Analyzing Amyloid-ß Peptide Modulation Profiles and Binding Sites of γ-Secretase Modulators.

γ-Secretase is a key player in the pathogenesis of Alzheimer's disease (AD). The intramembrane-cleaving enzyme initially cleaves a C-terminal fragment of the amyloid precursor protein (APP) at the ɛ-site within its transmembrane domain to release the APP intracellular domain. Subsequent stepwise carboxy-terminal trimming cleavages eventually release amyloid-ß (Aß) peptides of 37-43 amino acids into the extracellular space. Aß42 as well as the much less abundant Aß43 species are highly aggregation prone and can deposit as plaques in the brains of affected patients, which are widely believed to be causative of AD. Disappointingly, due to lack of efficacy and side effects likely attributable to the inhibition of the crucial substrate Notch, inhibitors of γ-secretase that lower Aß generation failed in clinical trials of AD. There is hope, however, that recently developed potent γ-secretase modulators (GSMs) provide a safer approach for disease modification. These compounds have the unique property of primarily shifting the generation of Aß42 toward that of shorter peptides without affecting the ɛ-site cleavage of Notch and other substrates. In this chapter, we describe methods to investigate how GSMs affect the activity of the enzyme as well as how their molecular targets are identified.

Amyloid-PET predicts inhibition of de novo plaque formation upon chronic γ-secretase modulator treatment.

In a positron-emission tomography (PET) study with the ß-amyloid (Aß) tracer [(18)F]-florbetaben, we previously showed that Aß deposition in transgenic mice expressing Swedish mutant APP (APP-Swe) mice can be tracked in vivo. γ-Secretase modulators (GSMs) are promising therapeutic agents by reducing generation of the aggregation prone Aß42 species without blocking general γ-secretase activity. We now aimed to investigate the effects of a novel GSM [8-(4-Fluoro-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-[1-(3-methyl-[1,2,4]thiadiazol-5-yl)-piperidin-4-yl]-amine (RO5506284) displaying high potency in vitro and in vivo on amyloid plaque burden and used longitudinal Aß-microPET to trace individual animals. Female transgenic (TG) APP-Swe mice aged 12 months (m) were assigned to vehicle (TG-VEH, n=12) and treatment groups (TG-GSM, n=12), which received daily RO5506284 (30 mg kg(-1)) treatment for 6 months. A total of 131 Aß-PET recordings were acquired at baseline (12 months), follow-up 1 (16 months) and follow-up 2 (18 months, termination scan), whereupon histological and biochemical analyses of Aß were performed. We analyzed the PET data as VOI-based cortical standard-uptake-value ratios (SUVR), using cerebellum as reference region. Individual plaque load assessed by PET remained nearly constant in the TG-GSM group during 6 months of RO5506284 treatment, whereas it increased progressively in the TG-VEH group. Baseline SUVR in TG-GSM mice correlated with Δ%-SUVR, indicating individual response prediction. Insoluble Aß42 was reduced by 56% in the TG-GSM versus the TG-VEH group relative to the individual baseline plaque load estimates. Furthermore, plaque size histograms showed differing distribution between groups of TG mice, with fewer small plaques in TG-GSM animals. Taken together, in the first Aß-PET study monitoring prolonged treatment with a potent GSM in an AD mouse model, we found clear attenuation of de novo amyloidogenesis. Moreover, longitudinal PET allows non-invasive assessment of individual plaque-load kinetics, thereby accommodating inter-animal variations.