Phenotypic down-regulation of glutamate receptor subunit GluR1 in Alzheimer’s disease☆

Abstract

Glutamate receptors play crucial roles in cognition and memory. We have quantitated the protein levels of α-amino-isoxazolepropionic acid (AMPA)-type (GluR1) and N-methyl-d-aspartate-type (NMDAR1) glutamate receptors in postmortem brain tissues of patients with Alzheimer’s disease and age-matched controls using western blotting. The bolts carrying fully denatured proteins were probed with antibodies specific to their carboxyl terminus of these receptors. In Alzheimer’s disease, GluR1 levels were significantly decreased in the entorhinal cortex and dentate gyrus, but not in the motor cortex. In contrast, levels of NMDAR1 were not altered in the dentate gyrus, suggesting that GluR1 expression was specifically diminished in this structure that is known to be preserved histologically in patients. However, the results of immunocytochemical examination confirmed a previous controversial report: GluR1-immunoreactive structures were labeled rather intensely in the molecular layer of the dentate gyrus of Alzheimer’s patients. Interestingly, levels of a postsynaptic density protein named SAP97, which recognizes and potentially masks the epitope region of GluR1, was positively correlated with those of GluR1 protein in the control group, but not in the patient group. Thus, the enhanced GluR1-like staining in Alzheimer’s disease might be ascribed to the hampered interaction between SAP97 and GluR1 leading to epitope unmasking of GluR1 on tissue sections. These findings indicate that abnormal expressions of the AMPA receptor and its interacting PSD molecule are associated with Alzheimer’s disease and implicated in pathophysiology of this disease.

Introduction

Alzheimer’s disease (AD) is a progressive dementing disorder characterized histologically by the presence of amyloid plaques and neurofibrillary tangles, accompanied by neuronal loss in certain neuronal populations of the brain. In particular, the entorhinal cortex, subiculum, and area CA1 of the hippocampus in AD are severely affected [13], and the neurofibrillary pathology correlates with atrophy of the hippocampal formation [4]. In contrast, the granule cells of the dentate gyrus are generally considered to be spared from the effects of the disease [15]. Whether neuronal loss, solely, accounts for the clinical manifestations of the disease remains a matter of controversy, however.

More than a dozen types of glutamate receptors have been identified by molecular cloning and have been classified into several subfamilies according to their structural homology and pharmacological profiles [14], [33], [44]. Among these, the N-methyl-d-aspartate (NMDA)R1 and GluR1/3 subunits are key subunits that form the physiological receptor complexes of the NMDA-type and α-amino-isoxazolepropionic acid (AMPA)-type channels, respectively [21], [28]. In the brain, amounts and properties of these glutamate receptors are known to be regulated in a plastic manner by various molecules as well as by neuronal activity [35], [36], [37]. Thus, abnormal functioning of these receptors is often implicated in many neurological and psychiatric diseases [2], [9], [23], [26], [27].

Previous autoradiographic studies have demonstrated that levels of AMPA and NMDA binding sites are reduced in the AD hippocampus [7], [19]. Recent immunocytochemical studies have shown that the numbers of GluR1- and GluR2/3-immunolabeled neurons decreased in the whole hippocampus and entorhinal cortex of patients with AD [3], [17]. More detailed immunocytochemistry, however, has revealed that GluR1-like immunoreactivity seems to be increased in the dentate gyrus of patients with AD [17]. Furthermore, such immunohistochemical studies on glutamate receptors had often been performed by using antibodies specific to their carboxyl-termini [31], [41], [42], even though it has been suggested that complete immunohistochemical detection of glutamate receptors seems to be difficult [12], [51] because epitopes on the receptors are often masked by many receptor-associated molecules such as the postsynaptic density proteins [6], [22], [38], [52].

These controversial findings and arguments raise a question of whether true levels of native AMPA receptors in the dentate gyrus increase or decrease with the progression of the disease.

In AD brain tissue the most severe neuronal degeneration was observed in the entorhinal cortex, whereas neuronal population is relatively preserved in the dentate gyrus and primary motor cortical area [13], [15]. Using Western blot analysis under a denatured condition, we therefore investigated the protein concentrations of GluR1 and NMDAR1 receptors in the cerebral neocortex (motor cortex), entorhinal cortex and dentate gyrus of postmortem tissues from patients with AD, and from normal subjects. To confirm the previous histological observations, we also immunostained tissue sections from the hippocampal-entorhinal formation with the same anti-GluR1 antibody. Furthermore, to evaluate contribution of receptor interacting molecules to the GluR1-like immunostaining, we determined the protein levels of a postsynaptic density protein named SAP97, which recognizes and potentially masks the epitope region of GluR1 [24]. Finally, we will discuss the reasons for the divergence of the biochemical and histological results.