New targets for anti-epileptic drugs

Epilepsy affects about 1-2 % of the population, accordingly 50 to 100 million people worldwide may suffer from epileptic disorders. The term epilepsy encompasses various syndromes whose cardinal feature is a predisposition to recurrent unprovoked seizures. Current anti-epileptic therapy is purely symptomatic, because the (largely unknown) pathomechanisms leading to epileptic seizures remain unaffected. If medication is discontinued, epileptic seizures frequently recur. Moreover, while suppression of seizures can be achieved in the majority of cases, seizures can not be controlled in about 30% of epilepsy patients. Thus, improved anti-epileptic drugs are needed. The neurological correlate of an epileptic seizure is an outburst of abnormal (ictal) discharge activity. Short and clinically silent epileptiform events also occur between seizures and appear as spikes in the EEG of epilepsy patients. These interictal spikes (IS) have become an important and reliable diagnostic indicator of the epileptic brain. Moreover, there is evidence that ISs are not merely a by-product of hyperexcitable, seizure prone neuronal networks, but contribute actively to generating and maintaining the epileptic condition. Thus, ISs appear to provide a promising target to counteract epileptogenesis and epileptic seizures. Besides IS, the transition of interictal to ictal discharge activity has also been considered recently as another novel strategic target to counteract epileptic seizures. Evidence suggests that transition can be induced by neurotransmitter systems, and cholinergic neurotransmission, for example, has frequently been reported to contribute to the physiopathogenesis of epileptic discharges. LTCCs contribute to IS and its epileptogenic consequences and are also involved in ictal discharge activity. Nicotinic acetylcholine receptors (nAChRs) on the other hand are known to cause presynaptic facilitation and, if functionally up-regulated, to cause epileptiform activity. Hence LTCCs and nAChRs are two potentials targets for novel AEDs. However, neuronal LTCCs come in two different subtypes, which differ considerably with respect to subcellular distribution, function and coupling to Ca2+ dependent signaling pathways. Which subtypes of LTCCs and in which role these LTCCs may contribute to interictal and ictal discharge activity, remained unknown. Moreover, the anti-epileptic potential of nAChR-antagonism has not been evaluated. In the proposed project I will investigate the role of LTCCs and nAChRs in epileptiform activity evoked in primary cultures of central neurons using LTCC-knock out mice on the one hand, and on the other hand agonists and antagonists of nAChRs. In particular, the following questions will be addressed: (i) How do neuronal LTCCs contribute to interictal and ictal discharge activity? (ii) Is there a pro- and an anti-epileptic role of the two neuronal LTCC subtypes? (iii) Do nAChRs provide suitable targets to counteract epileptiform activity? Experiments addressing these questions will be complemented by investigation of a novel LTCC blocker that I identified in the venom of the hunting spider Cupiennius salei. Present data suggest that this toxin, CSTX-1, discriminates between different LTCC subtypes. Therefore, I will evaluate whether CSTX-1 can be used as a lead for the development of therapeutical subtype- selective LTCC antagonists. Moreover, I obtained evidence that retigabine, a novel anti-epileptic drug currently being tested in clinical trials, inhibits central nAChRs at therapeutic concentrations. So far, retigabine was thought to act predominately by opening M-type potassium channels. Hence, retigabine will be used to further evaluate the anti-epileptic potential of nAChR-antagonism. The ultimate goal in epileptic therapy is to cure the disease, rather than solely suppressing seizures. Realization of the proposed project will provide essential knowledge that will aid in the development of novel, improved AEDs.

The aim of this study was to gather - via a basic-science approach and on an in vitro system - information on the targetability of ion channels that may kindle interest and serve in the development of a newer generation of anti- epileptic drugs. From initial explorative investigations on various pathways, L-type voltage gated calcium channels (LTCCs) emerged as particularly promising candidates. Hence our focus was directed to this group of channel proteins. As outlined in detail below, we acquired several lines of evidence in this project that targeting neuronal LTCCs could be exploited to suppress putatively epileptogenic abnormal electrical signals. In primary cultures of hippocampal neurons, pharmacological potentiation of LTCCs was shown to evoke electrical signals, that do not normally occur under physiological conditions. In the literature, these events are known as paroxysmal depolarization shifts (PDS). An exception where similar signals occur is the neuronal development in the first weeks after birth, which involves neuronal growth, arborization and circuitry formation. On the other hand, in the mature brain, PDS are considered neuropathogenic, at least according to one school of thought. However, other researchers suggested that PDS represent an attempt of neuronal circuits to suppress seizures. We showed in the present project that not only does potentiation of LTCCs by means of pharmacological up-regulation lead to PDS, but that PDS can also be evoked by oxidative stress, and that this process depends crucially on the availability of LTCCs. Oxidative stress can occur as a result of insufficient blood supply, for example in the course of brain damage. Because brain insult, particularly when associated with ischemia, is a known cause of acquired forms of epilepsies, the relationship between oxidative stress, LTCC potentiation and PDS formation is of particular interest for the development of future prophylactic approaches. LTCCs have been recognized as essential elements in mechanisms of structural and functional neuronal plasticity. Hence, the role of LTCCs in triggering PDS appears to indicate that the later do indeed act in a pro-epileptic manner. This does not rule out the possibility that PDS-like events form in the course of or after seizures. However, data obtained in this project demonstrate that LTCCs have little involvement in such "post-ictal" events, although these later events may indeed have anti-epileptic potential. Within recent years evidence has accumulated that PDS, besides in epileptogenesis, may play a role in other neurological diseases, such as age-related memory deficits and Morbus Alzheimer. Therefore, besides epileptic therapy, the implication of LTCCs in PDS may be of considerable interest also for other neurological applications.