MUTATIONS IN MUNC-18 AS A CAUSE OF SEVERE EPILEPSY AND MENTAL RETARDATION

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MUTATIONS IN MUNC-18 AS A CAUSE OF SEVERE EPILEPSY AND MENTAL RETARDATION

in summary

Most of the research activities in epilepsy studies focus on 'channelopathies', which may lead to increased excitability of neurons. The studies in this research area examine the role of a different disease mechanism for epilepsy.

the details

Funded through the Seed Grant program of the Vice-President of Research, OUHSC

SUMMARY

Most of the research activities in epilepsy studies focus on 'channelopathies', which may lead to increased excitability of neurons. The studies in this research area examine the role of a different disease mechanism for epilepsy. Neuronal firing is translated into a chemical signal at the synapse and the level of amplification at this step is regulated through the exocytotic release machinery. Therefore abnormal synaptic communication is possible in spite of maintained excitability. Neurons from munc18 KO animals exhibit normal action potentials but no synaptic release and represent the most extreme form of abnormal synaptic communication. Here we propose the first characterization of the functional significance of the new munc18-1 mutations found in the human epilepsy disorder: Early Infantile Epileptic Encephalopathy (EIEE), an extremely debilitating form of epilepsy that has no effective therapy. Our experimental approach allows us to study the effect of these mutations on synaptic transmission with high resolution on a genetically clean knock-out background. To make this study possible we successfully combined the genetically modified mouse model with a lentiviral gene delivery system, neuronal cultures and patch-clamp electrophysiology. Furthermore, we use live fluorescence imaging to measure synaptic vesicle release kinetics in identified individual synapses. Results from this project will provide new insight into the molecular mechanism of synaptic exocytosis and how point mutations in munc18-1 cause epilepsy. This work can have a significant impact on the field as it will clarify the cause of EIEE, including the genetic basis and pathological mechanism of the disease, thus shedding new light on molecular mechanisms of seizure disorders via pathological regulation of presynaptic neurotransmitter release. The proposal has excellent potential for translational research because of the success of levetiracetam, a recently developed drug targeting synaptic vesicle protein-2a, which is effective in treating specific types of seizures. Finally, this study will result in the establishment of a new model of EIEE that assesses the epileptic effects of other munc18-1 mutations and facilitates the development of effective drug therapies.

Epilepsy is a very common disorder and almost three million American patients live with the disease. About 200,000 new cases of seizure disorders and epilepsy are diagnosed each year. Early Infantile Epileptic Encephalopathy (EIEE) with suppression-burst pattern on EEG is one of the most severe and earliest forms of epilepsy. This neurological disorder, characterized by seizures affecting newborns, usually starts within the first three months of life. Infants have primarily tonic seizures, and EEG records display an inter-ictal suppression-burst pattern. EIEE is also referred to as Ohtahara syndrome after the individual that first described the syndrome. Antiepileptic drugs are rarely effective, and EIEE is progressive and has a poor prognosis. Seizures become more frequent, accompanied by physical and mental handicaps. Some children will die in infancy, others will survive but be profoundly handicapped. Genetic screening has identified STXBP1, a gene that encodes munc18-1 (also known as syntaxin binding protein1), as a potential contributor to the disease. Importantly mis-sense point mutations of munc18-1 were reported in several patients as the possible cause of EIEE. However, the functional consequence of these mutations is unknown. The conclusion from our previous studies on the physiological role of munc18-1 in synaptic transmitter release was that munc18-1 binding to the exocytotic core complex in the neuron is essential for priming synaptic vesicles to release their neurotransmitter cargo. In this research project we investigate the pathophysiological function of genetic point mutations of munc18-1 in EIEE. A knock-out mouse model lacking munc18-1 expression is used along with sophisticated viral gene transfer methods. We record neuronal network activity of cortical neurons with patch-clamp electrophysiology and visualize synaptic vesicular transmitter release with live fluorescence imaging. Goals of this project are as follows: 1. Characterize the effect of expression level and mis-sense mutations of munc18-1 in EIEE on excitatory and inhibitory synaptic neurotransmission and 2. Assess the effects of mutations in munc18-1 on the regulation of neuronal network function and seizure threshold. Using state-of-the-art methods combined with results from these experiments we will provide novel information on the mechanism of the synaptic defect in EIEE.