Neuron-specific apolipoprotein e4 proteolysis is associated with increased tau phosphorylation in brains of transgenic mice

Abstract

Apolipoprotein E (apoE) is found in amyloid plaques and neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) brains, but its role in their pathogenesis is unclear. Previously, we found C-terminal-truncated fragments of apoE in AD brains and showed that such fragments can cause neurodegeneration and can induce NFT-like inclusions in cultured neuronal cells and in transgenic mice. Here, we analyzed apoE fragmentation in brain tissue homogenates from transgenic mice expressing apoE3 or apoE4 in neurons [neuron-specific enolase (NSE)-apoE] or astrocytes [glial fibrillary acidic protein (GFAP)-apoE] by Western blotting. The C-terminal-truncated fragments of apoE accumulated, in an age-dependent manner, in the brains of NSE-apoE4 and, to a significantly lesser extent, NSE-apoE3 mice; however, no fragments were detected in GFAP-apoE3 or GFAP-apoE4 mice. In NSE-apoE mice, the pattern of apoE fragmentation resembled that seen in AD brains, and the fragmentation was specific for certain brain regions, occurring in the neocortex and hippocampus, which are vulnerable to AD-related neurodegeneration, but not in the less vulnerable cerebellum. Excitotoxic challenge with kainic acid significantly increased apoE fragmentation in NSE-apoE4 but not NSE-apoE3 mice. Phosphorylated tau (p-tau) also accumulated in an age-dependent manner in NSE-apoE4 mice and, to a much lesser extent, in NSE-apoE3 mice but not in GFAP-apoE3 or GFAP-apoE4 mice. Intraneuronal p-tau inclusions in the hippocampus were prominent in 21-month-old NSE-apoE4 mice but barely detectable in NSE-apoE3 mice. Thus, the accumulation of potentially pathogenic C-terminal-truncated fragments of apoE depends on both the isoform and the cellular source of apoE. Neuron-specific proteolytic cleavage of apoE4 is associated with increased phosphorylation of tau and may play a key role in the development of AD-related neuronal deficits.

Figure 1.

ApoE fragmentation occurs in brains of humans and NSE-apoE mice but not in brains of GFAP-apoE mice. Brain homogenates from a nondemented human control (age 73 years) and a case with AD (age 71 years; A), NSE-apoE mice (age 7-9 months; B), and GFAP-apoE mice (age 8-10 months; C) were analyzed by Western blotting with antibodies against full-length apoE or C-terminal apoE. In C, apoE in brains of GFAP-apoE mice expressing apoE at levels similar to (2 right lanes) or sixfold higher than (3 left lanes) those in NSE-apoE mice was analyzed.

Figure 2.

Region specificity of apoE fragmentation in NSE-apoE4 mouse brains. Brain tissues from NSE-apoE4 mice (n = 4; age 4-5 months) were dissected into neocortex, hippocampus, and cerebellum and homogenized. ApoE in the homogenates was detected by Western blotting with a polyclonal antibody against full-length apoE. A, Representative Western blot. B, The apoE fragmentation was quantified by measuring the ratio of apoE fragments to full-length apoE. Note that the apoE fragmentation is much more prominent in the neocortex and hippocampus than in the cerebellum (p < 0.001, neocortex or hippocampus vs cerebellum).

Figure 3.

ApoE fragmentation in NSE-apoE mice challenged with kainic acid. At 7-8 months of age, NSE-apoE3 and NSE-apoE4 mice were injected intraperitoneally with saline or kainic acid (KA; n = 4 per genotype and treatment). They were analyzed 6 d later. ApoE in brain homogenates (150 μg of total proteins) was subjected to SDS-PAGE and detected by Western blotting with a polyclonal antibody against full-length apoE. A, The apoE fragmentation was quantified by measuring the ratios of all apoE fragments (14-30 kDa) to the full-length apoE (p < 0.01, KA vs saline for NSE-apoE4 mice). B, The total apoE levels were determined by comparing the bands of samples (full-length apoE plus fragments of apoE) with standards of various amounts of recombinant human apoE3 or apoE4 by densitometric analysis.

Figure 4.

Age-dependent accumulation of C-terminal-truncated fragments of apoE in brains of NSE-apoE4 mice. A, ApoE in brain homogenates of NSE-apoE3 or NSE-apoE4 mice was detected by Western blotting with antibodies against full-length apoE. B, Quantitative analysis of the ratios of the C-terminal-truncated fragments of apoE to the full-length apoE in brain homogenates of NSE-apoE3 and NSE-apoE4 mice (n = 4-6 per genotype and age; p < 0.01, apoE3 vs apoE4 mice at all ages).

Figure 5.

Age-dependent accumulation of p-tau in brains of NSE-apoE4 mice. p-Tau in brain homogenates of NSE-apoE mice (A, B) and GFAP-apoE mice (C) at different ages was analyzed by Western blotting with monoclonal antibody AT8. Note the higher levels of SDS-resistant p-tau aggregates at the top of the gel in aged NSE-apoE4 mice compared with aged NSE-apoE3 mice. D, Quantitative analysis of the SDS-resistant p-tau aggregates in brain homogenates of NSE-apoE3 and NSE-apoE4 mice (n = 4-6 per genotype and age; p < 0.01, apoE3 vs apoE4 mice at 7-21 months). mE, mouse apoE.

Figure 6.

Formation of intraneuronal p-tau-containing inclusions in the hippocampus of aged NSE-apoE4 mice. Brain sections from 21-month-old NSE-apoE4 (A) and NSE-apoE3 (B) mice were immunostained with monoclonal antibody AT8. p-tau-positive intraneuronal inclusions were abundant in the CA3 region of NSE-apoE4 mice (A) but barely detected in the CA3 region of NSE-apoE3 mice (B). C, Quantitative analysis of neurons containing p-tau-positive inclusions in 21-month-old NSE-apoE4 and NSE-apoE3 mice (n = 5 per genotype; p < 0.01, apoE3 vs apoE4 mice). D-F, Colocalization of apoE4 and p-tau in intraneuronal inclusions in the hippocampus of a 21-month-old NSE-apoE4 mouse as determined by anti-apoE and anti-p-tau double immunofluorescent staining. G-I, Colocalization of apoE4 and phosphorylated neurofilament (p-NF) in intraneuronal inclusions in the hippocampus of a 21-month-old NSE-apoE4 mouse as determined by anti-apoE and anti-p-NF double immunofluorescence staining. J, Intraneuronal straight filaments with diameters of 17 ± 3 nm visualized by electron microscopy in the hippocampus of the NSE-apoE4 transgenic mice (magnification, 30,000×).