Accumulation of intraneuronal Abeta correlates with ApoE4 genotype

Abstract

In contrast to extracellular plaque and intracellular tangle pathology, the presence and relevance of intraneuronal Abeta in Alzheimer's disease (AD) is still a matter of debate. Human brain tissue offers technical challenges such as post-mortem delay and uneven or prolonged tissue fixation that might affect immunohistochemical staining. In addition, previous studies on intracellular Abeta accumulation in human brain often used antibodies targeting the C-terminus of Abeta and differed strongly in the pretreatments used. To overcome these inconsistencies, we performed extensive parametrical testing using a highly specific N-terminal Abeta antibody detecting the aspartate at position 1, before developing an optimal staining protocol for intraneuronal Abeta detection in paraffin-embedded sections from AD patients. To rule out that this antibody also detects the beta-cleaved APP C-terminal fragment (beta-CTF, C99) bearing the same epitope, paraffin-sections of transgenic mice overexpressing the C99-fragment were stained without any evidence for cross-reactivity in our staining protocol. The staining intensity of intraneuronal Abeta in cortex and hippocampal tissue of 10 controls and 20 sporadic AD cases was then correlated to patient data including sex, Braak stage, plaque load, and apolipoprotein E (ApoE) genotype. In particular, the presence of one or two ApoE4 alleles strongly correlated with an increased accumulation of intraneuronal Abeta peptides. Given that ApoE4 is a major genetic risk factor for AD and is involved in neuronal cholesterol transport, it is tempting to speculate that perturbed intracellular trafficking is involved in the increased intraneuronal Abeta aggregation in AD.

Fig. 1

Optimization for intraneuronal Aβ_1−x staining in the hippocampal formation of sporadic AD cases using the Aβ[N] antibody in paraffin-embedded sections. A faint homogenous intraneuronal Aβ_1−x staining could be detected even without any antigen retrieval (a). However, 10-min heat pretreatment in citric acid buffer pH 6 dramatically increased the intraneuronal Aβ_1−x staining that at higher magnification showed granularity and concentration around the nucleus (b). Compared to no treatment, 3-min formic acid (FA) pretreatment failed to improve intraneuronal Aβ_1−x staining (c). In the combined treatment of heat and FA, FA actually counteracted the enhancing effect of the heat pretreatment (d). Besides the intraneuronal Aβ_1−x staining, smaller nuclei surrounded by a highly granular Aβ staining pattern were observed with all the protocols (c, arrowheads). Scale bar 50 μm

Fig. 2

Double labeling of Aβ_1−x in reddish brown (DAB) using Aβ[N] antibody and astrocytes in blue (Histogreen, black arrowheads) using a GFAP antibody in paraffin-embedded sections. The highly granular staining pattern surrounding the smaller nuclei was due to astrocytes accumulating Aβ and was found in CA4 (a) as well as in CA3 (b) of many sporadic AD cases. However, in some AD cases, astrocytes without granular staining were found close to neurons in both CA4 (c) and CA3 (d). Scale bar 33 μm

Fig. 3

Intraneuronal staining detected by OC and 4G8 antibodies in a sporadic AD brain. The OC antibody disclosed intraneuronal Aβ staining much like that of Aβ[N] with both 10-min heat pretreatment alone (a, b) and combined heat and 3-min formic acid (FA) treatment (c, d). With 10-min heat pretreatment, the 4G8 antibody produced a different, highly abundant and granular intracellular staining (e, f). Bottom left corners of a, c, and e show intracellular staining at a high magnification. Scale bar 50 μm

Fig. 4

Rating of intraneuronal Aβ_1−x staining intensity. +++ was assigned to cases with very strong intraneuronal Aβ staining in CA4, CA3, as well as in CA1 (a–c). ++ was assigned to cases with weaker but still obvious intracellular Aβ staining in CA4 and CA3 and low staining in CA1 (d–f). + was assigned to cases with a very faint intracellular Aβ staining in CA4 and CA3 and apparently no staining in CA1 (g–i). 0 was assigned to cases showing no intracellular Aβ staining either in CA4, CA3, or CA1 (j–l). Scale bar 50 μm