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* , *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 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 substi- tutions at residues 112 and 158 (Weisgraber, 1994). A growing body of evidence suggests a role for endog- enous 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 out- comes (Laskowitz et al., 1997; Sheng et al., 1999; Hors- burgh et al., 2000). These effects appear to be isoform- specific, with the presence of the APOE4 allele associ- ated with worse outcome than that of the APOE3 allele (Sheng et al., 1998). These observations are con- sistent with clinical data implicating the presence of the APOE4 allele with increased neurological morbid- ity following stroke, intracranial hemorrhage, and car- diac 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 pro- tective role in the injured CNS, as suggested by prior in vitro data demonstrating isoform-specific antioxi- dant (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 modu- lation 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-meth- yl-D-aspartate (NMDA) receptor is believed to be pri- marily responsible for mediating excitotoxicity in a variety of neuron types (Meldrum & Garthwaite, Neurobiology of Disease 11, 214–220 (2002) doi:10.1006/nbdi.2002.0541 0969-9961/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved. 214