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

Background: The form(s) of amyloid-beta peptide (A beta) associated with the pathology characteristic of Alzheimer's disease (AD) remains unclear. In particular, the neurotoxicity of intraneuronal A beta accumulation is an issue of considerable controversy; even the existence of A beta 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 A beta. To further address this issue, an anti-A beta antibody was developed (MOAB-2) that specifically detects A beta, but not APP. This antibody allows for the further evaluation of the early accumulation of intraneuronal A beta in transgenic mice with increased levels of human A beta in 5xFAD and 3xTg mice. Results: MOAB-2 (mouse IgG(2b)) is a pan-specific, high-titer antibody to A beta residues 1-4 as demonstrated by biochemical and immunohistochemical analyses (IHC), particularly compared to 6E10 (a commonly used commercial antibody to A beta residues 3-8). MOAB-2 did not detect APP or APP-CTFs in cell culture media/lysates (HEK-APP(Swe) or HEK-APP(Swe)/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 A beta 40 and A beta 42. 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 A beta, distinct from A beta associated with the cell membrane. MOAB-2 demonstrated strong intraneuronal and extra-cellular immunoreactivity in 5xFAD and 3xTg mouse brain tissues. Conclusions: Both intraneuronal A beta accumulation and extracellular A beta deposition was demonstrated in 5xFAD mice and 3xTg mice with MOAB-2, an antibody that will help differentiate intracellular A beta from APP. However, further investigation is required to determine whether a molecular mechanism links the presence of intraneuronal A beta with neurotoxicity. As well, understanding the relevance of these observations to human AD patients is critical.