Abstracts

Rationale: Tuberous sclerosis complex (TSC) and focal cortical dysplasia type II (FCDII) are devastating neurological disorders caused by mutations in mTOR pathway genes (e.g., TSC1 and TSC2) leading to mTOR complex 1 (mTORC1) hyperactivity and focal malformations of cortical development. These abnormalities cause drug-resistant epileptic seizures in ~90% of patients. There is thus a critical need to identify molecular alterations that underlie disease pathogenesis and that offer new therapeutic opportunities. Several rodent models have been used to model TSC and FCDII. However, evaluating the efficacy of drugs or molecular interventions on seizure activity in murine models is time-consuming and expensive, restricting our capacity to efficiently test multiple drugs and molecular targets. We thus directed our attention to the fruit fly Drosophila melanogaster, which has served as a model organism in epilepsy research for over half a century since the discovery of “bang-sensitive” mutants (exhibiting stereotypical seizures in response to mechanical stimulation). Drosophila genetics can be utilized to study the basic mechanisms with the modeling of complex genetics of TSC comprising of germline mutations (heterozygosity) and second-hit somatic mutations. Further, Drosophila share conservation with 81% of human epilepsy genes. Surprisingly, there is no Drosophila model of TSC and FCDII related to brain abnormalities. We thus propose to develop Drosophila models of TSC and FCDII using diverse genetic strategies, with a focus on studying associated brain abnormalities and seizures.

Methods: To induce spatiotemporal RNAi-mediated knockdown in Drosophila, we used the Gal4/UAS system where cell/tissue-specific Gal4 transgenes drive co-expression of hairpin RNAs under UAS control. These hairpin transgenes are available as short hairpin RNAs (shRNAs) embedded within a miR-1 microRNA backbone. To assess seizures induced by mechanical stimulation, the vials were placed on a standard laboratory vortexer at maximum speed for 10 s. Seizure duration was measured as time required to regain posture and climbing ability for each fly.


Results: The preliminary data suggests that the flies with neuronal- specific gigas (homolog of human TSC2) knockdown exhibit seizures upon challenge with a sensory stimulus, most notably mechanical stimulation. In addition to these phenotypes, we have found climbing-related mobility dysfunctions. The knockdown flies display an increased mean recovery time from seizure, and decreased climbing ability as compared to isogenic w¹¹¹⁸ control lines.

Conclusions: These results imply that TSC2 neuronal-specific knockdown in drosophila leads to a strong seizure phenotype. Given that the TSC-mTORC1 signaling pathway is conserved between human and Drosophila, this new model can be used for screening drug candidates for seizure treatment and studying the molecular basis underlying the pathogenesis of TSC.

Funding: NIH