Biochemical and immunohistochemical analysis of an Alzheimer's disease mouse model reveals the presence of multiple cerebral Aβ assembly forms throughout life

Abstract

The amyloid β-protein (Aβ) is believed to play a causal role in Alzheimer's disease, however, the mechanism by which Aβ mediates its effect and the assembly form(s) of Aβ responsible remain unclear. Several APP transgenic mice have been shown to accumulate Aβ and to develop cognitive deficits. We have studied one such model, the J20 mouse. Using an immunoprecipitation/Western blotting technique we find an age-dependent increase in Aβ monomer and SDS-stable dimer. But prior to the earliest detection of Aβ dimers, immunohistochemical analysis revealed an increase in oligomer immunoreactivity that was coincident with reduced hippocampal MAP2 and synaptophysin staining. Moreover, biochemical fractionation and ELISA analysis revealed evidence of TBS and triton-insoluble sedimentable Aβ aggregates at the earliest ages studied. These data demonstrate the presence of multiple assembly forms of Aβ throughout the life of J20 mice and highlight the difficulty in attributing synaptotoxicity to a single Aβ species.

Introduction

Alzheimer's disease (AD) histopathology of the cerebral cortex is characterized by an accumulation of amyloid plaques containing the amyloid β-protein (Aβ) and neurofibrillary tangles composed of hyperphosphorylated tau (Mirra et al., 1991). Yet plaques and tangles are not unique to AD (Terry et al., 1987, 2001) and while the number and distribution of tangles show a moderate correlation with severity of disease (Braak and Braak, 1997, Schonheit et al., 2004), amyloid burden is a less robust indicator (Dickson, 1997; 2001; Bennett et al., 2006). Nonetheless, considerable experimental data suggests that Aβ plays an important role in AD pathogenesis (Selkoe, 2001). For instance, in the brains of humans affected by AD the concentration of aqueous-soluble Aβ predicts the clinical severity of dementia better than either amyloid plaque or neurofibrillary tangle density (Kuo et al., 1996, McLean et al., 1999, Lemere et al., 2002). Parallel in vitro studies have revealed that soluble oligomeric forms of synthetic Aβ perturb synaptic structure and activity and impair learning and memory whereas Aβ monomer has no adverse effect (Lambert et al., 1998, Hartley et al., 1999, Wang et al., 2002, Klyubin et al., 2004, Wang et al., 2004, Lacor et al., 2007, Puzzo et al., 2008). Together, these ex vivo and in vitro studies have led to a revision of the amyloid cascade hypothesis; wherein, soluble Aβ oligomers are the primary neurotoxic agents in AD (Klein et al., 2001, Hardy and Selkoe, 2002). However, the precise identity of these species and their relationship with amyloid plaques still remains unclear (Walsh and Selkoe, 2007).

Transgenic mouse models over-expressing various forms of human APP develop amyloid pathology, certain synaptic changes, electrophysiological deficits and impairment of learning and memory relevant to AD (Ashe, 2001, Games et al., 2006). Consequently, such mice have been studied in an effort to identify toxic Aβ assemblies (Westerman et al., 2002, Kawarabayashi et al., 2004, Lesne et al., 2006, Cheng et al., 2007). The J20 mouse used here expresses APP bearing the Swedish and Indiana mutations, promoting β-secretase cleavage and increasing the Aβ₄₂/Aβ₄₀ ratio, respectively (Mucke et al., 2000). These mice show an age-dependent deposition of Aβ beginning around ∼ 4–5 months together with various physiological changes that occur both before (Palop et al., 2007) and after the onset of plaque formation (Palop et al., 2003, Moreno et al., 2007).

Using a sensitive ELISA and a serial extraction procedure to isolate TBS-soluble, triton-soluble and GuHCl-soluble fractions we find that Aβ_1–40 is the predominant Aβ isoform detected in the aqueous extract, whereas Aβ_1–42 is the major species detected in the GuHCl extract. Importantly, detection of aggregated Aβ (i.e. Aβ sedimented by centrifugation and subsequently solubilized in GuHCl) preceded immunohistochemical (IHC) detection of amyloid deposits, but at all subsequent intervals the concentration of this GuHCl-solubilized Aβ strongly correlated with the extent of amyloid plaque burden. Assessment of non-fibrillar Aβ assemblies was accomplished using an immunoprecipitation (IP)/Western blot (WB) technique that detects Aβ monomer and SDS-stable low-n oligomers (Walsh et al., 2000, Shankar et al., 2008) and an anti-oligomer antibody, A11, reported to detect non-fibrillar Aβ oligomers larger than pentamer (Kayed et al., 2003). IHC analysis using the A11 antibody revealed the presence of oligomers at a time coincident with reduced hippocampal MAP2 and synaptophysin immunoreactivity; however, this was evident only at intervals after aggregated Aβ was first detected. Similarly, initial detection of TBS-soluble SDS-stable Aβ dimers occurred several months after the appearance of water-insoluble Aβ aggregates. These findings demonstrate the presence of several different Aβ assemblies in the cerebrum of J20 mice, before, coincident with, and after the onset of detectable synapto-dendritic compromise.