Seizure control by adding on other anti-seizure medication on seizure during levetiracetam administration in patients with glioma-related epilepsy.

BACKGROUND: Epilepsy is a major symptom in patients with glioma. Levetiracetam (LEV) is recognized as a first-line treatment for glioma-related epilepsy. Increasing the LEV dose is allowed into patients with seizure occurrence against its initial dose. However, the therapeutic efficacy of increasing the LEV dose in response to seizure occurrence remains unclear. METHODS: We retrospectively analyzed 236 glioma patients who were treated with antiseizure medications (ASMs) internally at our institute between September 2010 and December 2017. Of these, the analysis focused on 156 patients treated with LEV who had a clear history of administration. RESULTS: Seizure occurrences were observed in 21 of 75 patients (26.7%) who received LEV as first-line therapy and in 33 of 81 patients (40.7%) who received LEV as non-first-line treatment. The seizure control rate for seizure occurrence with LEV as first-line treatment was significantly higher in patients treated with addition of other ASMs (72.7%) than in those treated with increasing dose of LEV (20.0%) (p = 0.016). The seizure control rate for seizure occurrence with LEV as non-first-line treatment did not differ significantly between patients with addition of other ASMs (58.3%) and those treated with increasing dose of LEV (47.6%) (p = 0.554). CONCLUSIONS: Adding other ASMs was more effective than increasing the LEV dose for seizure control in patients treated with LEV as first-line treatment, but they demonstrated comparable efficacy in patients treated with LEV as non-first-line treatment.

Possible regulation of ganglioside GD3 synthase gene expression with DNA methylation in human glioma cells.

Gangliosides are expressed in nervous systems and some neuroectoderm-derived tumors at high levels and play pivotal roles. However, mechanisms for the regulation of glycosyltransferase genes responsible for the ganglioside synthesis are not well understood. In this study, we analyzed DNA methylation patterns of promoter regions of GD3 synthase (ST8SIA1) as well as mRNA levels and ganglioside expression using human glioma cell lines. Among 5 cell lines examined, 4 lines showed changes in the expression levels of related genes after treatment with 5-aza-dC. LN319 showed up-regulation of St8sia1 and increased b-series gangliosides after 5-aza-dC treatment, and an astrocytoma cell line, AS showed high expression of ST8SIA1 and b-series gangliosides persistently before and after 5-Aza-2'-deoxycytidine treatment. Using these 2 cell lines, DNA methylation patterns of the promoter regions of the gene were analyzed by bisulfite-sequencing. Consequently, 2 regions that were methylated before 5-Aza-2'-deoxycytidine treatment were demethylated in LN319 after the treatment, while those regions were persistently demethylated in AS. These 2 regions corresponded with sites defined as promoter regions by Luciferase assay. Taken together, it was suggested that ST8SIA1 gene is regulated by DNA methylation at the promoter regions, leading to the regulation of tumor phenotypes.

Signaling domains of cancer-associated glycolipids.

Immunotherapy of malignant cancers is now becoming one of representative approaches to overcome cancers. To construct strategies for immunotherapy, presence of tumor-specific antigens should be a major promise. A number of cancer specific- or cancer-associated antigens have been reported based on various experimental sets and various animal systems. The most reasonable strategy to define tumor-specific antigens might be "autologous typing" performed by Old's group, proposing three classes of tumor-antigens recognized by host immune systems of cancer patients. Namely, class 1, individual antigens that is present only in the patient's sample analyzed; class 2, shared antigens that can be found only in some group of cancers in some patients, but not in normal cells and tissues; class 3, universal antigens that are present in some cancers but also in normal cells and tissues with different densities. Sen Hakomori reported there were novel carbohydrates in cancers that could not be detected in normal cells mainly by biochemical approaches. Consequently, many of class 2 cancer-specific antigens have been revealed to be carbohydrate antigens, and been used for cancer diagnosis and treatment. Not only as cancer markers, but roles of those cancer-associated carbohydrates have also been recognized as functional molecules in cancer cells. In particular, roles of complex carbohydrates in the regulation of cell signaling on the cell surface microdomains, glycolipid-enriched microdomain (GEM)/rafts have been reported by Hakomori and many other researchers including us. The processes and present status of these studies on cancer-associated glycolipids were summarized.

Functional validation of novel variants in B4GALNT1 associated with early-onset complex hereditary spastic paraplegia with impaired ganglioside synthesis.

Childhood-onset forms of hereditary spastic paraplegia are ultra-rare diseases and often present with complex features. Next-generation-sequencing allows for an accurate diagnosis in many cases but the interpretation of novel variants remains challenging, particularly for missense mutations. Where sufficient knowledge of the protein function and/or downstream pathways exists, functional studies in patient-derived cells can aid the interpretation of molecular findings. We here illustrate the case of a 13-year-old female who presented with global developmental delay and later mild intellectual disability, progressive spastic diplegia, spastic-ataxic gait, dysarthria, urinary urgency, and loss of deep tendon reflexes of the lower extremities. Exome sequencing showed a novel splice-site variant in trans with a novel missense variant in B4GALNT1 [NM_001478.5: c.532-1G>C/c.1556G>C (p.Arg519Pro)]. Functional studies in patient-derived fibroblasts and cell models of GM2 synthase deficiency confirmed a loss of B4GALNT1 function with no synthesis of GM2 and other downstream gangliosides. Collectively these results established the diagnosis of B4GALNT1-associated HSP (SPG26). Our approach illustrates the importance of careful phenotyping and functional characterization of novel gene variants, particularly in the setting of ultra-rare diseases, and expands the clinical and molecular spectrum of SPG26, a disorder of complex ganglioside biosynthesis.

Majority of alpha2,6-sialylated glycans in the adult mouse brain exist in O-glycans: SALSA-MS analysis for knockout mice of alpha2,6-sialyltransferase genes.

Sialic acids are unique sugars with negative charge and exert various biological functions such as regulation of immune systems, maintenance of nerve tissues and expression of malignant properties of cancers. Alpha 2,6 sialylated N-glycans, one of representative sialylation forms, are synthesized by St6gal1 or St6gal2 gene products in humans and mice. Previously, it has been reported that St6gal1 gene is ubiquitously expressed in almost all tissues. On the other hand, St6gal2 gene is expressed mainly in the embryonic and perinatal stages of brain tissues. However, roles of St6gal2 gene have not been clarified. Expression profiles of N-glycans with terminal α2,6 sialic acid generated by St6gal gene products in the brain have never been directly studied. Using conventional lectin blotting and novel sialic acid linkage-specific alkylamidationmass spectrometry method (SALSA-MS), we investigated the function and expression of St6gal genes and profiles of their products in the adult mouse brain by establishing KO mice lacking St6gal1 gene, St6gal2 gene, or both of them (double knockout). Consequently, α2,6-sialylated N-glycans were scarcely detected in adult mouse brain tissues, and a majority of α2,6-sialylated glycans found in the mouse brain were O-linked glycans. The majority of these α2,6-sialylated O-glycans were shown to be disialyl-T antigen and sialyl-(6)T antigen by mass spectrometry analysis. Moreover, it was revealed that a few α2,6-sialylated N-glycans were produced by the action of St6gal1 gene, despite both St6gal1 and St6gal2 genes being expressed in the adult mouse brain. In the future, where and how sialylated O-linked glycoproteins function in the brain tissue remains to be clarified.

Lack of GD3 synthase (St8sia1) attenuates malignant properties of gliomas in genetically engineered mouse model.

High expression of gangliosides GD3 and GD2 is observed in human gliomas. The functions of GD3 and GD2 in malignant properties have been reported in glioma cells in vitro, but those functions have not yet been investigated in vivo. In this study, we showed that deficiency of GD3 synthase (GD3S, St8sia1) attenuated glioma progression and clinical and pathological features in a platelet-derived growth factor B-driven murine glioma model. Lack of GD3S resulted in the prolonged lifespan of glioma-bearing mice and low-grade pathology in generated gliomas. Correspondingly, they showed reduced phosphorylation levels of Akt, Erks, and Src family kinases in glioma tissues. A DNA microarray study revealed marked alteration in the expression of various genes, particularly in MMP family genes, in GD3S-deficient gliomas. Re-expression of GD3S restored expression of MMP9 in primary-cultured glioma cells. We also identified a transcription factor, Ap2α, expressed in parallel with GD3S expression, and showed that Ap2α was critical for the induction of MMP9 by transfection of its cDNA and luciferase reporter genes, and a ChIP assay. These findings suggest that GD3S enhances the progression of gliomas by enhancement of the Ap2α-MMP9 axis. This is the first report to describe the tumor-enhancing functions of GD3S in vivo.

Genome-wide CRISPR screens for Shiga toxins and ricin reveal Golgi proteins critical for glycosylation.

Glycosylation is a fundamental modification of proteins and membrane lipids. Toxins that utilize glycans as their receptors have served as powerful tools to identify key players in glycosylation processes. Here, we carried out Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated genome-wide loss-of-function screens using two related bacterial toxins, Shiga-like toxins (Stxs) 1 and 2, which use a specific glycolipid, globotriaosylceramide (Gb3), as receptors, and the plant toxin ricin, which recognizes a broad range of glycans. The Stxs screens identified major glycosyltransferases (GTs) and transporters involved in Gb3 biosynthesis, while the ricin screen identified GTs and transporters involved in N-linked protein glycosylation and fucosylation. The screens also identified lysosomal-associated protein transmembrane 4 alpha (LAPTM4A), a poorly characterized four-pass membrane protein, as a factor specifically required for Stxs. Mass spectrometry analysis of glycolipids and their precursors demonstrates that LAPTM4A knockout (KO) cells lack Gb3 biosynthesis. This requirement of LAPTM4A for Gb3 synthesis is not shared by its homolog lysosomal-associated protein transmembrane 4 beta (LAPTM4B), and switching the domains between them determined that the second luminal domain of LAPTM4A is required, potentially acting as a specific "activator" for the GT that synthesizes Gb3. These screens also revealed two Golgi proteins, Transmembrane protein 165 (TMEM165) and Transmembrane 9 superfamily member 2 (TM9SF2), as shared factors required for both Stxs and ricin. TMEM165 KO and TM9SF2 KO cells both showed a reduction in not only Gb3 but also other glycosphingolipids, suggesting that they are required for maintaining proper levels of glycosylation in general in the Golgi. In addition, TM9SF2 KO cells also showed defective endosomal trafficking. These studies reveal key Golgi proteins critical for regulating glycosylation and glycolipid synthesis and provide novel therapeutic targets for blocking Stxs and ricin toxicity.

Lactosylceramide synthases encoded by B4galt5 and 6 genes are pivotal for neuronal generation and myelin formation in mice.

It is uncertain which ß4-galactosyltransferase (ß4GalT; gene name, B4galt), ß4GalT-5 and/or ß4GalT-6, is responsible for the production of lactosylceramide (LacCer) synthase, which functions in the initial step of ganglioside biosynthesis. Here, we generated conditional B4galt5 knockout (B4galt5 cKO) mice, using Nestin-Cre mice, and crossed these with B4galt6 KO mice to generate B4galt5 and 6 double KO (DKO) mice in the central nervous system (CNS). LacCer synthase activity and major brain gangliosides were completely absent in brain homogenates from the DKO mice, although LacCer synthase activity was about half its normal level in B4galt5 cKO mice and B4galt6 KO mice. The DKO mice were born normally but they showed growth retardation and motor deficits at 2 weeks and died by 4 weeks of age. Histological analyses showed that myelin-associated proteins were rarely found localized in axons in the cerebral cortex, and axonal and myelin formation were remarkably impaired in the spinal cords of the DKO mice. Neuronal cells, differentiated from neurospheres that were prepared from the DKO mice, showed impairments in neurite outgrowth and branch formation, which can be explained by the fact that neurospheres from DKO mice could weakly interact with laminin due to lack of gangliosides, such as GM1a. Furthermore, the neurons were immature and perineuronal nets (PNNs) were poorly formed in DKO cerebral cortices. Our results indicate that LacCer synthase is encoded by B4galt5 and 6 genes in the CNS, and that gangliosides are indispensable for neuronal maturation, PNN formation, and axonal and myelin formation.

Ganglioside GD2 Enhances the Malignant Phenotypes of Melanoma Cells by Cooperating with Integrins.

Gangliosides have been considered to modulate cell signals in the microdomain of the cell membrane, lipid/rafts, or glycolipid-enriched microdomain/rafts (GEM/rafts). In particular, cancer-associated gangliosides were reported to enhance the malignant properties of cancer cells. In fact, GD2-positive (GD2+) cells showed increased proliferation, invasion, and adhesion, compared with GD2-negative (GD2-) cells. However, the precise mechanisms by which gangliosides regulate cell signaling in GEM/rafts are not well understood. In order to analyze the roles of ganglioside GD2 in the malignant properties of melanoma cells, we searched for GD2-associating molecules on the cell membrane using the enzyme-mediated activation of radical sources combined with mass spectrometry, and integrin ß1 was identified as a representative GD2-associating molecule. Then, we showed the physical association of GD2 and integrin ß1 by immunoprecipitation/immunoblotting. Close localization was also shown by immuno-cytostaining and the proximity ligation assay. During cell adhesion, GD2+ cells showed multiple phospho-tyrosine bands, i.e., the epithelial growth factor receptor and focal adhesion kinase. The knockdown of integrin ß1 revealed that the increased malignant phenotypes in GD2+ cells were clearly cancelled. Furthermore, the phosphor-tyrosine bands detected during the adhesion of GD2+ cells almost completely disappeared after the knockdown of integrin ß1. Finally, immunoblotting to examine the intracellular distribution of integrins during cell adhesion revealed that large amounts of integrin ß1 were localized in GEM/raft fractions in GD2+ cells before and just after cell adhesion, with the majority being localized in the non-raft fractions in GD2- cells. All these results suggest that GD2 and integrin ß1 cooperate in GEM/rafts, leading to enhanced malignant phenotypes of melanomas.

The ceramide moiety of disialoganglioside (GD3) is essential for GD3 recognition by the sialic acid-binding lectin SIGLEC7 on the cell surface.

To analyze the binding specificity of a sialic acid-recognizing lectin, sialic acid-binding Ig-like lectin 7 (SIGLEC7), to disialyl gangliosides (GD3s), here we established GD3-expressing cells by introducing GD3 synthase (GD3S or ST8SIA1) cDNA into a colon cancer cell line, DLD-1, that expresses no ligands for the recombinant protein SIGLEC7-Fc. SIGLEC7-Fc did not recognize newly-expressed GD3 on DLD-1 cells, even though GD3 was highly expressed, as detected by an anti-GD3 antibody. Because milk-derived GD3 could be recognized by this fusion protein when incorporated onto the surface of DLD-1 cells, we compared the ceramides in DLD-1-generated and milk-derived GD3s to identify the SIGLEC7-specific GD3 structures on the cell membrane, revealing that SIGLEC7 recognizes only GD3-containing regular ceramides but not phytoceramides. This was confirmed by knockdown/knockout of the sphingolipid delta(4)-desaturase/C4-monooxygenase (DES2) gene, involved in phytoceramide synthesis, disclosing that DES2 inhibition confers SIGLEC7 binding. Furthermore, knocking out fatty acid 2-hydroxylase also resulted in the emergence of SIGLEC7 binding to the cell surface. To analyze the effects of binding between SIGLEC7 and various GD3 species on natural killer function, we investigated cytotoxicity of peripheral blood mononuclear cells from healthy donors toward GD3S-transfected DLD-1 (DLD-1-GD3S) cells and DLD-1-GD3S cells with modified ceramides. We found that cytotoxicity is suppressed in DLD-1-GD3S cells with dehydroxylated GD3s. These results indicate that the ceramide structures in glycosphingolipids affect SIGLEC7 binding and distribution on the cell surface and influence cell sensitivity to killing by SIGLEC7-expressing effector cells.