Changes in glutamate receptor subunit composition in hippocampus and cortex in patients with refractory epilepsy

Abstract

An assessment of glutamate receptor subunit profiles was made in hippocampus and temporal lobe cortex of patients with refractory epilepsy. Molecular biological analyses using reverse transcription reaction (RT) followed by polymerase chain reaction (PCR) revealed changes in the distribution profile of the transcripts of AMPA/KA glutamate receptor subunits in hippocampal and cortical tissue from patients with refractory epilepsy when compared to similar tissue from six human and four non-human primate samples with no history of seizures or seizure medication. A severe mean decrease (38% of control) in mRNA for the GluR1 subunit was found in 400 mm cross-sections of hippocampus from patients with epilepsy. Less severe but significant reductions in that GluR1 subunit expression (54% of control) were exhibited in samples of excised temporal pole cortex from the same subjects. Message for the GluR4 subunit was also significantly decreased in hippocampus (68% of control), but in contrast to GluR1, GluR4 mRNA level was not decreased in temporal cortex. Levels of GluR2 mRNA were not significantly changed in epileptic hippocampal and cortical tissue relative to control samples. Protein levels of the GluR1 and GluR4 subunits quantified by Western blot analysis were also reduced in hippocampal and cortical tissue from epilepsy patients. Two other kainate subunit transcripts, GluR6 and KA1 also showed significant changes compared to non-epileptic tissue (136% and 71% of control, respectively). Results are discussed in terms of possible mechanisms by which protracted seizures could produce selective loss of certain AMPA/KA subunits.

Introduction

The neural processes responsible for human epilepsy have been investigated experimentally at various levels in rodent, monkey and man (McNamara, 1994, Schwartzkroin, 1994, Spencer and Spencer, 1994). Although seizure-induced changes expressed as specific hippocampal abnormalities have been described in experimental models (Friedman et al., 1994, Kamphius et al., 1994, Kamphius et al., 1995, Krestchatisky et al., 1995), associated alterations in excitatory synaptic receptor complexes in the hippocampus of patients with a long history of refractory epilepsy are still not understood. In patients with complex partial seizures, unilateral removal of the hippocampus and surrounding tissue is often an effective treatment for alleviation or reduction of refractory seizures. Autoradiographic studies of tissue from epilepsy patients following anterior temporal lobectomy show changes in the AMPA/KA receptor subunit (GluR1 and GluR2/3) protein expression associated with altered distribution of AMPA/KA receptor subtypes within hippocampus (Geddes et al., 1990, Hosford et al., 1991, Lynd-Balta et al., 1996). However, a detailed analysis of changes in AMPA/KA receptor composition with respect to alteration in the complement of subunit expression profiles has not been described in hippocampus or cortex removed from patients with long histories of refractory epilepsy.

The availability of molecular biological techniques for characterization of differences in neural tissue, makes it possible to examine brain tissue from epilepsy patients for changes similar to those reported in animal models of the disease (Vernet et al., 1991, Tishler et al., 1995; for rev. Glass and Dragunow, 1995). Application of these techniques has extended our understanding of epilepsy with regard to changes in the organization and distribution of subtypes of synaptic receptors within structures whose surgical removal leads to a decrease in mesial temporal lobe seizures. Molecular cloning has revealed a large degree of heterogeneity in AMPA/KA glutamate receptor subunit composition (Hollmann and Heinemann, 1994). Currently at least four α-amino-3-hydroxy-5-methylxazole-4-propionic acid (AMPA) receptor subunits have been cloned, designated: GluR1–GluR4 respectively (Hollmann et al., 1989, Boulter et al., 1990, Keinanen et al., 1990, Sommer et al., 1990). Five kainate (KA) receptor subunits designated GluR5–7 and KA1-2 have also been identified (Bettler et al., 1990, Egebjerg et al., 1991, Werner et al., 1991, Herb et al., 1992, Kamboj et al., 1992). Immunoprecipitation and mRNA expression studies provide evidence that AMPA/KA receptors likely form pentameric ligand-gated channels consisting of different subunit types, each with its own functional and pharmacological profile (Boulter et al., 1990, Keinanen et al., 1990, Nakanishi et al., 1990). Each AMPA/KA subunit combination appears with different densities in temporal lobe structures (cortex and hippocampus) in rat, non-human primate and human brain (Wisden and Seeburg, 1993, Bahn et al., 1994). Although exposure to exogenous kainate has frequently been used as a rodent model of epilepsy (Coyle, 1983, Westbrook and Lothman, 1983Ben-Ari, Y., 1985Friedman et al., 1994), comparison of the full complement of expression profile of AMPA/KA receptors from patients with epilepsy has not been published.

A recent report of changes in the distribution of GluR1 and GluR2/3 subunits in hippocampal tissue taken from epilepsy patients detected via immunohistochemistry (Lynd-Balta et al., 1996) suggests marked reorganization of glutamatergic pathways in human epileptic tissue, providing a basis for altered excitability and seizure susceptibility. In this study we examine gene and protein expression profiles of AMPA/KA receptor subunits in hippocampus and temporal pole cortex excised from patients with intractable seizures and compared them to similar samples from (1) non-epileptic human control tissue and (2) tissue from non-human primates, with no history of seizures. Using RT–PCR, we analyzed and characterized changes in AMPA/KA subunit distribution in tissue from epileptic patients vs non-epileptic human control samples and then verified those changes using commercially available antibodies directed toward specific subunit proteins. The results indicate that tissue from patients with a long history of epilepsy is associated with significant alteration in expression of a subset of AMPA/KA receptor subunits in hippocampus and adjacent temporal pole cortex.