Dynamics of β-Amyloid Reductions in Brain, Cerebrospinal Fluid, and Plasma of β-Amyloid Precursor Protein Transgenic Mice Treated with a γ-Secretase Inhibitor

Abstract

␥-Secretase inhibitors are one promising approach to the development of a therapeutic for Alzheimer's disease (AD). ␥-Secretase inhibitors reduce brain ␤-amyloid peptide (A␤), which is believed to be a major contributor in the etiology of AD. Transgenic mice overexpressing the human ␤-amyloid precursor protein (APP) are valuable models to examine the dynamics of A␤ changes with ␥-secretase inhibitors in plaque-free and plaque-bearing animals. BMS-299897 2-[(1R)-1-[[(4-chlorophenyl)sulfony](2,5-difluorophenyl)amino]ethyl]-5-fluorobenzenepropanoic acid, a ␥-secretase inhibitor, showed doseand time dependent reductions of A␤ in brain, cerebrospinal fluid (CSF), and plasma in young transgenic mice, with a significant correlation between brain and CSF A␤ levels. Because CSF and brain interstitial fluid are distinct compartments in composition and location, this correlation could not be assumed. In contrast, aged transgenic mice with large accumulations of A␤ in plaques showed reductions in CSF A␤ in the absence of measurable changes in plaque A␤ in the brain after up to 2 weeks of treatment. Hence, CSF A␤ levels were a valuable measure of ␥-secretase activity in the central nervous system in either the presence or absence of plaques. Transgenic mice were also used to examine potential side effects due to Notch inhibition. BMS-299897 was 15-fold more effective at preventing the cleavage of APP than of Notch in vitro. No changes in the maturation of CD8ϩ thymocytes or of intestinal goblet cells were observed in mice treated with BMS-299897, showing that it is possible for ␥-secretase inhibitors to reduce brain A␤ without causing Notch-mediated toxicity.Diagnosis of AD is based on key pathological features at autopsy in the presence of dementia: loss of neuronal mass, intracellular tangles, and extracellular plaques. Plaques are composed predominantly of A␤. A␤ has both N-and C-terminal heterogeneity, with the C terminus usually ending at residues 40 or 42. Although A␤40 is 80 to 90% of the total, A␤42 is most associated with disease and is predominant in plaques. A␤ is produced by two cleavage events in APP (Wolfe, 2001). ␤-Secretase, now identified as ␤-site APP cleaving enzyme, makes the initial N-terminal cleavage, releasing a soluble N-terminal form of APP. The remaining, membrane-bound C-terminal fragment (CTF) of APP is cleaved by ␥-secretase to release soluble A␤ along with the APP intracellular domain. Alternatively, APP can This research was supported by Bristol-Myers Squibb and SIBIA Neurosciences, Inc.Article, publication date, and citation information can be found at

Low-density lipoprotein receptor overexpression enhances the rate of brain-to-blood Aβ clearance in a mouse model of β-amyloidosis

“…These results suggest the LDLR overexpression in neurons and astrocytes (18) likely increases BBB-mediated Aβ clearance through an indirect mechanism involving other apoE receptors at the BBB, such as LRP1-a receptor shown to directly mediate Aβ clearance across the BBB (14,15,26). To test this hypothesis, we delivered an anti-LRP1 antibody (N20) or vehicle into the brains of TG mice immediately before coinjection of 12 nM [ The rapid degradation of Aβ in the periphery (t 1/2 = 2-3 min) precludes a direct and sensitive measurement of the rate of Aβ appearance from brain into blood (27,28). To more directly assess the rate of brain-to-blood Aβ clearance, we first cross-bred LDLR-TG mice with the PDAPP (APP-V717F) mouse model of β-amyloidosis.…”

“…Acknowledging these limitations, we sought to verify our observations with an independent method to assess brain-to-blood Aβ elimination. The rapid degradation of Aβ once it enters the blood from brain precludes direct and reliable measurement of its influx rate by conventional means (27,28). Thus, we reasoned that an anti-Aβ antibody would bind CNS-derived Aβ within the blood, preventing its degradation to allow direct measurement of its appearance rate.…”

“…Immunoprecipitated Aβ was trypsin-digested, and tryptic peptides were analyzed by mass spectrometry. The ratio of unlabeled to labeled Aβ [17][18][19][20][21][22][23][24][25][26][27][28] peptide was normalized against a SISAQ standard curve, allowing quantification of Aβ in the original plasma samples.…”

“…Plasma accumulation experiments were performed by administering 250 μg HJ5.1 (anti-Aβ [17][18][19][20][21][22][23][24][25][26][27][28] antibody generated in-house) by intrajugular injection under brief isoflurane exposure. Following injection, blood was sampled at various time points (20-240 min) by serial retro-orbital bleeding with heparinized capillary tubes (Chase Scientific Glass) under brief isoflurane exposure as described previously (30,32).…”

Moderate Reduction of γ-Secretase Attenuates Amyloid Burden and Limits Mechanism-Based Liabilities

“…Although several highly potent ␥-secretase inhibitors are available, reports of significant toxicity associated with pharmacological inhibition of ␥-secretase using these compounds (Milano et al, 2004;Wong et al, 2004;Barten et al, 2005) raised concerns regarding the value of ␥-secretase for AD therapy. However, it is unclear whether toxicities associated with these ␥-secretase inhibitors are attributable to mechanism-based side effects or are related to specific off-target side effects of these compounds.…”