N-methyl-d-aspartate receptor subunit NR2A and NR2B messenger RNA levels are altered in the hippocampus and entorhinal cortex in Alzheimer's disease

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Abstract

The N-methyl-d-aspartate (NMDA) receptor is a subtype of ionotropic glutamate receptor that is involved in synaptic mechanisms of learning and memory, and mediates excitotoxic neuronal injury. In this study, we tested the hypothesis that NMDA receptor subunit gene expression is altered in Alzheimer's disease (AD), especially in brain regions known to be important in memory. Quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) was used to determine the messenger RNA (mRNA) levels of the NMDA receptor subunits NR1, NR2A, and NR2B in the hippocampus and entorhinal cortex of postmortem brain samples from nine clinically well-characterized AD patients and nine aged controls. Cerebellum, a site minimally affected by AD, was also chosen for comparison assessment. Results showed decreased levels of the NR2 mRNAs in AD brains compared to controls. Reductions of NR2A (46.2%, p<0.01) and NR2B (43.2%, p<0.0001) mRNA levels were identified in the entorhinal cortex. Reductions of NR2A (41.4%, p<0.05) and NR2B (40.6%, p=0.058) mRNA levels were found in the hippocampus. NR1 mRNA levels were similar in all three brain regions in both AD and controls. No significant changes of subunit NR2A and NR2B mRNA levels were identified in the cerebellum. Postmortem delay (PMD), tissue storage time, brain weight, or age of the subjects did not affect these changes. These data suggest that alterations in NMDA receptor subunits, especially the NR2A and NR2B, may be important in AD, particularly in neuronal populations that underlie impaired learning and memory.

Introduction

The N-methyl-d-aspartate (NMDA) receptor is a subtype of the ionotropic glutamate receptor that plays a pivotal role in synaptic mechanisms of learning and memory [1]. Previous studies have suggested that glutamate-induced excitotoxic injury may disturb normal glutamate neurotransmission and glutamate receptor activation in a variety of neurological disorders including Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, and epileptic disorders [2], [3], [4]. It has also been reported that glutamate receptors can interact with amyloid precursor protein or β-amyloid, which can lead to either beneficial or detrimental effects on neurons [5], [6]. These observations suggest that the NMDA receptor could play a significant role in the neurobiology of Alzheimer's disease (AD).

The NMDA receptors are heteromeric complexes composed of the NR1 subunit combined with various types of NR2 subunit (A, B, C, or D) in different brain regions. In addition, alternative splicing and messenger RNA (mRNA) editing creates more variants, so that at least eight isoforms of the NMDA receptor exist. The different heteromeric forms of the NMDA receptor exhibit distinct physiological and pharmacological properties, which likely reflect the function of receptors in different areas of the brain [7]. While NR1 mRNA and protein are ubiquitously distributed in the brain [30], the NR2A and NR2B subunit mRNAs predominantly reside in certain forebrain regions of normal brain, such as the neocortex and hippocampus. In contrast, NR2C and NR2D subunits are mainly expressed in the nondiseased cerebellum and mid- and hindbrains of humans and animals [31], [32]. In contrast, limited information exists in the literature regarding the levels of these same NMDA receptor subunits in diseased brain, particularly in AD brain. Previous investigations have shown conflicting results regarding the levels of these various NMDA receptors in AD brain. Either reduced [8] or stable NMDA receptor ligand binding sites [9], [11] have been reported, but these studies focused on the protein, not on the mRNA. Studies focusing on the mRNAs of the NMDA receptors in AD are equally limited. One study, using hybridization of an antisense oligonucleotide probe in situ to estimate the level of receptor mRNA, reported that NMDA receptor gene expression is decreased in the AD brain [10]. However, this study was limited to the investigation of the NR1 subunit and levels of the NR2 subunit protein, or its mRNA levels were not assessed.

We had previously reported that NMDA receptor subunit proteins and their phosphorylation status, especially the NR2A and NR2B receptor subunits, were altered selectively in AD [19]. We were particularly interested in studying the NR2A and NR2B receptors because of their apparently critical role in memory. It has been demonstrated that overexpression of the NR2 subunit in a transgenic animal model enhances learning and memory [12]. Because it is not clear whether these protein alterations were due to impaired gene expression of these receptor subunit proteins, and also because there are limited studies on NR2A and NR2B mRNA levels in AD, we are now extending our original work. Reverse transcriptase–polymerase chain reaction (RT–PCR) was chosen as a reproducible and easily quantifiable method to analyze mRNA levels of the NMDA receptor subunit in AD using tissues from brain regions that are known to be important in memory. We also chose to assess these receptor types in cerebellum for comparison, a region minimally affected by AD.

Section snippets

Subjects and RT–PCR

Postmortem frozen brain tissue from nine pathologically confirmed AD patients and nine aged control subjects obtained from the Brain Bank at the Department of Pathology, University of Colorado Health Sciences Center (UCHSC) were used for this study (Table 1). The mean age±standard deviation were 84±10 and 69±9 for AD and control groups, respectively. The diagnoses of definite AD were formulated based on the criteria from the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) [13]

Results

The results of the semiquantitative RT–PCR to estimate the mRNA levels of NMDA receptor subunits NR1, NR2A, and NR2B in specific regions of the brain from AD patients and those of age-matched normal individuals are detailed in Table 3 and will be summarized here. A statistically significant reduction of NMDA receptor subunit NR2A and NR2B mRNA levels in the entorhinal cortex and hippocampus was found in Alzheimer's disease compared to normal-aged controls. Compared to normal controls, the NR2A

Discussion

We have identified a reduction in the expression of the NMDA receptors NR2A and NR2B subunit mRNA, but not in the NR1 subunit mRNA, in the hippocampus and entorhinal cortex in AD brains. These results support our previous observations using immunoblotting assays that NR2A and NR2B receptor subunit protein levels were selectively reduced in AD [19]. Our results also confirm the work of the investigations that NMDA receptors are abnormal in AD [8], [19] in these critical brain areas. This current

Acknowledgments

We thank Dr. Robert Low and Dr. Bette K. Kleinschmidt-DeMasters for helpful discussions and suggestions of the manuscript, Mr. Bob McCullough for assistance with photographs and figures, and Mrs. Ginger Woodward for assisting in manuscript preparation. This work was supported in part by funds from the Maria Teresa Jones Alzheimer's Disease Research Award, UCHSC (CIS).

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