upon the presence of NaCl, was enhanced by α-helical-promoting solvents, but was abolished when the peptide stock solution was stored frozen. The Aβ aggregates induced by zinc were reversible by chelation, but could then be reprecipitated by zinc for several cycles, indicating that the peptide's conformation is probably preserved in the zinc-mediated assembly. In contrast, Aβ aggregates induced by low pH (5.5) were not resolubilized by returning the pH milieu to 7.4. The zinc-Aβ interaction exhibits features resembling the gelation process of zinc-mediated fibrin assembly, suggesting that, in events such as clot formation or injury, reversible Aβ assembly could be physiologically purposive. Such a mechanism is contemplated in the early evolution of diffuse plaques in Alzheimer's disease and suggests a possible therapeutic strategy for the resolubilization of some forms of Aβ deposit in the disease.