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An isogenic panel of App knock-in mouse models: Profiling β-secretase inhibition and endosomal abnormalities.

ABSTRACT: We previously developed single App knock-in mouse models of Alzheimer's disease (AD) that harbor the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (App^NL-G-F and App^NL-F mice). We have now generated App knock-in mice devoid of the Swedish mutations (App^G-F mice) and evaluated its characteristics. Amyloid β peptide (Aβ) pathology was exhibited by App^G-F mice from 6 to 8 months of age and was accompanied by neuroinflammation. Aβ-secretase inhibitor, verubecestat, attenuated Aβ production in App^G-F mice, but not in App^NL-G-F mice, indicating that the App^G-F mice are more suitable for preclinical studies of β-secretase inhibition given that most patients with AD do not carry the Swedish mutations. Comparison of isogenic App knock-in lines revealed that multiple factors, including elevated C-terminal fragment β (CTF-β) and humanization of Aβ might influence endosomal alterations in vivo. Thus, experimental comparisons between different isogenic App, knock-in mouse lines will provide previously unidentified insights into our understanding of the etiology of AD.

SUBMITTER: Watamura N

PROVIDER: S-EPMC9177067 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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An isogenic panel of App knock-in mouse models: Profiling β-secretase inhibition and endosomal abnormalities.

Watamura Naoto N Sato Kaori K Shiihashi Gen G Iwasaki Ayami A Kamano Naoko N Takahashi Mika M Sekiguchi Misaki M Mihira Naomi N Fujioka Ryo R Nagata Kenichi K Hashimoto Shoko S Saito Takashi T Ohshima Toshio T Saido Takaomi C TC Sasaguri Hiroki H

Science advances 20220608 23

We previously developed single App knock-in mouse models of Alzheimer's disease (AD) that harbor the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL-G-F and AppNL-F mice). We have now generated App knock-in mice devoid of the Swedish mutations (AppG-F mice) and evaluated its characteristics. Amyloid β peptide (Aβ) pathology was exhibited by AppG-F mice from 6 to 8 months of age and ...[more]

PMID: 35675411

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Project description:BackgroundAlzheimer's disease (AD) is the most frequent and costly neurodegenerative disorder. Although diverse lines of evidence suggest that the amyloid precursor protein (APP) is involved in its causation, the precise mechanisms remain unknown and no treatments are available to prevent or halt the disease. A favorite hypothesis has been that APP contributes to AD pathogenesis through the cerebral accumulation of the amyloid-β peptide (Aβ), which is derived from APP through sequential proteolytic cleavage by BACE1 and γ-secretase. However, inhibitors of these enzymes have failed in clinical trials despite clear evidence for target engagement.MethodsTo further elucidate the roles of APP and its metabolites in AD pathogenesis, we analyzed transgenic mice overexpressing wildtype human APP (hAPP) or hAPP carrying mutations that cause autosomal dominant familial AD (FAD), as well as App knock-in mice that do not overexpress hAPP but have two mouse App alleles with FAD mutations and a humanized Aβ sequence.ResultsAlthough these lines of mice had marked differences in cortical and hippocampal levels of APP, APP C-terminal fragments, soluble Aβ, Aβ oligomers and age-dependent amyloid deposition, they all developed cognitive deficits as well as non-convulsive epileptiform activity, a type of network dysfunction that also occurs in a substantive proportion of humans with AD. Pharmacological inhibition of BACE1 effectively reduced levels of amyloidogenic APP C-terminal fragments (C99), soluble Aβ, Aβ oligomers, and amyloid deposits in transgenic mice expressing FAD-mutant hAPP, but did not improve their network dysfunction and behavioral abnormalities, even when initiated at early stages before amyloid deposits were detectable.ConclusionshAPP transgenic and App knock-in mice develop similar pathophysiological alterations. APP and its metabolites contribute to AD-related functional alterations through complex combinatorial mechanisms that may be difficult to block with BACE inhibitors and, possibly, also with other anti-Aβ treatments.

Project description:Proteolytic processing of amyloid precursor protein (APP) plays a critical role in the pathogenesis of Alzheimer's disease (AD). Sequential cleavage of APP by β and γ secretases leads to the generation of Aβ40 (non-amyloidogenic) and Aβ42 (amyloidogenic) peptides. Presenilin-1 (PS1) or presenilin-2 (PS2) play the role of a catalytic subunit of γ-secretase. Multiple familial AD (FAD) mutations in APP, PS1, or PS2 result in an increased Aβ42:Aβ40 ratio and the accumulation of toxic Aβ42 oligomers and plaques in patient brains. In this study, we perform molecular modeling of the APP complex with γ-secretase and analyze potential effects of FAD mutations in APP and PS1. We noticed that all FAD mutations in the APP transmembrane domain are predicted to cause an increase in the local disorder of its secondary structure. Based on structural analysis of known γ-secretase structures, we propose that APP can form a complex with γ-secretase in 2 potential conformations-M1 and M2. In conformation, the M1 transmembrane domain of APP forms a contact with the perimembrane domain that follows transmembrane domain 6 (TM6) in the PS1 structure. In conformation, the M2 transmembrane domain of APP forms a contact with transmembrane domain 7 (TM7) in the PS1 structure. By analyzing the effects of PS1-FAD mutations on the local protein disorder index, we discovered that these mutations increase the conformational flexibility of M2 and reduce the conformational flexibility of M1. Based on these results, we propose that M2 conformation, but not M1 conformation, of the γ secretase complex with APP leads to the amyloidogenic (Aβ42-generating) processing of APP. Our model predicts that APP processing in M1 conformation is favored by curved membranes, such as the membranes of early endosomes. In contrast, APP processing in M2 conformation is likely to be favored by relatively flat membranes, such as membranes of late endosomes and plasma membranes. These predictions are consistent with published biochemical analyses of APP processing at different subcellular locations. Our results also suggest that specific inhibitors of Aβ42 production could be potentially developed by selectively targeting the M2 conformation of the γ secretase complex with APP.

Project description:BackgroundAlzheimer's disease (AD), the most common cause of dementia, is characterized by the progressive deposition of amyloid-β (Aβ) peptides and neurofibrillary tangles. Mouse models of Aβ amyloidosis generated by knock-in (KI) of a humanized Aβ sequence provide distinct advantages over traditional transgenic models that rely on overexpression of amyloid precursor protein (APP). In App-KI mice, three familial AD-associated mutations were introduced into the endogenous mouse App locus to recapitulate Aβ pathology observed in AD: the Swedish (NL) mutation, which elevates total Aβ production; the Beyreuther/Iberian (F) mutation, which increases the Aβ42/Aβ40 ratio; and the Arctic (G) mutation, which promotes Aβ aggregation. AppNL-G-F mice harbor all three mutations and develop progressive Aβ amyloidosis and neuroinflammatory response in broader brain areas, whereas AppNL mice carrying only the Swedish mutation exhibit no overt AD-related pathological changes. To identify behavioral alterations associated with Aβ pathology, we assessed emotional and cognitive domains of AppNL-G-F and AppNL mice at different time points, using the elevated plus maze, contextual fear conditioning, and Barnes maze tasks.ResultsAssessments of emotional domains revealed that, in comparison with wild-type (WT) C57BL/6J mice, AppNL-G-F/NL-G-F mice exhibited anxiolytic-like behavior that was detectable from 6 months of age. By contrast, AppNL/NL mice exhibited anxiogenic-like behavior from 15 months of age. In the contextual fear conditioning task, both AppNL/NL and AppNL-G-F/NL-G-F mice exhibited intact learning and memory up to 15-18 months of age, whereas AppNL-G-F/NL-G-F mice exhibited hyper-reactivity to painful stimuli. In the Barnes maze task, AppNL-G-F/NL-G-F mice exhibited a subtle decline in spatial learning ability at 8 months of age, but retained normal memory functions.ConclusionAppNL/NL and AppNL-G-F/NL-G-F mice exhibit behavioral changes associated with non-cognitive, emotional domains before the onset of definitive cognitive deficits. Our observations consistently indicate that AppNL-G-F/NL-G-F mice represent a model for preclinical AD. These mice are useful for the study of AD prevention rather than treatment after neurodegeneration.

Dataset Information

An isogenic panel of App knock-in mouse models: Profiling β-secretase inhibition and endosomal abnormalities.

Publications

An isogenic panel of <i>App</i> knock-in mouse models: Profiling β-secretase inhibition and endosomal abnormalities.

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