Abstracts

One-third of children with epilepsy experience drug-resistant seizures, but treating these children is a challenge due to an incomplete understanding of the underlying mechanisms of epileptogenesis. Interestingly, loss-of-function somatic variants in the nucleotide sugar transporter gene SLC35A2 have been implicated in epilepsy in both human patients and rodent models; however, the exact molecular mechanisms driving seizures are unclear. As seen in congenital disorders of glycosylation, SLC35A2 loss leads to impaired glycosylation. Glycosylation defects can further induce the accumulation of misfolded proteins, or endoplasmic reticulum (ER) stress. Importantly, increased ER stress and activation of the compensatory unfolded protein response (UPR) can disrupt key neurodevelopmental processes. Taken together, these findings point to a potential role for ER stress in SLC35A2-related epilepsy, but this relationship remains unexplored. Here, we hypothesized that Slc35a2 loss drives ER stress in the cortex.

To test our hypothesis, we used our recently published Emx1-Cre-mediated Slc35a2 conditional knockout mouse model, which recapitulates spontaneous seizures. Cortex was collected from Slc35a2-floxed (control) and Emx1-Cre-Slc35a2 cKO (cKO) male and female mice at postnatal day 21 for RT-qPCR analysis. In another cohort of age-matched control and cKO male mice, 10X Genomics Visium Spatial Gene expression analysis was used to compare ER stress marker expression and localization. To maintain the scope of our Visium experiment, we chose to focus on males given that the cKO phenotype is more severe than in females.

RT-qPCR analysis revealed a 75% decrease in Slc35a2 expression in the cortex of cKO male mice compared to controls. cKO females displayed a milder reduction in Slc35a2 expression in the cortex relative to cKO males, which is expected given that Slc35a2 is an X-linked gene. Interestingly, cKO males exhibited ~40% decrease in ER chaperone Grp78 and an increase in UPR marker Pdi. When examining cKO females, sexual dimorphism in ER stress was evident. Specifically, UPR indicator Perk was upregulated in the cortex of cKO females while no change was observed in Grp78 or Pdi. We next aimed to examine the exact localization of ER stress marker expression related to Slc35a2 loss using 10X Genomics Visium. Spatial transcriptomic analysis paralleled our RT-qPCR findings, demonstrating downregulation of Grp78 in both the cortex and white matter of cKO males compared to controls. In line with this, gene expression of several UPR markers (Atf6, p58ipk, Pdi) was upregulated in cKOs throughout cortex and white matter. Intriguingly, cKO males also displayed downregulation of UPR markers (Perk, Ire1α) in the cortex and white matter, which may indicate negative feedback regulation of the UPR.

Our findings suggest 1) Slc35a2 loss elicits ER stress in the cortex and white matter, and 2) Slc35a2-related ER stress is sexually dimorphic. Collectively, our data may point to ER stress as a potential mediator for SLC35A2-related epilepsy.