Abstracts

Rationale: Tuberous Sclerosis is a genetic cause of epilepsy characterized by cortical dysplasia thought to result, in part, from defective neuronal migration during development. However, the pathogenesis of cortical tubers is poorly understood and the role of the TSC gene products, tuberin and hamartin, in neuronal migration remains unknown. Methods: In utero injection and electroporation of short hairpin RNAs into the developing cortices of embryonic rats on the 16th day of gestation; time-lapse microscopy of cultured cortical sectionsResults: To determine the effects of acute tuberin knockdown on migrating neurons, we used in utero injection and electroporation to introduce RNA interference (RNAi) targeting the TSC2 transcript into embryonic rat cortices. Whereas labeled neurons in the control conditions were observed to enter the developing cortex in large numbers by the fourth day following electroporation, tuberin deficient neurons appeared unable to penetrate the cortical plate. Moreover, neurons in the TSC2 RNAi condition ultimately failed to achieve their proper laminar fate in the adult brain, often coming to reside in the white matter. Thus loss of tuberin function led to a neuronal migration defect and replicated aspects of cortical tubers. To learn why tuberin is required for neuronal migration, we examined the morphology of tuberin-deficient cells en route to the cortex three days following electroporation. Bipolar neurons in the TSC2 RNAi condition were found to have longer leading processes and more spherical cell somas than controls. Additionally, the point at which the leading process joins the cell soma was constricted in these cells, while the proximal leading process was swollen relative to control neurons. Most of these attributes are exaggerations of features manifested by migrating neurons just prior to movement of their cell soma into the leading process, suggesting that loss of tuberin specifically disrupts this aspect of migration, known as “somal translocation.” We used time-lapse imaging to examine dynamic features of migration and to confirm that tuberin is required for somal translocation. Acute sections of electroporated brains were cultured and imaged hourly for nine to twelve hours on the third day. While control cells were observed to undergo periodic saltatory displacements of their somas, tuberin deficient cells displayed disrupted somal translocation. Whereas contractions in the control condition served to propel the cell soma forward, contractions in the TSC2 RNAi condition often resulted in reduced or even negative displacement of the cell soma. In addition, the membranes of TSC2 RNAi neurons were frequently stretched by isolated contractions, occasionally resulting in cytoplasmic amputations. Conclusions: We propose that tuberin, likely in conjunction with hamartin, regulates the position and/or timing of contractile events required for somal translocation of migrating cortical neurons.