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

Department of Anatomy and Cell Biology, University of Illinois at Chicago, IL 60612, USA.
Molecular Neurodegeneration (Impact Factor: 5.29). 03/2012; 7:8. DOI: 10.1186/1750-1326-7-8
Source: PubMed

ABSTRACT The 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.
MOAB-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-immunoreactivity 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.
Both 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.

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    • "ic pathway are already observed in the pre - symptomatic stage of AD . The resulting increase in Ab production may explain the observations made by us and others that Ab ac - cumulates inside neurons in the pre - AD and Down syndrome brain and in several AD mouse models , before the onset of plaque pa - thology ( e . g . , Bossers et al . , 2010 ; Youmans et al . , 2012 , reviewed in LaFerla et al . , 2007 ) . Whether neuronal hyperactivity is causally linked to disturbances in clathrin - mediated endocytosis ( e . g . , by overloading the system ) , or that it amplifies the effects of alter - ations already present in the endocytic machinery , is unclear at this moment . In any case , the interaction "
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    ABSTRACT: Neuronal activity directly promotes the production and secretion of amyloid β (Aβ). Interestingly, neuronal hyperactivity can be observed in presymptomatic stages of both sporadic and familial Alzheimer's disease (AD) and in several AD mouse models. In this review, we will highlight the recent evidence for neuronal hyperactivity before or during the onset of cognitive defects in mild cognitive impairment. Furthermore, we review specific molecular mechanisms through which neuronal hyperactivity affects Aβ production and degradation. With these data, we will provide more insight into the 2-faced nature of neuronal hyperactivity: does enhanced neuronal activity during the presymptomatic stages of AD provide protection against the earliest disease processes or is it a pathogenic contributor to AD? Copyright © 2014 Elsevier Inc. All rights reserved.
    Neurobiology of Aging 09/2014; 36(1). DOI:10.1016/j.neurobiolaging.2014.08.014 · 4.85 Impact Factor
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    • "Furthermore, presence of Aβ peptides in Tg+ve NS lysates suggest the presence of intracellular Aβ peptides which is well supported by the results from conformation dependent immunocytochemistry on cells and NS sections. Intraneuronal accumulation of Aβ peptide has been associated with cellular pathology related to cognitive malfunction in AD brain (Takahashi et al. 2002; Walsh et al. 2000) and in the brain from mouse models of AD (Eimer and Vassar 2013; Oddo et al. 2006; Wirths et al. 2001; Youmans et al. 2012). Neurotoxic effect (Deshpande et al. 2006) and synaptotoxic effect (Walsh et al. 2002) of oligomeric forms of Aβ have also been reported. "
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    SpringerPlus 03/2014; 3:161. DOI:10.1186/2193-1801-3-161
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    • "Predominantly, Ab peptides of 40 (Ab 40 ) or 42 amino acids (Ab 42 ) are generated, with Ab 42 being more aggregation prone (Thinakaran & Koo, 2008). While extracellular Ab aggregation has long been considered as the primary cause of AD, intracellular Ab accumulation is detected in neurons prior to the appearance of extracellular deposits (Wirths et al., 2001; Youmans et al., 2012) and is associated with cytotoxicity, dysfunction of organelles, and neurodegeneration (Bayer & Wirths, 2010). Translocation of Ab peptides into the cytoplasm could occur via various routes (Li et al., 2007), including transport of ER-generated Ab peptides into the cytoplasm via systems related to ER-associated degradation (ERAD) (B€ uckig et al., 2002; Schmitz et al., 2004), passive leakage of Ab along the secretory pathway, or by membrane permeability of lysosomes containing internalized Ab (Yang et al., 1998). "
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    ABSTRACT: Alzheimer's disease is hallmarked by amyloid β peptides accumulation and aggregation in extracellular plaques, preceded by intracellular accumulation. We examined whether intracellular amyloid β can be cleared by cytosolic peptidases, and whether this capacity is affected during progression of sporadic Alzheimer's disease in humans and in the commonly used APPswePS1dE9 and 3xTg-AD mouse models. A quenched amyloid β peptide that becomes fluorescent upon degradation was used to screen for amyloid β-degrading cytoplasmic peptidases cleaving the aggregation-prone KLVFF region of the peptide. In addition, this quenched peptide was used to analyze amyloid β degrading capacity in the hippocampus of sporadic Alzheimer's disease patients with different Braak stages as well as APPswePS1dE9 and 3xTg-AD mice. Insulin degrading enzyme (IDE) was found to be the main peptidase that degrades cytoplasmic, monomeric amyloid β. Oligomerisation of amyloid β prevents its clearance by IDE. Intriguingly, the amyloid β degrading capacity decreases already during the earliest Braak stages of sporadic Alzheimer's disease, and this decline correlates with IDE protein levels, but not with mRNA levels. This suggests that decreased IDE levels could contribute to early sporadic Alzheimer's disease. In contrast to the human data, the commonly used APPswePS1dE9 and 3xTg-AD mouse models do not show altered amyloid β degradation and IDE levels with Alzheimer's disease progression, raising doubts whether mouse models that overproduce amyloid β peptides are representative for human sporadic Alzheimer's disease. © 2013 The Authors Aging Cell © 2013 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.
    Aging cell 03/2013; 12(3). DOI:10.1111/acel.12074 · 5.94 Impact Factor
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