Apolipoprotein E Protects against NMDA Excitotoxicity

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Neurobiology of Disease 11, 214 –220 (2002) doi:10.1006/nbdi.2002.0541

Apolipoprotein E Protects against NMDA Excitotoxicity Mitsuo Aono,* ,† Yoonki Lee,* ,† Elfrida R. Grant, ‡ Robert A. Zivin, ‡ Robert D. Pearlstein,* ,§ David S. Warner,* ,† Ellen R. Bennett,* ,¶ and Daniel T. Laskowitz* ,储 *Multidisciplinary Neuroprotection Laboratory, †Department of Anesthesiology, §Department of Surgery, ¶Department of Pathology, and Department of Medicine (Neurology), Duke University Medical Center, Durham, North Carolina 27710; and ‡Drug Discovery, R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey 08869 Received August 21, 2001; revised April 23, 2002; accepted for publication August 1, 2002

Preclinical and clinical evidence implicates a role for endogenous apolipoprotein E in modifying the response of the brain to focal and global ischemia. To investigate whether apoE modulates the neuronal response to glutamate excitotoxicity, we exposed primary neuronal glial cultures and a neuronal cell line to biologically relevant concentrations of apolipoprotein E prior to NMDA exposure. In both of these paradigms, apolipoprotein E exerted partial protective effects. At neuroprotective concentrations, however, apolipoprotein E failed to block NMDA-induced calcium influx to the same magnitude as the NMDA receptor antagonist MK-801. These results suggest that one mechanism by which apolipoprotein E modifies the central nervous system response to ischemia may be by reducing glutamate-induced excitotoxicity. © 2002 Elsevier Science (USA) Key Words: excitotoxicity; NMDA; apolipoprotein E; cell culture; calcium influx; neuroprotection; RAP; cerebral ischemia.

INTRODUCTION

ity following stroke, intracranial hemorrhage, and cardiac arrest (Alberts et al., 1995; Laskowitz et al., 1998; McArron et al., 1998). Despite this compelling clinical and preclinical data, the molecular basis by which apoE modifies the response of the brain to ischemia remains poorly defined. There are likely multiple mechanisms by which endogenous apoE plays a protective role in the injured CNS, as suggested by prior in vitro data demonstrating isoform-specific antioxidant (Miyata & Smith, 1996) and neurotrophic effects of apoE (Holtzman et al., 1995). An additional mechanism by which apoE might influence the CNS response to ischemia is via modulation of glutamate excitotoxicity. Glutamate toxicity is believed to contribute to neuronal injury in the setting of ischemia, and of the different classes of glutamate channels, specific activation of the N-methyl-d-aspartate (NMDA) receptor is believed to be primarily responsible for mediating excitotoxicity in a variety of neuron types (Meldrum & Garthwaite,

Apolipoprotein E (apoE) is a 299-amino-acid protein with multiple biological properties. There are three common human isoforms of apoE, designated apoE2, E3, and E4, which differ by single amino acid substitutions at residues 112 and 158 (Weisgraber, 1994). A growing body of evidence suggests a role for endogenous apoE in modifying the central nervous system (CNS) response to acute injury (Laskowitz et al., 1998). In preclinical models of focal and global ischemia, the absence of endogenous apoE has been associated with a worsening of both histological and functional outcomes (Laskowitz et al., 1997; Sheng et al., 1999; Horsburgh et al., 2000). These effects appear to be isoformspecific, with the presence of the APOE4 allele associated with worse outcome than that of the APOE3 allele (Sheng et al., 1998). These observations are consistent with clinical data implicating the presence of the APOE4 allele with increased neurological morbid-

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1990). Toward this end, we used a model of NMDA toxicity in both primary neuronal– glial culture and a well-characterized neuronal cell line to evaluate the role of apoE in modulating excitotoxic cell death.

MATERIALS AND METHODS All animal procedures were approved by the Duke University Animal Care and Use Committee. Preparation of Primary Neuronal–Glial Cultures and P19 Neuronal Cell Lines Primary neuronal cultures were prepared from fetal Sprague–Dawley rat brains (Harlan Sprague Dawley, Inc., Indianapolis, IN) at 18 days of gestation as previously described (Pearlstein et al., 1998). Briefly, cortices were dissected from the brains of 10 –15 animals, pooled, and minced into 2-mm 3 pieces in a buffered salt solution. Following trituration, the tissue suspension was centrifuged at 50g for 10 min and the pellet was resuspended in growth medium (MEM supplemented with 15 mM glucose, 5% fetal bovine serum, and 5% horse serum). The dissociated cells were plated to achieve a confluent monolayer on poly-dlysine-coated, 24- or 96-well culture plates and maintained undisturbed at 37°C in a humidified 5% CO 2/ 95% room air atmosphere for 13–16 days prior to use. Preparation and characterization of these cultures, which contain approximately 54% neurons and 46% glia, have been described in detail elsewhere (Pearlstein et al., 1998; Kudo et al., 2001). The P19 neuronal cell line was obtained from American Type Culture Collection (Rockville, MD). Maintenance and differentiation of this cell line have been described in detail elsewhere (Grant et al., 2001; Jones-Velleneuve et al., 1982). Expression and Purification of Recombinant Receptor-Associated Protein (RAP) Expression and purification of RAP were performed as previously described (Herz et al., 1991; Laskowitz et al., 2001). Briefly, rat cDNA sequences corresponding to the coding region of the mature 39-kDa RAP were subcloned into a 6⫻His-tag bacterial expression vector (Novagen, Madison, WI) and induced in Bl21 Escherichia coli cells. Bacterial lysate was applied to a nickel– nitrilotriacetic metal-affinity chromatography column which bound the 6⫻His-tagged RAP. RAP was puri-

fied under native conditions using the QIAexpressionist kit (Qiagen, Valencia CA). Exposure to NMDA Mature cultures (13–16 days in vitro) were washed with Mg 2⫹-free BSS containing 20 mM Hepes buffer and 1.8 mM CaCl 2, pH 7.4, prior to the addition of 100 ␮M NMDA. The exposed cultures were maintained at 37°C for 30 min prior to removal of the medium containing NMDA. Medium was then replaced with MEM supplemented with 20 mM glucose ⫹ 1.8 mM CaCl 2 ⫹ 20 mM Hepes, pH 7.4. The cultures were then returned to the incubator and maintained at 37°C for an additional 24 h for determination of NMDA-induced lactate dehydrogenase (LDH) release as described below. Assessing Effect of ApoE on NMDA Toxicity To avoid any confounding effects of the ammonium bicarbonate vehicle in which the human recombinant apoE (Pan Vera Corp., Madison, WI) was manufactured, apoE was resuspended in isotonic PBS by dialyzing with Slide-A-Lyzer dialysis cassettes (Pierce Chemical Co., Rockland, IL). The effect of human recombinant apoE2 on NMDA-induced neuronal excitotoxicity was assessed by preincubating apoE with mixed glial cultures 30 min prior to the addition of 100 ␮M NMDA. To assess the effect of apoE preparation (i.e., lipid-poor human recombinant apoE vs lipid bound apoE) apoE–VLDL purified from human plasma (Calbiochem, La Jolla, CA) was compared directly to an equimolar concentration of human recombinant apoE. This apoE preparation was not isoformspecific. To determine whether isoform-specific effects existed, two different concentrations (300 nM and 3 ␮M) of apoE2, apoE3, and apoE4 were examined. To assess whether the protective effects of apoE were mediated by glia, a glutamate excitotoxicity assay was performed on the P19 neuronal cell line as previously described (Grant et al., 2001). This assay involves preincubating P19 cells with increasing concentrations of the different apoE isoforms for 30 min prior to exposure with glutamate and glycine (3 and 1 mM, respectively). The effect of pretreatment of apoE timing on NMDA toxicity was assessed by pretreating cultures with 3 ␮M apoE2 for 24 h and removing it 30 min prior to NMDA exposure. In a separate set of sister cultures, 3 ␮M apoE2 was added concurrent with or immediately ©

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following NMDA exposure. In all cases, the cultures were exposed to 100 ␮M NMDA for 30 min at 37°C, and the effect of treatment on cell viability was examined 24 h after NMDA exposure.

for post hoc multiple comparisons against the control group. Significance was assumed at P ⬍ 0.05. Values for LDH release are reported as means ⫾ standard error.

Determination of Cell Viability

RESULTS

Cell viability was assessed in primary mixed neuronal– glial cultures by quantifying the amount of LDH released into medium by injured cell as previously described (Pearlstein et al., 1998; Amador et al., 1963). A 200-␮l sample of culture medium was added to a polystyrene cuvette containing 10 mM lactate and 5 ␮mol of nicotinamide adenine dinucleotide in 2.75 ml of 50 mM glycine buffer, pH 9.2, at 24°C. LDH activity was determined from the initial rate of reduction of nicotinamide adenine dinucleotide as calculated using a linear least-square curve fit of the temporal changes in fluorescence signal from the cuvette (340 nM excitation, 450 nM emission) and expressed in units of enzymatic activity (nanomoles of lactate converted to pyruvate per minute). Analysis was performed on a fluorescence spectrophotometer (Perkin– Elmer Model LS50B, Uberlinger, Germany). The validity of this assay as a marker for cell death was periodically checked by staining cells with 5 ␮g/ml propidium iodide and manually counting viable cells by fluorescence microscopy. Cell viability in the neuronal cell line was assessed with alamar blue fluorescence as previously described (Grant et al., 2001; White et al., 1996). Effect of ApoE on Cellular Calcium Uptake Cellular Ca 2⫹ uptake from extracellular space was assessed using 45CaCl 2. After cultures were incubated for 30 min with 3 ␮M apoE or 10 ␮M MK-801 in Mg 2⫹-free BSS containing 20 mM Hepes buffer, 45Ca 2⫹ was added to each well (0.28 ␮Ci/ml, 0.9 ␮Ci/well) and NMDA was added to a final concentration of 100 ␮M. The cultures were returned to the incubator and maintained at 37°C. Thirty minutes later, the exposure medium was removed and each well was washed three times with ice cold Mg 2⫹-free BSS containing 20 mM Hepes buffer. Afterward, the cells were lysed by addition of 0.2% sodium dodecyl sulfate, and radioactivity was determined by scintillation counting. Statistical Analysis Group comparisons were performed using analysis of variance followed by Dunnet’s method to correct 2002 Elsevier Science (USA) All rights reserved.

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To ensure that apoE2 did not interfere with the quantification of LDH activity, duplicate standards were run in the presence and absence of apoE. Results were identical in both groups, demonstrating that the presence of the apoE protein did not interfere with the assay for LDH activity. Preincubation of apoE2 in primary rat mixed neuronal– glial cultures 30 min prior to exposure with100 ␮m NMDA resulted in a dose-dependent reduction in cytotoxicity (Fig. 1A). At concentrations of 100 nM to 3 ␮M, a modest yet significant reduction in cell death was observed following NMDA exposure compared to vehicle control. To address the question of whether lipid binding influenced the ability of apoE to protect against NMDA excitotoxicity, we compared preincubation of cultures with 3 ␮M human recombinant apoE2 vs an equimolar concentration of apoE–VLDL purified from human serum. Preincubation with either apoE preparation prior to NMDA exposure reduced LDH release compared to NMDA alone, but there was no significant difference between the lipid-bound vs the lipidpoor apoE preparation (Fig. 1B). In addition to the LDH assay, cytotoxicity was assessed by staining cells with propidium iodide and manually counting viable neurons by fluorescence microscopy. This method of assessing cell viability paralleled the LDH assay. We found a significant protective effect of preincubation with 3 ␮M apoE vs NMDA alone (48% ⫾ 15 dead neurons in apoE preincubation vs 74% ⫾ 13 dead neurons in NMDA group; P ⬍ 0.05). However, there was no significant difference between preincubation with human recombinant vs lipid-bound apoE (48% ⫾ 15 vs 63% ⫾ 8 dead neurons in human recombinant apoE vs apoE–VLDL, respectively). We next addressed the question of whether apoE exerted its neuroprotective effect in an isoform-specific fashion. All three isoforms (apoE2, apoE3, and apoE4) provided partial protection from NMDA excitotoxicity at both 300 nM and 3 ␮M, but there were no differences among isoforms (Fig. 1C). When these experiments were repeated in the P19 neuronal cell line, apoE exerted a partial neuroprotective effect without isoform specificity similar to that observed with primary mixed cultures (data not shown). This suggests

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FIG. 2. Treatment with 3 ␮M apoE2 conferred benefit against 100 ␮M NMDA toxicity in primary mixed neuronal– glial cultures when apoE was added prior to, concurrent with, or following NMDA exposure. Preincubation of 10 ␮M MK-801 30 min prior to NMDA exposure conferred complete protection from excitotoxicity (*P ⬍ 0.05, **P ⬍ 0.001 versus NMDA alone, ANOVA with Dunnet’s test for multiple comparisons against control). Each data point represents 11 wells and is presented as the mean ⫾ SEM.

that the neuroprotective effect of apoE is independent of the presence of glia. ApoE neuroprotection was detected when apoE2 was added prior to, concurrent with, or following exposure of primary mixed neuronal– glial cultures with NMDA (Fig. 2). The fact that the neuroprotective activity of apoE was retained post-exposure to NMDA suggests that the role of apoE extends beyond NMDA receptor antagonism. We next investigated the role of apoE in modulating cellular Ca 2⫹ influx following exposure to 100 ␮M

FIG. 1. (A) Incubation of mixed neuron– glial cultures with 100 ␮M NMDA caused cell death, as measured by LDH assay and verified by propidium iodide assay. LDH release is expressed as nmol of lactate converted to pyruvate per min. Pretreatment with apoE2 for 30 min prior to NMDA exposure resulted in a partial

neuroprotective effect at an apoE concentration range of 100 nM to 3 ␮M. Higher concentrations of apoE2 were not tested, as they were not felt to represent a biologically relevant concentration. Values are means ⫾ SEM of 6 wells per condition (*P ⬍ 0.05 vs control; ANOVA with Dunnet’s post hoc test to compare each concentration vs control). (B) To address whether lipid binding modulates the neuroprotective effects of apoE, we compared 3 ␮M concentrations of human recombinant apoE with apoE–VLDL complex. Both apoE preparations protected neuronal/glia cultures from NMDA excitotoxicity. There was no significant difference between the neuroprotective effects of lipid-poor (human recombinant) apoE and lipid bound apoE–VLDL. (C) At both high (3 ␮M) and low (300 nM) concentrations, apoE resulted in significant neuroprotection from NMDA excitotoxicity in neuronal– glial cultures (*P ⬍ 0.05; ANOVA with Dunnet’s post hoc test to compare each condition vs NMDA alone). No isoform-specific effects of apoE were observed. Each data point represents the mean ⫾ SEM from 48 wells.

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NMDA excitotoxicity (Fig. 4). The RAP used in these experiments was determined to be biologically active by its ability to compete with ␣2 macroglobulin for the LRP/␣2M receptor of murine peritoneal macrophages (data not shown).

DISCUSSION

FIG. 3. Exposure of primary cultures to 100 ␮M NMDA for 20 min resulted in a significant influx of Ca 2⫹ compared to controls; this effect was completely reversed by pretreating cells with 10 ␮M MK-801 (*P ⬍ 0.05). Pretreatment of cells with 3 ␮M apoE for 30 min prior to NMDA did not reduce Ca 2⫹ influx vs NMDA alone, although there was a significant protective effect on cell viability as quantified by LDH release in sister cultures studied simultaneously. Data are presented as mean counts/min/well ⫾ SEM; each data point represents the mean of a minimum of four wells.

NMDA. As a control, we first demonstrated that 3 ␮M apoE2 alone had no direct effect on Ca 2⫹ influx in mixed neuronal– glial cultures (data not shown). Following 30 min exposure to 100 ␮M NMDA, increased Ca 2⫹ influx was detected relative to control, an effect that was completely reversed by preincubation with 10 ␮M MK-801 (Fig. 3). Interestingly, preincubation with 3 ␮M apoE2 30 min prior to NMDA exposure failed to reduce Ca 2⫹ influx, despite the fact that apoEmediated neuroprotection was observed in sister cultures at the same concentration. One possible mechanism to explain the apoE effect on reducing excitotoxic injury would be by initiating a signaling cascade downstream of the NMDA receptor via interaction with a cell surface receptor. ApoE is known to bind with a high affinity to a family of receptors, including the low-density lipoprotein receptor-related protein/␣2 macroglobulin receptor (LRP/␣2M), which is present on both neurons and microglia. To address this question, primary mixed cultures were preincubated with RAP, which is known to block all functional ligand–receptor interactions with the LRP/␣2M receptor (Herz et al., 1991). Preincubation of cells with 10 ␮M RAP alone had no effect on cell viability. Preincubation of cultures with 10 ␮M RAP for 1 h prior to addition of 3 ␮M apoE failed to reverse the neuroprotective effects of apoE on 2002 Elsevier Science (USA) All rights reserved.

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Although apoE has been demonstrated to play a protective role in both clinical and experimental models of brain ischemia, the mechanisms by which this occurs remain poorly defined. In this study, biologically relevant concentrations of apoE exerted a modest protective effect against NMDA-induced excitotoxicity. This protection was not associated with inhibition of NMDA-induced Ca 2⫹ influx, as was observed with MK-801, a noncompetitive NMDA receptor antagonist. In addition to the protective effect observed when administered concurrently with NMDA, apoE also protected neurons when added transiently prior to NMDA exposure and salvaged neurons when added subsequent to NMDA exposure. Although these results do not preclude the possibility that apoE functions as an NMDA receptor antagonist, the possibility

FIG. 4. Exposure of cells to 1 ␮M RAP had no significant effect on cell viability. Exposure of cells to 100 ␮M NMDA resulted in substantial cytotoxicity, an effect that was attenuated by pretreatment with 3 ␮M apoE for 30 min prior to NMDA (*P ⬍ 0.05; ANOVA with Dunnet’s post hoc test for multiple comparisons). Pretreatment with RAP for 1 h prior to treatment with apoE did not reverse the protective effects of apoE, suggesting that these effects were not mediated via the LRP receptor. LDH release is expressed as units of enzyme activity, defined as nmol of lactate converted to pyruvate per minute.

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is raised that apoE may be exerting neuroprotection by other mechanisms as well. Given evidence that apoE directly modulates glial function and activation (Barger & Harmon, 1997; Hu et al., 1998; Laskowitz et al., 2001; Lynch et al., 2001) one potential mechanism to account for our results is that that apoE mediates its neuroprotective effect indirectly through glia, rather than interacting directly with neurons. To address this possibility, we examined the role of apoE in modulating NMDA excitotoxicity in the neuronal P19 cell line. This cell line has neuronal morphology, expresses the NMDA receptor, and is sensitive to NMDA-induced excitotoxicity (Grant et al., 2001). The observation that apoE exerted a similar neuroprotective effect in the absence of glia suggests that apoE elicits protection by acting directly on neurons. Despite preclinical and clinical data demonstrating that apoE isoform affects neurological recovery following brain injury (Alberts et al., 1995; McArron et al., 1998; Laskowitz et al., 1998; Sheng et al., 1998; Horsburgh et al., 2000; Schiefermeier et al., 2000) we observed no significant isoform-specific effect of apoE at any of the concentrations tested in either primary mixed neuronal– glial cultures or the neuronal cell line. This does not preclude the possibility that there might be significant isoform-specific differences in vivo, as an in vitro model employing the addition of exogenous apoE may be insensitive to subtle isoformspecific differences in synthesis, secretion, or metabolism of apoE that may be biologically relevant in situ. For example, apoE3 and apoE4 may differ in their susceptibility to being modified by the oxidizing environment present in areas of tissue inflammation or may be differentially incorporated into biologically active lipoprotein particles. For these reasons, we are currently examining isoform-specific effects of apoE in protecting from excitotoxicity in vivo. In addition to direct receptor antagonism, another mechanism that might explain the neuroprotective effects of apoE is interaction with a specific cell surface receptor to promote cell viability downstream of the NMDA receptor. For example, the LRP/␣2M receptor, which is present on both microglia and neurons, initiates a typical signaling cascade upon binding of apoE (Misra et al., 2001). A recent report has demonstrated that the LRP receptor is capable of initiating a calcium signaling response mediated by the NMDA receptor (Bacskai et al., 2000). Although the exact mechanism by which this occurs remains undefined, the authors speculate on the presence of a neuronspecific intracellular adaptor protein that modulates

NMDA activity following ligand binding and dimerization of the LRP receptor. In this study, we found that preincubation with RAP, a protein that blocks all known binding to this receptor (Herz et al., 1991), failed to reverse the protective effects of apoE on NMDA excitotoxicity, indicating that specific apoE– LRP interactions are unlikely to mediate neuronal protection from excitotoxic stress. It is likely that apoE performs this function via interactions with other neuronal receptors. Additional receptors expressed in brain and known to bind apoE are LR8 (Novak et al., 1996) and apoER2 (Kim et al., 1997). Alternatively, it has been suggested that apoE can mediate biological activity via heparan sulfate proteoglycans (Ji et al., 1997). Our finding that apoE protects against NMDA excitotoxicity is in contrast to other in vitro observations. In a recent series of experiments, Lendon et al. (2000) examined modulation of the NMDA receptor by apoE. Exogenous administration of high-density lipoprotein (20 ␮g/ml) exerted neuroprotective effects against NMDA toxicity in primary neuronal cultures. However, the additional administration of 5 ␮g/ml apoE failed to provide further protection. Unfortunately, the experimental design did not include the effects of apoE alone on NMDA-induced neuronal death. Using rat hippocampal cultures exposed to apoE purified from the conditioned medium of HEK-293 cells stably expressing human apoE3 and apoE4, Jordan et al. similarly observed that apoE failed to modulate NMDAinduced calcium influx (Jordan et al., 1998). However, in these experiments, apoE was added 5 days prior to NMDA exposure, and the one concentration of apoE that was reported (10 ␮g/ml, or approximately 0.3 ␮M) was less than the concentration of human recombinant apoE with which we observed peak neuroprotective effects, which occurred at 3 ␮M. In summary, we demonstrate that apoE confers partial protection against excitotoxic neuronal cell death induced by exposure to NMDA. This neuroprotective effect can occur in the absence of glia and is similar in potency for each of the three human isoforms. This observation has direct relevance for the accumulating clinical and experimental data implicating a role for endogenous apoE in affecting histological and functional outcome following global and focal cerebral ischemia. Although the specific mechanism(s) for this protective effect remains to be elucidated, our results suggest that protection is not restricted to NMDA receptor antagonism. Insight into the role of apoE in the injured CNS may lead to novel therapeutic strategies following stroke and acute brain injury. ©

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ACKNOWLEDGMENTS This work was funded by NIH 1K08NS01949, R01NS37235, and 1R01NS368087 and the Paul Beeson Physician Faculty Award (D.T.L.).

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