Combined gene overexpression of neuropeptide Y and its receptor Y5 in the hippocampus suppresses seizures.

We recently demonstrated that recombinant adeno-associated viral vector-induced hippocampal overexpression of neuropeptide Y receptor, Y2, exerts a seizure-suppressant effect in kindling and kainate-induced models of epilepsy in rats. Interestingly, additional overexpression of neuropeptide Y in the hippocampus strengthened the seizure-suppressant effect of transgene Y2 receptors. Here we show for the first time that another neuropeptide Y receptor, Y5, can also be overexpressed in the hippocampus. However, unlike Y2 receptor overexpression, transgene Y5 receptors in the hippocampus had no effect on kainate-induced motor seizures in rats. However, combined overexpression of Y5 receptors and neuropeptide Y exerted prominent suppression of seizures. This seizure-suppressant effect of combination gene therapy with Y5 receptors and neuropeptide Y was significantly stronger as compared to neuropeptide Y overexpression alone. These results suggest that overexpression of Y5 receptors in combination with neuropeptide Y could be an alternative approach for more effective suppression of hippocampal seizures.

Adeno-associated viral vector-induced overexpression of neuropeptide Y Y2 receptors in the hippocampus suppresses seizures.

Gene therapy using recombinant adeno-associated viral vectors overexpressing neuropeptide Y in the hippocampus exerts seizure-suppressant effects in rodent epilepsy models and is currently considered for clinical application in patients with intractable mesial temporal lobe epilepsy. Seizure suppression by neuropeptide Y in the hippocampus is predominantly mediated by Y2 receptors, which, together with neuropeptide Y, are upregulated after seizures as a compensatory mechanism. To explore whether such upregulation could prevent seizures, we overexpressed Y2 receptors in the hippocampus using recombinant adeno-associated viral vectors. In two temporal lobe epilepsy models, electrical kindling and kainate-induced seizures, vector-based transduction of Y2 receptor complementary DNA in the hippocampus of adult rats exerted seizure-suppressant effects. Simultaneous overexpression of Y2 and neuropeptide Y had a more pronounced seizure-suppressant effect. These results demonstrate that overexpression of Y2 receptors (alone or in combination with neuropeptide Y) could be an alternative strategy for epilepsy treatment.

GDNF released from encapsulated cells suppresses seizure activity in the epileptic hippocampus.

To date, a variety of pharmacological treatments exists for patients suffering epilepsy, but systemically administered drugs offer only symptomatic relief and often cause unwanted side effects. Moreover, available drugs are not effective in one third of the patients. Thus, more local and more effective treatment strategies need to be developed. Gene therapy-based expression of endogenous anti-epileptic agents represents a novel approach that could interfere with the disease process and result in stable and long-term suppression of seizures in epilepsy patients. We have reported earlier that direct in vivo viral vector-mediated overexpression of the glial cell line-derived neurotrophic factor (GDNF) in the rat hippocampus suppressed seizures in different animal models of epilepsy. Here we explored whether transplantation of encapsulated cells that release GDNF in the hippocampus could also exert a seizure-suppressant effect. Such ex vivo gene therapy approach represents a novel, more clinically safe approach, since the treatment could be terminated by retrieving the transplants from the brain. We demonstrate here that encapsulated cells, which are genetically modified to produce and release GDNF, can suppress recurrent generalized seizures when implanted into the hippocampus of kindled rats.

Topiramate reduces AMPA-induced Ca(2+) transients and inhibits GluR1 subunit phosphorylation in astrocytes from primary cultures.

Topiramate (TPM) is a structurally novel broad spectrum anticonvulsant known to have a negative modulatory effect on the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate subtypes of glutamate receptors and some types of voltage-gated Na(+) and Ca(2+) channels, and a positive modulatory effect on some types of gamma-aminobutyric acid(A) (GABA(A)) receptors and at least one type of K(+) channels in neurons. In an earlier work, we showed that the negative modulatory effect of TPM (100 mum) on AMPA/kainate receptors in neurons is dependent on TPM modulation of the phosphorylation state of these receptors. In this work, we investigated the effect of TPM on AMPA-induced intracellular calcium ([Ca(2+)](i)) responses in cultured rat cortical astrocytes, with special interest in intracellular mechanisms. Here, we report that the ability of TPM (1-100 mum) to inhibit AMPA-induced accumulation of Ca(2+) in astrocytes is inversely related to the level of protein kinase A (PKA) -mediated phosphorylation of channels activated by AMPA. The level of receptor phosphorylation was further determined with western blot using phosphorylation specific antibodies that recognize the glutamate receptor 1 (GluR1) subunit phosphorylated on Ser845. These results demonstrated that, even in cultured cortical astrocytes, TPM significantly reduced the phophorylation level of GluR1 subunits. Furthermore, it was shown that TPM binds to AMPA receptors in the dephosphorylated state and thereby exerts an allosteric modulatory effect on the ion channel.

Topiramate modulation of kainate-induced calcium currents is inversely related to channel phosphorylation level.

Topiramate (TPM) is a structurally novel broad-spectrum anticonvulsant known to modulate the activity of several ligand- and voltage-gated ion channels in neurons. These include an inhibitory effect on the AMPA and kainate subtypes of glutamate receptors, mixed modulatory effects (usually positive) on some types of GABAA receptors, negative modulatory effects on some types of voltage-gated Na+ and Ca2+ channels, and a positive modulatory effect on at least one type of K+ channel. The nature of these effects at the molecular level has not been established, but two previous studies have implicated the phosphorylation state of these receptor/channel complexes as an influencing factor in the activity of TPM. Here, we report that the ability of TPM to inhibit a kainate-induced accumulation of free Ca2+ in cultured neurons from rat cerebral cortex is inversely related to the level of cAMP-dependent protein kinase (cAPK) mediated phosphorylation of kainate-activated receptors/channels. Specifically, when cell cultures were pre-treated with forskolin or dibutyryl cAMP, indirect activators of cAPK, the activity of TPM was abolished, whereas when the cells were pre-treated with H89, an inhibitor of cAPK, the relative activity of TPM was enhanced. The results of this study support the hypothesis that TPM binds to phosphorylation sites on AMPA and kainate receptors, but only in the dephosphorylated state and thereby exerts an allosteric modulatory effect on channel conductance.

Topiramate protects against glutamate- and kainate-induced neurotoxicity in primary neuronal-astroglial cultures.

Potential neuroprotective effects of the antiepileptic drug (AED) topiramate (TPM) were evaluated using primary neuronal-astroglial cultures or astroglial-enriched cultures from newborn rats exposed to excitotoxic concentrations of glutamate (Glu) or kainate. Neurons expressed functional Glu receptors of the NMDA and AMPA/kainate types as evaluated by immunocytochemistry and Ca(2+) imaging. When Glu (10 mM) was added to 9-10-day cultures incubated with the fluorescent dye calcein/AM for 5h, there was a marked cell loss in both culture types, but was more pronounced in the neuronal-astroglial cultures. When TPM (5-10 microM) was included in the medium together with Glu, the amount of surviving cells was significantly higher in the neuronal-astroglial cultures, but not in the astroglial-enriched cultures. Immuno-labeling of the cultures revealed an enhanced survival of MAP positive neuronal cells when TPM was included in the Glu containing medium. As TPM has a proven negative modulatory effect on kainate activated receptors, neuronal-astroglial cultures were further exposed to excitotoxic concentrations of kainate (100 microM) and analyzed immunohistochemically. Significantly more MAP positive neurons survived in the TPM containing medium and showed a morphology similar to untreated cells. Valproate and phenytoin were used as reference AEDs. In conclusion, our results demonstrate a protective effect of TPM upon neuronal cells in primary culture, exposed to excitotoxic levels of Glu or kainate.

Levetiracetam reduces caffeine-induced Ca2+ transients and epileptiform potentials in hippocampal neurons.

The effect of the novel antiepileptic drug levetiracetam on caffeine (10 mM)-induced intracellular calcium ([Ca2+]i) response was investigated in rat hippocampal neurons in culture, with the aim of exploring the cellular mechanisms of this new drug. Levetiracetam significantly reduced caffeine-induced [Ca2+]i) response, with maximum inhibition at 32 microM. The R-enantiomer of levetiracetam, ucb L060, which is devoid of anticonvulsant activity, at 32 microM had no effect on caffeine-induced [Ca2+]i) response. Caffeine 10 mM also induced epileptiform field potentials in rat hippocampal slices : single stimuli evoked repetitive population spikes and spontaneous field bursts regularly occurred. Levetiracetam (32 microM) significantly inhibited the amplitudes and the number of caffeine-induced repeated population spikes and delayed the appearance of spontaneous bursts, while ucb L060 (32 microM) completely lacked anti-caffeine activity. These results suggest that the inhibition of caffeine-induced Ca release from intra-neuronal stores might be an excitability-reducing effect of levetiracetam, contributing to its antiepileptic activity.

Novel mechanisms of action of three antiepileptic drugs, vigabatrin, tiagabine, and topiramate.

Epilepsy, a functional disturbance of the CNS and induced by abnormal electrical discharges, manifests by recurrent seizures. Although new antiepileptic drugs have been developed during recent years, still more than one third of patients with epilepsy are refractory to treatment. Therefore, the search for new mechanisms that can regulate cellular excitability are of utmost importance. Three currently available drugs are of special interest because they have novel mechanisms of action and are especially effective for partial onset seizures. Vigabatrin is a selective and irreversible GABA-transaminase inhibitor that greatly increases whole-brain levels of GABA. Tiagabine is a potent inhibitor of GABA uptake into neurons and glial cells. Topiramate is considered to produce its antiepileptic effect through several mechanisms, including modification of Na(+)-and/or Ca(2+)-dependent action potentials, enhancement of GABA-mediated Cl- fluxes into neurons, and inhibition of kainate-mediated conductance at glutamate receptors of the AMPA/kainate type. This review will discuss these mechanisms of action at the cellular and molecular levels.