Biochemical and molecular studies of NMDA receptor subunits NR1/ 2A/2B in hippocampal subregions throughout progression of Alzheimer’s disease pathology Amanda J. Mishizen-Eberz, a,b, * Robert A. Rissman, a,b Troy L. Carter, a Milos D. Ikonomovic, c Barry B. Wolfe, d and David M. Armstrong a a Laboratory of Neuronal Vulnerability and Aging, The Lankenau Institute for Medical Research, Jefferson Health System, Wynnewood, PA, USA b Department of Neurobiology and Anatomy, MCP Hahnemann University School of Medicine, Philadelphia, PA, USA c Departments of Neurology and Psychiatry, Alzheimer’s Disease Research Center, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA d Department of Pharmacology, Georgetown University Medical Center, Washington, DC, USA Received 24 February 2003; revised 30 July 2003; accepted 16 September 2003 Alzheimer’s disease (AD) is characterized by loss of specific cell populations within selective subregions of the hippocampus. Excitotox- icity, mediated via ionotropic glutamate receptors, may play a crucial role in this selective neuronal vulnerability. We investigated whether alterations in NMDA receptor subunits occurred during AD pro- gression. Employing biochemical and in situ hybridization techniques in subjects with a broad range of AD pathology, protein levels, and mRNA expression of NR1/2A/2B subunits were assayed. With increasing AD neuropathology, protein levels and mRNA expression for NR1/2B subunits were significantly reduced, while the NR2A subunit mRNA expression and protein levels were unchanged. Cellular analysis of neuronal mRNA expression revealed a significant increase in the NR2A subunit in subjects with moderate neurofibrillary tangle neuropathology. This investigation supports the hypothesis that alterations occur in the expression of specific NMDA receptor subunits with increasing AD pathologic severity, which is hypothesized to contribute to the vulnerability of these neurons. D 2003 Elsevier Inc. All rights reserved. Keywords: NMDA receptor; Alzheimer’s disease; Hippocampus; In situ hybridization; Western blot; Neuropathology Introduction N-methyl-D-aspartate (NMDA) glutamate receptors are com- posed of a combination of at least four subunits from two gene families: NR1 and NR2 (NR2A-D) (McBain and Mayer, 1994). The NR1 subunit is present in all native NMDA receptors and two different NR2 subunits can exist within the same receptor complex (Chazot et al., 1994; Hawkins et al., 1999; Luo et al., 1997). NMDA receptors are highly expressed in the hippocampus, a region of the brain known to subserve several key learning and memory activities (Mayer and Westbrook, 1987). In AD, specific subregions of the hippocampus have been found to be selectively vulnerable to degeneration. For example, the subiculum and CA1 region are affected early in the disease process and display extensive pathological changes and cell loss while other subregions (CA3, CA4, dentate gyrus) are much less affected (Ball, 1977). Of particular interest in AD is the observation that within vulnerable hippocampal regions, specific cell populations (i.e., pyramidal neurons) are selectively affected. Although the mechanism under- lying the neuronal degeneration of selective cell populations has not been clearly elucidated, it is hypothesized that the abusive stimulation of NMDA glutamate receptors permeable to calcium ions (Ca 2+ ) may contribute to the process of excitotoxic cell death (Olney, 1990). Excitotoxicity is thought to be highly dependent on the diver- sity of glutamate receptors expressed by a cell. In addition, numerous studies demonstrate that subunit composition of indi- vidual NMDA receptors affects the kinetic properties of the intrinsic ion channel. For example, electrophysiological and phar- macological studies indicate that inclusion of specific NR2 sub- units within heteromeric NMDA receptors alters both receptor affinity for glutamatergic agonists and antagonists as well as changes the response of the receptor to various pharmacological agents, including glutamate (Ishii et al., 1993; Kutsuwada et al., 1992; Monyer et al., 1992; Priestley et al., 1995; Vicini et al., 1998). Therefore, the subunit composition of NMDA glutamate receptors likely defines the response of the receptor to glutamate activation, which subsequently affects neuronal susceptibility to excitotoxic insult. Numerous studies have relied upon autoradiographic techni- ques to examine ionotropic glutamate receptors in the AD brain (for review, see Mishizen et al., 2001). While these studies have been valuable in documenting NMDA receptor binding in AD, 0969-9961/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2003.09.016 * Corresponding author. Department of Neurology, School of Medi- cine, University of Pennsylvania, Philadelphia, PA 19104. Fax: +1-215- 590-3779. E-mail address: aeberz@mail.med.upenn.edu (A.J. Mishizen-Eberz). Available online on ScienceDirect (www.sciencedirect.com.) www.elsevier.com/locate/ynbdi Neurobiology of Disease 15 (2004) 80 – 92