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Analyzing microglial-associated A? in Alzheimer's disease transgenic mice with a novel mid-domain A?-antibody.

ABSTRACT: The mechanisms of amyloid-? (A?)-degradation and clearance in Alzheimer's disease (AD) pathogenesis have been relatively little studied. Short A?-fragments form by enzymatic cleavage and alternate amyloid-beta precursor protein (APP)-processing. Here we characterized a novel polyclonal A?-antibody raised against an A? mid-domain and used it to investigate microglial A?-uptake in situ by microscopy at the light- and ultrastructural levels. The rabbit A?-mid-domain antibody (ab338), raised against the mid-domain amino acids 21-34 (A?_21-34), was characterized with biochemical and histological techniques. To identify the epitope in A? recognized by ab338, solid phase and solution binding data were compared with peptide folding scores as calculated with the Tango software. The ab338 antibody displayed high average affinity (K_D: 6.2?×?10^-10 M) and showed preference for C-terminal truncated A?-peptides ending at amino acid 34 and A?-mid domain peptides with high scores of ?-turn structure. In transgenic APP-mouse brain, ab338 labelled amyloid plaques and detected A?-fragments in microglia at the ultra- and light microscopic levels. This reinforces a role of microglia/macrophages in A?-clearance in vivo. The ab338 antibody might be a valuable tool to study A?-clearance by microglial uptake and A?-mid-domain peptides generated by enzymatic degradation and alternate production.

SUBMITTER: Henjum K

PROVIDER: S-EPMC7324359 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Analyzing microglial-associated Aβ in Alzheimer's disease transgenic mice with a novel mid-domain Aβ-antibody.

Henjum Kristi K Årskog Vibeke V Jendresen Charlotte B CB Fladby Tormod T Torp Reidun R Nilsson Lars N G LNG

Scientific reports 20200629 1

The mechanisms of amyloid-β (Aβ)-degradation and clearance in Alzheimer's disease (AD) pathogenesis have been relatively little studied. Short Aβ-fragments form by enzymatic cleavage and alternate amyloid-beta precursor protein (APP)-processing. Here we characterized a novel polyclonal Aβ-antibody raised against an Aβ mid-domain and used it to investigate microglial Aβ-uptake in situ by microscopy at the light- and ultrastructural levels. The rabbit Aβ-mid-domain antibody (ab338), raised against ...[more]

PMID: 32601313

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Project description:BackgroundAmyloid β (Aβ)-directed immunotherapy has shown promising results in preclinical and early clinical Alzheimer's disease (AD) trials, but successful translation to late clinics has failed so far. Compelling evidence suggests that post-translationally modified Aβ peptides might play a decisive role in onset and progression of AD and first clinical trials targeting such Aβ variants have been initiated. Modified Aβ represents a small fraction of deposited material in plaques compared to pan-Aβ epitopes, opening up pathways for tailored approaches of immunotherapy. Here, we generated the first monoclonal antibodies that recognize L-isoaspartate-modified Aβ (isoD7-Aβ) and tested a lead antibody molecule in 5xFAD mice.MethodsThis work comprises a combination of chemical and biochemical techniques as well as behavioral analyses. Aβ peptides, containing L-isoaspartate at position 7, were chemically synthesized and used for immunization of mice and antibody screening methods. Biochemical methods included anti-isoD7-Aβ monoclonal antibody characterization by surface plasmon resonance, immunohistochemical staining of human and transgenic mouse brain, and the development and application of isoD7-Aβ ELISA as well as different non-modified Aβ ELISA. For antibody treatment studies, 12 mg/kg anti-isoD7-Aβ antibody K11_IgG2a was applied intraperitoneally to 5xFAD mice for 38 weeks. Treatment controls implemented were IgG2a isotype as negative and 3D6_IgG2a, the parent molecule of bapineuzumab, as positive control antibodies. Behavioral studies included elevated plus maze, pole test, and Morris water maze.ResultsOur advanced antibody K11 showed a KD in the low nM range and > 400fold selectivity for isoD7-Aβ compared to other Aβ variants. By using this antibody, we demonstrated that formation of isoD7-Aβ may occur after formation of aggregates; hence, the presence of the isoD7-modification differentiates aged Aβ from newly formed peptides. Importantly, we also show that the Tottori mutation responsible for early-onset AD in a Japanese pedigree is characterized by massively accelerated formation of isoD7-Aβ in cell culture. The presence of isoD7-Aβ was verified by K11 in post mortem human cortex and 5xFAD mouse brain tissue. Passive immunization of 5xFAD mice resulted in a significant reduction of isoD7-Aβ and total Aβ in brain. Amelioration of cognitive impairment was demonstrated by Morris water maze, elevated plus maze, pole, and contextual fear conditioning tests. Interestingly, despite the lower abundance of the isoD7-Aβ epitope, the application of anti-isoD7-Aβ antibodies showed comparable treatment efficacy in terms of reduction of brain amyloid and spatial learning but did not result in an increase of plasma Aβ concentration as observed with 3D6 treatment.ConclusionsThe present study demonstrates, for the first time, that the antibody-mediated targeting of isoD7-modified Aβ peptides leads to attenuation of AD-like amyloid pathology. In conjunction with previously published data on antibodies directed against pGlu-modified Aβ, the results highlight the crucial role of modified Aβ peptides in AD pathophysiology. Hence, the results also underscore the therapeutic potential of targeting modified amyloid species for defining tailored approaches in AD therapy.

Project description:Background: The deposition of β-sheet rich amyloid in senile plaques is a pathological hallmark of Alzheimer's disease (AD), which is thought to cause neuronal dysfunction. Previous studies have strongly implicated that intracerebral infusion of brain extract containing aggregated β-amyloid (Aβ) is able to induce cerebral amyloidosis thus causing neuronal damage and clinical abnormalities in rodents and nonhuman primates, which are reminiscent of a prion-like mechanism. Prion disease has been documented in cases of prion-contaminated food consumption. Methods: We investigated whether cerebral transmission of Aβ was possible via oral administration of Aβ-rich brain extract in non-susceptible and susceptible host mice by immunohistochemistry, western blotting and behavior tests. Also brain extracts were supplied to AD transgenic Caenorhabditis elegans, and paralysis curve were conducted, following detection of Aβ amyloid. RNA sequencing of nematodes was applied then inhibitors for relevant dysregulated genes were used in the paralysis induction. Results: The oral treatment of AD brain extract or normal brain extract neither aggravated nor mitigated the Aβ load, glial activation or the abnormal behaviors in recipient Amyloid precursor protein/presenilin 1 (APP/PS1) mice. Whereas, a significant improvement of AD pathology was detected in worms treated with Aβ-rich or normal brain extracts, which was attributable to the heat-sensitive components of brain extracts. Transcriptome sequencing of CL4176 nematodes suggested that brain extracts could delay worm paralysis through multiple pathways, including ubiquitin mediated proteolysis and Transforming growth factor β (TGF-β) signaling pathway. Inhibitors of the ubiquitin proteasome system and the TGF-β signaling pathway significantly blocked the suppressive effects of brain extracts on worm paralysis. Conclusions: Our results suggest that systemic transmissible mechanisms of prion proteopathy may not apply to β amyloid, at least in terms of oral administration. However, brain extracts strongly ameliorated AD pathology in AD transgenic nematodes partially through TGF-β signaling pathway and ubiquitin mediated proteolysis, which indicated that some natural endogenous components in the mammalian tissues could resist Aβ toxicity.

Dataset Information

Analyzing microglial-associated A? in Alzheimer's disease transgenic mice with a novel mid-domain A?-antibody.

Publications

Analyzing microglial-associated Aβ in Alzheimer's disease transgenic mice with a novel mid-domain Aβ-antibody.

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