Abstracts

Rationale: Malformations of cortical development (MCD) are commonly associated with drug-resistant epilepsy in pediatric patients, often requiring alternative treatment such as surgical resection of affected brain tissue. Recent developments in genetic sequencing technologies have enabled the identification of brain-restricted somatic variants as a prominent cause of MCD. Recently, somatic loss-of-function variants in SLC35A2 have been reported in several cases of MCD. This X-linked gene encodes a UDP-galactose translocator necessary for galactosylation of macromolecules that are critical for cellular processes during neurodevelopment. Interestingly, brain restricted somatic SLC35A2 variants at allele frequencies as low as 2% are sufficient to cause developmental delay, cognitive deficits, and seizures. This mosaic distribution creates a challenging environment to model and study such somatic conditions. As a result, the mechanisms by which SLC35A2 variants contribute to developmental delays and drug-resistant epilepsy remain unknown. Therefore, we created a novel conditional knockout (cKO) mouse model to investigate Slc35a2-associated disease pathology. Here we hypothesized that both primary neuron and primary derived-neural progenitor cell (NPC) cultures would recapitulate several aspects of the disease, yielding a tractable model for future mechanistic studies.

Methods: NPCs were isolated from Slc35a2 floxed mice at embryonic day 14.5 and pooled based on genotype. After passaging, NPCs were transfected with a plasmid either encoding GFP and Cre Recombinase or GFP only. The proliferation capacity of transfected cells was assessed by EdU labeling. To assess developmental trajectories, the cells were assessed over 14 days of differentiation by immunofluorescent staining for developmental markers: SOX2, PAX6, DCX, and MAP2. To assess physiology, primary neural cells were collected from P0 EMX1-Cre Slc35a2 cKO mice and cultured on 3Brain High Density Microelectrode Array (HD-MEA). Both spontaneous and network activity of cKO neurons were characterized with respect to floxed controls. Parallel cultures were characterized for amounts of inhibitory and excitatory neurons using immunofluorescent staining with antibodies against VGLUT1 and GAD1.

Results: Preliminary data suggest that Cre-mediated knockout of Slc35a2 may increase the rate of neuronal maturation. When assessed on HD-MEA, cKO cultures displayed increased spontaneous activity and connectivity advanced for their developmental state; but at the same time, demonstrated aberrant firing frequencies. By investigating the timing of developmental marker expression as well as proliferation capacity, we expect to demonstrate if this physiological state is due to changes in NPC differentiation or proliferation. Moreover, we expect to further characterize any electrophysiological deficits, including the inhibitory and excitatory composition of early in vitro neural circuitry.


Conclusions: Understanding the trajectory of neural development in Slc35a2 knockout cells will identify potential mechanisms of disease as well as provide key targets for future treatment and therapeutic studies.


Funding: Funding by NIH-NINDS (R01NS129784) to TAB.