Abstracts

Rationale: Approximately 1/3 of focal epilepsy patients show no structural abnormality on MRI (non-lesional focal epilepsy, NLFE). Recently, somatic variants in SLC35A2 (predicted to be loss-of-function, LOF) have emerged as a common cause of NLFE. SLC35A2 encodes for the protein UGT-1, a UDP-galactose transporter that transports UDP-galactose from the cytosol to the lumen of the Golgi apparatus. Brain somatic mutations in SLC35A2 are also associated with focal cortical dysplasia type I. Although the clinical phenotypes associated with somatic SLC35A2 mutations are well defined, the molecular mechanisms leading to enhanced excitability and cortical laminar disruption are not well understood. UGT-1 is a solute carrier transporter (SLC) shown to support dendritic growth and is a known modulator of protein glycosylation; SLC35A2 variants are associated with aberrant N-glycosylation of proteins. We hypothesize that Slc35a2 knockout (KO) results in disrupted neuronal dendritic growth, aberrant Golgi structure, and altered glycosylation profiles in vitro.

Methods: We designed a CRISPR/CAS9 construct (targeting Slc35a2 exons 2 and 3) to create an Slc35a2 KO plasmid. Mouse Neuro2a cells (N2aC) were transfected using Lipofectamine in serum free media with either the Slc35a2 KO plasmid or a Scramble (Scr) plasmid to create KO and Scr cell lines. KO was validated using Western blot (Sigma Aldrich) and qPCR analysis. KO, Scr, and wild type (WT) cells were used to assay the effects of Slc35a2 KO on Golgi structure, dendritic architecture, and protein glycosylation. Immunocytochemistry (ICC) was used to visualize localization of UGT-1 (Biorbyt,1:100), Golgi structure (GM1301:500), and dendritic architecture (MAP2 1:500). Fluorescent secondary antibodies (alexa 488, 594, 750) were incubated for 2 hours at room temperature. ICC images were captured on a spinning disk confocal microscope. Golgi structure was assayed by measuring the degree of the crescentic angle of GM130 fluorescence on FIJI (degree to which GM130 fluorescence was spread). Loss of organelle structure is defined by an obtuse angle representing organelle disarray. We assessed dendrites using an automated Sholl analysis software (NeuronJ-ImageJ). Glycosylation profiles were analyzed using cell lysates of cell lines (3 biological replicates, 3 technical replicates per condition) and probing with biotinylated Maackia Amurensis Lectin I (a biotinylated lectin ideal for examining glycoconjugates)

Results: Slc35a2 KO showed no mRNA expression by qPCR and no UGT-1 protein expression (WB). KO also showed a reduction in dendritic intersections on Sholl analysis when compared to controls. KO cells showed less colocalization of UGT-1 with the Golgi as well as disrupted Golgi organellar structure evidenced by larger measured crescentic angles than controls. Slc35a2 KO cells showed an alteration in Lectin/streptavidin binding compared to controls, indicating disrupted protein glycosylation.

Conclusions: Slc35a2 KO in N2aC leads to loss of Golgi integrity, aberrant dendritic structure, and altered glycosylation profiles. These results provide new insights into how loss of UGT-1 may lead to establishment of an epileptic network.

Funding: Please list any funding that was received in support of this abstract.: R01NS115017-01A1 to PBC.