Profiling glycosphingolipid changes in mouse and human cellular models of lysosomal free sialic acid storage disorder

Abstract

Free sialic acid storage disorder (FSASD) is an autosomal recessive lysosomal storage disease caused by biallelic pathogenic variants in SLC17A5, which encodes the lysosomal sialic acid transporter, sialin. FSASD is characterized by excessive lysosomal free sialic acid accumulation, leading to either a severe, early-onset lethal phenotype or a progressive neurodegenerative course. To characterize biochemical alterations in FSASD models, we performed comprehensive profiling of glycosphingolipids (GSLs), including sialylated species (i.e., gangliosides), in mouse embryonic fibroblasts (MEFs) derived from Slc17a5-R39C/R39C and Slc17a5-KO/KO mouse models, as well as in human SLC17A5-deficient HEK-293 T cells generated via CRISPR-Cas9-mediated non-homologous end joining. HPLC-based analyses demonstrated GM3 ganglioside accumulation in MEFs and significant reductions in a-series GSLs-including GM2, GM1a, and GD1a-in SLC17A5-deficient HEK-293 T cells. Analysis of neuraminidase 1/3/4 activities revealed consistently elevated activity across all cell models, while cytosolic neuraminidase 2 showed only a modest increase in Slc17a5-R39C/R39C MEFs. Preliminary quantification showed elevated free sialic acid across all models, consistent with the characteristic biochemical defect observed in FSASD and supporting their relevance for mechanistic studies. These findings highlight that free sialic acid storage leads to changes in GSL homeostasis in FSASD mouse (MEFs) and human (SLC17A5-deficient HEK-293T) cellular models, underscoring their utility as models for studying FSASD pathogenesis.

Keywords: Gangliosides; HEK-293 T; Mouse embryonic fibroblasts; Neuraminidase; Salla disease; Sialin.

Fig. 1

Schematic overview of study. Mouse embryonic fibroblasts (MEFs) from two FSASD mouse models, as well as HEK-293T wild-type and SLC17A5-deficient cells, were used in this study. MEFs and HEK-293T cells were profiled using several assays, including quantification of glycosphingolipid (GSL) levels, neuraminidase and β-hexosaminidase activity screening, and sialic acid levels. Figure created using BioRender.com.

Fig. 2

Levels of total GSLs and individual GSL species in MEFs. (A) Total GSLs (addition of individual species listed in (A)) and several individual GSL species labelled with glucose units (GU) and provisional GSL assignments. (B) Glucosylceramide (Glc) levels in knock-out MEFs only. Each point denotes one replicate. Mean ± SEM; ordinary one-way ANOVA with Fisher's LSD test in (A) or unpaired t-test in (B) with p-value <0.05 (*), <0.0099 (**), <0.0001 (****), and ns = not significant.

Fig. 3

Levels of total GSLs and individual GSL species in HEK-293T cells. Total GSLs (addition of individual species listed) and several individual GSL species labelled with glucose units (GU) and provisional GSL assignments. Each point denotes one replicate. Mean ± SEM; unpaired t-test with p-value <0.05 (*), <0.002 (**), and ns = not significant.

Fig. 4

Activity levels of enzymes involved in GSL catabolism in MEFs and HEK-293T cells. The following enzyme activities were measured in MEFs: neuraminidase (Neu1/3/4 and Neu2) and total β-hexosaminidase (Total Hex); (A) passage 8 cells for knock-in model and (B) passage 13 cells for knock-out model. (C) Only neuraminidase (NEU1/3/4 and NEU2) was measured in HEK-293 T cells. Activity represented as nmol per hour per mg total protein content. Each point denotes one replicate; Mean ± SEM; unpaired t-test p-value <0.05 (*), <0.002 (**), <0.0002 (***), and ns = not significant.

Fig. 5

Summary of (a-series) GSLs and GlcCer in addition to enzyme activity levels across each cellular model. Comparison of cells harboring Slc17a5/SLC17A5 mutations versus respective wild-type cells. Up arrows denote significantly elevated GSL species or enzyme activities, while down arrows indicate significantly decreased levels or activities. An equal sign signifies no significant difference between wild-type and mutant groups. NA* indicates that the GlcCer was assayed but was not detectable above baseline. NM (not measured) means that no additional enzyme activity assays were conducted for that cell type.

Fig. 6

Schematic overview of the intersection between sialic acid and ganglioside metabolism in the context of SLC17A5 (sialin) deficiency. (1) Sialic acid (Neu5Ac) is synthesized from glucose in the cytosol through sequential enzymatic reactions and subsequently activated in the nucleus to CMP-Neu5Ac. CMP-Neu5Ac is then transported into the Golgi by the CMP-sialic acid transporter SLC35A1, where sialylstransferases use it to sialylate glycoproteins and glycolipids. In this figure, GM1a synthesis is shown to illustrate the role of sialic acid in glycolipid biosynthesis. (2) Newly synthesized glycosphingolipids (GSLs, including gangliosides) are trafficked via vesicles to the plasma membrane. After functional turnover, GSLs (e.g., GM1a) are endocytosed and delivered to the lysosome. (3) In lysosomes, gangliosides in intraluminal vesicles are stepwise degraded by glycosidases, including neuraminidases (e.g., Neu1) which releases free Neu5Ac. Under normal conditions, SLC17A5 (sialin) exports free Neu5Ac from lysosomes to the cytosol, a process that contributes to the sialic acid salvage (recycling) pathway. Free Neu5Ac then is either reactivated to CMP-Neu5Ac to support ongoing sialylation, or cleaved to ManNAc + pyruvate by the cytosolic enzyme N-acetylneuraminate pyruvate lyase (NPL), with ManNAc re-entering the de novo sialic acid biosynthesis. In FSASD, loss of sialin function impairs lysosomal efflux of free Neu5Ac, causing intralysosomal accumulation and curtailing the salvage contribution to the CMP-Neu5Ac pool. This Neu5Ac accumulation has been reported to inhibit neuraminidases involved in ganglioside catabolism [13,45], resulting in secondary gangliosides storage. Although global cellular sialylation can often be maintained by de novo synthesis, reduced salvage likely can, in some contexts, limit sialylation of specific substrates under high demand. Created with BioRender.com.