Despite the emergence of new drugs and surgical methods, the control of seizures remains inadequate for many patients with epilepsy. Pharmacological treatment is often unsatisfactory due to side effects of anti-convulsant drugs. Surgical treatment can sometimes remove a focal area of epileptogenic tissue, but in the process, the normal functions of that portion of the brain are lost. We propose a genetic engineering technique that could be used to modify the ionic channels in localized areas of the brain. This novel approach could circumvent the drawbacks inherent in current therapeutic modalities. The pharmacological modification of voltage-gated ionic channels is a cornerstone of epilepsy treatment. The major anti-convulsant drugs phenytoin and carbamazepine act by promoting sodium channel inactivation. Potassium channels act in opposition to sodium channels and are responsible for the repolarization phase of the action potential. We hypothesize that enhancement of potassium channel function by blockade of inactivation should provide an anti-epileptic effect. We propose to test this hypothesis by reducing the inactivation of potassium channels in cultured rat hippocampal neurons by antisense knockdown of the beta1 subunit which is necessary for channel inactivation. The antisense knockdown will be carried by direct exposure to oligonucleotides and by transfection with the herpes simplex viral vector system. The viral vector will be directly applicable to future in vivo studies of efficiency in experimental models of epilepsy. The potassium channels produced by neurons unable to fully express the beta1 subunit are expected to functional normally, but will have a slower inactivation properties, thereby enhancing potassium efflux from the cell. We hypothesize that this change will render the neuron less likely to sustain rapid repetitive firing: the altered neurons will have a longer interspike interval and a slower maximum frequency of firing. The introduction of these changes in even a small proportion of a pool of neurons could be sufficient to prevent the rapid synchronous firing that constitutes the neurons basis of a seizure. The successful completion of this project might set the stage of the development of a genetic therapy for focal epilepsies.
The specific aims of this proposal are to: (1) Develop methods for the quantification of specific mRNA message and protein expression of the beta1 subunit in primary cultures of pyramidal neurons from rat hippocampus. (2) Develop methods of introducing antisense DNA into cultured pyramidal neurons using oligonucleotides, and the replication- deficient herpes viral vector system. (3) Demonstrate the effect of these antisense knockdown techniques on the transcription and translation of the beta1 subunit. (4) Study the effect of loss of the beta1 subunit on the action potential shape and firing patterns of pyramidal neurons using patch clamp techniques, thereby testing our hypothesis that inhibition of inactivation will limit the maximal neuronal firing rate.
