Truncated apolipoprotein E (ApoE) causes increased intracellular calcium and may mediate ApoE neurotoxicity

Abstract

Apolipoprotein E (apoE)-related synthetic peptides, the 22 kDa N-terminal thrombin-cleavage fragment of apoE (truncated apoE), and full-length apoE have all been shown to exhibit neurotoxic activity under certain culture conditions. In the present study, protease inhibitors reduced the neurotoxicity and proteolysis of full-length apoE but did not block the toxicity of truncated apoE or a synthetic apoE peptide, suggesting that fragments of apoE may account for its toxicity. Additional experiments demonstrated that both truncated apoE and the apoE peptide elicit an increase in intracellular calcium levels and subsequent death of embryonic rat hippocampal neurons in culture. Similar effects on calcium were found when the apoE peptide was applied to chick sympathetic neurons. The rise in intracellular calcium and the hippocampal cell death caused by the apoE peptide were significantly reduced by receptor-associated protein, removal of extracellular calcium, or administration of the specific NMDA glutamate receptor antagonist MK-801. These results suggest that apoE may be a source of both neurotoxicity and calcium influx that involves cell surface receptors. Such findings strengthen the hypothesis that apoE plays a direct role in the pathology of Alzheimer's disease.

Fig. 1.

ApoE neurotoxicity may involve proteolysis. Dissociated chick sympathetic neurons were exposed to the apoE peptide (ApoE_p) or full-length apoE4 (apoE4) in the presence or absence of protease inhibitors (PI). Controls included cells not receiving any experimental treatment (Control), receiving protease inhibitors alone (PI), or treated with an antibody against mouse IgG (IgG) that had been subjected to the same purification procedure as that used for apoE4. Significant toxicity was obtained with the apoE peptide and apoE4 but not with the control treatments. Protease inhibitors prevented the toxic effects of apoE4 but not of the apoE peptide. Medium from the cultures treated with apoE4 with or without PI was subjected to Western blotting using an antibody to apoE. The results are shown below the corresponding treatments in the bar graph. PI treatment reduced the number and intensity of bands representing low molecular weight apoE fragments in the medium. The approximate location of the molecular weight standards is indicated to the left of the Western blot.

Fig. 2.

ApoE peptide is neurotoxic in hippocampal cell cultures. A, Cultures were exposed for the indicated times to saline (Control), 2 μmapoE peptide (ApoE_p), or 4 μm RAP plus 2 μm apoE peptide (RAP+ApoE_p). The percentage of neurons surviving at each time point was quantified; values are the mean and SEM of determinations made in four separate cultures. *p < 0.05, **p < 0.01 compared with corresponding values in control and RAP + apoE cultures (ANOVA with Scheffe’s post hoc test). B, Cultures were exposed for 24 hr to the indicated concentrations of apoE peptide (ApoE_p) in the absence (Control) or presence of 4 μmRAP. The percentage of neurons was quantified, and values are the mean and SEM of determinations made in four separate cultures. *p < 0.05, **p < 0.01 compared with corresponding values in RAP-treated cultures (ANOVA with Scheffe’s post hoc test). C, Phase-contrast micrographs of hippocampal neurons in a culture before treatment (A1), and 6 hr (A2) and 24 hr (A3) after exposure to 0.5 μm apoE peptide. B1 and B2 are micrographs of neurons in a culture before and 24 hr after exposure to saline, respectively. Note the degeneration of neuronal cell bodies (arrow) and neurites (arrowhead) in the culture exposed to the apoE peptide. (Figure 2continues.)

Fig. 2.

ApoE peptide is neurotoxic in hippocampal cell cultures. A, Cultures were exposed for the indicated times to saline (Control), 2 μmapoE peptide (ApoE_p), or 4 μm RAP plus 2 μm apoE peptide (RAP+ApoE_p). The percentage of neurons surviving at each time point was quantified; values are the mean and SEM of determinations made in four separate cultures. *p < 0.05, **p < 0.01 compared with corresponding values in control and RAP + apoE cultures (ANOVA with Scheffe’s post hoc test). B, Cultures were exposed for 24 hr to the indicated concentrations of apoE peptide (ApoE_p) in the absence (Control) or presence of 4 μmRAP. The percentage of neurons was quantified, and values are the mean and SEM of determinations made in four separate cultures. *p < 0.05, **p < 0.01 compared with corresponding values in RAP-treated cultures (ANOVA with Scheffe’s post hoc test). C, Phase-contrast micrographs of hippocampal neurons in a culture before treatment (A1), and 6 hr (A2) and 24 hr (A3) after exposure to 0.5 μm apoE peptide. B1 and B2 are micrographs of neurons in a culture before and 24 hr after exposure to saline, respectively. Note the degeneration of neuronal cell bodies (arrow) and neurites (arrowhead) in the culture exposed to the apoE peptide. (Figure 2continues.)

Fig. 3.

Truncated apoE4 is neurotoxic and increases [Ca²⁺]_i in cultured rat hippocampal neurons. A, Cultures were exposed to the indicated concentrations of truncated apoE4 for 24 hr, and neuronal survival was quantified. Values are the mean and SEM of determinations made in three separate cultures. B, Cultures were exposed to the indicated concentrations of truncated apoE4 for 2 hr, and the [Ca²⁺]_i was measured in 12–17 neurons/culture. Values are the mean and SEM of determinations made in three separate cultures.

Fig. 4.

ApoE peptide induces a rapid and sustained elevation of [Ca²⁺]_i in chick sympathetic neurons that is inhibited by MK-801. Pretreatment of chick sympathetic neurons with 200 μm MK-801 for 30 min attenuates the peptide-induced increase in [Ca²⁺]_i. MK-801 pretreatment also resulted in an increase in the resting [Ca²⁺]_i. The [Ca²⁺]_i was measured by imaging the fluorescent calcium indicator dye fura-2 AM in neurons before and after exposure to 8 μm of the peptide at 80 sec (AEP, arrow). Values are the mean of determinations made in 70–80 cells in 10–13 separate cultures in either Neurobasal or F-12 medium.

Fig. 5.

ApoE peptide induces rapid and sustained elevations of [Ca²⁺]_i in rat hippocampal neurons. The [Ca²⁺]_i was monitored by fluorescence ratio imaging of the calcium indicator dye fura-2 AM in neuronal somata before and after exposure to 2 μm apoE peptide (arrow denotes time of addition of the apoE peptide) in the absence (A) or presence (B) of 1 μm RAP. Values represent mean [Ca²⁺]_i in 10–15 neurons and are representative of results obtained in at least six separate experiments. C, Images of intracellular free calcium levels in hippocampal neurons obtained by imaging of the calcium indicator dye fura-2 AM. The [Ca²⁺]_i is represented on acolor scale with values in nanomoles as indicated on the scale bar. Images were taken immediately before treatment and 1.5, 3, and 10 min after exposure to 2 μm apoE peptide. Note the rapid and sustained elevation of [Ca²⁺]_i in neuronal somata and in neurites, as well as swelling of the neuronal cell bodies and fragmentation of neurites (arrowheads). (Figure 5continues.)

Fig. 5.

ApoE peptide induces rapid and sustained elevations of [Ca²⁺]_i in rat hippocampal neurons. The [Ca²⁺]_i was monitored by fluorescence ratio imaging of the calcium indicator dye fura-2 AM in neuronal somata before and after exposure to 2 μm apoE peptide (arrow denotes time of addition of the apoE peptide) in the absence (A) or presence (B) of 1 μm RAP. Values represent mean [Ca²⁺]_i in 10–15 neurons and are representative of results obtained in at least six separate experiments. C, Images of intracellular free calcium levels in hippocampal neurons obtained by imaging of the calcium indicator dye fura-2 AM. The [Ca²⁺]_i is represented on acolor scale with values in nanomoles as indicated on the scale bar. Images were taken immediately before treatment and 1.5, 3, and 10 min after exposure to 2 μm apoE peptide. Note the rapid and sustained elevation of [Ca²⁺]_i in neuronal somata and in neurites, as well as swelling of the neuronal cell bodies and fragmentation of neurites (arrowheads). (Figure 5continues.)

Fig. 6.

ApoE-induced elevation of [Ca²⁺]_i involves calcium release from intracellular stores and is attenuated by MK-801. The [Ca²⁺]_i was monitored before and after exposure of rat hippocampal neurons to 2 μm apoE peptide (added at the time point indicated by arrow) in a control culture (A), a culture incubated in medium lacking Ca²⁺ and containing 0.5 mm EGTA (B), and a culture pretreated with 200 μm MK-801 (C). Values represent the mean [Ca²⁺]_i in at least 10 neurons and are representative of results obtained in at least six separate experiments.

Fig. 7.

The neurotoxic action of the apoE peptide is attenuated by MK-801 or removal of extracellular calcium. Hippocampal cultures were exposed for 24 hr to saline (Cont), 2 μm apoE peptide (ApoE_p), 200 μm MK-801 (MK8) plus 2 μm apoE peptide, or medium lacking calcium plus 2 μm apoE peptide. The percentage of neurons surviving was quantified, and values are the mean and SEM of determinations made in four separate cultures. *p < 0.05 compared with values for cultures exposed to apoE peptide alone. **p < 0.01 compared with values in control cultures and cultures exposed to MK-801, and MK-801 + apoE peptide (ANOVA with Scheffe’s post hoc test).