Berkley Lynch

The antiepileptic drug levetiracetam selectively modifies kindling‐induced alterations in gene expression in the temporal lobe of rats

Gene expression profiling by microarrays is a powerful tool for identification of genes that may encode key proteins involved in molecular mechanisms underlying epileptogenesis. Using the Affymetrix oligonucleotide microarray, we have surveyed the expression levels of more than 26,000 genes and expressed sequence tags (ESTs) in the amygdala‐kindling model of temporal lobe epilepsy. Furthermore, the effect of the antiepileptic drug levetiracetam (LEV) on kindling‐induced alterations of gene expression was studied. Treatment of rats with LEV during kindling acquisition significantly suppressed kindling development. For gene expression profiling, six groups of rats were included in the present study: (i) and (ii) sham‐operated rats treated with saline or LEV; (iii) and (iv) electrode‐implanted but non‐kindled rats treated with saline or LEV; (v) and (vi) kindled rats treated with saline or LEV. Treatment was terminated after 11 or 12 daily amygdala stimulations, when all vehicle‐treated rats had reached kindling criterion, i.e. a stage 5 seizure. Twenty‐four hours later, the ipsilateral temporal lobe was dissected for mRNA preparation. Six temporal lobe preparations from each group were analysed for differential gene expression. In control (non‐kindled) rats, LEV treatment was devoid of any significant effect on gene expression. In saline‐treated kindled rats, a large number of genes were observed to display mRNA expression alterations compared with non‐kindled rats. LEV treatment induced marked effects on gene expression from kindled rats. Previously described epilepsy‐related genes, such as neuropeptide Y (NPY), thyrotropin‐releasing hormone (TRH) and glial fibrillary acidic protein (GFAP) were confirmed to be up‐regulated by kindling and partially normalized by LEV treatment. Real‐time quantitative polymerase chain reaction confirmed NPY, TRH and GFAP expression data from chip experiments. Furthermore, a number of novel genes were identified from the gene chip experiments. A subgroup of these genes demonstrated correlation between expression changes and kindled phenotype measurements. In summary, this study identified many genes with potentially important roles in epileptogenesis and highlighted several important issues in using the gene chip technology for the study of animal models of CNS disorders.

Visualization of SV2A conformations in situ by the use of Protein Tomography.

The synaptic vesicle protein 2A (SV2A), the brain-binding site of the anti-epileptic drug levetiracetam (LEV), has been characterized by Protein Tomography. We identified two major conformations of SV2A in mouse brain tissue: first, a compact, funnel-structure with a pore-like opening towards the cytoplasm; second, a more open, V-shaped structure with a cleft-like opening towards the intravesicular space. The large differences between these conformations suggest a high degree of flexibility and support a valve-like mechanism consistent with the postulated transporter role of SV2A. These two conformations are represented both in samples treated with LEV, and in saline-treated samples, which indicates that LEV binding does not cause a large-scale conformational change of SV2A, or lock a specific conformational state of the protein. This study provides the first direct structural data on SV2A, and supports a transporter function suggested by sequence homology to MFS class of transporter proteins.

Combining modelling and mutagenesis studies of synaptic vesicle protein 2A to identify a series of residues involved in racetam binding

LEV (levetiracetam), an antiepileptic drug which possesses a unique profile in animal models of seizure and epilepsy, has as its unique binding site in brain, SV2A (synaptic vesicle protein 2A). Previous studies have used a chimaeric and site-specific mutagenesis approach to identify three residues in the putative tenth transmembrane helix of SV2A that, when mutated, alter binding of LEV and related racetam derivatives to SV2A. In the present paper, we report a combined modelling and mutagenesis study that successfully identifies another 11 residues in SV2A that appear to be involved in ligand binding. Sequence analysis and modelling of SV2A suggested residues equivalent to critical functional residues of other MFS (major facilitator superfamily) transporters. Alanine scanning of these and other SV2A residues resulted in the identification of residues affecting racetam binding, including Ile273 which differentiated between racetam analogues, when mutated to alanine. Integrating mutagenesis results with docking analysis led to the construction of a mutant in which six SV2A residues were replaced with corresponding SV2B residues. This mutant showed racetam ligand-binding affinity intermediate to the affinities observed for SV2A and SV2B.