Calcium Signalling and Endogenous Opioids in Hippocampal Neurons of Patients withTemporal Lobe Epilepsy

Many patients suffering from temporal lobe epilepsy (TLE) do not respond satisfactorily to drug treatment. Some of these patients may benefit from a surgical removal of the hippocampus as this structure often contains the epileptogenic focus and is the main site of pathological changes. The aim of this project is to study the molecular basis of TLE using hippocampal tissue resected from patients with TLE for therapeutic reasons. We will investigate a number of issues that have arisen from cell culture or animal studies but so far have not been tested in human tissue. We will concentrate on two closely linked signalling systems that appear to play a prominent role in the pathogenesis of TLE, that is (i) the calcium signalling pathway and (ii) the endogenous opioid transmitter system. We intend to use immunohistochemical techniques (combined with confocal laser scanning microscopy for higher resolution) to study the tissue localisation, cellular and subcellular distribution of a range of molecules participating in these signalling systems. In particular, we want to investigate the distribution of voltage gated calcium channel subunits (a l-A, a l-B, a l-C, a l-D, b 1, a 2), of calcium-dependent enzymes (calcium/calmodulin-dependent protein kinase II, protein kinase C and calcineurin), of the endogenous opioids dynorphin and enkephalin and finally of opioid receptors (d -, k - and m -subforms). In addition to comparing staining results to post-mortem non- epileptic tissue, we will place special emphasis on correlating staining patterns with a number of parameters of the clinical course of the disease and also with results from genetic studies investigating the polymorphism of the prodynorphin gene in our patients.

The aim of this FWF funded project was to study genetic and molecular mechanisms underlying temporal lobe epilepsy, the most common epilepsy syndrome. We obtained two major results. Altered Expression of calcium channel alpha1 subunits in temporal lobe epilepsy (Neuroscience, 2002, 111:57-69) Summary: Voltage dependent calcium channels are calcium ion selective pores in the cell membrane, composed of several subunits, with regulatory functions on a wide range of cellular processes. We found an altered expression of calcium channel alpha1 subunits in patients with temporal lobe epilepsy. These results suggest a functional role for calcium channels in the disease process of temporal lobe epilepsy. Methods: In medically refractory forms of temporal lobe epilepsy surgically removing the area of the seizure origin, the hippocampus, is often the best therapeutic option. Using the technique of immunohistochemistry we selectively labelled calcium channel alpha1 subunits (A to E) in these surgical specimens and compared the pattern with normal tissue obtained from autopsies. Findings: We observed an up-regulation of most calcium channel subunits (A, B, D, E) in the dentate gyrus, an area with a central role in generating the increased excitability. In dying pyramidal cells in the hippocampus there was an increased labelling for the A but a decreased labelling for the C subunit. We could further demonstrate, for the first time in humans, that astrocytes, non-neuronal support cells, express calcium channels (C subunit specific) under the stressful conditions of epilepsy. These results will help clarifying the molecular pathways leading to temporal lobe epilepsy. A functional polymorphism in the prodynorphin gene is associated with temporal lobe epilepsy (Annals of Neurology, 2002, 51:260-263) Summary: The prodynorphin gene encodes the endogenous morphine like molecule dynorphin, an important regulatory transmitter substance in the brain, with a probable anti-epileptic function. 10% of the population display low activity levels in both inherited copies of this gene. Given a positive family history for seizures such individuals are more likely to develop temporal lobe epilepsy. Irrespective of the family background these patients suffer from a more severe form of epilepsy. Methods: There are two variations (alleles) of the epilepsy candidate gene prodynorphin, that either show high or low levels of dynorphin expression (H or L alleles). To reveal a possible influence of this genetic polymorphism on temporal lobe epilepsy we studied the allele frequencies in patients with temporal lobe epilepsy and in healthy volunteers. We further assessed the severity of the disease in L homozygous patients (two L alleles) versus patients with at least one H allele. Findings: Patients with a familial background for epilepsy are more as twice as likely to be L homozygous (two L alleles) than healthy volunteers (23% vs. 9%). Irrespective of the familial risk L homozygotes are two to three times as likely to develop a status epilepticus (a life threatening prolonged seizure), frequent generalised seizures and childhood febrile convulsions.