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.

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.

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.

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.

A Comparative Study of Serum Exosome Isolation Using Differential Ultracentrifugation and Three Commercial Reagents.

Exosomes play a role in cell-to-cell signaling and serve as possible biomarkers. Isolating exosomes with reliable quality and substantial concentration is a major challenge. Our purpose is to compare the exosomes extracted by three different exosome isolation kits (miRCURY, ExoQuick, and Invitrogen Total Exosome Isolation Reagent) and differential ultracentrifugation (UC) using six different volumes of a non-cancerous human serum (5 ml, 1 ml, 500 µl, 250 µl, 100 µl, and 50 µl) and three different volumes (1 ml, 500 µl and 100 µl) of six individual commercial serum samples collected from human donors. The smaller starting volumes (100 µl and 50 µl) are used to mimic conditions of limited availability of heterogeneous biological samples. The isolated exosomes were characterized based upon size, quantity, zeta potential, CD63 and CD9 protein expression, and exosomal RNA (exRNA) quality and quantity using several complementary methods: nanoparticle tracking analysis (NTA) with ZetaView, western blot, transmission electron microscopy (TEM), the Agilent Bioanalyzer system, and droplet digital PCR (ddPCR). Our NTA results showed that all isolation techniques produced exosomes within the expected size range (40-150 nm). The three kits, though, produced a significantly higher yield (80-300 fold) of exosomes as compared to UC for all serum volumes, except 5 mL. We also found that exosomes isolated by the different techniques and serum volumes had similar zeta potentials to previous studies. Western blot analysis and TEM immunogold labelling confirmed the expression of two common exosomal protein markers, CD63 and CD9, in samples isolated by all techniques. All exosome isolations yielded high quality exRNA, containing mostly small RNA with a peak between 25 and 200 nucleotides in size. ddPCR results indicated that exosomes isolated from similar serum volumes but different isolation techniques rendered similar concentrations of two selected exRNA: hsa-miR-16 and hsa-miR-451. In summary, the three commercial exosome isolation kits are viable alternatives to UC, even when limited amounts of biological samples are available.

Sphingolipid-Enriched Extracellular Vesicles and Alzheimer's Disease: A Decade of Research.

Extracellular vesicles (EVs), particularly exosomes, have emerged in the last 10 years as a new player in the progression of Alzheimer's disease (AD) with high potential for being useful as a diagnostic and treatment tool. Exosomes and other EVs are enriched with the sphingolipid ceramide as well as other more complex glycosphingolipids such as gangliosides. At least a subpopulation of exosomes requires neutral sphingomyelinase activity for their biogenesis and secretion. As ceramide is often elevated in AD, exosome secretion may be affected as well. Here, we review the available data showing that exosomes regulate the aggregation and clearance of amyloid-beta (Aß) and discuss the differences in data from laboratories regarding Aß binding, induction of aggregation, and glial clearance. We also summarize available data on the role of exosomes in extracellular tau propagation, AD-related exosomal mRNA/miRNA cargo, and the use of exosomes as biomarker and gene therapy vehicles for diagnosis and potential treatment.

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.

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.

The 5XFAD Mouse Model of Alzheimer's Disease Exhibits an Age-Dependent Increase in Anti-Ceramide IgG and Exogenous Administration of Ceramide Further Increases Anti-Ceramide Titers and Amyloid Plaque Burden.

We present evidence that 5XFAD Alzheimer's disease model mice develop an age-dependent increase in antibodies against ceramide, suggesting involvement of autoimmunity against ceramide in Alzheimer's disease pathology. To test this, we increased serum anti-ceramide IgG (2-fold) by ceramide administration and analyzed amyloid plaque formation in 5XFAD mice. There were no differences in soluble or total amyloid-ß levels. However, females receiving ceramide had increased plaque burden (number, area, and size) compared to controls. Ceramide-treated mice showed an increase of serum exosomes (up to 3-fold using Alix as marker), suggesting that systemic anti-ceramide IgG and exosome levels are correlated with enhanced plaque formation.

Exosome reduction in vivo is associated with lower amyloid plaque load in the 5XFAD mouse model of Alzheimer's disease.

We present evidence here that exosomes stimulate aggregation of amyloid beta (Aß)1-42 in vitro and in vivo and interfere with uptake of Aß by primary cultured astrocytes and microglia in vitro. Exosome secretion is prevented by the inhibition of neutral sphingomyelinase 2 (nSMase2), a key regulatory enzyme generating ceramide from sphingomyelin, with GW4869. Using the 5XFAD mouse, we show that intraperitoneal injection of GW4869 reduces the levels of brain and serum exosomes, brain ceramide, and Aß1-42 plaque load. Reduction of total Aß1-42 as well as number of plaques in brain sections was significantly greater (40% reduction) in male than female mice. Our results suggest that GW4869 reduces amyloid plaque formation in vivo by preventing exosome secretion and identifies nSMase2 as a potential drug target in AD by interfering with exosome secretion.

Integrin alpha chains exhibit distinct temporal and spatial localization patterns in epithelial cells of the Drosophila ovary.

Integrins are heterodimeric transmembrane receptors that modulate cell adhesion, migration, and signaling. Multiple integrin chains contribute to development and morphogenesis of a given tissue. Here, we analyze the expression of Drosophila integrin alpha chains in the ovarian follicular epithelium, a model for tissue morphogenesis and cell migration. We find expression throughout development of the beta chain, betaPS. Alpha chains, however, exhibit both spatial and temporal expression differences. alphaPS1 and alphaPS2 integrins are detected during early and mid-oogenesis on apical, lateral, and basal membranes with the betaPS chain, whereas alphaPS3-family integrins (alphaPS3, alphaPS4, alphaPS5) are expressed in anterior cells late in oogenesis. Surprisingly, we find that alphaPS3-family integrins are dispensable for dorsal appendage morphogenesis but play a role in the final length of the egg, suggesting redundant functions of integrins in a simple tissue. We also demonstrate roles for alphaPS3betaPS integrin in border cell migration and in stretch cells.

Further characterization of embryonic stem cell-derived radial glial cells.

Previously, we showed that radial glia-like (RG) cells differentiated from embryonic stem (ES) cells after retinoic acid induction (Liour and Yu, 2003: Glia 42:109-117). In the present study, we demonstrate that the production of RG cells from ES cells is independent of the neural differentiation protocol used. These ES cell-derived RG (ES-RG) cells are similar in morphology to RG cells in vivo and express several characteristic RG cell markers. The processes of these ES-RG cells are organized into radial arrays similar to the RG scaffold in developing CNS. Expression of Pax6, along with other circumstantial data, suggests that at least some of these ES-RG cells are neural progenitors. The progression of neurogenesis into gliogenesis during the in vitro neural differentiation of ES cells recapitulates the in vivo developmental process. The identification of two cell surface markers, SSEA-1 and GM1, on both the native embryonic RG cells and ES-RG cells, may facilitate purification of radial glial cells for future studies and cell therapy. Overall, our study suggests that differentiation of radial glial cells is a common pathway during the neural differentiation of ES cells.

Spatiotemporal expression of GM1 in murine medial pallial neural progenitor cells.

The expression of gangliosides is developmentally regulated in the central nervous system. The expression of GM1 in the neural progenitor cells of the telencephalonic ventricular zone (VZ) has been reported in several studies. However, information on the spatial and temporal regulation of GM1 expression in the VZ is still lacking. In this study, we characterized the expression of GM1 in the developing mouse telencephalon. At E13, GM1 is expressed in neuronal cells as well as in the VZ. The initial expression of GM1 in the VZ is restricted to regions close to the medial pallium. Fluorescence-activated cell sorting (FACS) analysis and characterization of E14 GM1-positive cells showed that they contain progenitor cells that proliferate in response to epidermal growth factor (EGF) and/or basic fibroblast growth factor (bFGF) stimulation. The results obtained from quantitative gene expression analysis of region-specific genes (Emx1, Lhx2, Ngn1, Ngn2, Pax6, Dlx2, Gsh2, Mash1, and Nkx2.1), using real-time polymerase chain reaction indicate that FACS of GM1-expressing cells in the fetal forebrain enriches for the medial pallial neural progenitor cells.