Neuronal Apoptosis by Apolipoprotein E4 through Low-Density Lipoprotein Receptor-Related Protein and Heterotrimeric GTPases

Abstract

The ⑀4 genotype of apolipoprotein E (apoE4) is the most established predisposing factor in Alzheimer's disease (AD); however, it remains unclear how apoE4 contributes to the pathophysiology. Here, we report that the apoE4 protein (ApoE4) evokes apoptosis in neuronal cells through the low-density lipoprotein receptor-related protein (LRP) and heterotrimeric GTPases. We examined neuron/neuroblastoma hybrid F11 cells and found that these cells were killed by 30 g/ml ApoE4, but not by 30 g/ml ApoE3. ApoE4-induced death occurred with typical features for apoptosis in time-and dose-dependent manners, and was observed in SH-SY5Y neuroblastomas, but not in glioblastomas or non-neuronal Chinese hamster ovary cells. Activated, but not native, ␣2-macroglobulin suppressed this ApoE4 toxicity. Suppression by the antisense oligonucleotide to LRP and inhibition by low nanomolar concentrations of LRP-associated protein RAP provided evidence for the involvement of LRP. The involvement of heterotrimeric GTPases was demonstrated by the findings that (1) ApoE4-induced death was suppressed by pertussis toxin (PTX), but not by heat-inactivated PTX; and (2) transfection with PTX-resistant mutant cDNAs of G␣ i restored the toxicity of ApoE4 restricted by PTX. We thus conclude that one of the neurotoxic mechanisms triggered by ApoE4 is to activate a cell type-specific apoptogenic program involving LRP and the G i class of GTPases and that the apoE4 gene may play a direct role in the pathogenesis of AD and other forms of dementia.

A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Aβ

“…To use HN peptides, cells were transfected with FAD genes in the absence of serum for 3 h, incubated with HF-18% for 2 h, and cultured with HF-10% and HN peptides (with or without inhibitors) for 67 h, and cell mortality and viability were measured. Cell mortality was assessed by Trypan blue and cell viability by WST-8 (Wako, Osaka), as described (12,14). F11/EcR cell experiments were performed as described (12).…”

Alsin, the Product of ALS2 Gene, Suppresses SOD1 Mutant Neurotoxicity through RhoGEF Domain by Interacting with SOD1 Mutants

“…Cell mortality assessed by this method thus represents the population of dead cells in total cells, including both adhesive and floating cells at the termination of experiments. It has been established by our reported experiments that our cell mortality assay by the trypan blue exclusion test gives precisely reciprocal results of WST-8 assay, the most established cell viability assay (20). WST-8 assay was performed with 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8), using Cell Counting kit-8 (Wako Pure Chemicals Industries, Tokyo, Japan).…”

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“…After two days the cells were starved in 1.25% serum medium for 17 h, and then pretreated with inhibitors for 1 h before exposure to DCA (200 μM) for 4 h before preparation of lysates and assays. Cell viability was measured by trypan blue exclusion assay (25). Data are presented graphically as the average of triplicates with statistical significance determined by Student's t-test.…”