Cellular source of apolipoprotein E4 determines neuronal susceptibility to excitotoxic injury in transgenic mice

Abstract

The lipid transport protein apolipoprotein E (apoE) is abundantly expressed in the brain. Its main isoforms in humans are apoE2, apoE3, and apoE4. ApoE4 is the major known genetic risk factor for Alzheimer's disease and also contributes to the pathogenesis of various other neurological conditions. In the central nervous system, apoE is synthesized by glial cells and neurons, but it is unclear whether the cellular source affects its biological activities. To address this issue, we induced excitotoxic injury by systemic kainic acid injection in transgenic Apoe knockout mice expressing human apoE isoforms in astrocytes or neurons. Regardless of its cellular source, apoE3 expression protected neuronal synapses and dendrites against the excitotoxicity seen in apoE-deficient mice. Astrocyte-derived apoE4, which has previously been shown to have detrimental effects in vitro, was as excitoprotective as apoE3 in vivo. In contrast, neuronal expression of apoE4 was not protective and resulted in loss of cortical neurons after excitotoxic challenge, indicating that neuronal apoE4 promotes excitotoxic cell death. Thus, an imbalance between astrocytic (excitoprotective) and neuronal (neurotoxic) apoE4 expression may increase susceptibility to diverse neurological diseases involving excitotoxic mechanisms.

Figure 1

ApoE levels in the neocortex and hippocampus of GFAP-apoE and NSE-apoE mice. All brain tissues were harvested and processed in parallel. Total protein (30 μg/lane) was separated by SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting with anti-apoE. A: Western blot shows apoE as a doublet in the 34 to 38 kDa range, as is typical for mouse and human brain tissues., The third lower-intensity band was probably due to partial degradation, which is often seen and was, on average, comparable across genotypes. B: ApoE levels were determined by densitometric analysis of Western blot signals, using human recombinant apoE as a standard (not shown). Results are means ± SEM; n = 4 to 5 mice per genotype.

Figure 2

Transgenic apoE expression in astrocytes of GFAP-apoE mice. A: Immunoperoxidase staining of brain sections with an anti-apoE antibody revealed widespread and comparable labeling of neocortex and hippocampus in GFAP-E3 and GFAP-E4 mice. Similar comparable staining was observed in other brain regions of these two lines (not shown). No labeling was detected in apoE-deficient mice (knockout). Scale bars: 80 μm (neocortex); 200 μm (hippocampus). B: Immunofluorescence for apoE and GFAP. Neocortical sections were double-immunolabeled for apoE (red) and GFAP (green) and imaged by confocal microscopy. In GFAP-E3 and GFAP-E4 mice, apoE was found only in astrocytes. Scale bar: 10 μm.

Figure 3

Neuronal integrity in the neocortex of GFAP-apoE versus NSE-apoE mice six days after intraperitoneal injection of KA (18 mg/kg) or saline (n = 4 to 13 mice per group). A: Neocortical brain sections were immunostained for synaptophysin, MAP-2, or NeuN, and immunoreactive structures were quantified as described in Materials and Methods. KA impaired synaptic and dendritic integrity in NSE-E4 mice and apoE-deficient mice, but not in GFAP-E3, NSE-E3, and GFAP-E4 mice. Loss of NeuN-positive pyramidal neurons was significant only in NSE-E4 mice. Thus, neuron-produced apoE4 increases vulnerability to excitotoxic injury, whereas astrocyte-produced apoE4 is as excitoprotective as apoE3. Results are means ± SEM; **P < 0.01; ***P < 0.001 versus control of the same genotype (Bonferroni post test). B: Photomicrographs depict examples of SYN-IR presynaptic terminals (top), MAR2-IR dendrites (middle), and NeuN-IR neuronal nuclei (bottom). Astrocyte-derived, but not neuron-derived, apoE4 protected neurons against excitotoxic injury. Apoe^−/− control mice showed no loss of pyramidal neurons, although their loss of cortical SYN-IR terminals and MAP-2 dendrites was similar to that seen in NSE-apoE4 mice. Scale bars: 40 μm (upper rows); 75 μm (bottom row).