Novel function of ceramide for regulation of mitochondrial ATP release in astrocytes
2018; Elsevier BV; Volume: 59; Issue: 3 Linguagem: Inglês
10.1194/jlr.m081877
ISSN1539-7262
AutoresJi-Na Kong, Zhihui Zhu, Yutaka Itokazu, Guanghu Wang, Michael B. Dinkins, Liansheng Zhong, Hsuan-Pei Lin, Ahmed Elsherbini, Silvia Leanhart, Xue Jiang, Haiyan Qin, Wenbo Zhi, Stefka D. Spassieva, Erhard Bieberich,
Tópico(s)Alzheimer's disease research and treatments
ResumoWe 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. 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. In addition to accumulating amyloid β peptide (Aβ42), elevation of ceramide and mitochondrial damage is observed in Alzheimer's disease (AD) (1.Kennedy M.A. Moffat T.C. Gable K. Ganesan S. Niewola-Staszkowska K. Johnston A. Nislow C. Giaever G. Harris L.J. Loewith R. A signaling lipid associated with Alzheimer's disease promotes mitochondrial dysfunction.Sci. Rep. 2016; 6: 19332Crossref PubMed Scopus (20) Google Scholar, 2.Chakrabarti S.S. Bir A. Poddar J. Sinha M. Ganguly A. Chakrabarti S. Ceramide and sphingosine-1-phosphate in cell death pathways: relevance to the pathogenesis of Alzheimer's disease.Curr. Alzheimer Res. 2016; 13: 1232-1248Crossref PubMed Scopus (17) Google Scholar). Our laboratory discovered that incubation of astrocytes with Aβ42 activates neutral SMase 2 (nSMase2), a bona fide signaling enzyme generating ceramide from sphingomyelin (3.Dinkins M.B. Enasko J. Hernandez C. Wang G. Kong J. Helwa I. Liu Y. Terry Jr., A.V. Bieberich E. Neutral sphingomyelinase-2 deficiency ameliorates Alzheimer's disease pathology and improves cognition in the 5XFAD mouse.J. Neurosci. 2016; 36: 8653-8667Crossref PubMed Scopus (144) Google Scholar, 4.Wang G. Dinkins M. He Q. Zhu G. Poirier C. Campbell A. Mayer-Proschel M. Bieberich E. Astrocytes secrete exosomes enriched with proapoptotic ceramide and prostate apoptosis response 4 (PAR-4): potential mechanism of apoptosis induction in Alzheimer disease (AD).J. Biol. Chem. 2012; 287: 21384-21395Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). While examining the effect of Aβ42 on astrocytes in time-lapse imaging, we discovered that mitochondria in nSMase2-deficient astrocytes were more resistant to Aβ42-induced damage, but they also moved more rapidly than those in wild-type astrocytes. nSMase2 has been shown to shuttle between the plasma membrane and Golgi and it is activated by mitochondrial lipids (5.Milhas D. Clarke C.J. Idkowiak-Baldys J. Canals D. Hannun Y.A. Anterograde and retrograde transport of neutral sphingomyelinase-2 between the Golgi and the plasma membrane.Biochim. Biophys. Acta. 2010; 1801: 1361-1374Crossref PubMed Scopus (44) Google Scholar, 6.Clarke C.J. Guthrie J.M. Hannun Y.A. Regulation of neutral sphingomyelinase-2 (nSMase2) by tumor necrosis factor-alpha involves protein kinase C-delta in lung epithelial cells.Mol. Pharmacol. 2008; 74: 1022-1032Crossref PubMed Scopus (58) Google Scholar, 7.Marchesini N. Luberto C. Hannun Y.A. Biochemical properties of mammalian neutral sphingomyelinase 2 and its role in sphingolipid metabolism.J. Biol. Chem. 2003; 278: 13775-13783Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 8.Airola M.V. Shanbhogue P. Shamseddine A.A. Guja K.E. Senkal C.E. Maini R. Bartke N. Wu B.X. Obeid L.M. Garcia-Diaz M. Structure of human nSMase2 reveals an interdomain allosteric activation mechanism for ceramide generation.Proc. Natl. Acad. Sci. USA. 2017; 114: E5549-E5558Crossref PubMed Scopus (51) Google Scholar), prompting us to test the effect of nSMase2-generated ceramide on mitochondrial motility and function. Ceramide is a sphingolipid the concentration of which is less than 1 mol% (compared with approximately 10 mol% sphingomyelin) in cellular membranes (9.Das A. Brown M.S. Anderson D.D. Goldstein J.L. Radhakrishnan A. Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis.eLife. 2014; 3: 02882Crossref Scopus (213) Google Scholar). It is mainly synthesized in the endoplasmic reticulum (ER) by ceramide synthases (CerSs), which attach a variety of fatty acid residues to long chain bases, such as sphingosine and dihydrosphingosine, or by hydrolysis of sphingomyelin that is catalyzed by different SMases in distinct cellular compartments, such as the plasma membrane, lysosomes, the Golgi apparatus, and mitochondria (10.Bartke N. Hannun Y.A. Bioactive sphingolipids: metabolism and function.J. Lipid Res. 2009; 50: S91-S96Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar, 11.Bieberich E. It's a lipid's world: bioactive lipid metabolism and signaling in neural stem cell differentiation.Neurochem. Res. 2012; 37: 1208-1229Crossref PubMed Scopus (72) Google Scholar, 12.Futerman A.H. Hannun Y.A. The complex life of simple sphingolipids.EMBO Rep. 2004; 5: 777-782Crossref PubMed Scopus (535) Google Scholar, 13.Gault C.R. Obeid L.M. Hannun Y.A. An overview of sphingolipid metabolism: from synthesis to breakdown.Adv. Exp. Med. Biol. 2010; 688: 1-23Crossref PubMed Scopus (665) Google Scholar, 14.Hannun Y.A. Obeid L.M. Many ceramides.J. Biol. Chem. 2011; 286: 27855-27862Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 15.Wu B.X. Clarke C.J. Hannun Y.A. Mammalian neutral sphingomyelinases: regulation and roles in cell signaling responses.Neuromolecular Med. 2010; 12: 320-330Crossref PubMed Scopus (111) Google Scholar). Increase of ceramide levels through upregulation of CerSs or SMases leads to induction of apoptosis and other cell signaling pathways critical for cell death, senescence, autophagy, or cell cycle arrest. We and others showed that the localized generation or distribution of ceramide in ceramide microdomains (rafts) or more extended ceramide-enriched compartments (CECs) may have additional cellular functions of ceramide (16.Zhang Y. Li X. Becker K.A. Gulbins E. Ceramide-enriched membrane domains–structure and function.Biochim. Biophys. Acta. 2009; 1788: 178-183Crossref PubMed Scopus (195) Google Scholar, 17.Gulbins E. Kolesnick R. Raft ceramide in molecular medicine.Oncogene. 2003; 22: 7070-7077Crossref PubMed Scopus (354) Google Scholar, 18.Burgert A. Schlegel J. Becam J. Doose S. Bieberich E. Schubert-Unkmeir A. Sauer M. Characterization of plasma membrane ceramides by super-resolution microscopy.Angew. Chem. Int. Ed. Engl. 2017; 56: 6131-6135Crossref PubMed Scopus (37) Google Scholar, 19.Kong J.N. Hardin K. Dinkins M. Wang G. He Q. Mujadzic T. Zhu G. Bielawski J. Spassieva S. Bieberich E. Regulation of Chlamydomonas flagella and ependymal cell motile cilia by ceramide-mediated translocation of GSK3.Mol. Biol. Cell. 2015; 26: 4451-4465Crossref PubMed Scopus (18) Google Scholar, 20.He Q. Wang G. Wakade S. Dasgupta S. Dinkins M. Kong J.N. Spassieva S.D. Bieberich E. Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide.Mol. Biol. Cell. 2014; 25: 1715-1729Crossref PubMed Google Scholar, 21.He Q. Wang G. Dasgupta S. Dinkins M. Zhu G. Bieberich E. Characterization of an apical ceramide-enriched compartment regulating ciliogenesis.Mol. Biol. Cell. 2012; 23: 3156-3166Crossref PubMed Google Scholar, 22.Bieberich E. Ceramide in stem cell differentiation and embryo development: novel functions of a topological cell-signaling lipid and the concept of ceramide compartments.J. Lipids. 2011; 2011: 610306Crossref PubMed Google Scholar, 23.Wang G. Krishnamurthy K. Bieberich E. Regulation of primary cilia formation by ceramide.J. Lipid Res. 2009; 50: 2103-2110Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Instrumental to our studies was the generation of a ceramide-specific antibody originally developed in our laboratory (24.Krishnamurthy K. Dasgupta S. Bieberich E. Development and characterization of a novel anti-ceramide antibody.J. Lipid Res. 2007; 48: 968-975Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). This antibody has been shared with several independent laboratories to reliably detect and visualize ceramide in cells and tissues and it is the only antibody applicable to super-resolution microscopy using stochastic optical reconstruction microscopy (STORM) (3.Dinkins M.B. Enasko J. Hernandez C. Wang G. Kong J. Helwa I. Liu Y. Terry Jr., A.V. Bieberich E. Neutral sphingomyelinase-2 deficiency ameliorates Alzheimer's disease pathology and improves cognition in the 5XFAD mouse.J. Neurosci. 2016; 36: 8653-8667Crossref PubMed Scopus (144) Google Scholar, 18.Burgert A. Schlegel J. Becam J. Doose S. Bieberich E. Schubert-Unkmeir A. Sauer M. Characterization of plasma membrane ceramides by super-resolution microscopy.Angew. Chem. Int. Ed. Engl. 2017; 56: 6131-6135Crossref PubMed Scopus (37) Google Scholar, 25.Walter T. Collenburg L. Japtok L. Kleuser B. Schneider-Schaulies S. Muller N. Becam J. Schubert-Unkmeir A. Kong J.N. Bieberich E. Incorporation and visualization of azido-functionalized N-oleoyl serinol in Jurkat cells, mouse brain astrocytes, 3T3 fibroblasts and human brain microvascular endothelial cells.Chem. Commun. (Camb.). 2016; 52: 8612-8614Crossref PubMed Google Scholar, 26.Kong J.N. Hardin K. Dinkins M. Wang G. He Q. Mujadzic T. Zhu G. Bielawski J. Spassieva S. Bieberich E. Regulation of Chlamydomonas flagella and ependymal cell motile cilia by ceramide-mediated translocation of GSK3.Mol. Biol Cell. 2015; 26: 4451-4465Crossref PubMed Scopus (23) Google Scholar, 27.Muscoli C. Doyle T. Dagostino C. Bryant L. Chen Z. Watkins L.R. Ryerse J. Bieberich E. Neumman W. Salvemini D. Counter-regulation of opioid analgesia by glial-derived bioactive sphingolipids.J. Neurosci. 2010; 30: 15400-15408Crossref PubMed Scopus (77) Google Scholar). In addition to the ceramide-specific antibody, we developed a novel imaging protocol based on cross-linking of photoactivatable ceramide analogs {bifunctional ceramide or N-[9-(3-pent-4-ynyl-3-H-diazirine-3-yl)-nonanoyl]-D-erythro-sphingosine (pacFACer)} that can be derivatized with fluorophores or biotin using click chemistry. We demonstrated that these ceramide analogs are distributed to CECs and can be used to visualize and identify proteins binding to ceramide (19.Kong J.N. Hardin K. Dinkins M. Wang G. He Q. Mujadzic T. Zhu G. Bielawski J. Spassieva S. Bieberich E. Regulation of Chlamydomonas flagella and ependymal cell motile cilia by ceramide-mediated translocation of GSK3.Mol. Biol. Cell. 2015; 26: 4451-4465Crossref PubMed Scopus (18) Google Scholar). Previous studies showed that ceramide analogs were cross-linked or bound to tubulin (28.Kota V. Szulc Z.M. Hama H. Identification of C(6)-ceramide-interacting proteins in D6P2T Schwannoma cells.Proteomics. 2012; 12: 2179-2184Crossref PubMed Scopus (15) Google Scholar, 29.Elsen L. Betz R. Schwarzmann G. Sandhoff K. van Echten-Deckert G. Identification of ceramide binding proteins in neuronal cells: a critical point of view.Neurochem. Res. 2002; 27: 717-727Crossref PubMed Scopus (22) Google Scholar). We showed that tubulin was coimmunoprecipitated when pulling down intracellular vesicles using anti-ceramide IgG (20.He Q. Wang G. Wakade S. Dasgupta S. Dinkins M. Kong J.N. Spassieva S.D. Bieberich E. Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide.Mol. Biol. Cell. 2014; 25: 1715-1729Crossref PubMed Google Scholar). These studies suggested that ceramide may bind to free tubulin dispersed in the cytosol, or to tubulin embedded into microtubules or associated with cellular membranes (membrane tubulin). Membrane tubulin has been detected in the plasma membrane, but also in other compartments and organelles, such as the ER and mitochondria (30.Wolff J. Plasma membrane tubulin.Biochim. Biophys. Acta. 2009; 1788: 1415-1433Crossref PubMed Scopus (59) Google Scholar, 31.Bernier-Valentin F. Aunis D. Rousset B. Evidence for tubulin-binding sites on cellular membranes: plasma membranes, mitochondrial membranes, and secretory granule membranes.J. Cell Biol. 1983; 97: 209-216Crossref PubMed Scopus (94) Google Scholar). Tubulin has been suggested to bind to lipids in the cytosolic leaflet of the outer mitochondrial membrane (OMM) to initiate closure of voltage-dependent anion channel 1 (VDAC1) (32.Rostovtseva T.K. Kazemi N. Weinrich M. Bezrukov S.M. Voltage gating of VDAC is regulated by nonlamellar lipids of mitochondrial membranes.J. Biol. Chem. 2006; 281: 37496-37506Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 33.Rostovtseva T.K. Bezrukov S.M. VDAC regulation: role of cytosolic proteins and mitochondrial lipids.J. Bioenerg. Biomembr. 2008; 40: 163-170Crossref PubMed Scopus (187) Google Scholar, 34.Rostovtseva T.K. Gurnev P.A. Chen M.Y. Bezrukov S.M. Membrane lipid composition regulates tubulin interaction with mitochondrial voltage-dependent anion channel.J. Biol. Chem. 2012; 287: 29589-29598Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), one of the key channels mediating ADP/ATP transport between mitochondria and the cytosol. Malfunction of mitochondria and dysregulated VDAC1 have been implicated in many neurodegenerative diseases, including AD, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, and frontotemporal dementia (35.Manczak M. Reddy P.H. Abnormal interaction of VDAC1 with amyloid beta and phosphorylated tau causes mitochondrial dysfunction in Alzheimer's disease.Hum. Mol. Genet. 2012; 21: 5131-5146Crossref PubMed Scopus (212) Google Scholar, 36.Manczak M. Sheiko T. Craigen W.J. Reddy P.H. Reduced VDAC1 protects against Alzheimer's disease, mitochondria, and synaptic deficiencies.J. Alzheimers Dis. 2013; 37: 679-690Crossref PubMed Scopus (47) Google Scholar, 37.Ben-Hail D. Begas-Shvartz R. Shalev M. Shteinfer-Kuzmine A. Gruzman A. Reina S. De Pinto V. Shoshan-Barmatz V. Novel compounds targeting the mitochondrial protein VDAC1 inhibit apoptosis and protect against mitochondrial dysfunction.J. Biol. Chem. 2016; 291: 24986-25003Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 38.Shoshan-Barmatz V. De Pinto V. Zweckstetter M. Raviv Z. Keinan N. Arbel N. VDAC, a multi-functional mitochondrial protein regulating cell life and death.Mol. Aspects Med. 2010; 31: 227-285Crossref PubMed Scopus (519) Google Scholar, 39.Reddy P.H. Amyloid beta-induced glycogen synthase kinase 3beta phosphorylated VDAC1 in Alzheimer's disease: implications for synaptic dysfunction and neuronal damage.Biochim. Biophys. Acta. 2013; 1832: 1913-1921Crossref PubMed Scopus (94) Google Scholar, 40.Geisler S. Holmstrom K.M. Skujat D. Fiesel F.C. Rothfuss O.C. Kahle P.J. Springer W. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1.Nat. Cell Biol. 2010; 12: 119-131Crossref PubMed Scopus (2018) Google Scholar, 41.Le Verche V. Przedborski S. Is amyotrophic lateral sclerosis a mitochondrial channelopathy?.Neuron. 2010; 67: 523-524Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 42.Johri A. Beal M.F. Mitochondrial dysfunction in neurodegenerative diseases.J. Pharmacol. Exp. Ther. 2012; 342: 619-630Crossref PubMed Scopus (432) Google Scholar). In AD, Aβ has been shown to bind to VDAC1, reduce ATP levels, and cause mitochondrial fragmentation during early stages of the disease (35.Manczak M. Reddy P.H. Abnormal interaction of VDAC1 with amyloid beta and phosphorylated tau causes mitochondrial dysfunction in Alzheimer's disease.Hum. Mol. Genet. 2012; 21: 5131-5146Crossref PubMed Scopus (212) Google Scholar, 43.Camara A.K. Lesnefsky E.J. Stowe D.F. Potential therapeutic benefits of strategies directed to mitochondria.Antioxid. Redox Signal. 2010; 13: 279-347Crossref PubMed Scopus (149) Google Scholar, 44.Rui Y. Zheng J.Q. Amyloid beta oligomers elicit mitochondrial transport defects and fragmentation in a time-dependent and pathway-specific manner.Mol. Brain. 2016; 9: 79Crossref PubMed Scopus (41) Google Scholar). VDAC1 downregulation has been reported to reduce cellular ATP levels and induce mitochondrial fragmentation (45.Fatima M. Prajapati B. Saleem K. Kumari R. Mohindar Singh Singal C. Seth P. Novel insights into role of miR-320a-VDAC1 axis in astrocyte-mediated neuronal damage in neuroAIDS.Glia. 2017; 65: 250-263Crossref PubMed Scopus (25) Google Scholar, 46.Arif T. Vasilkovsky L. Refaely Y. Konson A. Shoshan-Barmatz V. Silencing VDAC1 expression by siRNA inhibits cancer cell proliferation and tumor growth in vivo.Mol. Ther. Nucleic Acids. 2014; 3: e159Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), eventually leading to neurodegeneration. In astrocytes, VDAC1 is critical for secretion of ATP, a gliotransmitter regulating synaptic plasticity and neuronal function (47.Butt A.M. ATP: a ubiquitous gliotransmitter integrating neuron-glial networks.Semin. Cell Dev. Biol. 2011; 22: 205-213Crossref PubMed Scopus (133) Google Scholar, 48.Rivera A. Vanzulli I. Butt A.M. A central role for ATP signalling in glial interactions in the CNS.Curr. Drug Targets. 2016; 17: 1829-1833Crossref PubMed Scopus (49) Google Scholar, 49.Harada K. Kamiya T. Tsuboi T. Gliotransmitter release from astrocytes: functional, developmental, and pathological implications in the brain.Front. Neurosci. 2016; 9: 499Crossref PubMed Scopus (119) Google Scholar, 50.Fields R.D. Stevens B. ATP: an extracellular signaling molecule between neurons and glia.Trends Neurosci. 2000; 23: 625-633Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Most recently, VDAC1 closure by tubulin has been suggested to be critical for the Warburg effect, a metabolic switch from mitochondria-mediated oxidative phosphorylation to glycolysis for ATP production, often observed in cancer cells (51.Maldonado E.N. Lemasters J.J. Warburg revisited: regulation of mitochondrial metabolism by voltage-dependent anion channels in cancer cells.J. Pharmacol. Exp. Ther. 2012; 342: 637-641Crossref PubMed Scopus (78) Google Scholar, 52.Maldonado E.N. Sheldon K.L. DeHart D.N. Patnaik J. Manevich Y. Townsend D.M. Bezrukov S.M. Rostovtseva T.K. Lemasters J.J. Voltage-dependent anion channels modulate mitochondrial metabolism in cancer cells: regulation by free tubulin and erastin.J. Biol. Chem. 2013; 288: 11920-11929Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 53.Maldonado E.N. VDAC-tubulin, an anti-Warburg pro-oxidant switch.Front. Oncol. 2017; 7: 4Crossref PubMed Scopus (49) Google Scholar). While assays with mitochondria showed that lipid-tubulin interaction is important for tubulin-mediated VDAC1 closure (52.Maldonado E.N. Sheldon K.L. DeHart D.N. Patnaik J. Manevich Y. Townsend D.M. Bezrukov S.M. Rostovtseva T.K. Lemasters J.J. Voltage-dependent anion channels modulate mitochondrial metabolism in cancer cells: regulation by free tubulin and erastin.J. Biol. Chem. 2013; 288: 11920-11929Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 54.Noskov S.Y. Rostovtseva T.K. Bezrukov S.M. ATP transport through VDAC and the VDAC-tubulin complex probed by equilibrium and nonequilibrium MD simulations.Biochemistry. 2013; 52: 9246-9256Crossref PubMed Scopus (40) Google Scholar, 55.Martel C. Wang Z. Brenner C. VDAC phosphorylation, a lipid sensor influencing the cell fate.Mitochondrion. 2014; 19 Pt A: 69-77Crossref PubMed Scopus (54) Google Scholar, 56.Hoogerheide D.P. Noskov S.Y. Jacobs D. Bergdoll L. Silin V. Worcester D.L. Abramson J. Nanda H. Rostovtseva T.K. Bezrukov S.M. Structural features and lipid binding domain of tubulin on biomimetic mitochondrial membranes.Proc. Natl. Acad. Sci. USA. 2017; 114: E3622-E3631Crossref PubMed Scopus (29) Google Scholar), the role of ceramide in this regulation has not been investigated yet. In addition, there is only little information available on the role of lipid-tubulin interaction for the function of mitochondria in intact cells. Studies from several laboratories found that ceramide is enriched in mitochondria and mitochondria-associated membranes (MAMs) (57.Bionda C. Portoukalian J. Schmitt D. Rodriguez-Lafrasse C. Ardail D. Subcellular compartmentalization of ceramide metabolism: MAM (mitochondria-associated membrane) and/or mitochondria?.Biochem. J. 2004; 382: 527-533Crossref PubMed Scopus (202) Google Scholar, 58.Hayashi T. Fujimoto M. Detergent-resistant microdomains determine the localization of sigma-1 receptors to the endoplasmic reticulum-mitochondria junction.Mol. Pharmacol. 2010; 77: 517-528Crossref PubMed Scopus (165) Google Scholar). MAMs are a subcompartment of the ER that forms contact sites with mitochondria and takes part in the cross-talk between mitochondria and microtubules. While CerSs and nSMases have been shown to generate ceramide in mitochondria involved in inducing apoptosis (59.Wu B.X. Rajagopalan V. Roddy P.L. Clarke C.J. Hannun Y.A. Identification and characterization of murine mitochondria-associated neutral sphingomyelinase (MA-nSMase), the mammalian sphingomyelin phosphodiesterase 5.J. Biol. Chem. 2010; 285: 17993-18002Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 60.Perera M.N. Ganesan V. Siskind L.J. Szulc Z.M. Bielawska A. Bittman R. Colombini M. Ceramide channel: structural basis for selective membrane targeting.Chem. Phys. Lipids. 2016; 194: 110-116Crossref PubMed Scopus (12) Google Scholar, 61.Birbes H. El Bawab S. Hannun Y.A. Obeid L.M. Selective hydrolysis of a mitochondrial pool of sphingomyelin induces apoptosis.FASEB J. 2001; 15: 2669-2679Crossref PubMed Scopus (221) Google Scholar, 62.Novgorodov S.A. Chudakova D.A. Wheeler B.W. Bielawski J. Kindy M.S. Obeid L.M. Gudz T.I. Developmentally regulated ceramide synthase 6 increases mitochondrial Ca2+ loading capacity and promotes apoptosis.J. Biol. Chem. 2011; 286: 4644-4658Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), it is not known whether ceramide regulates the function of MAMs and whether this involves ceramide-associated tubulin (CAT) and its effect on VDAC1. To test a potential role of CAT located at MAMs for the regulation of mitochondria, we used several complementary experimental approaches: 1) determining the effect of ceramide depletion by nSMase2 deficiency or the CerS inhibitor, fumonisin B1 (FB1), on mitochondrial morphology and motility; 2) confocal microscopy and STORM using anti-ceramide IgG for testing the association of ceramide with mitochondria and microtubules; 3) cross-linking of pacFACer and binding to ceramide agarose beads to test ceramide association to tubulin; 4) proximity ligation assays (PLAs) and coimmunoprecipitation assays to quantify CAT and CAT-VDAC1 complexes in primary cultured astrocytes and test the effect of ceramide depletion on complex formation; and 5) measurement of ATP levels in nSMase2-deficient or FB1-treated astrocytes to determine the effect of ceramide depletion on CAT-induced VDAC1 closure. Our results suggest that CAT localized at ceramide-enriched MAMs (CEMAMs) induces interaction of tubulin with VDAC1, a novel regulatory mechanism critical for mitochondria and, potentially, pathophysiology in AD. MitoTracker Red CM-H2Xros (M-7513), Duolink PLA probe anti-rabbit plus (DUO92002), Duolink PLA probe anti-mouse minus (DUO92004), Duolink PLA probe anti-goat minus (DUO92006), Duolink detection reagent red (DUO92008), Duolink detection reagent green (DUO92014), Alexa Fluor 647 azide (A10277), Invitrogen Click-iT cell reaction buffer kit (C10269), and Click-iT protein reaction buffer kit (C10276) were purchased from Thermo Fisher Scientific (West Columbia, SC); CytoPainter mitochondrial staining kit-green fluorescence (ab112143) and ATP photometric/fluorimetric assay (ab83355) were from Abcam (Cambridge, MA); pacFACer was obtained from Avanti Polar Lipids (Alabaster, AL); TAMRA-azide-desthiobiotin (1110-5) was from Click Chemistry Tools (Scottsdale, AZ); cyanine 7.5 azide (A6030) was purchased from Lumiprobe (Hallandale Beach, FL); 2-mercaptoethanol (636890), cysteamine (30070), glucose oxidase from Aspergillus niger type VII (G2133-250 KU), and catalase from bovine liver (C40) were purchased from Sigma-Aldrich (St. Louis, MO). Aβ1-42 was from Anaspec (Freemont, CA) dissolved in 1% ammonia and diluted to 1 mg/ml and neutralized in PBS prior to use. All experiments were carried out according to an Animal Use Protocol approved by the Institutional Animal Care and Use Committee at Augusta University and the University of Kentucky. Primary glial cells were isolated from brains of P0-P1 (day at birth or next day) C57BL/6 wild-type or nSMase2-deficient fro/fro mouse pups. Brains were dissociated in PBS containing 0.1 M glucose, passed through a 40 μm filter, and plated in T-25 flasks in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum, and 1% penicillin/streptomycin solution at 37°C in a humidified atmosphere containing 5% CO2. After 7 days, adherent cells were passed to 24-well plates containing uncoated glass coverslips and cultured in DMEM as described above. Cells were fixed with 4% p-formaldehyde/0.5% glutaraldehyde/PBS for 15 min and then permeabilized by incubation with 0.2% Triton X-100 in PBS for 5 min at room temperature. Nonspecific binding sites were blocked with 3% ovalbumin/PBS for 1 h at 37°C. The primary antibodies used were: anti-acetylated tubulin mouse IgG (1:3,000, Sigma-Aldrich, clone 6-1113-1, T6793), anti-ceramide rabbit IgG (1:100, our laboratory) (20.He Q. Wang G. Wakade S. Dasgupta S. Dinkins M. Kong J.N. Spassieva S.D. Bieberich E. Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide.Mol. Biol. Cell. 2014; 25: 1715-1729Crossref PubMed Google Scholar, 24.Krishnamurthy K. Dasgupta S. Bieberich E. Development and characterization of a novel anti-ceramide antibody.J. Lipid Res. 2007; 48: 968-975Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), anti-ceramide mouse IgM (1:100, MAB0014, Glycobiotech GmbH), anti-sigma receptor 1 goat IgG (1:200, Santa Cruz, clone S-18, sc-22948), anti-α-tubulin mouse monoclonal IgG (1:200, Santa Cruz, clone B-7, sc-5286), anti-β-tubulin goat IgG (1:200, Santa Cruz, clone N-20, sc-9935), anti-β-tubulin mouse monoclonal IgG (1:200, Santa Cruz, clone D-10, sc-5274), anti-calnexin goat IgG (1:200, Santa Cruz, sc-6465), anti-Tom 20 rabbit IgG (1:200, Santa Cruz, sc-11415), anti-tubb4 mouse IgG (1:200, Invitrogen, 1-20247), anti-IP3 receptor mouse IgG (1:200, University of California Davis/National Institutes of Health NeuroMab facility, clone L2418), anti-VDAC1 rabbit IgG (1:500, Abcam, ab15895). Secondary antibodies (Alexa Fluor 546-conjugated donkey anti-rabbit IgG, Cy5-conjugated donkey anti-mouse IgM μ-chain specific, Alexa Fluor 647-conjugated goat anti-mouse IgG γ-chain specific (all Jackson ImmunoResearch, West Grove, PA) were diluted 1:300 in 0.1% ovalbumin/PBS and samples incubated for 2 h at 37°C. After washing, coverslips were mounted using Fluoroshield supplemented with DAPI (Sigma-Aldrich) to visualize the nuclei. Confocal fluorescence microscopy was performed using a Zeiss LSM780 upright confocal laser scanning microscope (Zeiss, Jena, Germany) equipped with
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