Novel Function of Ceramide for Regulation of Mitochondrial ATP Release in Astrocytes

We reported that amyloid β peptide (Aβ42) activated neutral SMase 2 (nSMase2), thereby increasing the concentration of the sphingolipid ceramide in astrocytes. Here, we show that Aβ42 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 Aβ42. 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

Redox Regulation of Mitochondrial Fission Protein Drp1 by Protein Disulfide Isomerase Limits Endothelial Senescence.

Mitochondrial dynamics are tightly controlled by fusion and fission, and their dysregulation and excess reactive oxygen species (ROS) contribute to endothelial cell (EC) dysfunction. How redox signals regulate coupling between mitochondrial dynamics and endothelial (dys)function remains unknown. Here, we identify protein disulfide isomerase A1 (PDIA1) as a thiol reductase for the mitochondrial fission protein Drp1. A biotin-labeled Cys-OH trapping probe and rescue experiments reveal that PDIA1 depletion in ECs induces sulfenylation of Drp1 at Cys644, promoting mitochondrial fragmentation and ROS elevation without inducing ER stress, which drives EC senescence. Mechanistically, PDIA1 associates with Drp1 to reduce its redox status and activity. Defective wound healing and angiogenesis in diabetic or PDIA1+/- mice are restored by EC-targeted PDIA1 or the Cys oxidation-defective mutant Drp1. Thus, this study uncovers a molecular link between PDIA1 and Drp1 oxidoreduction, which maintains normal mitochondrial dynamics and limits endothelial senescence with potential translational implications for vascular diseases associated with diabetes or aging.This research was supported by NIH R01HL135584 (to M.U.-F.), NIH R21HL112293 (to M.U.-F.), NIH R01HL133613 (to T.F. and M.U.-F.), NIH R01HL116976 (to T.F. and M.U.-F.), NIH R01HL070187 (to T.F.), NIH R01HL112626 (to J.K.), Department of Veterans Affairs Merit Review Grant 2I01BX001232 (to T.F.), AHA 16GRNT31390032 (to M.U.-F.), AHA 15SDG25700406 (to S.V.), AHA 16POST27790038 (to A.D.), and NIH T32HL07829 (to R.C.). We thank Mr. Kyle Taylor at Keyence Corporation for assisting with taking images using the Keyence microscope; Dr. John O’Bryan at UIC for assisting with the BiFC assays; Dr. Leslie Poole at Wake Forest University for providing DCP-Bio1, as well as Dr. Jody Martin and the Center for Cardiovascular Research-supported Vector Core Facility at UIC for amplifying adenoviruses.S

Reduction of T Cell Receptor Diversity in NOD Mice Prevents Development of Type 1 Diabetes but Not Sjögren’s Syndrome

<div><p>Non-obese diabetic (NOD) mice are well-established models of independently developing spontaneous autoimmune diseases, Sjögren’s syndrome (SS) and type 1 diabetes (T1D). The key determining factor for T1D is the strong association with particular MHCII molecule and recognition by diabetogenic T cell receptor (TCR) of an insulin peptide presented in the context of I-A^g7 molecule. For SS the association with MHCII polymorphism is weaker and TCR diversity involved in the onset of the autoimmune phase of SS remains poorly understood. To compare the impact of TCR diversity reduction on the development of both diseases we generated two lines of TCR transgenic NOD mice. One line expresses transgenic TCRβ chain originated from a pathogenically irrelevant TCR, and the second line additionally expresses transgenic TCRα^mini locus. Analysis of TCR sequences on NOD background reveals lower TCR diversity on Treg cells not only in the thymus, but also in the periphery. This reduction in diversity does not affect conventional CD4⁺ T cells, as compared to the TCR^mini repertoire on B6 background. Interestingly, neither transgenic TCRβ nor TCR^mini mice develop diabetes, which we show is due to lack of insulin B:9–23 specific T cells in the periphery. Conversely SS develops in both lines, with full glandular infiltration, production of autoantibodies and hyposalivation. It shows that SS development is not as sensitive to limited availability of TCR specificities as T1D, which suggests wider range of possible TCR/peptide/MHC interactions driving autoimmunity in SS.</p></div

Lack of response to insulin B:9–23 peptide in transgenic NOD mice.

<p>(A) 5×10⁴ of CD4⁺ T cells sorted from lymph nodes of NOD, NOD^βTg and NOD^mini mice were cultured in the presence of 5×10⁵ splenocytes from NOD.TCRα^−/− (A^g7) or B6.TCRα^−/− (A^b) mice and soluble anti-CD3 (1 µg/ml) or insulin B:9–23 peptide (50 µg/ml), as indicated. Proliferation of cells was measured after 3 days by MTT assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112467#pone.0112467-Pacholczyk2" target="_blank">[38]</a>. Experiments were done twice with 3 mice per group. (B) T-cell hybridomas specific to allo-antigens or insulin B:9–23 peptide were generated from indicated mice. For generation of B:9–23 specific hybridomas, mice were immunized with the peptide 7 days prior to isolation of lymph nodes for <i>in vitro</i> blasts generation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112467#pone.0112467-Pacholczyk2" target="_blank">[38]</a>. Generated hybridomas were tested for their ability to respond to syngeneic (NOD.TCRα^−/−) or allogeneic (B6.TCRα^−/−) splenocytes with or without B:9–23 peptide or anti-CD3. Table shows numbers of identified hybridomas specific to indicated antigens and numbers of hybridomas responding to anti-CD3 stimulation. Table is representative of 3 independent experiments with two mice per group.</p

Efficient selection of CD4⁺ T lymphocytes in NOD^mini and NOD^βTg mice.

<p>Lymphocytes isolated from thymii (A) and lymph nodes (B) of indicated mice were stained with monoclonal antibodies and analyzed by flow cytometry. Numbers in quadrants are representative percentages of at least six mice (6 week old) per group. The numbers of thymocytes and lymphocytes ± SD recovered from NOD^mini, NOD^βTg and NOD mice were: from thymii 78.5±16.7×10⁶, 72.05±13.8×10⁶ and 70.1±14.0×10⁶, and from lymph nodes (axillary, brachial and inguinal) 17.2±5.8×10⁶, 14.1±2.5×10⁶, and 13.2±3.0×10⁶, respectively. (C) Percentages (top) and total numbers (bottom) of CD4⁺Foxp3⁺ T cells in peripheral lymph nodes of 6 week old mice; each circle represents individual mouse. (D) Expression of mRNA of TCRVα genes in sorted CD4⁺ T cells isolated from NOD and NOD^βTg mice. Analysis was done by RT-PCR using primers specific to indicated Vα segments and Cα region. (E) Comparison of CD4⁺Foxp3⁺ T (Treg) cells from thymii and peripheral lymph nodes of transgenic and wild type B6 and NOD mice. Mean percentage and SD of six young mice per group are shown.</p

TCR repertoire of naïve and regulatory T cells in NOD^mini mice.

<p>(A) Similarity between indicated populations (first 289 single cell sequences per each population) was estimated based on Morisita-Horn index (MH). T_N - naïve CD4⁺Foxp3⁻CD45RB⁺CD62L⁺ T cells, T_R - regulatory CD4⁺Foxp3⁺ T cells, TH - thymus, LN - lymph nodes. (B) Ratio of richness of TCRs on T_N and T_R cells between NOD^mini and B6^mini mice. Abundance coverage estimator (ACE) was calculated based on 578 sequences for each population combined from thymus and peripheral lymph nodes. (C) Evenness and effective number of species (ENS) for analyzed TCR repertoires. Shannon evenness index was calculated as Shannon entropy (H_s) divided by maximum diversity D_max, where D_max equals natural logarithm of number of unique sequences in analyzed population. ENS (true diversity) was calculated as exponential of Shannon entropy. (D) Comparison of frequency of 20 most dominant unique protein CDR3 clonotypes found in each population indicated on the right of the heat map. Table indicates analyzed populations from lymph nodes and thymii by single cell analysis (SC) and high throughput sequencing (HT). Experimental mouse 1 and 2 are marked as m1 and m2. Numbers next to each population indicate total numbers of DNA sequences analyzed. All 86 unique CDR3α protein sequences in the heat map are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112467#pone.0112467.s001" target="_blank">Table S1</a>. (E) Accumulation curve of unique DNA clonotypes observed after accumulation of 124,730 sequences for T_N cells and 124,696 for T_R cells. (A–C) All indices were computed based on DNA sequences for each population using software SPADE and EstimateS8.2.</p

Detection of autoantibodies and hyposalivation in NOD^mini and NOD^βTg mice.

<p>(A) ELISA assay was performed using mouse serum (1∶100) from indicated age groups. Each graph represents mean value of OD₄₅₀ and standard deviation for indicated antigens. At least six mice were used per each group. *p<0.05, **p<0.005. (B) Detection of ANAs pattern using Hep-2 cell line. Sera from mice were diluted 1∶40, incubated with HEp-2-fixed slides and evaluated under fluorescent microscope at x20 magnifications. Representative images are shown. (C) Quantitative ELISA analysis of IgG1 levels in sera (1∶50,000) from indicated mice. Each bar represents mean value and standard deviation from six mice per experimental groups. (D) Salivary flow rates after pilocarpine injection in indicated mice at 10 and 20 wks of age. Double congenic B6.NODIdd3.NODIdd5 (B6.DC) mice, that develop SS on B6 genetic background, were used as a control. Saliva volume was measured and calculated in mg per mouse body mass. Each circle represents one mouse and horizontal lines indicate mean values of the experimental groups. T-test was used to calculate differences between groups. *p<0.002, **p<0.0002.</p