N-Methyl-d-aspartate receptor subunit proteins and their phosphorylation status are altered selectively in Alzheimer’s disease

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Abstract

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. We tested the hypothesis that NMDA receptor protein levels are abnormal in Alzheimer’s disease (AD). By immunoblotting, we assessed levels of both non-phosphorylated and phosphorylated receptor subunit proteins from four separate regions of 16 post-mortem brains. Three patient groups with thorough pre-mortem neuropsychological testing were evaluated, including AD, early AD (p-AD), and control patients. Protein levels and phosphorylation status of NMDA receptor subunits NR1, NR2A and NR2B were correlated with measurements of cognitive performance. Selective regional reductions in NMDA receptor subunit protein levels were found in AD compared to controls, but protein levels in the p-AD group were similar to controls. Reductions of NR1 (53%, P<0.05) and NR2B (40%, P<0.05) were identified in hippocampus. Reductions of NR2A (39%, P<0.05) and NR2B (31%, P<0.01) were found in entorhinal cortex. No reductions were noted in occipital cortex and caudate. Phosphorylated NR2A (30%, P<0.05) and NR2B (56%, P<0.01) were selectively reduced in entorhinal cortex in AD when compared to controls. Both phosphorylated and non-phosphorylated NMDA receptor protein levels in entorhinal cortex correlated with Mini-Mental Status Examination (MMSE) and Blessed (BIMC) scores. The losses of phosphorylated and non-phosphorylated NMDA receptor subunit proteins correlated with changes in synaptobrevin levels (a presynaptic protein), but not with age or post-mortem interval. Our results demonstrate that NMDA receptor subunits are selectively and differentially reduced in areas of AD brain, and these abnormalities correlate with presynaptic alterations and cognitive deficits in AD.

Introduction

Glutamate is the major excitatory neurotransmitter in the central nervous system and functions in synaptic neurotransmission by activating glutamate receptors. One subtype of glutamate receptor that is critical for synaptic function is the N-methyl-d-aspartate (NMDA) receptor. Activation of NMDA receptors participates in the mechanism of long-term potentiation (LTP), the best-understood synaptic model of learning and memory [1]. However, perturbations in glutamate neurotransmission and glutamate receptor activation may be fundamental to a variety of neurological disorders [2], [3], [4]. Excessive release of glutamate contributing to excitotoxic neuronal death has been implicated as an etiologic mechanism in anoxic-ischemic injury, epilepsy, and chronic neurodegenerative disorders, including amyotrophic lateral sclerosis and Alzheimer’s disease (AD) [5], [6], [7], [8].

Progressive impairments in memory and cognition are the clinical hallmarks of AD. These functional deficits correlate with the neuropathologic features of the disease, including neuronal loss, senile plaque and neurofibrillary tangle formation, and loss of synaptic integrity. Previous studies have shown neuronal degeneration and synaptic loss in the hippocampus and entorhinal cortex occur in AD, and these deficits correlate with neuropsychological scores that reflect impaired memory and cognitive function in AD [18], [19], [26], [29], [30].

Recent studies on AD have suggested that amyloid precursor protein may play an important role in regulating glutamate levels at synapses by modulating the activity of glutamate receptors [9]. Furthermore, β-amyloid protein, a key component of senile plaques, can enhance the neurotoxicity of glutamate [10], [11]. In this study we evaluated one major subtype of glutamate receptor, the NMDA receptor, because of its integral involvement in learning and memory, processes that are strikingly compromised in AD, and because of its important role in excitotoxic neuronal death.

Previous studies using autoradiographic ligand binding assays to measure NMDA receptors in AD brain have shown conflicting results. Some studies have reported that NMDA receptors are lost from cortical and hippocampal areas [12], [13], while others have found hippocampal NMDA receptors to be relatively stable [14], [15]. These studies provide valuable information on NMDA binding site localization in the human brain. Several NMDA receptor subunits have been identified. Some subunits appear to have a greater role in learning and memory [16]. Little is known in AD, however, about the levels of the various NR1, NR2A, and NR2B subunits that comprise the NMDA receptor, or whether alterations in these subunits of the receptor have any clinical significance. In addition, the phosphorylation status of NMDA receptor subunits is important in ischemic neuronal injury [17]. We therefore tested the hypothesis that NMDA receptor subunits and their phosphorylation status are selectively and differentially affected in AD patients who have variable cognitive impairments.

Section snippets

Subjects and clinical evaluation

The subjects selected for this study were described previously [18], [19]. The groups of subjects were characterized as definite AD (n=6), possible AD or early AD (n=4), and normal controls (n=6) (Table 1) [20] using the criteria from the Braak and Braak staging scheme and the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) [21], [22]. Subjects without clinical manifestation of dementia but with sufficient numbers of neuritic plaques in neocortex for the diagnosis of AD by

Results

The quantitative densitometric results of the regional levels of NMDA receptor subunit protein levels in immunoblots (Fig. 1) are summarized (Table 2). Western blots of AD versus controls showed that the percentage (%) reductions compared to controls among receptor subunits are differentially and regionally selective in AD. Specifically, reductions in NR1 (53%, P<0.05) and NR2B (40%, P<0.05) were found in hippocampus, and reductions in NR2A (39%, P<0.05) and NR2B (31%, P<0.01) were noted in

Discussion

We found that specific subunits of NMDA receptor are abnormal in individuals with definite AD, but not in individuals with early AD. Levels of NR1 and NR2B were profoundly reduced in the hippocampus, and levels of NR2A and NR2B were decreased in the entorhinal cortex. Interestingly, presynaptic defects have previously been shown to occur in these regions [18], [19] and in the caudate nucleus [19] early in AD, but in this study no statistically significant changes were detected in NMDA receptor

Acknowledgements

We thank Drs. Claudia Kawas, Juan Troncoso, the ADRC and the BLSA staff, and the ADRC and BLSA participants for the clinical information. We gratefully acknowledge Mr. Bob McCullough for assistance with photographs and figures and Ginger Woodward for secretarial support. This work was supported in part by funds from the Maria Teresa Jones Alzheimer’s Disease Research Award, UCHSC (CIS) and by a NIH-NIA grant (LJM, AG16282).

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