Isolation of Lipid Rafts (Detergent-Resistant Microdomains) and Comparison to Extracellular Vesicles (Exosomes).

Lipid rafts (LRs) represent cellular microdomains enriched in sphingolipids and cholesterol which may fuse to form platforms in which signaling molecules can be organized and regulated (Simons and Ikonen, Nature 387:569-572, 1997; Pike, Biochem J 378:281-292, 2004; Grassme et al., J Immunol 168: 300-307, 2002; Cheng et al., J Exp Med 190:1549-1550, 1999; Kilkus et al., J Neurosci Res 72(1) 62-75, 2003). In a proposed Model 1 (Cheng et al., J Exp Med 190:1549-1550, 1999) the LR has a well-ordered central core composed mainly of cholesterol and sphingolipids that is surrounded by a zone of decreasing lipid order. Detergents such as Triton X-100 can solubilize the core (and a significant amount of phosphoglyceride), but the LRs will be insoluble at 4 °C and be enriched in a well-characterized set of biomarkers. Model 2 proposes that the LRs are homogeneous, but there is selectivity in the lipids (and proteins) extracted by the 1% Triton X-100. Model 3 proposes LRs with distinct lipid compositions are highly structured and can be destroyed by binding molecules such as beta-methylcyclodextrin or filipin. These may be Caveolin in some cell types but not in brain. Since it is unlikely that two LR preparations will be exactly the same this review will concentrate on LRs defined as "small (50 nm) membranous particles which are insoluble in 1% Triton X-100 at 4 °C and have a low buoyant density (Simons and Ikonen, Nature 387:569-572, 1997; Pike, Biochem J 378:281-292, 2004; Grassme et al., J Immunol 168: 300-307, 2002; Cheng et al., J Exp Med 190:1549-1550, 1999; Kilkus et al., J Neurosci Res 72(1):62-75, 2003; Testai et al., J Neurochem 89:636-644, 2004). We will present a generic method for isolating LRs for both lipidomic, proteomic, and cellular signaling analysis [1-6].

Global Brain Transcriptome Analysis of a Tpp1 Neuronal Ceroid Lipofuscinoses Mouse Model.

In humans, homozygous mutations in the TPP1 gene results in loss of tripeptidyl peptidase 1 (TPP1) enzymatic activity, leading to late infantile neuronal ceroid lipofuscinoses disease. Using a mouse model that targets the Tpp1 gene and recapitulates the pathology and clinical features of the human disease, we analyzed end-stage (4 months) transcriptional changes associated with lack of TPP1 activity. Using RNA sequencing technology, Tpp1 expression changes in the forebrain/midbrain and cerebellum of 4-month-old homozygotes were compared with strain-related controls. Transcriptional changes were found in 510 and 1,550 gene transcripts in forebrain/midbrain and cerebellum, respectively, from Tpp1-deficient brain tissues when compared with age-matched controls. Analysis of the differentially expressed genes using the Ingenuity™ pathway software, revealed increased neuroinflammation activity in microglia and astrocytes that could lead to neuronal dysfunction, particularly in the cerebellum. We also observed upregulation in the production of nitric oxide and reactive oxygen species; activation of leukocyte extravasation signals and complement pathways; and downregulation of major transcription factors involved in control of circadian rhythm. Several of these expression changes were confirmed by independent quantitative polymerase chain reaction and histological analysis by mRNA in situ hybridization, which allowed for an in-depth anatomical analysis of the pathology and provided independent confirmation of at least two of the major networks affected in this model. The identification of differentially expressed genes has revealed new lines of investigation for this complex disorder that may lead to novel therapeutic targets.

Connecting Exosomes and Connexins.

Intercellular communication is accomplished by passage of ions and small molecules through gap junction channels in directly contacting cells or by secretion and response to transmitters, hormones and extracellular vesicles in cells that are distant from each other. Recent studies have suggested that there may be overlap of these processes; specifically, small extracellular vesicles may contain subunit gap junction proteins, connexins. We isolated and analyzed extracellular vesicles secreted by cultured microvascular endothelial cells. These vesicles had a diameter of ~120 nm. They contained four exosomal proteins (flotillin-1, CD63, CD81 and Alix) and the gap junction protein, connexin43. They did not contain an endoplasmic reticulum protein (Grp94) or an adherens junction protein (VE-cadherin). Secretion of vesicles was increased by treatment of the cells with staurosporine. Our data confirm that the gap junction protein, connexin43, can be secreted in vesicles with the properties of exosomes. Although the role of vesicular connexin is not clearly known, we speculate that it might participate in docking/fusion of the exosomes with the recipient cell, transmission of vesicular contents, or cellular signaling.

The cross roles of sphingosine kinase 1/2 and ceramide glucosyltransferase in cell growth and death.

Sphingosine-1-phosphate is synthesized by two sphingosine kinases, cytosolic SK1 and nuclear SK2 but SK2 expression was much higher than SK1in mouse skin fibroblasts. However, in SK2-/- cells, SK1 expression was markedly increased to SK2 levels whereas in SK1-/- cells, SK2 expression was unaffected. Ceramide, glucosylceramide and sphingosine levels were all increased in SK1-/- but less so in SK2-/- cells and S1P levels were not significantly reduced in either SK1-/- or SK2-/- cells. Greatly increased Ceramide glucosyltransferase expression was observed in SK1-/- cells but less so in SK2-/- cells suggested a role in drug resistance. SK2-/- cells grew faster than control and SK1-/-. The cell division gene PCNA was significantly overexpressed in SK2-/- cells, suggesting a cross regulation between SKs and Ceramide glucosyltransferase.

Quantum dots and potential therapy for Krabbe's disease.

Enzyme replacement therapy and substrate reduction therapy have proved useful in reversing many pathological consequences of many nonneural lysosomal storage diseases but have not yet reversed pathology or influenced disease outcome in Krabbe's disease (KD). This Review discusses the relative merits of stem cell therapy, molecular chaperone therapy, gene therapy, substrate reduction therapy, enzyme replacement therapy, and combination therapy. Given the limitations of these approaches, this Review introduces the idea of using tiny, 6-nm, intensely fluorescent quantum dots (QDs) to deliver a cell-penetrating peptide and 6 histidine residue-tagged ß-D-galactocerebrosidase across the blood-brain barrier. We can therefore follow the fate of injected material and ensure that all targets are reached and that accumulated material is degraded. Uptake of lysosomal hydrolases is a complex process, and the cell-penetrating peptide JB577 is uniquely able to promote endosomal egress of the QD cargo. This Review further shows that uptake may depend on the charge of the coating of the QD, specifically, that negative charge directs the cargo to neurons. Because KD involves primarily glia, specifically oligodendroglia, we experiment with many coatings and discover a coating (polyethylene glycol 600 amino) that has a positive charge and targets oligodendrocytes. A similar effect is achieved by treating with chondroitinase ABC to degrade the extracellular matrix, indicating that enzyme replacement has several hurdles to overcome before it can become a routine CNS therapy. © 2016 Wiley Periodicals, Inc.

Quantum dot-mediated delivery of siRNA to inhibit sphingomyelinase activities in brain-derived cells.

The use of RNAi to suppress protein synthesis offers a potential way of reducing the level of enzymes or the synthesis of mutant toxic proteins but there are few tools currently available for their delivery. To address this problem, bioconjugated quantum dots (QDs) containing a hydrophobic component (N-palmitate) and a sequence VKIKK designed to traverse across cell membranes and visualize drug delivery were developed and tested on cell lines of brain origin. We used the Zn outer shell of the QD to bind HIS6 in JB577 (W•G•Dap(N-Palmitoyl)•VKIKK•P9 •G2 •H6 ) and by a gel-shift assay showed that siRNAs would bind to the positively charged KIKK sequence. By comparing many peptides and QD coatings, we showed that the QD-JB577-siRNA construct was taken up by cells of nervous system origin, distributed throughout the cytosol, and inhibited protein synthesis, implying that JB577 was also promoting endosome egress. By attaching siRNA for luciferase in a cell line over-expressing luciferase, we showed 70% inhibition of mRNA after 24-48 h. To show more specific effects, we synthesized siRNA for neutral (NSMase2), acid (lysosomal ASMase) sphingomyelinase, and sphingosine kinase 1 (SK1), we demonstrated a dose-dependent inhibition of activity. These data suggest that QDs are a useful siRNA delivery tool and QD-siRNA could be a potential theranostic for a variety of diseases.

Neuronal ceroid lipofuscinosis with DNAJC5/CSPα mutation has PPT1 pathology and exhibit aberrant protein palmitoylation.

Neuronal ceroid lipofuscinoses (NCL) are a group of inherited neurodegenerative disorders with lysosomal pathology (CLN1-14). Recently, mutations in the DNAJC5/CLN4 gene, which encodes the presynaptic co-chaperone CSPα were shown to cause autosomal-dominant NCL. Although 14 NCL genes have been identified, it is unknown if they act in common disease pathways. Here we show that two disease-associated proteins, CSPα and the depalmitoylating enzyme palmitoyl-protein thioesterase 1 (PPT1/CLN1) are biochemically linked. We find that in DNAJC5/CLN4 patient brains, PPT1 is massively increased and mis-localized. Surprisingly, the specific enzymatic activity of PPT1 is dramatically reduced. Notably, we demonstrate that CSPα is depalmitoylated by PPT1 and hence its substrate. To determine the consequences of PPT1 accumulation, we compared the palmitomes from control and DNAJC5/CLN4 patient brains by quantitative proteomics. We discovered global changes in protein palmitoylation, mainly involving lysosomal and synaptic proteins. Our findings establish a functional link between two forms of NCL and serve as a springboard for investigations of NCL disease pathways.

The hyaluronic acid inhibitor 4-methylumbelliferone is an NSMase2 activator-role of Ceramide in MU anti-tumor activity.

Increased synthesis of hyaluronic acid (HA) is often associated with increased metastatic potential and invasivity of tumor cells. 4-Methylumbelliferone (MU) is an inhibitor of HA synthesis, and has been studied as a potential anti-tumor drug to inhibit the growth of primary tumors and distant metastasis of tumor cells. Although several studies reported that the anticancer effects of MU are mediated by inhibition of HA signaling, the mechanism still needs to be clarified. In a previous study we demonstrated the regulation of HA synthesis by ceramide, and now show how MU activated neutral sphingomyelinase2 (NSMase2) generates ceramides and mediates MU induced inhibition of HA synthesis, cell migration and invasion, and apoptosis of tumor cells. Using a HA enriched mouse oligodendroglioma cell line G26-24 we found that MU elevated the activity of NSMase2 and increased ceramide levels, which in turn increased phosphatase PP2A activity. Further, the activated PP2A reduced phosphorylation of Akt, decreased activities of HA synthase2 (HAS2) and calpains, and inhibited both the synthesis of HA, and the migration and invasion of G26-24 tumor cells. In addition, MU mediated ceramide stimulated activation of p53 and caspase-3, reduced SIRT1 expression and decreased G26-24 viability. The mechanism of the MU anticancer therefore initially involves NSMase2/ceramide/PP2A/AKT/HAS2/caspase-3/p53/SIRT1 and the calpain signaling pathway, suggesting that ceramides play a key role in the ability of a tumor to become aggressively metastatic and grow.

The Role of Negative Charge in the Delivery of Quantum Dots to Neurons.

Despite our extensive knowledge of the structure of negatively charged cell surface proteoglycans and sialoglycoconjugates in the brain, we have little understanding of how their negative charge contributes to brain function. We have previously shown that intensely photoluminescent 9-nm diameter quantum dots (QDs) with a CdSe core, a ZnS shell, and a negatively charged compact molecular ligand coating (CL4) selectively target neurons rather than glia. We now provide an explanation for this selective neuronal delivery. In this study, we compared three zwitterionic QD coatings differing only in their regions of positive or negative charge, as well as a positively charged (NH2) polyethylene glycol (PEG) coat, for their ability to deliver the cell-membrane-penetrating chaperone lipopeptide JB577 (WG(Palmitoyl)VKIKKP9G2H6) to individual cells in neonatal rat hippocampal slices. We confirm both that preferential uptake in neurons, and the lack of uptake in glia, is strongly associated with having a region of greater negative charge on the QD coating. In addition, the role of negatively charged chondroitin sulfate of the extracellular matrix (ECM) in restricting uptake was further suggested by digesting neonatal rat hippocampal slices with chondroitinase ABC and showing increased uptake of QDs by oligodendrocytes. Treatment still did not affect uptake in astrocytes or microglia. Finally, the future potential of using QDs as vehicles for trafficking proteins into cells continues to show promise, as we show that by administering a histidine-tagged green fluorescent protein (eGFP-His6) to hippocampal slices, we can observe neuronal uptake of GFP.

Delivery and tracking of quantum dot peptide bioconjugates in an intact developing avian brain.

Luminescent semiconductor ∼9.5 nm nanoparticles (quantum dots: QDs) have intrinsic physiochemical and optical properties which enable us to begin to understand the mechanisms of nanoparticle mediated chemical/drug delivery. Here, we demonstrate the ability of CdSe/ZnS core/shell QDs surface functionalized with a zwitterionic compact ligand to deliver a cell-penetrating lipopeptide to the developing chick embryo brain without any apparent toxicity. Functionalized QDs were conjugated to the palmitoylated peptide WGDap(Palmitoyl)VKIKKP9GGH6, previously shown to uniquely facilitate endosomal escape, and microinjected into the embryonic chick spinal cord canal at embryo day 4 (E4). We were subsequently able to follow the labeling of spinal cord extension into the ventricles, migratory neuroblasts, maturing brain cells, and complex structures such as the choroid plexus. QD intensity extended throughout the brain, and peaked between E8 and E11 when fluorescence was concentrated in the choroid plexus before declining to hatching (E21/P0). We observed no abnormalities in embryonic patterning or embryo survival, and mRNA in situ hybridization confirmed that, at key developmental stages, the expression pattern of genes associated with different brain cell types (brain lipid binding protein, Sox-2, proteolipid protein and Class III-ß-Tubulin) all showed a normal labeling pattern and intensity. Our findings suggest that we can use chemically modified QDs to identify and track neural stem cells as they migrate, that the choroid plexus clears these injected QDs/nanoparticles from the brain after E15, and that they can deliver drugs and peptides to the developing brain.

Measuring brain lipids.

The rapid development of analytical technology has made lipidomics an exciting new area and this review will focus more on modern approaches to lipidomics than on earlier technology. Although not fully comprehensive for all possible brain lipids, the intent is to at least provide a reference for the analysis of classes of lipids found in brain and nervous tissue. We will discuss problems posed by the brain because of its structural and functional heterogeneity, the development changes it undergoes (myelination, aging, pathology etc.) and its cellular heterogeneity (neurons, glia etc.). Section 2 will discuss the various ways in which brain tissue can be extracted to yield lipids for analysis and section 3 will cover a wide range of techniques used to analyze brain lipids such as chromatography and mass-spectrometry. In Section 4 we will discuss ways of analyzing some of the specific biologically active brain lipids found in very small amounts except in pathological conditions and section 5 looks to the future of experimental lipidomic modification in the brain. This article is part of a Special Issue entitled Brain Lipids.

Changes in the metabolism of sphingolipids after subarachnoid hemorrhage.

We previously described how ceramide (Cer), a mediator of cell death, increases in the cerebrospinal fluid (CSF) of subarachnoid hemorrhage (SAH) patients. This study investigates the alterations of biochemical pathways involved in Cer homeostasis in SAH. Cer, dihydroceramide (DHC), sphingosine-1-phosphate (S1P), and the activities of acid sphingomyelinase (ASMase), neutral sphingomyelinase (NSMase), sphingomyelinase synthase (SMS), S1P-lyase, and glucosylceramide synthase (GCS) were determined in the CSF of SAH subjects and in brain homogenate of SAH rats. Compared with controls (n = 8), SAH patients (n = 26) had higher ASMase activity (10.0 ± 3.5 IF/µl· min vs. 15.0 ± 4.6 IF/µl • min; P = 0.009) and elevated levels of Cer (11.4 ± 8.8 pmol/ml vs. 33.3 ± 48.3 pmol/ml; P = 0.001) and DHC (1.3 ± 1.1 pmol/ml vs. 3.8 ± 3.4 pmol/ml; P = 0.001) in the CSF. The activities of GCS, NSMase, and SMS in the CSF were undetectable. Brain homogenates from SAH animals had increased ASMase activity (control: 9.7 ± 1.2 IF/µg • min; SAH: 16.8 ± 1.6 IF/µg • min; P < 0.05) and Cer levels (control: 3,422 ± 26 fmol/nmol of total lipid P; SAH: 7,073 ± 2,467 fmol/nmol of total lipid P; P < 0.05) compared with controls. In addition, SAH was associated with a reduction of 60% in S1P levels, a 40% increase in S1P-lyase activity, and a twofold increase in the activity of GCS. In comparison, NSMase and SMS activities were similar to controls and SMS activities similar to controls. In conclusion, our results show an activation of ASMase, S1P-lyase, and GCS resulting in a shift in the production of protective (S1P) in favor of deleterious (Cer) sphingolipids after SAH. Additional studies are needed to determine the effect of modulators of the pathways described here in SAH.

Glycosignaling: a general review.

The concept of glycosignaling, in which neural cell-surface glycoconjugates form microdomains (Lipid Rafts) to facilitate the recruitment of signaling molecule components to form a transient signaling unit, is helping us understand the reason for glycoheterogeneity in the brain and is leadings to important translational efforts in medicine. In this review we first describe the origins of the concept of glycomicrodomains, how lipid heterogeneity might have relevance for the brain development, pathology and how the glycocalyx acts as a barrier in glia. After a discussion of how such microdomains are isolated and studied using modern technology such as nanoparticle labeling and molecular microscopy, we will present examples of how glycosignaling can function in such brain-specific situations as axonal growth and protein phosphorylation-mediated signaling.

Multiple sphingolipid abnormalities following cerebral microendothelial hypoxia.

Hypoxia has been previously shown to inhibit the dihydroceramide (DHC) desaturase, leading to the accumulation of DHC. In this study, we used metabolic labeling with [3H]-palmitate, HPLC/MS/MS analysis, and specific inhibitors to show numerous sphingolipid changes after oxygen deprivation in cerebral microendothelial cells. The increased DHC, particularly long-chain forms, was observed in both whole cells and detergent-resistant membranes. This was reversed by reoxygenation and blocked by the de novo sphingolipid synthesis inhibitor myriocin, but not by the neutral sphingomyelinase inhibitor GW-4869. Furthermore, oxygen deprivation of microendothelial cells increased levels of dihydro-sphingosine (DH-Sph), DH-sphingosine1-phosphate (DH-S1P), DH-sphingomyelin (DH-SM), DH-glucosylceramide (DH-GlcCer), and S1P levels. In vitro assays revealed no changes in the activity of sphingomyelinases or sphingomyelin synthase, but resulted in reduced S1P lyase activity and 40% increase in glucosylceramide synthase (GCS) activity, which was reversed by reoxygenation. Inhibition of the de novo sphingolipid pathway (myriocin) or GCS (EtPoD4) induced endothelial barrier dysfunction and increased caspase 3-mediated cell death in response to hypoxia. Our findings suggest that hypoxia induces synthesis of S1P and multiple dihydro-sphingolipids, including DHC, DH-SM, DH-GlcCer, DH-Sph and DH-S1P, which may be involved in ameliorating the effects of stroke . Progressive hypoxia leads to the accumulation of several dihydrosphingolipids in cerebral microendothelial cells. Hypoxia also increases sphingosine-1-phosphate and the activity of glucosylceramide (Glc-Cer) synthase. These changes reverse by inhibiting the de novo sphingolipid synthesis, which worsens hypoxia-induced endothelial barrier dysfunction and apoptosis, suggesting that the identified sphingolipids may be vasculoprotective.

Selecting improved peptidyl motifs for cytosolic delivery of disparate protein and nanoparticle materials.

Cell penetrating peptides facilitate efficient intracellular uptake of diverse materials ranging from small contrast agents to larger proteins and nanoparticles. However, a significant impediment remains in the subsequent compartmentalization/endosomal sequestration of most of these cargoes. Previous functional screening suggested that a modular peptide originally designed to deliver palmitoyl-protein thioesterase inhibitors to neurons could mediate endosomal escape in cultured cells. Here, we detail properties relevant to this peptide's ability to mediate cytosolic delivery of quantum dots (QDs) to a wide range of cell-types, brain tissue culture and a developing chick embryo in a remarkably nontoxic manner. The peptide further facilitated efficient endosomal escape of large proteins, dendrimers and other nanoparticle materials. We undertook an iterative structure-activity relationship analysis of the peptide by discretely modifying key components including length, charge, fatty acid content and their order using a comparative, semiquantitative assay. This approach allowed us to define the key motifs required for endosomal escape, to select more efficient escape sequences, along with unexpectedly identifying a sequence modified by one methylene group that specifically targeted QDs to cellular membranes. We interpret our results within a model of peptide function and highlight implications for in vivo labeling and nanoparticle-mediated drug delivery by using different peptides to co-deliver cargoes to cells and engage in multifunctional labeling.

Evidence for coordination of lysosomal (ASMase) and plasma membrane (NSMase2) forms of sphingomyelinase from mutant mice.

NSMase2 is associated to the plasma membrane, whereas ASMase is predominantly lysosomal; both hydrolyze sphingomyelin (SM) to ceramide and phosphocholine. Although SM accumulated in both ASMase(-/-) and fro/fro (NSMase2(-/-)) fibroblasts, the reduction of ceramides was more dramatic in fro/fro cells. ASMase mRNA, protein and enzyme activity were substantially elevated in fro/fro fibroblasts. In contrast, NSMase2 activity was unaffected in ASMase(-/-) fibroblasts. ASMase(-/-) cells showed normal cell cycling whereas fro/fro cells grew slowly and were arrested in G1/G0 and could be corrected by transfection with smpd3 gene. This suggests two distinct subcellular pathways for SM catabolism with distinct functions.

Nanoparticle targeting to neurons in a rat hippocampal slice culture model.

We have previously shown that CdSe/ZnS core/shell luminescent semiconductor nanocrystals or QDs (quantum dots) coated with PEG [poly(ethylene glycol)]-appended DHLA (dihydrolipoic acid) can bind AcWG(Pal)VKIKKP(9)GGH(6) (Palm1) through the histidine residues. The coating on the QD provides colloidal stability and this peptide complex uniquely allows the QDs to be taken up by cultured cells and readily exit the endosome into the soma. We now show that use of a polyampholyte coating [in which the neutral PEG is replaced by the negatively heterocharged CL4 (compact ligand)], results in the specific targeting of the palmitoylated peptide to neurons in mature rat hippocampal slice cultures. There was no noticeable uptake by astrocytes, oligodendrocytes or microglia (identified by immunocytochemistry), demonstrating neuronal specificity to the overall negatively charged CL4 coating. In addition, EM (electron microscopy) images confirm the endosomal egress ability of the Palm1 peptide by showing a much more disperse cytosolic distribution of the CL4 QDs conjugated to Palm1 compared with CL4 QDs alone. This suggests a novel and robust way of delivering neurotherapeutics to neurons.

Changes in the cerebrospinal fluid ceramide profile after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: The purpose of this study was to investigate changes in the cerebrospinal fluid sphingolipid profile in patients with subarachnoid hemorrhage in relation to the occurrence of symptomatic vasospasm and outcome at hospital discharge. METHODS: The ceramide profile in the cerebrospinal fluid was determined by mass spectrometry in control subjects and patients with Fisher 3 grade subarachnoid hemorrhage within 48 hours of the bleed. Patients were prospectively followed and subcategorized based on the occurrence of symptomatic vasospasm and modified Rankin Scale at discharge. RESULTS: Compared to control subjects, patients with subarachnoid hemorrhage had higher cerebrospinal fluid levels of total ceramide (12.4±8.8 versus 54.6±49.3 pmol/mL; P<0.001). In the subgroup analysis, total ceramide levels in individuals with symptomatic vasospasm (104.2±57.0 pmol/mL) were higher than in those with asymptomatic vasospasm (32.4±25.7 pmol/mL; P=0.006) and no vasospasm (30.9±15.7 pmol/mL; P=0.003). In addition, compared to patients with a good outcome (modified Rankin Scale ≤3), individuals with poor outcome (modified Rankin Scale ≥4) had higher cerebrospinal fluid levels of total ceramide (79±25 versus 23±6 pmol/mL; P=0.008). When the relative contributions of the different ceramide species were calculated, a higher relative concentration of C(18:0) ceramide was observed in individuals with symptomatic vasospasm (P=0.018) and poor outcome (P=0.028). CONCLUSIONS: Ceramide profile changes occur in subarachnoid hemorrhage. In this small case-based series elevation of levels of this sphingolipid, particularly C(18:0), was associated with the occurrence of symptomatic vasospasm and poor neurological outcome after subarachnoid hemorrhage.

Neutral sphingomyelinase 2 deficiency increases hyaluronan synthesis by up-regulation of Hyaluronan synthase 2 through decreased ceramide production and activation of Akt.

Fibroblasts from the fro/fro mouse, with a deletion in the Smpd3 gene coding for the active site of neutral sphingomyelinase 2 (NSMase2), secreted increased amounts of hyaluronan (HA). This was reversed by transfection with the Smpd3 gene, suggesting a connection between sphingolipid and glycosaminoglycan metabolism. The deficiency of NSMase2 resulted in storage of sphingomyelin (SM) and cholesterol with a 50% reduction in ceramides (Cer). RT-PCR and Western blot analysis showed that increased HA secretion resulted from increased hyaluronan synthase 2 (HAS2) activity localized to sphingolipid-enriched lipid rafts. Although cholesterol levels were also elevated in lipid rafts from mouse fibroblasts deficient in lysosomal acid SMase activity (deletion of the Smpd1(-/-) gene), there was no increase in HA secretion. We then showed that in fro/fro fibroblasts, the reduced ceramide was associated with decreased phosphorylation of protein phosphatase 2A (PP2A) and increased phosphorylation of its substrate Akt-p, together with PI3K, PDK1, mTOR (mammalian target of rapamycin), and p70S6K, although PTEN was unaffected. Exogenous ceramide, as well as inhibitors of Akt (Akt inhibitor VIII), PI 3-kinase (LY294002 and wortmannin), and mTOR (rapamycin) reduced secretion of HA, whereas the NSMase2 inhibitor GW4869 increased HA synthesis and secretion. We propose that NSMase2/Cer are the key mediators of the regulation of HA synthesis, via microdomains and the Akt/mTOR pathway.

American ginseng suppresses Western diet-promoted tumorigenesis in model of inflammation-associated colon cancer: role of EGFR.

BACKGROUND: Western diets increase colon cancer risk. Epidemiological evidence and experimental studies suggest that ginseng can inhibit colon cancer development. In this study we asked if ginseng could inhibit Western diet (20% fat) promoted colonic tumorigenesis and if compound K, a microbial metabolite of ginseng could suppress colon cancer xenograft growth. METHODS: Mice were initiated with azoxymethane (AOM) and, two weeks later fed a Western diet (WD, 20% fat) alone, or WD supplemented with 250-ppm ginseng. After 1 wk, mice received 2.5% dextran sulfate sodium (DSS) for 5 days and were sacrificed 12 wks after AOM. Tumors were harvested and cell proliferation measured by Ki67 staining and apoptosis by TUNEL assay. Levels of EGF-related signaling molecules and apoptosis regulators were determined by Western blotting. Anti-tumor effects of intraperitoneal compound K were examined using a tumor xenograft model and compound K absorption measured following oral ginseng gavage by UPLC-mass spectrometry. Effects of dietary ginseng on microbial diversity were measured by analysis of bacterial 16S rRNA. RESULTS: Ginseng significantly inhibited colonic inflammation and tumorigenesis and concomitantly reduced proliferation and increased apoptosis. The EGFR cascade was up-regulated in colonic tumors and ginseng significantly reduced EGFR and ErbB2 activation and Cox-2 expression. Dietary ginseng altered colonic microbial diversity, and bacterial suppression with metronidazole reduced serum compound K following ginseng gavage. Furthermore, compound K significantly inhibited tumor xenograft growth. CONCLUSIONS: Ginseng inhibited colonic inflammation and tumorigenesis promoted by Western diet. We speculate that the ginseng metabolite compound K contributes to the chemopreventive effects of this agent in colonic tumorigenesis.