Single-Cell RNA Sequencing Analysis Reveals Metabolic Changes in Epithelial Glycosphingolipids and Establishes a Prognostic Risk Model for Pancreatic Cancer.

OBJECTIVE: Metabolic reprogramming serves as a distinctive feature of cancer, impacting proliferation and metastasis, with aberrant glycosphingolipid expression playing a crucial role in malignancy. Nevertheless, limited research has investigated the connection between glycosphingolipid metabolism and pancreatic cancer. METHODS: This study utilized a single-cell sequencing dataset to analyze the cell composition in pancreatic cancer tissues and quantified single-cell metabolism using a newly developed computational pipeline called scMetabolism. A gene signature developed from the differential expressed genes (DEGs), related to epithelial cell glycosphingolipid metabolism, was established to forecast patient survival, immune response, mutation status, and reaction to chemotherapy with pancreatic adenocarcinoma (PAAD). RESULTS: The single-cell sequencing analysis revealed a significant increase in epithelial cell proportions in PAAD, with high glycosphingolipid metabolism occurring in the cancerous tissue. A six-gene signature prognostic model based on abnormal epithelial glycosphingolipid metabolism was created and confirmed using publicly available databases. Patients with PAAD were divided into high- and low-risk categories according to the median risk score, with those in the high-risk group demonstrating a more unfavorable survival outcome in all three cohorts, with higher rates of gene mutations (e.g., KRAS, CDKN2A), increased levels of immunosuppressive cells (macrophages, Th2 cells, regulatory T cells), and heightened sensitivity to Acetalax and Selumetinlb. CONCLUSIONS: Abnormal metabolism of glycosphingolipids in epithelial cells may promote the development of PAAD. A model utilizing a gene signature associated with epithelial glycosphingolipids metabolism has been established, serving as a valuable indicator for the prognostic stratification of patients with PAAD.

Surface Glycans of Microvesicles Derived from Endothelial Cells, as Probed Using Plant Lectins.

Glycans of MVs are proposed to be candidates for mediating targeting specificity or at least promoting it. In contrast to exosomes, glycomic studies of MVs are largely absent. We studied the glycoprofile of endothelial cell-derived MVs using 21 plant lectins, and the results show the dominance of oligolactosamines and their α2-6-sialylated forms as N-glycans and low levels of α2-3-sialylated glycans. The low levels of α2-3-sialosides could not be explained by the action of extracellular glycosidases. Additionally, the level of some Man-containing glycans was also decreased in MVs. Spatial masking as the causative relationship between these low level glycans (as glycosphingolipids) by integral proteins or proteoglycans (thus, their lack of interaction with lectins) seems unlikely. The results suggest that integral proteins do not pass randomly into MVs, but instead only some types, differing in terms of their specific glycosylation, are integrated into MVs.

Increased glucosylsphingosine levels and Gaucher disease in GBA1-associated Parkinson's disease.

INTRODUCTION: Gaucher's disease (GD) is caused by biallelic mutations in the GBA1 gene, leading to reduced glucocerebrosidase (GCase) activity and substrate (glucosylceramide and glucosylsphingosine, GlcSph) accumulation. GBA1 variant carriers are at risk of Parkinson's disease (PD), but only those with biallelic mutations cross the threshold of GCase reduction, leading to substrate accumulation and GD. The link between GBA1 mutations, GD and PD is not fully understood. Here we aimed at reporting the results of a large PD population screening with dried blood spot tests for GD. METHODS: We measured GCase activity and GlcSph levels in 1344 PD patients with dried blood spot tests, and performed GBA1 genetic sequencing. RESULTS: While the GCase activity was reduced in GBA1-PD carriers compared to wild type PD, GlcSph was increased in GBA1-PD compared to GBA1-controls, regardless of the underlying type of GBA1 variant. 13.6 % and 0.4 % of PD patients had mono- or biallelic GBA1 mutations respectively. GCase deficiency, lipid accumulation and clinical manifestations of GD was detected in five PD patients with biallelic GBA1 mutations, of whom four had a risk combined with a GD causing variant. CONCLUSIONS: GlcSph appearing higher in PD may represent a reliable biomarker of the disease and deserves to be further investigated. This study highlights the importance of screening PD patients for possible underlying GD, which is a treatable condition that should not be missed. We diagnosed GD cases carrying a "risk" variant in one allele, which is an unprecedented finding deserving further investigation.

Bifunctional glycosphingolipid (GSL) probes to investigate GSL-interacting proteins in cell membranes.

Glycosphingolipids (GSLs) are abundant glycolipids on cells and essential for cell recognition, adhesion, signal transduction, and so on. However, their lipid anchors are not long enough to cross the membrane bilayer. To transduce transmembrane signals, GSLs must interact with other membrane components, whereas such interactions are difficult to investigate. To overcome this difficulty, bifunctional derivatives of II3-ß-N-acetyl-D-galactosamine-GA2 (GalNAc-GA2) and ß-N-acetyl-D-glucosamine-ceramide (GlcNAc-Cer) were synthesized as probes to explore GSL-interacting membrane proteins in live cells. Both probes contain photoreactive diazirine in the lipid moiety, which can crosslink with proximal membrane proteins upon photoactivation, and clickable alkyne in the glycan to facilitate affinity tag addition for crosslinked protein pull-down and characterization. The synthesis is highlighted by the efficient assembly of simple glycolipid precursors followed by on-site lipid remodeling. These probes were employed to profile GSL-interacting membrane proteins in HEK293 cells. The GalNAc-GA2 probe revealed 312 distinct proteins, with GlcNAc-Cer probe-crosslinked proteins as controls, suggesting the potential influence of the glycan on GSL functions. Many of the proteins identified with the GalNAc-GA2 probe are associated with GSLs, and some have been validated as being specific to this probe. The versatile probe design and experimental protocols are anticipated to be widely applicable to GSL research.

Glycosphingolipids in Osteoarthritis and Cartilage-Regeneration Therapy: Mechanisms and Therapeutic Prospects Based on a Narrative Review of the Literature.

Glycosphingolipids (GSLs), a subtype of glycolipids containing sphingosine, are critical components of vertebrate plasma membranes, playing a pivotal role in cellular signaling and interactions. In human articular cartilage in osteoarthritis (OA), GSL expression is known notably to decrease. This review focuses on the roles of gangliosides, a specific type of GSL, in cartilage degeneration and regeneration, emphasizing their regulatory function in signal transduction. The expression of gangliosides, whether endogenous or augmented exogenously, is regulated at the enzymatic level, targeting specific glycosyltransferases. This regulation has significant implications for the composition of cell-surface gangliosides and their impact on signal transduction in chondrocytes and progenitor cells. Different levels of ganglioside expression can influence signaling pathways in various ways, potentially affecting cell properties, including malignancy. Moreover, gene manipulations against gangliosides have been shown to regulate cartilage metabolisms and chondrocyte differentiation in vivo and in vitro. This review highlights the potential of targeting gangliosides in the development of therapeutic strategies for osteoarthritis and cartilage injury and addresses promising directions for future research and treatment.

Limited impact of cancer-derived gangliosides on anti-tumor immunity in colorectal cancer.

Aberrant glycosylation is a key mechanism employed by cancer cells to evade immune surveillance, induce angiogenesis and metastasis, among other hallmarks of cancer. Sialic acids, distinctive terminal glycan structures located on glycoproteins or glycolipids, are prominently upregulated across various tumor types, including colorectal cancer (CRC). Sialylated glycans modulate anti-tumor immune responses through their interactions with Siglecs, a family of glycan-binding receptors with specificity for sialic acid-containing glycoconjugates, often resulting in immunosuppression. In this paper, we investigated the immunomodulatory function of ST3Gal5, a sialyltransferase that catalyzes the addition of α2-3 sialic acids to glycosphingolipids, since lower expression of ST3Gal5 is associated with better survival of CRC patients. We employed CRISPR/Cas9 to knock out the ST3Gal5 gene in two murine CRC cell lines MC38 and CT26. Glycomics analysis confirmed the removal of sialic acids on glycolipids, with no discernible impact on glycoprotein sialylation. Although knocking out ST3Gal5 in both cell lines did not affect in vivo tumor growth, we observed enhanced levels of regulatory T cells in CT26 tumors lacking ST3Gal5. Moreover, we demonstrate that the absence of ST3Gal5 affected size and blood vessel density only in MC38 tumors. In summary, we ascertain that sialylation of glycosphingolipids has a limited influence on the anti-tumor immune response in CRC, despite detecting alterations in the tumor microenvironment, possibly due to a shift in ganglioside abundance.

Clinical and biochemical footprints of congenital disorders of glycosylation: Proposed nosology.

We have identified 200 congenital disorders of glycosylation (CDG) caused by 189 different gene defects and have proposed a classification system for CDG based on the mode of action. This classification includes 8 categories: 1. Disorders of monosaccharide synthesis and interconversion, 2. Disorders of nucleotide sugar synthesis and transport, 3. Disorders of N-linked protein glycosylation, 4. Disorders of O-linked protein glycosylation, 5. Disorders of lipid glycosylation, 6. Disorders of vesicular trafficking, 7. Disorders of multiple glycosylation pathways and 8. Disorders of glycoprotein/glycan degradation. Additionally, using information from IEMbase, we have described the clinical involvement of 19 organs and systems, as well as essential laboratory investigations for each type of CDG. Neurological, dysmorphic, skeletal, and ocular manifestations were the most prevalent, occurring in 81%, 56%, 53%, and 46% of CDG, respectively. This was followed by digestive, cardiovascular, dermatological, endocrine, and hematological symptoms (17-34%). Immunological, genitourinary, respiratory, psychiatric, and renal symptoms were less frequently reported (8-12%), with hair and dental abnormalities present in only 4-7% of CDG. The information provided in this study, including our proposed classification system for CDG, may be beneficial for healthcare providers caring for individuals with metabolic conditions associated with CDG.

(Sialyl)Lewis Antigen Expression on Glycosphingolipids, N-, and O-Glycans in Colorectal Cancer Cell Lines is Linked to a Colon-Like Differentiation Program.

Alterations in the glycomic profile are a hallmark of cancer, including colorectal cancer (CRC). While, the glycosylation of glycoproteins and glycolipids has been widely studied for CRC cell lines and tissues, a comprehensive overview of CRC glycomics is still lacking due to the usage of different samples and analytical methods. In this study, we compared glycosylation features of N-, O-glycans, and glycosphingolipid glycans for a set of 22 CRC cell lines, all measured by porous graphitized carbon nano-liquid chromatography-tandem mass spectrometry. An overall, high abundance of (sialyl)Lewis antigens for colon-like cell lines was found, while undifferentiated cell lines showed high expression of H blood group antigens and α2-3/6 sialylation. Moreover, significant associations of glycosylation features were found between the three classes of glycans, such as (sialyl)Lewis and H blood group antigens. Integration of the datasets with transcriptomics data revealed positive correlations between (sialyl)Lewis antigens, the corresponding glycosyltransferase FUT3 and transcription factors CDX1, ETS, HNF1/4A, MECOM, and MYB. This indicates a possible role of these transcription factors in the upregulation of (sialyl)Lewis antigens, particularly on glycosphingolipid glycans, via FUT3/4 expression in colon-like cell lines. In conclusion, our study provides insights into the possible regulation of glycans in CRC and can serve as a guide for the development of diagnostic and therapeutic biomarkers.

Regulation of Glycosylation in Bone Metabolism.

Glycosylation plays a crucial role in the maintenance of homeostasis in the body and at the onset of diseases such as inflammation, neurodegeneration, infection, diabetes, and cancer. It is also involved in bone metabolism. N- and O-glycans have been shown to regulate osteoblast and osteoclast differentiation. We recently demonstrated that ganglio-series and globo-series glycosphingolipids were essential for regulating the proliferation and differentiation of osteoblasts and osteoclasts in glycosyltransferase-knockout mice. Herein, we reviewed the importance of the regulation of bone metabolism by glycoconjugates, such as glycolipids and glycoproteins, including our recent results.

Wide-Targeted Semi-Quantitative Analysis of Acidic Glycosphingolipids in Cell Lines and Urine to Develop Potential Screening Biomarkers for Renal Cell Carcinoma.

Glycosphingolipids (GSLs), mainly located in the cell membrane, play various roles in cancer cell function. GSLs have potential as renal cell carcinoma (RCC) biomarkers; however, their analysis in body fluids is challenging because of the complexity of numerous glycans and ceramides. Therefore, we applied wide-targeted lipidomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with selected reaction monitoring (SRM) based on theoretical mass to perform a comprehensive measurement of GSLs and evaluate their potency as urinary biomarkers. In semi-quantitative lipidomics, 240 SRM transitions were set based on the reported/speculated structures. We verified the feasibility of measuring GSLs in cells and medium and found that disialosyl globopentaosylceramide (DSGb5 (d18:1/16:0)) increased GSL in the ACHN medium. LC-MS/MS analysis of urine samples from clear cell RCC (ccRCC) patients and healthy controls showed a significant increase in the peak intensity of urinary DSGb5 (d18:1/16:0) in the ccRCC group compared with that in the control group. Receiver operating characteristic analysis indicated that urinary DSGb5 could serve as a sensitive and specific marker for RCC screening, with an AUC of 0.89. This study demonstrated the possibility of urinary screening using DSGb5 (d18:1/16:0). In conclusion, urinary DSGb5 (d18:1/16:0) was a potential biomarker for cancer screening, which could contribute to the treatment of RCC patients.

Chromatographic Separation and Quantitation of Sphingolipids from the Central Nervous System or Any Other Biological Tissue.

Chromatographic separation and purification of an individual lipid to homogeneity have long been introduced. Using this concept, a more precise method has been developed to identify and characterize the sphingolipid composition(s) using a small amount (30 mg) of biological sample. Sphingolipids (lipids containing sphingosine or dihydrosphingosine) are well-known regulators of the central nervous system development and play a critical role in neurodegenerative diseases. Introducing a silicic acid column chromatography, sphingolipid components have been separated to individual fractions such as ceramide, glucosyl/galactosylceramide, other neutral and acidic glycosphingolipids, including (dihydro)sphingosine and psychosine; as well as phospholipids from which individual components are quantified employing a single or combination of other advanced chromatography procedures such as thin-layer chromatography, gas chromatography-mass spectrometry, and high-performance liquid chromatography-mass spectrometry.

Glycosphingolipids in congenital disorders of glycosylation (CDG).

Congenital disorders of glycosylation (CDG) are a large family of rare disorders affecting the different glycosylation pathways. Defective glycosylation can affect any organ, with varying symptoms among the different CDG. Even between individuals with the same CDG there is quite variable severity. Associating specific symptoms to deficiencies of certain glycoproteins or glycolipids is thus a challenging task. In this review, we focus on the glycosphingolipid (GSL) synthesis pathway, which is still rather unexplored in the context of CDG, and outline the functions of the main GSLs, including gangliosides, and their role in the central nervous system. We provide an overview of GSL studies that have been performed in CDG and show that abnormal GSL levels are not only observed in CDG directly affecting GSL synthesis, but also in better known CDG, such as PMM2-CDG. We highlight the importance of studying GSLs in CDG in order to better understand the pathophysiology of these disorders.

Methodological Pitfalls of Investigating Lipid Rafts in the Brain: What Are We Still Missing?

The purpose of this review is to succinctly examine the methodologies used in lipid raft research in the brain and to highlight the drawbacks of some investigative approaches. Lipid rafts are biochemically and biophysically different from the bulk membrane. A specific lipid environment within membrane domains provides a harbor for distinct raftophilic proteins, all of which in concert create a specialized platform orchestrating various cellular processes. Studying lipid rafts has proved to be arduous due to their elusive nature, mobility, and constant dynamic reorganization to meet the cellular needs. Studying neuronal lipid rafts is particularly cumbersome due to the immensely complex regional molecular architecture of the central nervous system. Biochemical fractionation, performed with or without detergents, is still the most widely used method to isolate lipid rafts. However, the differences in solubilization when various detergents are used has exposed a dire need to find more reliable methods to study particular rafts. Biochemical methods need to be complemented with other approaches such as live-cell microscopy, imaging mass spectrometry, and the development of specific non-invasive fluorescent probes to obtain a more complete image of raft dynamics and to study the spatio-temporal expression of rafts in live cells.

The Sphinx and the egg: Evolutionary enigmas of the (glyco)sphingolipid biosynthetic pathway.

In eukaryotes, the de novo synthesis of sphingolipids (SLs) consists of multiple sequential steps which are compartmentalized between the endoplasmic reticulum and the Golgi apparatus. Studies over many decades have identified the enzymes in the pathway, their localization, topology and an array of regulatory mechanisms. However, little is known about the evolutionary forces that underly the generation of this complex pathway or of its anteome, i.e., the metabolic pathways that converge on the SL biosynthetic pathway and are essential for its activity. After briefly describing the pathway, we discuss the mechanisms by which the enzymes of the SL biosynthetic pathway are targeted to their different subcellular locations, how the pathway per se may have evolved, including its compartmentalization, and the relationship of the pathway to eukaryogenesis. We discuss the circular interdependence of the evolution of the SL pathway, and comment on whether current Darwinian evolutionary models are able to provide genuine mechanistic insight into how the pathway came into being.

Recent progress in the synthesis of glycosphingolipids.

To accelerate the biological study and application of the diverse functions of glycosphingolipids (GSLs), the production of structurally defined GSLs has been greatly demanded. In this review, we focus on the recent developments in the chemical and chemoenzymatic synthesis of GSLs. In the chemical synthesis section, the syntheses based on glucosyl ceramide cassette, late-stage sialylation, and diversity-oriented strategies for GSLs or ganglioside synthesis are highlighted, which delivered terpioside B, fluorescent sialyl lactotetraosyl ceramide, and analogs of lacto-ganglio-series GSLs, respectively. In the chemoenzymatic synthesis section, the synthesis of ganglioside GM1 by multistep one-pot multienzyme method and the total synthesis of highly complex ganglioside LLG-5 using a water-soluble lactosyl ceramide as a key substrate for enzymatic sialylation are described.

Impact of Nontreatment Duration and Keratopathy on Major Adverse Cardiovascular Events in Fabry Disease: A Nationwide Cohort Study.

Fabry disease (FD) is a rare inherited X-linked lysosomal storage disorder that results in the progressive accumulation of glycosphingolipids in multiple organs. Early FD-specific treatments may improve clinical outcomes; however, clinical evidence about early FD treatment is limited. We aimed to determine the cardiovascular outcomes of patients with FD who received enzyme replacement therapy. This nationwide observational study was conducted using the National Health Claims database of the Korean population with FD. The primary outcome was major adverse cardiovascular events (MACEs). MACE risk factors in FD were evaluated using time-dependent Cox regression. Between January 2007 and April 2022, 188 patients with FD were analyzed. Among them, 22 (11.7%) experienced MACE (males: 14/95 [14.7%]; females: 8/93 [8.6%]). The mean age at MACE diagnosis was 53.5 ± 11.0 years in all patients with FD, which was lower in males compared with in females (49.7 ± 9.6 vs. 60.0 ± 10.7 years, p = 0.030). Multivariate analysis (HR, 95% CI) revealed that age (1.042; 1.004-1.082) and duration of FD nontreatment (1.040; 1.003-1.078) were significant MACE risk factors in all patients. In males, age (1.080; 1.032-1.131), FD nontreatment duration (1.099; 1.048-1.152), and keratopathy (18.920; 4.174-85.749) were significant MACE risk factors in multivariate analysis. In females, the only significant MACE risk factor was a high Charlson comorbidity index score (1.795; 1.229-2.622). In conclusion, duration of FD nontreatment and keratopathy are significant MACE risk factors in males with FD. These findings suggest the importance of early initiation of FD-specific treatment and careful evaluation of keratopathy in males with FD.

Galactosylceramide Upregulates the Expression of the BCL2 Gene and Downregulates the Expression of TNFRSF1B and TNFRSF9 Genes, Acting as an Anti-Apoptotic Molecule in Breast Cancer Cells.

Galactosylceramide (GalCer) increases the resistance of breast cancer cells to doxorubicin, paclitaxel, and cisplatin by acting as an anti-apoptotic molecule. GalCer was found to specifically downregulate the levels of the pro-apoptotic TNFRSF1B and TNFRSF9 genes and upregulate the levels of the anti-apoptotic BCL2 gene, suggesting that this glycosphingolipid regulates their expression at the transcriptional level. Consistent with this hypothesis, MDA-MB-231 and MCF7 breast cancer cells with high levels of GalCer showed lower activity of the TNFRSF1B and TNFRSF9 promoters than cells lacking GalCer. In contrast, the activity of the BCL2 promoter was higher in MCF7 cells overproducing GalCer than in MCF7 cells without GalCer. However, no difference in BCL2 promoter activity was observed between MDA-MB-231 cells with high and no GalCer content. Instead, we found that high levels of GalCer increased the stability of Bcl-2 mRNA. Subsequent studies showed that breast cancer cells with high levels of GalCer are characterized by significantly lower expression of P53. Importantly, inhibition of P53 expression by siRNA in MCF7 and MDA-MB-231 cells lacking GalCer resulted in decreased expression and promoter activity of the TNFRS1B and TNFRSF9 genes. On the other hand, increased expression and promoter activity of the BCL2 gene was found in such MCF7 cells, and increased stability of Bcl-2 transcripts was observed in such MDA-MB-231 cells. Taken together, these data strongly suggest that the regulatory protein that simultaneously increases the expression of the TNFRSF1B and TNFRSF9 genes and decreases the expression of the BCL2 gene and the stability of Bcl-2 transcripts is most likely P53, the expression of which is GalCer dependent.

Globo-series Gb4 activates ERK and promotes the proliferation of osteoblasts.

OBJECTIVES: Globo-series Gb4 (globoside) is involved in the immune system and disease pathogenesis. We recently reported that systemic Gb4 deficiency in mice led to decreased bone formation due to a reduction in osteoblast number. However, it remains unclear whether Gb4 expressed in osteoblasts promotes their proliferation. Therefore, we investigated the role of Gb4 in osteoblast proliferation in vitro. METHODS: We examined osteoblast proliferation in Gb3 synthase knockout mice lacking Gb4. We investigated the effects of Gb4 synthase knockdown in the mouse osteoblast cell line MC3T3-E1 on its proliferation. Furthermore, we administered Gb4 to MC3T3-E1 cells in which Gb4 was suppressed by a glucosylceramide synthase (GCS) inhibitor and evaluated its effects on their proliferation. To elucidate the mechanisms by which Gb4 promotes osteoblast proliferation, the phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2) levels were measured in MC3T3-E1 cells. RESULTS: Osteoblast proliferation was lower in Gb3 synthase knockout mice lacking Gb4 than in wild-type mice. Proliferation was inhibited by Gb4 synthase knockdown in MC3T3-E1 cells. Furthermore, the administration of Gb4 to MC3T3-E1 cells, in which a GCS inhibitor suppressed Gb4, promoted their proliferation. Moreover, it increased the phosphorylated ERK1/2 levels in MC3T3-E1 cells. CONCLUSIONS: Our results suggest that Gb4 expressed in osteoblasts promotes their proliferation through ERK1/2 activation.

Lysoglycosphingolipids have the ability to induce cell death through direct PI3K inhibition.

Sphingolipidoses are inherited metabolic disorders associated with glycosphingolipids accumulation, neurodegeneration, and neuroinflammation leading to severe neurological symptoms. Lysoglycosphingolipids (lysoGSLs), also known to accumulate in the tissues of sphingolipidosis patients, exhibit cytotoxicity. LysoGSLs are the possible pathogenic cause, but the mechanisms are still unknown in detail. Here, we first show that lysoGSLs are potential inhibitors of phosphoinositide 3-kinase (PI3K) to reduce cell survival signaling. We found that phosphorylated Akt was commonly reduced in fibroblasts from patients with sphingolipidoses, including GM1/GM2 gangliosidoses and Gaucher's disease, suggesting the contribution of lysoGSLs to the pathogenesis. LysoGSLs caused cell death and decreased the level of phosphorylated Akt as in the patient fibroblasts. Extracellularly administered lysoGM1 permeated the cell membrane to diffusely distribute in the cytoplasm. LysoGM1 and lysoGM2 also inhibited the production of phosphatidylinositol-(3,4,5)-triphosphate and the translocation of Akt from the cytoplasm to the plasma membrane. We also predicted that lysoGSLs could directly bind to the catalytic domain of PI3K by in silico docking study, suggesting that lysoGSLs could inhibit PI3K by directly interacting with PI3K in the cytoplasm. Furthermore, we revealed that the increment of lysoGSLs amounts in the brain of sphingolipidosis model mice correlated with the neurodegenerative progression. Our findings suggest that the down-regulation of PI3K/Akt signaling by direct interaction of lysoGSLs with PI3K in the brains is a neurodegenerative mechanism in sphingolipidoses. Moreover, we could propose the intracellular PI3K activation or inhibition of lysoGSLs biosynthesis as novel therapeutic approaches for sphingolipidoses because lysoGSLs should be cell death mediators by directly inhibiting PI3K, especially in neurons.