Abstracts

Rationale: Focal cortical dysplasia (FCD) is a developmental brain malformation characterized by the abnormal organization of the cerebral cortex. Notably, FCD is a leading cause of intractable pediatric epilepsy. A mosaic loss of function in the gene coding for a UDP-galactose translocator, SLC35A2, has been implicated in over one third of Type I FCD cases (Baldassari et al., 2019), however, the mechanisms by which this mutation leads to epileptogenesis have yet to be defined. In this investigation, we aim to establish an animal model of mosaic Slc35a2 knockdown to delineate the mechanisms by which loss of SLC35A2 function gives rise to the cortical dysplasia and epilepsy phenotypes.

Methods: Using in utero electroporation, we transfected neural progenitor cells of embryonic day 15.5 mice with plasmids expressing either an shRNA targeted against the Slc35a2 transcript, or a scrambled sequence used as an experimental control. Both plasmids also expressed green fluorescent protein (GFP) to allow for identification of transfected cells. Tissue was collected from GFP positive mice at post-natal day 1 or post-natal day 8, coronally sectioned, and stained for cortical layer markers. Analysis was performed to quantify expression levels of layer markers and to measure the migration of the GFP expressing cells throughout the cortical layers. Tissue was also used to quantify RNA expression using single-cell RNA-sequencing.

Results: Fluorescence microscopy revealed that GFP labeled cells in the Slc35a2 knockdown condition were significantly retained in the white matter and lower layers of the mouse cortex at post-natal day 1 and 8 when compared to the control condition, in which a higher fraction of GFP+ cells migrated to the outer layers.  Single-cell RNA-sequencing revealed changes in the proportions of Satb2- and Tbr1-positive cells, which suggests a shift in the fate of cells lacking SLC35A2 function.

Conclusions: Here, we describe migration defects and changes to cortical lamination in our Slc35a2 mosaic knockdown animal model which mirror hallmark phenotypes observed in Type I FCD patients. These findings help establish the in utero electroporation method of Slc35a2 knockdown as a valid technique for studying SLC35A2-associated malformations of cortical development, enabling further investigations into the molecular mechanisms by which loss of the UDP-galactose translocator affects cortical development and gives rise to epilepsy.

Funding: This work is funded by Internal Startup funds for Tracy Bedrosian (Nationwide Children’s Hospital, Columbus, Ohio).