Investigations on Y-receptor function in Epilepsy

With a prevalence of 1-2 % epilepsy is one of the most frequent neurological disorders. About 70 % of all epilepsy patients suffer from focal seizures. The mesial temporal lobe epilepsy is probably the most frequent type of focal epilepsy at all. Although patients can be pharmaceutically treated well in the beginning, about 30 - 50 % of patients turn refractory to medical treatment after some years. To date, surgical resection of the epileptic focus remains as a final treatment for these patients. The presurgical investigations as well as the surgery itself require enormous effort, and can not guarantee success. Thus, the introduction of new pharmaceutical approaches to treat epilepsy would be of high value. In recent years, neuropeptide Y (NPY) has become the focus of much attention for its possible protective role in epileptogenesis and epilepsy. NPY is one of the most abundant neuropeptides in the mammalian brain, where it is involved in numerous functions like feeding behavior, anxiety, memory processing and cognition, fertility, circadian rythms and blood pressure regulation. Although a lot of work has been done, the function of NPY on its complex receptor family has not been entirely understood. One major reason for this is the limited availability of selective agonists and antagonists for the pharmacologically very similar Y-receptors. Which is further complicated by the overlapping expression patterns of Y-receptors especially in brain areas playing crucial roles in epilepsy. In order to enlight the rational basis for a pharmacological approach to target the NPY system for antiepileptic treatment, we intend to investigate the involvement of NPY in epilepsy in various inducible Y-receptor knockout animal models. Our strategy will focus on the use of conditional Y-receptor knockouts crossed with transgenic mice, expressing Cre-recombinase under the control of the prodynorphin promoter. Such mice allow for deletion of distinct Y-receptors in a defined subset of neurons within key structures of the brain important in epilepsy. This strategy circumvents the drawbacks of pharmacological as well as classical gentechnological techniques, which effect all cells of the animal already during developement. Applying this system, we will be able to identify the function of distinct Y-receptors in various stages of epilepsy.

Several functions have been described for neuropeptides since their discovery over 25 years ago. Thus, neuropetide Y is involved in physiological and pathophysiological functions like epilepsy, feeding behaviour, memory processing and cognition, anxiety, fertility, alcohol consumption, circadian rhythm and blood pressure regulation. Similarly complex are the functions of the opioide system. The diverse activities of NPY are mediated by at least five G-protein coupled receptors (Y1, Y2, Y4, Y5 and y6), while opioide peptides act mainly through delta, kappa and mu receptors. Due to the complexity of the systems and restricted pharmacological tools, knockout mice have gained significant importance. The main objective of this project was the characterisation of conditional Y receptor knockout mice carrying an inducible system, designed to allow spatial and temporal restricted Y receptor knockouts. The inducible Y receptor knockout system consists of tet-on regulated expression of Cre-recombinase under control of the prodynorpin gene promoter. Due to the chosen strategy, homozygous Cre-mice are prodynorphin deficient. Using PCR we were able to show correct recombination of the Y2 receptor gene in hippocampal granule cells of mice carrying homozygous loxP site flanked Y2 genes and heterozygous the Cre construct when induced by doxycyclin added to drinking water. However, we were not able to reach significant reduction of Y2 receptor binding in these mice, although we tried several application protocols for doxycyclin, including local injection into the brain. Although negative, our results are in line with recent experience reported from other labs, showing that the classical tet-on system is not suitable for use in the central nerve system. On the other hand, mice carrying the Cre construct were very interesting as they are prodynorphin deficient. Testing of homozygous Cre mice in various models of epilepsy allowed us to access the importance of endogenous prodynorphin derived peptides in seizure control. Prodynophin deficient mice showed a significantly reduced seizure threshold and faster onset. Together with this, mice displayed seizures over a much longer period once initiated. Applying receptor specific agonists and antagonists, we were able to show that dynorphin almost exclusively acts via kappa receptors in these functions. Dynorphin deficient mice also showed significantly increased neurodegeneration in a model of kainic acid induced epilepsy. Noteworthy, in a model of epileptogenesis (classical kindling), dynorphin deficient mice developed generalized seizures much faster than their wild-type littermates. This fits well to the observation of increased seizure susceptibility in human beings expressing lower levels of dynorphin mRNA. Thus our findings potentially offer diagnostic as well as therapeutic strategies. However, detailed analysis of all potentially involved mechanisms will help to provide an even better understanding and more precise approach to temporal lobe epilepsy.