Abstracts

Rationale: Germline genetic variants in SLC35A2, which encodes a UDP-galactose transporter (UDPGalT) critical for cellular glycosylation, have been implicated in one type of congenital disorder of glycosylation associated with intractable seizures and a rare X-linked developmental and epileptic encephalopathy (Kodera H, et al. Hum Mut 2013;34:1708-14,2; Ng BG, et al. Am J Hum Genet 2013;92:632-6). Our lab was the first to identify post-zygotically-acquired, de novo, loss-of-function (LOF) SLC35A2 variants in brain tissue in focal neocortical epilepsy (Winawer MR, et al. Ann Neurol 2018;83:1133-46). Despite this clear link to epilepsy, the mechanism of SLC35A2 associated epilepsy remain unknown. This study explores how LOF variants in SLC35A2 contribute to the epileptogenic phenotype using a human induced pluripotent stem cell (iPSC)-derived neuron model.

Methods: Healthy male iPSCs (isogenic control) were CRIPSR-edited to harbor a patient identified missense variant (SLC35A2^S304P/Y) or a frameshift indel (SLC35A2^-/Y). To investigate the effects of these variants on neurodevelopment and neural network formation, we applied the dual Smad inhibition protocol to generate dorsal forebrain glutamatergic neurons. Early neurodevelopmental phenotypes were characterized using immunofluorescent (IF) staining and qRT-PCR. Neural network activity was evaluated using the multielectrode array (MEA) system. A MALI lectin binding assay and Western blot (WB) was performed to assess cellular glycosylation and protein expression, respectively.

Results: SLC35A2^S304P/Y and SLC35A2^-/Y encode LOF proteins as shown by loss of UDPGalT expression on WB and by IF, leading to reduced migration of highly glycosylated proteins on WB and reduced MAL-I lectin binding compared to the isogenic control. When undergoing forebrain directed neural differentiation, lumen formation and ß3tubulin+ neurons were detected at earlier timepoints in SLC35A2 variant neurons than the isogenic control suggesting precocious neurodevelopment. To evaluate whether SLC35A2-variants recapitulate the seizure phenotype, we characterized neural network activity on MEA. Neural networks formed by the isogenic control develop clear, synchronous activity, which is largely absent in the SLC35A2-variant networks. The addition of bicuculline, a GABAergic antagonist, partially rescued the synchrony phenotype in SLC35A2-variant harboring networks suggesting a possible imbalance in the proportion of glutamatergic/GABAergic neurons. Indeed, we detected an increase in GABAergic-specific markers in SLC35A2-variant harboring networks compared to the isogenic control by qRT-PCR.

Conclusions: Our data show that LOF SLC35A2-variants increase neurogenesis and influence the differentiation trajectory towards a GABAergic fate, thereby disrupting the glutamatergic/GABAergic balance and resulting in asynchronous, hypoactive networks. We are further exploring the underlying cell signaling pathways affected, single cell RNA sequencing to classify cell types, and the possibility of galactose therapy on reversing the phenotypes.

Funding: R01NS115017