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

Katherine L Youmans 0 Leon M Tai 0 Takahisa Kanekiyo W Blaine Stine Jr Sara-Claude Michon Evelyn Nwabuisi-Heath 0 Arlene M Manelli Yifan Fu Sean Riordan William A Eimer Lester Binder Guojun Bu Chunjiang Yu 0 Dean M Hartley Mary Jo LaDu 0 0 Department of Anatomy and Cell Biology, University of Illinois at Chicago , Chicago IL 60612 , USA Background: The form(s) of amyloid-b peptide (Ab) associated with the pathology characteristic of Alzheimer's disease (AD) remains unclear. In particular, the neurotoxicity of intraneuronal Ab accumulation is an issue of considerable controversy; even the existence of Ab 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 Ab. To further address this issue, an anti-Ab antibody was developed (MOAB2) that specifically detects Ab, but not APP. This antibody allows for the further evaluation of the early accumulation of intraneuronal Ab in transgenic mice with increased levels of human Ab in 5xFAD and 3xTg mice. Results: MOAB-2 (mouse IgG2b) is a pan-specific, high-titer antibody to Ab residues 1-4 as demonstrated by biochemical and immunohistochemical analyses (IHC), particularly compared to 6E10 (a commonly used commercial antibody to Ab 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 Ab40 and Ab42. 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 Ab, distinct from Ab associated with the cell membrane. MOAB-2 demonstrated strong intraneuronal and extra-cellular immunoreactivity in 5xFAD and 3xTg mouse brain tissues. Conclusions: Both intraneuronal Ab accumulation and extracellular Ab deposition was demonstrated in 5xFAD mice and 3xTg mice with MOAB-2, an antibody that will help differentiate intracellular Ab from APP. However, further investigation is required to determine whether a molecular mechanism links the presence of intraneuronal Ab with neurotoxicity. As well, understanding the relevance of these observations to human AD patients is critical. - Background The form(s) of amyloid-b peptide (Ab), particularly the 42 amino acid form (Ab42), associated with the neurotoxicity characteristic of Alzheimers disease (AD) remains unclear. The potential toxic assemblies of the peptide include soluble Ab [1], oligomeric Ab [2], intraneuronal Ab [3] and specific plaque morphology [4]. Evidence indicates that intraneuronal Ab accumulation may be an important proximal neurotoxic event in AD pathogenesis (reviewed in [5,6]). Studies suggest intraneuronal Ab accumulation in AD [7-9] and Downs Syndrome patients [10,11]. However, the relationship between intraneuronal Ab and plaque deposition remains unclear. Evidence suggests that intraneuronal Ab may precede extracellular plaque deposition in the brains of AD patients [12,13]. In particular, intraneuronal Ab42 accumulates in AD susceptible brain regions and precedes both extracellular amyloid deposition and neurofibrillar tangle formation [3]. The inside-out hypothesis posits that the intraneuronal Ab remaining after neuronal apoptosis serves as seeds for amyloid plaques. This is supported by several human studies demonstrating that increasing plaque deposition corresponds to decreased intraneuronal Ab staining [8,9]. However, beyond this temporal sequence, the functional connection between the deposition of Ab in neurons and the parenchyma has not been established in human brain. To further investigate intraneuronal Ab, attention has focused on analysis of transgenic mice with increased levels of human Ab (Ab-Tg mice). In accordance with data from AD patients, intraneuronal Ab precedes plaque deposition in multiple Ab-Tg mouse models ([14-23]) and may decrease as plaque deposition increases ([17,19,22,24]). Importantly, clearance of intraneuronal Ab via immunotherapy reversed cognitive deficits in triple-transgenic (3xTg mice) mice that harbor the PS1M146V, APPSwe and tauP301L transgenes [14,19]. Furthermore, after termination of immunotherapy, intraneuronal Ab re-appears prior to extracellular plaque deposition [20]. Intraneuronal Ab is also associated with impaired long-term potentiation (LTP), cognitive deficits and eventual neuronal loss in Ab-Tg mouse models ([14,15,17-19]). However, the neurotoxicity of intraneuronal Ab accumulation is an issue of considerable controversy; indeed even the existence of Ab deposits within neurons is currently subject to debate and interpretation http://www. alzforum.org/res/for/journal/detail.asp?liveID=193. Concern centers on whether the detected intraneuronal immunoreactivity is the result of Ab antibodies binding to APP [16]. Recently, Winton and co-workers used 3xTg mice to demonstrate intraneuronal immunodetection with the commonly used commercial antibodies 6E10 (residues 3-8 of Ab), 4G8 (residues 17-24 of Ab) and 22C11 (N-terminal APP residues 66-81), but not with C-terminal Ab40- and 42-specific antibodies [25]. This staining pattern was unchanged in the absence of Ab (3xTg/b-secretase (BACE)-/- mice), suggesting the intraneuronal staining represents APP and not Ab. These data are in stark contrast to multiple publications demonstrating intraneuronal Ab staining in 3xTg mice and other Ab-Tg mice [14,19,20,26]. These issues highlight experimental considerations that need to be addressed in order to investigate intraneuronal Ab accumulation in vivo. First, as the conformation or conformations of intraneuronal Ab is not known, the detection of intraneuronal Ab it is likely to be optimal with a pan-specific antibody that detects different conformations of Ab. Second, antibodies must be specific for Ab and not detect APP. Thus, intraneuronal Ab cannot be specifically identified by antibodies directed against residues 3-8 (e.g. 6E10), and residues 17-24 (e.g. 4G8) of Ab because these antibodies also recognize full length APP [16] and APP C-terminal fragments (APP-CTFs) [27-30]. This is particularly relevant for Ab-Tg mouse models that express high levels of the APP transgene (e.g. 2 and ~5 fold higher in the brains of the hemizygous and homozygous 3xFAD mice than endogenous APP in wildtype (WT) mice [19]). Third, the detection of intraneuronal Ab in Ab-Tg mouse models can be confirmed by genetic or pharmacological approaches. For example, in Tg-ArcSwe/BACE1-/- (...truncated)