Abstracts

Diacylglycerol kinase epsilon (DGK[epsilon]), a member of the DGK gene family that catalyzes phosphorylation of the second messenger diacylglycerol to phosphatidic acid, is known to contribute to the modulation of synaptic activity, neuronal plasticity, and epileptogenesis (PNAS 98:4740-4745, 2001). In these experiments we have analyzed changes in gene transcription patterns in a mouse kindling model of epileptogenesis using control and DGK[epsilon] knockout mice. Two month old control DGK[epsilon] (+/+) or DGK[epsilon] knockout (-/-) mice were implanted with electrode units (Tripolar, Plastic One Inc., Roanoke, VA) in the right dorsal hippocampus. At 10 days post surgery, kindling was accomplished after sub-convulsive electrical stimulation (6 times daily for 4 days, using a 10 second train of 50-Hz biphasic pulses of 75-100-[mu]A amplitude). Seizures were graded according to the Racine Scale. Total brain RNA was isolated using Trizol reagent (Invitrogen Life Sciences, Carlsbad, CA), screened using RNA LabChips (Caliper Technologies, Mountain View, CA), and labeled using the Enzo Bioarray High Yield RNA Transcript Labeling system (Affymetrix, Santa Clara, CA). DNA-array data analysis and feature extraction were performed using Murine Genome MG430 2.0 GeneChip DNA probe arrays (Affymetrix) and GeneSpring 7.2 (Silicon Genetics, Redwood City, CA) analytical algorithms. In agreement with our previous studies using the Murine Genome U74Av2 GeneChip array (12,000 gene targets), a surprisingly small number of genes reached a significance of 2-fold or greater (either up- or down-regulated) with an ANOVA ([italic]p) [/italic][lt]0.05 under the experimental conditions described. The most significantly up-regulated genes included those encoding transthyretin (TTR), a 55-kDa homotetrameric thyroxine (T4) transport protein, suggesting the involvement of retinol (vitamin A) and/or thyroxine (T4) signaling in kindling epilepsy in both control and DGK[epsilon] knockout mice. DNA arrays containing entire genomes and genomic activity profiling provide new insights into the genetics of complex neurological disorders. Previous studies have shown that DGK[epsilon] is abundantly expressed in neurons, and that DGK[epsilon] knockout mice exhibit an elevated resistance to electroconvulsive shock when compared to control DGK[epsilon] mice. Significant elevations in TTR suggest the mobilization of retinol (vitamin A) and/or thyroxine (T4) within the phosphatidylinositol 4,5-bisphosphate-signaling pathway in kindling epileptogenesis. Interestingly, elevations in TTR have been observed clinically in the choroid plexus and cerebrospinal fluid of patients with intractable epilepsy. These data provide new insights into the basic mechanisms of epileptogenesis and may provide novel pathways for anti-epileptogenic drug development. (Supported by NIH NS22002, NIH AG18031 and COBRE NIH P29RR16816-02.)