Palmitoylation of acetylated tubulin and association with ceramide-rich platforms is critical for ciliogenesis.

Microtubules are polymers composed of αß-tubulin subunits that provide structure to cells and play a crucial role in in the development and function of neuronal processes and cilia, microtubule-driven extensions of the plasma membrane that have sensory (primary cilia) or motor (motile cilia) functions. To stabilize microtubules in neuronal processes and cilia, α tubulin is modified by the posttranslational addition of an acetyl group, or acetylation. We discovered that acetylated tubulin in microtubules interacts with the membrane sphingolipid, ceramide. However, the molecular mechanism and function of this interaction are not understood. Here, we show that in human induced pluripotent stem cell-derived neurons, ceramide stabilizes microtubules, which indicates a similar function in cilia. Using proximity ligation assays, we detected complex formation of ceramide with acetylated tubulin in Chlamydomonas reinhardtii flagella and cilia of human embryonic kidney (HEK293T) cells, primary cultured mouse astrocytes, and ependymal cells. Using incorporation of palmitic azide and click chemistry-mediated addition of fluorophores, we show that a portion of acetylated tubulin is S-palmitoylated. S-palmitoylated acetylated tubulin is colocalized with ceramide-rich platforms in the ciliary membrane, and it is coimmunoprecipitated with Arl13b, a GTPase that mediates transport of proteins into cilia. Inhibition of S-palmitoylation with 2-bromo palmitic acid or inhibition of ceramide biosynthesis with fumonisin B1 reduces formation of the Arl13b-acetylated tubulin complex and its transport into cilia, concurrent with impairment of ciliogenesis. Together, these data show, for the first time, that ceramide-rich platforms mediate membrane anchoring and interaction of S-palmitoylated proteins that are critical for cilium formation, stabilization, and function.

Lipid transporter Spns2 promotes microglia pro-inflammatory activation in response to amyloid-beta peptide.

Accumulating evidence indicates that neuroinflammation contributes to the pathogenesis and exacerbation of neurodegenerative disorders, such as Alzheimer's disease (AD). Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid that regulates many pathophysiological processes including inflammation. We present evidence here that the spinster homolog 2 (Spns2), a S1P transporter, promotes microglia pro-inflammatory activation in vitro and in vivo. Spns2 knockout (Spns2KO) in primary cultured microglia resulted in significantly reduced levels of pro-inflammatory cytokines induced by lipopolysaccharide (LPS) and amyloid-beta peptide 1-42 oligomers (Aß42) when compared with littermate controls. Fingolimod (FTY720), a S1P receptor 1 (S1PR1) functional antagonist and FDA approved drug for relapsing-remitting multiple sclerosis, partially blunted Aß42-induced pro-inflammatory cytokine generation, suggesting that Spns2 promotes microglia pro-inflammatory activation through S1P-signaling. Spns2KO significantly reduced Aß42-induced nuclear factor kappa B (NFκB) activity. S1P increased, while FTY720 dampened, Aß42-induced NFκB activity, suggesting that Spns2 activates microglia inflammation through, at least partially, NFκB pathway. Spns2KO mouse brains showed significantly reduced Aß42-induced microglia activation/accumulation and reduced levels of pro-inflammatory cytokines when compared with age-matched controls. More interestingly, Spns2KO ameliorated Aß42-induced working memory deficit detected by Y-Maze. In summary, these results suggest that Spns2 promotes pro-inflammatory polarization of microglia and may play a crucial role in AD pathogenesis.

Novel function of ceramide for regulation of mitochondrial ATP release in astrocytes.

We reported that amyloid ß peptide (Aß42) activated neutral SMase 2 (nSMase2), thereby increasing the concentration of the sphingolipid ceramide in astrocytes. Here, we show that Aß42 induced mitochondrial fragmentation in wild-type astrocytes, but not in nSMase2-deficient cells or astrocytes treated with fumonisin B1 (FB1), an inhibitor of ceramide synthases. Unexpectedly, ceramide depletion was concurrent with rapid movements of mitochondria, indicating an unknown function of ceramide for mitochondria. Using immunocytochemistry and super-resolution microscopy, we detected ceramide-enriched and mitochondria-associated membranes (CEMAMs) that were codistributed with microtubules. Interaction of ceramide with tubulin was confirmed by cross-linking to N-[9-(3-pent-4-ynyl-3-H-diazirine-3-yl)-nonanoyl]-D-erythro-sphingosine (pacFACer), a bifunctional ceramide analog, and binding of tubulin to ceramide-linked agarose beads. Ceramide-associated tubulin (CAT) translocated from the perinuclear region to peripheral CEMAMs and mitochondria, which was prevented in nSMase2-deficient or FB1-treated astrocytes. Proximity ligation and coimmunoprecipitation assays showed that ceramide depletion reduced association of tubulin with voltage-dependent anion channel 1 (VDAC1), an interaction known to block mitochondrial ADP/ATP transport. Ceramide-depleted astrocytes contained higher levels of ATP, suggesting that ceramide-induced CAT formation leads to VDAC1 closure, thereby reducing mitochondrial ATP release, and potentially motility and resistance to Aß42 Our data also indicate that inhibiting ceramide generation may protect mitochondria in Alzheimer's disease.

Increased liver tumor formation in neutral sphingomyelinase-2-deficient mice.

Sphingolipids are key signaling lipids in cancer. Genome-wide studies have identified neutral SMase-2 (nSMase2), an enzyme generating ceramide from SM, as a potential repressor for hepatocellular carcinoma. However, little is known about the sphingolipids regulated by nSMase2 and their roles in liver tumor development. We discovered growth of spontaneous liver tumors in 27.3% (9 of 33) of aged male nSMase2-deficient (fro/fro) mice. Lipidomics analysis showed a marked increase of SM in the tumor. Unexpectedly, tumor tissues presented with more than a 7-fold increase of C16-ceramide, concurrent with upregulation of ceramide synthase 5. The fro/fro liver tumor, but not adjacent tissue, exhibited substantial accumulation of lipid droplets, suggesting that nSMase2 deficiency is associated with tumor growth and increased neutral lipid generation in the tumor. Tumor tissue expressed significantly increased levels of CD133 and EpCAM mRNA, two markers of liver cancer stem-like cells (CSCs) and higher levels of phosphorylated signal transducer and activator of transcription 3, an essential regulator of stemness. CD133(+) cells showed strong labeling for SM and ceramide. In conclusion, these results suggest that SMase-2 deficiency plays a role in the survival or proliferation of CSCs, leading to spontaneous tumors, which is associated with tumor-specific effects on lipid homeostasis.

Neutral Sphingomyelinase-2 Deficiency Ameliorates Alzheimer's Disease Pathology and Improves Cognition in the 5XFAD Mouse.

UNLABELLED: Recent evidence implicates exosomes in the aggregation of Aß and spreading of tau in Alzheimer's disease. In neural cells, exosome formation can be blocked by inhibition or silencing of neutral sphingomyelinase-2 (nSMase2). We generated genetically nSMase2-deficient 5XFAD mice (fro;5XFAD) to assess AD-related pathology in a mouse model with consistently reduced ceramide generation. We conducted in vitro assays to assess Aß42 aggregation and glial clearance with and without exosomes isolated by ultracentrifugation and determined exosome-induced amyloid aggregation by particle counting. We analyzed brain exosome content, amyloid plaque formation, neuronal degeneration, sphingolipid, Aß42 and phospho-tau levels, and memory-related behaviors in 5XFAD versus fro;5XFAD mice using contextual and cued fear conditioning. Astrocyte-derived exosomes accelerated aggregation of Aß42 and blocked glial clearance of Aß42 in vitro Aß42 aggregates were colocalized with extracellular ceramide in vitro using a bifunctional ceramide analog preloaded into exosomes and in vivo using anticeramide IgG, implicating ceramide-enriched exosomes in plaque formation. Compared with 5XFAD mice, the fro;5XFAD mice had reduced brain exosomes, ceramide levels, serum anticeramide IgG, glial activation, total Aß42 and plaque burden, tau phosphorylation, and improved cognition in a fear-conditioned learning task. Ceramide-enriched exosomes appear to exacerbate AD-related brain pathology by promoting the aggregation of Aß. Reduction of exosome secretion by nSMase2 loss of function improves pathology and cognition in the 5XFAD mouse model. SIGNIFICANCE STATEMENT: We present for the first time evidence, using Alzheimer's disease (AD) model mice deficient in neural exosome secretion due to lack of neutral sphingomyelinase-2 function, that ceramide-enriched exosomes exacerbate AD-related pathologies and cognitive deficits. Our results provide rationale to pursue a means of inhibiting exosome secretion as a potential therapy for individuals at risk for developing AD.

Sphingosine Toxicity in EAE and MS: Evidence for Ceramide Generation via Serine-Palmitoyltransferase Activation.

Multiple sclerosis (MS) is a demyelinating disorder characterized by massive neurodegeneration and profound axonal loss. Since myelin is enriched with sphingolipids and some of them display toxicity, biological function of sphingolipids in demyelination has been investigated in MS brain tissues. An elevation of sphingosine with a decrease in monoglycosylceramide and psychosine (myelin markers) was observed in MS white matter and plaque compared to normal brain tissue. This indicated that sphingosine toxicity might mediate oligodendrocyte degeneration. To explain the source of sphingosine accumulation, total sphingolipid profile was investigated in Lewis rats after inducing experimental autoimmune encephalomyelitis (EAE) and also in human oligodendrocytes in culture. An intermittent increase in ceramide followed by sphingosine accumulation in EAE spinal cord along with a stimulation of serine-palmitoyltransferase (SPT) activity was observed. Apoptosis was identified in the lumbar spinal cord, the most prominent demyelinating area, in the EAE rats. TNFα and IFNγ stimulation of oligodendrocytes in culture also led to an accumulation of ceramide with an elevation of sphingosine. Ceramide elevation was drastically blocked by myriocin, an inhibitor of SPT, and also by FTY720. Myriocin treatment also protected oligodendrocytes from cytokine mediated apoptosis or programmed cell death. Hence, we propose that sphingosine toxicity may contribute to demyelination in both EAE and MS, and the intermittent ceramide accumulation in EAE may, at least partly, be mediated via SPT activation, which is a novel observation that has not been previously reported.

Guggulsterone and bexarotene induce secretion of exosome-associated breast cancer resistance protein and reduce doxorubicin resistance in MDA-MB-231 cells.

Many breast cancer cells acquire multidrug resistance (MDR) mediated by ABC transporters such as breast cancer resistance protein (BCRP/ABCG2). Here we show that incubation of human breast cancer MDA-MB-231 cells with farnesoid X receptor antagonist guggulsterone (gug) and retinoid X receptor agonist bexarotene (bex) elevated ceramide, a sphingolipid known to induce exosome secretion. The gug+bex combination reduced cellular levels of BCRP to 20% of control cells by inducing its association and secretion with exosomes. Exogenous C6 ceramide also induced secretion of BCRP-associated exosomes, while siRNA-mediated knockdown or GW4869-mediated inhibition of neutral sphingomyelinase 2 (nSMase2), an enzyme generating ceramide, restored cellular BCRP. Immunocytochemistry showed that ceramide elevation and concurrent loss of cellular BCRP was prominent in Aldefluor-labeled breast cancer stem-like cells. These cells no longer excluded the BCRP substrate Hoechst 33342 and showed caspase activation and apoptosis induction. Consistent with reduced BCRP, ABC transporter assays showed that gug+bex increased doxorubicin retention and that the combination of gug+bex with doxorubicin enhanced cell death by more than fivefold. Taken together, our results suggest a novel mechanism by which ceramide induces BCRP secretion and reduces MDR, which may be useful as adjuvant drug treatment for sensitizing breast cancer cells and cancer stem cells to chemotherapy.

Astrocytes secrete exosomes enriched with proapoptotic ceramide and prostate apoptosis response 4 (PAR-4): potential mechanism of apoptosis induction in Alzheimer disease (AD).

Amyloid protein is well known to induce neuronal cell death, whereas only little is known about its effect on astrocytes. We found that amyloid peptides activated caspase 3 and induced apoptosis in primary cultured astrocytes, which was prevented by caspase 3 inhibition. Apoptosis was also prevented by shRNA-mediated down-regulation of PAR-4, a protein sensitizing cells to the sphingolipid ceramide. Consistent with a potentially proapoptotic effect of PAR-4 and ceramide, astrocytes surrounding amyloid plaques in brain sections of the 5xFAD mouse (and Alzheimer disease patient brain) showed caspase 3 activation and were apoptotic when co-expressing PAR-4 and ceramide. Apoptosis was not observed in astrocytes with deficient neutral sphingomyelinase 2 (nSMase2), indicating that ceramide generated by nSMase2 is critical for amyloid-induced apoptosis. Antibodies against PAR-4 and ceramide prevented amyloid-induced apoptosis in vitro and in vivo, suggesting that apoptosis was mediated by exogenous PAR-4 and ceramide, potentially associated with secreted lipid vesicles. This was confirmed by the analysis of lipid vesicles from conditioned medium showing that amyloid peptide induced the secretion of PAR-4 and C18 ceramide-enriched exosomes. Exosomes were not secreted by nSMase2-deficient astrocytes, indicating that ceramide generated by nSMase2 is critical for exosome secretion. Consistent with the ceramide composition in amyloid-induced exosomes, exogenously added C18 ceramide restored PAR-4-containing exosome secretion in nSMase2-deficient astrocytes. Moreover, isolated PAR-4/ceramide-enriched exosomes were taken up by astrocytes and induced apoptosis in the absence of amyloid peptide. Taken together, we report a novel mechanism of apoptosis induction by PAR-4/ceramide-enriched exosomes, which may critically contribute to Alzheimer disease.

Regulation of embryonic stem cell pluripotency by heat shock protein 90.

Deciphering the molecular basis of stem cell pluripotency is fundamental to the understanding of stem cell biology, early embryonic development, and to the clinical application of regenerative medicine. We report here that the molecular chaperone heat shock protein 90 (Hsp90) is essential for mouse embryonic stem cell (ESC) pluripotency through regulating multiple pluripotency factors, including Oct4, Nanog, and signal transducer and activator of transcription 3. Inhibition of Hsp90 by either 17-N-Allylamino-17-demethoxygeldanamycin or miRNA led to ESC differentiation. Overexpression of Hsp90ß partially rescued the phenotype; in particular, the levels of Oct4 and Nanog were restored. Notably, Hsp90 associated with Oct4 and Nanog in the same cellular complex and protected them from degradation by the ubiquitin proteasome pathway, suggesting that Oct4 and Nanog are potential novel Hsp90 client proteins. In addition, Hsp90 inhibition reduced the mRNA level of Oct4, but not that of Nanog, indicating that Hsp90 participates in Oct4 mRNA processing or maturation. Hsp90 inhibition also increased expression of some protein markers for mesodermal lineages, implying that Hsp90 suppresses mesodermal differentiation from ESCs. These findings support a new role for Hsp90 in maintaining ESC pluripotency by sustaining the level of multiple pluripotency factors, particularly Oct4 and Nanog.

Protection of murine neural progenitor cells by the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin in the low nanomolar concentration range.

Stem cell-based approaches provide hope as a potential therapy for neurodegenerative diseases and stroke. One of the major scientific hurdles for stem cell therapy is the poor survival rate of the newly formed or transplanted neural stem cells. In this study, we found that low-dose treatment with the Heat shock protein 90 (Hsp90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), a heavily investigated anti-cancer drug, prevented neural progenitor cells from either naturally-occurring or stress-induced apoptosis, although it induced apoptosis at higher doses. This stress adaptation effect mediated by low-dose 17-AAG is accompanied by activation of multiple cell survival pathways, including the stress response pathway (induction of Hsp70), the MAPK pathway, and the PI3K/Akt pathway. When administered in vivo, 17-AAG led to Akt and glycogen synthase kinase 3ß phosphorylation, and more 5-bromo-2'-deoxyuridine positive cells in the mouse brain. These findings could have profound implications in stem cell therapy for neurodegenerative diseases and stroke.

Sulfoglucuronosyl paragloboside promotes endothelial cell apoptosis in inflammation: elucidation of a novel glycosphingolipid-signaling pathway.

Sulfoglucuronosyl paragloboside (SGPG), a minor glycosphingolipid of endothelial cells, is a ligand for L-selectin and has been implicated in neuro-inflammatory diseases, such as Guillian-Barré syndrome. Inflammatory cytokines, such as TNFα and IL-1ß, up-regulate SGPG expression by stimulating gene expression for glucuronosyltransferases, both P and S forms (GlcATp and GlcATs), and the human natural killer antigen (HNK-1) sulfotransferase (HNK-1 ST). Transfection of a human cerebromicrovascular endothelial cell (SV-HCEC) line with HNK-1 ST siRNA down-regulated SGPG expression, inhibited cytokine-stimulated T-cell adhesion, and offered protection against apoptosis. However, the precise mechanisms of SGPG elevation in endothelial cell apoptosis and the maintenance of blood-brain or blood-nerve barrier integrity in inflammation have not been elucidated. Blocking SGPG expression inhibited cytokine-mediated stimulation of NF-κB activity but stimulated MAP kinase activity. Furthermore, elevation of SGPG by over-expression of GlcATp and GlcATs triggered endothelial cell apoptosis, with GlcATs being more potent than GlcATp. Although SGPG-mediated endothelial cell apoptosis was preceded by inhibiting the intracellular NF-κB activity, interfering with Akt and ERK activation and stimulating caspase 3 in SV-HCECs, HNK-1ST siRNA transfection also interfered with IκB phosphorylation but stimulated ERK activation. Our data indicate that SGPG is a critical regulatory molecule for maintaining endothelial cell survival and blood-brain or blood-nerve barrier function.

The temporal and spatial expression pattern of the LGI1 epilepsy predisposition gene during mouse embryonic cranial development.

BACKGROUND: Mutations in the LGI1 gene predispose to a rare, hereditary form of temporal epilepsy. Currently, little is known about the temporal and spatial expression pattern of Lgi1 during normal embryogenesis and so to define this more clearly we used a transgenic mouse line that expresses GFP under the control of Lgi1 cis-regulatory elements. RESULTS: During embryonic brain growth, high levels of Lgi1 expression were found in the surface ectoderm, the neuroepithelium, mesenchymal connective tissue, hippocampus, and sensory organs, such as eye, tongue, and the olfactory bulb. Lgi1 was also found in the cranial nerve nuclei and ganglia, such as vestibular, trigeminal, and dorsal ganglia. Expression of Lgi1 followed an orchestrated pattern during mouse development becoming more subdued in areas of the neocortex of the mid- and hind-brain in early postnatal animals, although high expression levels were retained in the choroid plexus and hippocampus. In late postnatal stages, Lgi1 expression continued to be detected in many areas in the brain including, hippocampus, paraventricular thalamic nuclei, inferior colliculus, and the cerebral aqueduct. We also showed that Lgi1-expressing cells co-express nestin, DCX, and beta-III tubulin suggesting that Lgi1-expressing cells are migratory neuroblasts. CONCLUSION: These observations imply that Lgi1 may have a role in establishing normal brain architecture and neuronal functions during brain development suggesting that it may be involved in neurogenesis and neuronal plasticity, which become more specifically defined in the adult animal.

Extracellular Vesicles Containing Ceramide-Rich Platforms: "Mobile Raft" Isolation and Analysis.

Extracellular vesicles (EVs) are secreted by eukaryotic cells and serve as carriers for a variety of cell signaling factors, including RNAs, proteins, and lipids. We described a unique population of EVs, the membrane of which is highly enriched with the sphingolipid ceramide. We suggested that ceramide in the EV membrane is organized in ceramide-rich platforms (CRPs), a type of lipid raft that mediates interaction of ceramide with ceramide-associated proteins (CAPs). Here, we describe methods using anti-ceramide antibody to isolate ceramide-enriched EVs and detect exosomes after uptake into recipient cells. In addition, we discuss methods for EV analysis using nanoparticle tracking and mass spectrometry. The methods can be extended to the isolation of other types of EVs and "mobile rafts" transported by EVs from donor to recipient cells using antibodies against lipids specific for these EVs.

Functional pre- mRNA trans-splicing of coactivator CoAA and corepressor RBM4 during stem/progenitor cell differentiation.

Alternative splicing yields functionally distinctive gene products, and their balance plays critical roles in cell differentiation and development. We have previously shown that tumor-associated enhancer loss in coactivator gene CoAA leads to its altered alternative splicing. Here we identified two intergenic splicing variants, a zinc finger-containing coactivator CoAZ and a non-coding transcript ncCoAZ, between CoAA and its downstream corepressor gene RBM4. During stem/progenitor cell neural differentiation, we found that the switched alternative splicing and trans-splicing between CoAA and RBM4 transcripts result in lineage-specific expression of wild type CoAA, RBM4, and their variants. Stable expression of CoAA, RBM4, or their variants prevents the switch and disrupts the embryoid body formation. In addition, CoAA and RBM4 counter-regulate the target gene Tau at exon 10, and their splicing activities are subjected to the control by each splice variant. Further phylogenetic analysis showed that mammalian CoAA and RBM4 genes share common ancestry with the Drosophila melanogaster gene Lark, which is known to regulate early development and circadian rhythms. Thus, the trans-splicing between CoAA and RBM4 transcripts may represent a required regulation preserved during evolution. Our results demonstrate that a linked splicing control of transcriptional coactivator and corepressor is involved in stem/progenitor cell differentiation. The alternative splicing imbalance of CoAA and RBM4, because of loss of their common enhancer in cancer, may deregulate stem/progenitor cell differentiation.

Association of Aß with ceramide-enriched astrosomes mediates Aß neurotoxicity.

Amyloid-ß (Aß) associates with extracellular vesicles termed exosomes. It is not clear whether and how exosomes modulate Aß neurotoxicity in Alzheimer's disease (AD). We show here that brain tissue and serum from the transgenic mouse model of familial AD (5xFAD) and serum from AD patients contains ceramide-enriched and astrocyte-derived exosomes (termed astrosomes) that are associated with Aß. In Neuro-2a cells, primary cultured neurons, and human induced pluripotent stem cell-derived neurons, Aß-associated astrosomes from 5xFAD mice and AD patient serum were specifically transported to mitochondria, induced mitochondrial clustering, and upregulated the fission protein Drp-1 at a concentration corresponding to 5 femtomoles Aß/L of medium. Aß-associated astrosomes, but not wild type or control human serum exosomes, mediated binding of Aß to voltage-dependent anion channel 1 (VDAC1) and subsequently, activated caspases. Aß-associated astrosomes induced neurite fragmentation and neuronal cell death, suggesting that association with astrosomes substantially enhances Aß neurotoxicity in AD and may comprise a novel target for therapy.

Regulation of primary cilia formation by ceramide.

The primary cilium is an important sensory organelle, the regulation of which is not fully understood. We found that in polarized Madin-Darby Canine Kidney cells, the sphingolipid ceramide is specifically distributed to a cis-Golgi compartment at the base of the primary cilium. This compartment immunostained for the centrosome marker gamma-tubulin, the Rho type GTPase cell division cycle 42 (Cdc42), and atypical protein kinase Czeta/lambda (aPKC), a kinase activated by ceramide and associated with a polarity protein complex consisting of partitioning defective (Par)6 and Cdc42. Inhibition of ceramide biosynthesis with Fumonisin B1 prevented codistribution of aPKC and Cdc42 in the centrosomal/pericentriolar compartment and severely impaired ciliogenesis. Cilium formation and codistribution of aPKC and Cdc42 were restored by incubation with N-acetyl or N-palmitoyl sphingosine (C2 or C16 ceramide), or the ceramide analog N-oleoyl serinol (S18). Cilium formation was also restored by the glycogen synthase kinase-3beta (GSK-3beta) inhibitor indirubin-3-monoxime, suggesting that regulation of ciliogenesis depends on the inhibition of GSK-3beta by ceramide-activated aPKC. Consistently, inhibition of aPKC with a pseudosubstrate inhibitor prevented restoration of ciliogenesis by C2 ceramide or S18. Our data show for the first time that ceramide is required for primary cilium formation.

Sulfoglucuronosyl paragloboside is a ligand for T cell adhesion: regulation of sulfoglucuronosyl paragloboside expression via nuclear factor kappaB signaling.

Inflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta stimulate glucuronosyltransferase genes (S and P) in endothelial cells (ECs) and up-regulate sulfoglucuronosyl paragloboside (SGPG) expression, which serves as a ligand for T cell adhesion. However, the mechanism of cytokine-mediated gene up-regulation has not been elucidated. To evaluate the precise mechanism of SGPG up-regulation, we have specifically inhibited the SGPG synthesis in the cerebromicrovascular EC line (SV-HCECs), a transformed brain ECs of human origin. SV-HCECs were transfected with small interfering RNA designed to mimic the human natural killer epitope-1 sulfotransferase (HNK-1ST), the ultimate enzyme that transfers the sulfate group to glucuronic acid for SGPG synthesis. An inhibition of SGPG expression along with a reduction of human CD4(+) cell adhesion was observed in siRNA HNK-1ST (siHNK-1)-transfected cells after TNFalpha stimulation. A thorough screening of the signaling system confirmed that TNFalpha/IL-1beta stimulation up-regulated nuclear factor kappaB (NFkappaB) signaling in SV-HCECs. siHNK-1 transfection interfered with the SGPG up-regulation after TNFalpha/IL-1beta stimulation in transfected cells and reduced the T cell adhesion. Hence, our study indicates that T cell-SGPG adhesion in SV-HCECs may proceed through NFkappaB activation. In addition, siHNK-1 transfection reduced the NFkappaB activity compared with cells that were transfected with scrambled siRNA, before and after TNFalpha/IL-1beta stimulation. This is the first report indicating that NFkappaB signaling is involved in SGPG gene expression in brain ECs by an unknown mechanism. Its down-regulation by inhibiting HNK-1ST expression may have a potential use in preventing the T cell invasion and consequently nerve damage during inflammation.

Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents teratoma formation and enriches for neural precursors in ES cell-derived neural transplants.

The formation of stem cell-derived tumors (teratomas) is observed when engrafting undifferentiated embryonic stem (ES) cells, embryoid body-derived cells (EBCs), or mammalian embryos and is a significant obstacle to stem cell therapy. We show that in tumors formed after engraftment of EBCs into mouse brain, expression of the pluripotency marker Oct-4 colocalized with that of prostate apoptosis response-4 (PAR-4), a protein mediating ceramide-induced apoptosis during neural differentiation of ES cells. We tested the ability of the novel ceramide analogue N-oleoyl serinol (S18) to eliminate mouse and human Oct-4(+)/PAR-4(+) cells and to increase the proportion of nestin(+) neuroprogenitors in EBC-derived cell cultures and grafts. S18-treated EBCs persisted in the hippocampal area and showed neuronal lineage differentiation as indicated by the expression of beta-tubulin III. However, untreated cells formed numerous teratomas that contained derivatives of endoderm, mesoderm, and ectoderm. Our results show for the first time that ceramide-induced apoptosis eliminates residual, pluripotent EBCs, prevents teratoma formation, and enriches the EBCs for cells that undergo neural differentiation after transplantation.

Visualization of Ceramide-Associated Proteins in Ceramide-Rich Platforms Using a Cross-Linkable Ceramide Analog and Proximity Ligation Assays With Anti-ceramide Antibody.

Ceramide-rich platforms (CRPs) mediate association of proteins with the sphingolipid ceramide and may regulate protein interaction in membrane contact sites to the cytoskeleton, organelles, and infectious pathogens. However, visualization of ceramide association to proteins is one of the greatest challenges in understanding the cell biology of ceramide. Here we introduce a novel labeling technique for ceramide-associated proteins (CAPs) by combining photoactivated cross-linking of a bioorthogonal and bifunctional ceramide analog, pacFACer with proximity ligation assays (PLAs). pacFACer cross-linked to CAPs is covalently attached to a fluorophore using click chemistry. PLAs use antibodies to: (1) the candidate CAP and the fluorophore (PLA1); and (2) the CAP and ceramide (PLA2). PLA1 shows the subcellular localization of a particular CAP that is cross-linked to pacFACer, while PLA2 tests if the cross-linked CAP forms a complex with endogenous ceramide. Two proteins, tubulin and voltage-dependent anion channel 1 (VDAC1), were cross-linked to pacFACer and showed PLA signals for a complex with ceramide and pacFACer, which were predominantly colocalized with microtubules and mitochondria, respectively. Binding of tubulin and VDAC1 to ceramide was confirmed by coimmunoprecipitation assays using anti ceramide antibody. Cross-linking to pacFACer was confirmed using click chemistry-mediated attachment of biotin and streptavidin pull-down assays. Inhibition of ceramide synthases with fumonisin B1 (FB1) reduced the degree of pacFACer cross-linking and complex formation with ceramide, while it was enhanced by amyloid beta peptide (Aß). Our results show that endogenous ceramide is critical for mediating cross-linking of CAPs to pacFACer and that a combination of cross-linking with PLAs (cross-link/PLA) is a novel tool to visualize CAPs and to understand the regulation of protein interaction with ceramide in CRPs.

Ceramide regulates interaction of Hsd17b4 with Pex5 and function of peroxisomes.

The sphingolipid ceramide regulates beta-oxidation of medium and long chain fatty acids in mitochondria. It is not known whether it also regulates oxidation of very long chain fatty acids (VLCFAs) in peroxisomes. Using affinity chromatography, co-immunoprecipitation, and proximity ligation assays we discovered that ceramide interacts with Hsd17b4, an enzyme critical for peroxisomal VLCFA oxidation and docosahexaenoic acid (DHA) generation. Immunocytochemistry showed that Hsd17b4 is distributed to ceramide-enriched mitochondria-associated membranes (CEMAMs). Molecular docking and in vitro mutagenesis experiments showed that ceramide binds to the sterol carrier protein 2-like domain in Hsd17b4 adjacent to peroxisome targeting signal 1 (PTS1), the C-terminal signal for interaction with peroxisomal biogenesis factor 5 (Pex5), a peroxin mediating transport of Hsd17b4 into peroxisomes. Inhibition of ceramide biosynthesis induced translocation of Hsd17b4 from CEMAMs to peroxisomes, interaction of Hsd17b4 with Pex5, and upregulation of DHA. This data indicates a novel role of ceramide as a molecular switch regulating interaction of Hsd17b4 with Pex5 and peroxisomal function.