Abstract
γ-Secretase is involved in the final processing of the amyloid precursor protein into a heterogeneous pool of β-amyloid (Aβ) peptides. Current Alzheimer’s disease drug discovery efforts include targeting γ-secretase activity in brain to attenuate production of the neurotoxic Aβ species. The resulting pharmacology may be affected by species-specific differences in the γ-secretase core complex or its associated proteins. Therefore, we utilized partially purified γ-secretase membranes derived from the brains of different species, including human cortex, to quantitatively assess the de novo production of both Aβ42 and Aβ40 following treatment with known γ-secretase inhibitors and modulators. We determined that the inhibitory activity of a Notch-1 sparing γ-secretase inhibitor and the modulatory activity of two classes of γ-secretase modulators were equipotent at affecting the production of Aβ across rodent and human brain membrane preparations. Additionally, the observed modulator-specific Aβ profile in isolated brain membranes across species was similar to that observed in HeLa cell membranes, and the brain and CSF of guinea pigs following oral administration. By utilizing rapidly purified γ-secretase, we were able to probe and compare the complex pharmacology of γ-secretase in the brain across common rodent species and human cortex.
Highlights
According to the amyloid cascade hypothesis of Alzheimer’s disease (AD) pathogenesis, deposition of β-amyloid peptide (Aβ) peptides in brain regions critical for learning and memory is causative to disease progression [1,2]
The levels of PS1NTF in each of the subcellular fractions isolated from HeLa cells and human brain was assessed as a surrogate of active γ-secretase complex levels, we did not observe a direct correlation between normalized PS1NTF levels and Aβ production (Figures 1(a) and (b))
We were able to demonstrate the presence of each of the γ-secretase core components, PS1-C-terminal fragment (CTF), PS1-N-terminal fragment (NTF), PS2-CTF, NIC, presenilin enhancer-2 (PEN-2), Aph-1A, and Aph-1B in the brain membranes from each species as well as from membranes prepared from HeLa cells (Figure 2)
Summary
According to the amyloid cascade hypothesis of Alzheimer’s disease (AD) pathogenesis, deposition of Aβ peptides in brain regions critical for learning and memory is causative to disease progression [1,2]. APP undergoes regulated intra-membrane proteolysis (RIP) initiated by the β-site APP cleavage enzyme (BACE), leaving a C-terminal membrane bound stub that undergoes further proteolysis by γ-secretase [3]. These cleavage events generate of a pool of Aβ peptides of various lengths, the predominant species of which is Aβ40. Aβ42 represents only a minor (~5%) proportion of the Aβ peptides generated via this “amyloidogenic pathway”, the hydrophobic nature of this particular peptide is such that even small increases in relative amounts can drive fibrillization and deposition in brain parenchyma [4,5].
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