Intraneuronal Aβ detection in 5xFAD mice by a new Aβ-specific antibody

Abstract

BackgroundThe form(s) of amyloid-β peptide (Aβ) associated with the pathology characteristic of Alzheimer's disease (AD) remains unclear. In particular, the neurotoxicity of intraneuronal Aβ accumulation is an issue of considerable controversy; even the existence of Aβ deposits within neurons has recently been challenged by Winton and co-workers. These authors purport that it is actually intraneuronal APP that is being detected by antibodies thought to be specific for Aβ. To further address this issue, an anti-Aβ antibody was developed (MOAB-2) that specifically detects Aβ, but not APP. This antibody allows for the further evaluation of the early accumulation of intraneuronal Aβ in transgenic mice with increased levels of human Aβ in 5xFAD and 3xTg mice.ResultsMOAB-2 (mouse IgG2b) is a pan-specific, high-titer antibody to Aβ residues 1-4 as demonstrated by biochemical and immunohistochemical analyses (IHC), particularly compared to 6E10 (a commonly used commercial antibody to Aβ residues 3-8). MOAB-2 did not detect APP or APP-CTFs in cell culture media/lysates (HEK-APPSwe or HEK-APPSwe/BACE1) or in brain homogenates from transgenic mice expressing 5 familial AD (FAD) mutation (5xFAD mice). Using IHC on 5xFAD brain tissue, MOAB-2 immunoreactivity co-localized with C-terminal antibodies specific for Aβ40 and Aβ42. MOAB-2 did not co-localize with either N- or C-terminal antibodies to APP. In addition, no MOAB-2-immunreactivity was observed in the brains of 5xFAD/BACE-/- mice, although significant amounts of APP were detected by N- and C-terminal antibodies to APP, as well as by 6E10. In both 5xFAD and 3xTg mouse brain tissue, MOAB-2 co-localized with cathepsin-D, a marker for acidic organelles, further evidence for intraneuronal Aβ, distinct from Aβ associated with the cell membrane. MOAB-2 demonstrated strong intraneuronal and extra-cellular immunoreactivity in 5xFAD and 3xTg mouse brain tissues.ConclusionsBoth intraneuronal Aβ accumulation and extracellular Aβ deposition was demonstrated in 5xFAD mice and 3xTg mice with MOAB-2, an antibody that will help differentiate intracellular Aβ from APP. However, further investigation is required to determine whether a molecular mechanism links the presence of intraneuronal Aβ with neurotoxicity. As well, understanding the relevance of these observations to human AD patients is critical.

Proteins that mediate protein aggregation and cytotoxicity distinguish Alzheimer's hippocampus from normal controls

“…Abundant PLA signal, indicating P‐tau contiguous to Aβ 1–42, was seen in LC3‐positive puncta of AD hippocampus (panels A, B, E, F) but only rarely in AMC (panels C, D). The antibody for these PLA reactions is specific to Aβ 1–42 residues 1–4 and does not bind to APP (Youmans et al ., 2012); it preferentially labels neurotoxic oligomers of Aβ 1–42 (Tai et al ., 2013). These data imply that P‐tau complexes with Aβ 1–42 form inside AD neurons and are sequestered in a subset of autophagosomes.…”

“…The ability to distinguish immunologically between aggregates cannot be taken for granted, given that Aβ aggregates contain some tau, and tau‐affinity aggregates (at least from AD tissue) contain a little Aβ [or APP, which may also be recognized by most antibodies to Aβ (Youmans et al ., 2012)]. Nevertheless, the amount of ‘crossover’ (based on data in Table 1) is relatively small: Tau was identified in Aβ aggregates at only 3% of the level in tau‐affinity aggregates (15 vs. 499 hits), whereas Aβ was also quite scarce in tau tangles compared to Aβ aggregates (2 vs. 15 hits).…”

“…Although amyloid beta peptides Aβ 1–42 and Aβ 1–40 are best known as components of extracellular plaque, they are also found within neurons (Youmans et al ., 2012) where neurofibrillary tangles reside. We consistently identified Aβ 1–42 or its precursor APP in tau‐IP fractions (Table 2, line 1).…”

PPARγ-coactivator-1α gene transfer reduces neuronal loss and amyloid-β generation by reducing β-secretase in an Alzheimer’s disease model

“…To quantify Αβ load by IHC, we used a monoclonal Aβ-specific antibody that does not cross-react with full-length APP or carboxy-terminus domains (CTFs) (15). Histopathological examination revealed significantly lower Αβ burden in cortex (19.1% reduction) and HIP (30% reduction) in APP23/LVhPGC-1α mice compared with animals injected with the control vector (Fig.…”

Synaptic Amyloid-β Oligomers Precede p-Tau and Differentiate High Pathology Control Cases

“…The bright-specific flow cytometric signal of 10G4 also contrasts with the poorer flow cytometric results with the use of 6E10, which does not label synaptosomes in situ above background, despite mapping to a similar epitope. Like 10G4, MOAB2, a new monoclonal antibody specific for intraneuronal Ab that does not label APP, 45 shows a strong specific signal for synaptic Ab with flow cytometric analysis (Supplemental Figure S2, CeE). Taken together with evidence from Western blot analysis that 10G4 strongly labels high molecular weight aggregates, SDS-stable oligomers, and monomer in a pattern that resembles labeling by an Ab42-specific antibody, 26 these results indicate that, for flow cytometry, 10G4 is selective for aggregated Ab in situ.…”