Analysis of Munc-18 Epilepsy-Associated Point Mutations in C. elegans

Friday, February 12, 2016
Olga Ivleva, Riverdale Country School, Bronx, NY
Infantile epilepsy is an untreatable neurological disorder, one cause for which can be directly associated with mutations of the Munc18 protein in the synapse. Because Munc18 is one of the key proteins in vesicle fusion, different mutations of the protein have varying effects on the synaptic vesicle, which transmits information between neurons in the brain. We used a model nervous system to study neurotransmitter secretion in order to connect disease-associated mutations in Munc18 with synaptic dysfunction. We hypothesized that the mutant Munc18 protein is misfolded and prone to aggregation, disrupting brain development as well as synapse function, and ultimately leading to epilepsy. Munc18 was originally discovered in the genetic model organism C. elegans. Several of the amino acids altered in human disease are conserved in the C. elegans ortholog, UNC-18.  In addition to the postulated aggregation of mutant Munc18, this process may also drive normal copies of Munc18 protein to collect within aggregates, thereby leading to a dominant phenotype where a single mutant allele leads to dysfunction. Consistent with the unfolded protein hypothesis, our heat shock assay preferentially disrupted nervous system function in transgenic animals expressing the disease mutation using either worm UNC-18 or human Munc18 protein. However, over-expressing mutant UNC-18/Munc18 in a wild-type animal failed to sensitize the transgenic animals to heat shock. The heat shock assay disproved our assumption, revealing that mutant UNC-18/Munc18 variants cannot act in a dominant fashion. We then imaged Munc18 tagged with the genetically-encoded fluorophore GFP in mammalian cell culture. Unlike wild-type Munc18, several disease mutations caused Munc18 to collect in a punctate distribution throughout the cell. We will next image GFP-tagged UNC-18 in living C. elegans' neurons, providing an in vivo corroboration of the cell culture findings. Furthermore, the degree of aggregation within worm neurons can be correlated to the severity of the heat-shock induced behavioral phenotype. The experiments described here will deepen our understanding of the relationship between mutant Munc18 protein aggregation and infantile epilepsy.