Metabolic gene expression changes in the hippocampus of obese epileptic male rats in the pilocarpine model of temporal lobe epilepsy.

Chronically epileptic male adult rats in the pilocarpine model of temporal lobe epilepsy (TLE), exhibited gross expansion of abdominal fat mass and significant weight gain several months after induction of status epilepticus (SE) when compared to control rats. We hypothesized that epileptogenesis can induce molecular changes in the hippocampus that may be associated with metabolism. We determined the expression levels of genes Hsd11b1, Nr3c1, Abcc8, Kcnj11, Mc4r, Npy, Lepr, Bdnf, and Drd2 that are involved in regulation of energy metabolism, in the hippocampus of age-matched control and chronic epileptic animals. Taqman-based quantitative real time polymerase chain reaction (qPCR) and the delta-delta cycle threshold (CT) methods were used for the gene expression assays. Gene expression of Hsd11b1 (cortisol generating enzyme) was significantly higher in epileptic versus control rats at 24h and 2 months, after induction of SE. Nr3c1 (glucocorticoid receptor) mRNA levels on the other hand were down-regulated at 24h, 10 days and 2 months, post SE. Abcc8 (Sur1; subunit of ATP-sensitive potassium (K(ATP)) channel) was significantly down-regulated at 10 days post SE. Kcnj11 (Kir6.2; subunit of ATP-sensitive potassium (K(ATP)) channel) was significantly up-regulated at 24h, 1 month and 2 months post SE. Thus, we demonstrated development of obesity and changes in the expression of metabolic genes in the hippocampus during epileptogenesis in male rats in the pilocarpine model of TLE.

Upregulation of STREX splice variant of the large conductance Ca2+-activated potassium (BK) channel in a rat model of mesial temporal lobe epilepsy.

Functional properties of large conductance Ca(2+) activated potassium (BK) channels are determined by complex alternative splicing of the Kcnma1 gene encoding the alpha pore-forming subunit. Inclusion of the STREX exon in a C-terminal splice site is dynamically regulated and confers enhanced Ca(2+) sensitivity and channel inhibition via cAMP-dependent phosphorylation. Here, we describe a real time quantitative PCR (qPCR) approach to investigate relative changes in the expression of STREX and ZERO splice variants using a newly designed set of probes and primers for TaqMan-based qPCR analysis of cDNA from the rat dentate gyrus at different time points following pilocarpine-induced status epilepticus. Reduction in Kcnma1 gene expression is associated with a relative increase of STREX splice variant. Relative expression of STREX variant mRNA was increased at 10 days and at more than 1 month following status epilepticus. The biological consequences of seizure-related changes in alternative splicing of Kcnma1 deserve additional investigation.

Chronic deficit in the expression of voltage-gated potassium channel Kv3.4 subunit in the hippocampus of pilocarpine-treated epileptic rats.

Voltage gated K(+) channels (Kv) are a highly diverse group of channels critical in determining neuronal excitability. Deficits of Kv channel subunit expression and function have been implicated in the pathogenesis of epilepsy. In this study, we investigate whether the expression of the specific subunit Kv3.4 is affected during epileptogenesis following pilocarpine-induced status epilepticus. For this purpose, we used immunohistochemistry, Western blotting assays and comparative analysis of gene expression using TaqMan-based probes and delta-delta cycle threshold (ΔΔCT) method of quantitative real-time polymerase chain reaction (qPCR) technique in samples obtained from age-matched control and epileptic rats. A marked down-regulation of Kv3.4 immunoreactivity was detected in the stratum lucidum and hilus of dentate gyrus in areas corresponding to the mossy fiber system of chronically epileptic rats. Correspondingly, a 20% reduction of Kv3.4 protein levels was detected in the hippocampus of chronic epileptic rats. Real-time quantitative PCR analysis of gene expression revealed that a significant 33% reduction of transcripts for Kv3.4 (gene Kcnc4) occurred after 1 month of pilocarpine-induced status epilepticus and persisted during the chronic phase of the model. These data indicate a reduced expression of Kv3.4 channels at protein and transcript levels in the epileptic hippocampus. Down-regulation of Kv3.4 in mossy fibers may contribute to enhanced presynaptic excitability leading to recurrent seizures in the pilocarpine model of temporal lobe epilepsy.

Altered expression and function of small-conductance (SK) Ca(2+)-activated K+ channels in pilocarpine-treated epileptic rats.

Small conductance calcium (Ca(2+)) activated SK channels are critical regulators of neuronal excitability in hippocampus. Accordingly, these channels are thought to play a key role in controlling neuronal activity in acute models of epilepsy. In this study, we investigate the expression and function of SK channels in the pilocarpine model of mesial temporal lobe epilepsy. For this purpose, protein expression was assessed using western blotting assays and gene expression was analyzed using TaqMan-based probes and the quantitative real-time polymerase chain reaction (qPCR) comparative method delta-delta cycle threshold ( big up tri, open big up tri, openCT) in samples extracted from control and epileptic rats. In addition, the effect of SK channel antagonist UCL1684 and agonist NS309 on CA1 evoked population spikes was studied in hippocampal slices. Western blotting analysis showed a significant reduction in the expression of SK1 and SK2 channels at 10days following status epilepticus (SE), but levels recovered at 1month and at more than 2months after SE. In contrast, a significant down-regulation of SK3 channels was detected after 10days of SE. Analysis of gene expression by qPCR revealed a significant reduction of transcripts for SK2 (Kcnn1) and SK3 (Kcnn3) channels as early as 10days following pilocarpine-induced SE and during the chronic phase of the pilocarpine model. Moreover, bath application of UCL1684 (100nM for 15min) induced a significant increase of the population spike amplitude and number of spikes in the hippocampal CA1 area of slices obtained control and chronic epileptic rats. This effect was obliterated by co-administration of UCL1684 with SK channel agonist NS309 (1microM). Application of NS309 failed to modify population spikes in the CA1 area of slices taken from control and epileptic rats. These data indicate an abnormal expression of SK channels and a possible dysfunction of these channels in experimental MTLE.

Impaired expression and function of group II metabotropic glutamate receptors in pilocarpine-treated chronically epileptic rats.

Group II metabotropic (mGlu II) receptor subtypes mGlu2 and mGlu3 are important modulators of synaptic plasticity and glutamate release in the brain. Accordingly, several pharmacological ligands have been designed to target these receptors for the treatment of neurological disorders characterized by anomalous glutamate regulation including epilepsy. In this study, we examine whether the expression level and function of mGlu2 and mGlu3 are altered in experimental epilepsy by using immunohistochemistry, Western blot analysis, RT-PCR and extracellular recordings. A down-regulation of mGlu2/3 protein expression at the mossy fiber pathway was associated with a significant reduction in mGlu2/3 protein expression in the hippocampus and cortex of chronically epileptic rats. Moreover, a reduction in mGlu2 and mGlu3 transcripts levels was noticed as early as 24 h after pilocarpine-induced status epilepticus (SE) and persisted during subsequent "latent" and chronic periods. In addition, a significant impairment of mGlu II-mediated depression of field excitatory postsynaptic potentials at mossy fiber-CA3 synapses was detected in chronically epileptic rats. Application of mGlu II agonists (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) induced a significant reduction of the fEPSP amplitude in control rats, but not in chronic epileptic rats. These data indicate a long-lasting impairment of mGlu2/3 expression that may contribute to abnormal presynaptic plasticity, exaggerate glutamate release and hyperexcitability in temporal lobe epilepsy.

Dinucleoside monophosphates containing AZT and 1-methyladenosine or 7-methylguanosine.

Dinucleoside monophosphates containing AZT and 1-methyladenosine or 7-methylguanosine were synthesized and their in vitro anti-HIV activity was determined.

Affinity modification of EcoRII DNA methyltransferase by the dialdehyde-substituted DNA duplexes: mapping the enzyme region that interacts with DNA.

Affinity modification of EcoRII DNA methyltransferase (M x EcoRII) by DNA duplexes containing oxidized 2'-O-beta-D-ribofuranosylcytidine (Crib*) or 1-(beta-D-galactopyranosyl)thymine (Tgal*) residues was performed. Cross-linking yields do not change irrespective of whether active Crib* replaces an outer or an inner (target) deoxycytidine within the EcoRII recognition site. Chemical hydrolysis of M x EcoRII in the covalent cross-linked complex with the Tgal*-substituted DNA indicates the region Gly268-Met391 of the methylase that is likely to interact with the DNA sugar-phosphate backbone. Both specific and non-specific DNA interact with the same M x EcoRII region. Our results support the theoretically predicted DNA binding region of M x EcoRII.