Partial Purification and Characterization of γ-Secretase from Post-mortem Human Brain

Abstract One characteristic feature of Alzheimer's disease is the deposition of amyloid β-peptide (Aβ) as amyloid plaques within specific regions of the human brain. Aβ is derived from the amyloid β-peptide precursor protein (β-APP) by the intramembranous cleavage activity of γ-secretase. Studies in cells have revealed that γ-secretase is a large multimeric membrane-bound protein complex that is functionally dependent on several proteins, including presenilin, nicastrin, Aph-1, and Pen-2. However, the precise biochemical and molecular nature of γ-secretase is as yet to be fully elucidated, and no investigations have analyzed γ-secretase in human brain. To address this we have developed a novel in vitro γ-secretase activity assay using detergent-solubilized cell membranes and a β-APP-derived fluorescent probe. We report that human brain-derived γ-secretase activity co-purifies with a high molecular weight protein complex comprising presenilin, nicastrin, Aph-1, and Pen-2. The inhibitor profile and solubility characteristics of brain-derived γ-secretase are similar to those described in cells, and proteolysis occurs at the Aβ40- and Aβ42-generating cleavage sites. The ability to isolate γ-secretase from post-mortem human brain may facilitate the identification of brain-specific modulators of β-APP processing and provide new insights into the biology of this important factor in the pathogenesis of Alzheimer's disease.