Summary:
Neuronal cell death causes progressive loss of brain tissue and function after acute brain injury and in chronic neurodegenerative diseases. Although the pathological features of stroke and brain trauma or Alzheimer’s and Parkinson’s disease differ greatly, the underlying neuronal damage shares common molecular and cellular mechanisms. Despite extensive research and increasing knowledge on the molecular pathology, no efficient therapy has been born from these efforts until today. As a promising concept to overcome this plight, it has been suggested to enhance endogenous survival signaling pathways like the transcription factor NF-κB and thus obtain neuroprotection.
Increasing neuroprotective NF-κB signaling can be achieved by blocking repressors of NF-κB transcriptional activity such as p53 and CYLD. Both factors may mediate cell death by mechanisms dependent on and independent of NF-ΚB signaling. Therefore, the major aim of this study was to explore the roles of p53 and CYLD in neuronal cell death and to connect their detrimental effects with NF-κB activity. This issue was addressed in immortalized mouse hippocampal HT-22 neurons and in primary neuronal cultures exposed to glutamate toxicity. Furthermore, an in vivo model system of traumatic brain injury was employed to compare infarct development after controlled cortical impact in wild-type and CYLD-/- mice.
The present study revealed that both approaches, inhibiting p53 and CYLD successfully preserved mitochondrial integrity and function, and significantly attenuated neuronal cell death. Surprisingly, however, the pronounced neuroprotective effect of the p53-inhibitor pifithrin-α occurred independently of enhanced NF-κB activity in HT-22 cells. In addition, neuroprotection induced by silencing of CYLD was completely independent of NF-κB, despite of the previously established role of CYLD as a negative regulator of NF-κB in keratinocytes.
In line with that notion, the NF-κB subunit expression and NF-κB transcriptional activity were not significantly altered in HT-22 neurons undergoing glutamate dependent cell death. In conclusion, these data suggested that the NF-κB pathway was neither significantly affected by glutamate dependent cell death, nor did it mediate the neuroprotective response of CYLD and p53 inhibition in this model system of glutamate toxicity. Interestingly, inhibiting p53 with pifithrin-α maintained mitochondrial morphology and mitochondrial membrane potential in HT-22 cells. This effect occurred independently of p53 dependent transcription.
Investigating the underlying cause of neuroprotection associated with CYLD depletion, it was unveiled that glutamate-induced oxytosis in HT-22 cells occurred through mechanisms of necroptosis. This conclusion is based on the detection of RIP1/RIP3 complexes as a hallmark of necroptotic cell death in HT-22 cells exposed to glutamate. Further, silencing either RIP-kinase provided strong protection of the cells. Repressing CYLD, in turn, prevented the formation of the RIP1/RIP3 necrosome, suggesting that inhibition of necroptosis was the underlying mechanism of neuroprotection after CYLD depletion.
In contrast, CYLD depletion had no effect on cell death in a model of glutamate excitotoxicity in primary cultured neurons, while inhibition of RIP1 kinase by necrostatin-1 significantly enhanced neuronal survival. These data suggest a CYLD independent but RIP1 dependent mechanism of glutamate toxicity in primary neurons, which requires further investigation.
In vivo, however, using a model of traumatic brain injury, CYLD knockout mice showed a significantly reduced infarct size compared to wild-type littermates suggesting a potent neuroprotective effect inherent with CYLD repression.
In summary the data from this thesis highlight a yet unknown role of CYLD in neuronal cell death and unravel CYLD and p53-dependent mechanisms of cell death as a putative therapeutic approach for the treatment of acute and chronic neurodegenerative diseases. Future research, however, is warranted to further elucidate the exact mechanisms leading to CYLD and RIP-kinase activation in neurons and to determine the exact molecular link to mitochondria.
Bibliographie / References
- Courtois G. Tumor suppressor CYLD: negative regulation of NF-kappa B signaling and more. Cell Mol Life Sci. 2008;65(7-8):1123–1132.
- Massoumi R, Chmielarska K, Hennecke K, Pfeifer A, Fässler R. Cyld Inhibits Tumor Cell Proliferation by Blocking Bcl-3-Dependent NF-κB Signaling. Cell. 115 2006;125(4):665–677.
- Hitomi J, Christofferson DE, Ng A, Yao J, Degterev A, Xavier RJ, et al. Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell. 2008;135(7):1311–1323.
- Feng S, Yang Y, Mei Y, Ma L, Zhu D-E, Hoti N, et al. Cleavage of RIP3 inactivates its caspase-independent apoptosis pathway by removal of kinase domain. Cell. Signal. 2007;19(10):2056–2067.
- Seiler A, Schneider M, Förster H, Roth S, Wirth EK, Culmsee C, et al. Glutathione peroxidase 4 senses and translates oxidative stress into 12/15- lipoxygenase dependent-and AIF-mediated cell death. Cell Metab. 2008;8(3):237–248.
- Simonson SJS, Wu Z-H, Miyamoto S. CYLD: a DUB with many talents. Developmental Cell. 2007;13(5):601–603.
- Tenev T, Bianchi K, Darding M, Broemer M, Langlais C, Wallberg F, et al. The Ripoptosome, a Signaling Platform that Assembles in Response to Genotoxic Stress and Loss of IAPs. Molecular Cell. 2011;43(3):432–448.
- Moquin D, Chan FK-M. The molecular regulation of programmed necrotic cell injury. Trends Biochem Sci. 2010;35(8):434–441.
- Sun S-C. Deubiquitylation and regulation of the immune response. Nature Reviews Immunology. 2008;8(7):501–511.
- Courtois G, Gilmore TD. Mutations in the NF-κB signaling pathway: implications for human disease. Oncogene. 2006;25(51):6831–6843.
- Vandenabeele P, Declercq W, Van Herreweghe F, Vanden Berghe T. The role of the kinases RIP1 and RIP3 in TNF-induced necrosis. Sci Signal. 2010;3(115):re4.
- Temkin V, Huang Q, Liu H, Osada H, Pope RM. Inhibition of ADP/ATP exchange in receptor-interacting protein-mediated necrosis. Mol Cell Biol. 2006;26(6):2215–2225.
- de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987;7(2):725–737.
- Manero F. The Small Organic Compound HA14-1 Prevents Bcl-2 Interaction with Bax to Sensitize Malignant Glioma Cells to Induction of Cell Death. Cancer Research. 2006;66(5):2757–2764.
- Knott AB, Bossy-Wetzel E. Impairing the mitochondrial fission and fusion balance: a new mechanism of neurodegeneration. Ann. N. Y. Acad. Sci. 2008;1147283–292.
- Martin LJ. Mitochondrial and Cell Death Mechanisms in Neurodegenerative Diseases. Pharmaceuticals (Basel). 2010;3(4):839–915.
- Vandenabeele P, Galluzzi L, Vanden Berghe T, Kroemer G. Molecular mechanisms of necroptosis: an ordered cellular explosion. Nat Rev Mol Cell Biol. 2010;11(10):700–714.
- Smith CCT, Davidson SM, Lim SY, Simpkin JC, Hothersall JS, Yellon DM. Necrostatin: a potentially novel cardioprotective agent? Cardiovasc Drugs Ther. 2007;21(4):227–233.
- Landshamer S, Hoehn M, Barth N, Duvezin-Caubet S, Schwake G, Tobaben S, et al. Bid-induced release of AIF from mitochondria causes immediate neuronal cell death. Cell Death Differ. 2008;15(10):1553–1563.
- Tobaben S, Grohm J, Seiler A, Conrad M, Plesnila N, Culmsee C. Bid- mediated mitochondrial damage is a key mechanism in glutamate-induced oxidative stress and AIF-dependent cell death in immortalized HT-22 hippocampal neurons. Cell Death Differ. 2010;18(2):282–292.
- S Diemert, Krieg, S W Kim, N Plesnila, C Culmsee; Implications of CYLD in neuronal cell death, 18th Euroconference on Apoptosis, Ghent, Belgium, 1.09.2010-4.09.2010
- S Diemert, S Krieg, S W Kim, N Plesnila, C Culmsee; Loss of CYLD protects neurons in vitro and in vivo, Jahrestagung der deutschen pharmazeutischen Gesellschaft, Jena, 28.9.2009-1.10.2009
- S Diemert, S Krieg, S W Kim, N Plesnila, C Culmsee; Neuroprotection through CYLD depletion in-vitro and in-vivo. Apoptosis (under revision)
- Publications 10.1. Original papers S Diemert, J Grohm, S Tobaben, A Dolga, C Culmsee; Real-Time Detection of Neuronal Cell Death by Impedance-Based Analysis using the xCELLigence System. Application Note, Issue 06, Roche Diagnostics GmbH, Roche Applied Science, Penzberg, Germany, 2010
- Sagara Y, Dargusch R, Chambers D, Davis J, Schubert D, Maher P. Cellular mechanisms of resistance to chronic oxidative stress. Free Radical Biology and Medicine. 1998;24(9):1375–1389.
- Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 2005;1(2):112–119.
- Vanlangenakker N, Vanden Berghe T, Bogaert P, Laukens B, Zobel K, Deshayes K, et al. cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent reactive oxygen species production. Cell Death Differ. 2011;18(4):656–665.
- Bhakar A, Tannis L, Zeindler C, Russo M, Jobin C, Park D, et al. Constitutive nuclear factor-kappa B activity is required for central neuron survival. J Neurosci. 2002;22(19):8466–8475.
- Schulze-Osthoff K, Bakker AC, Vanhaesebroeck B, Beyaert R, Jacob WA, Fiers W. Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation. J Biol Chem. 1992;267(8):5317–5323.
- Morimoto BH, Koshland DE. Excitatory amino acid uptake and N-methyl-D- aspartate-mediated secretion in a neural cell line. Proc Natl Acad Sci USA. 1990;87(9):3518–3521.
- Welz P-S, Wullaert A, Vlantis K, Kondylis V, Fernández-Majada V, Ermolaeva M, et al. FADD prevents RIP3-mediated epithelial cell necrosis and chronic intestinal inflammation. Nature. 2011;477(7364):330–334.
- Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, et al. Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol. 2000;1(6):489–495.
- Degterev A, Hitomi J, Germscheid M, Ch'en IL, Korkina O, Teng X, et al. Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol. 2008;4(5):313–321.
- Bignell GR, Warren W, Seal S, Takahashi M, Rapley E, Barfoot R, et al. Identification of the familial cylindromatosis tumour-suppressor gene. Nat. Genet. 2000;25(2):160–165.
- Wang X, Su B, Lee H-G, Li X, Perry G, Smith MA, et al. Impaired balance of mitochondrial fission and fusion in Alzheimer's disease. J Neurosci. 2009;29(28):9090–9103.
- Zhang J, Stirling B, Temmerman ST, Ma CA, Fuss IJ, Derry JMJ, et al. Impaired regulation of NF-kappaB and increased susceptibility to colitis- associated tumorigenesis in CYLD-deficient mice. J Clin Invest. 2006;116(11):3042–3049.
- Diemert S, Dolga AM, Tobaben S, Grohm J, Pfeifer S, Oexler E, et al. Impedance measurement for real time detection of neuronal cell death. Journal of Neuroscience Methods. 2012;203(1):69–77.
- Oral Presentations and Poster presentations S Diemert, J Grohm, R Hartmannsgruber, C Culmsee; Inhibition of p53 preserves mitochondrial morphology and function and prevents glutamate-induced cell death in neurons, 50. Jahrestagung der Deutschen Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie Mainz, 10.3.2009-12.3.2009
- Eguchi Y, Shimizu S, Tsujimoto Y. Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Research. 1997;57(10):1835– 1840.
- Culmsee C, Krieglstein J. Ischaemic brain damage after stroke: new insights into efficient therapeutic strategies. International Symposium on Neurodegeneration and Neuroprotection. EMBO Rep. 2007;8(2):129–133. 113
- Gerlach B, Cordier SM, Schmukle AC, Emmerich CH, Rieser E, Haas TL, et al. Linear ubiquitination prevents inflammation and regulates immune signalling. Nature. 2011;471(7340):591–596.
- Brummelkamp TR, Nijman SMB, Dirac AMG, Bernards R. Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-κB. Nature. 2003;424(6950):797–801.
- Bradbury D, Simmons T, Slater K, Crouch S. Measurement of the ADP: ATP ratio in human leukaemic cell lines can be used as an indicator of cell 118 viability, necrosis and apoptosis. Journal of immunological methods. 2000;240(1):79–92.
- Liu Y, Peterson D, Kimura H, Schubert D. Mechanism of cellular 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. J Neurochem. 1997;69(2):581–593.
- Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature. 2006;443(7113):787–795.
- Culmsee C, Landshamer S. Molecular insights into mechanisms of the cell death program: role in the progression of neurodegenerative disorders. Curr Alzheimer Res. 2006;3(4):269–283.
- Arundine M, Tymianski M. Molecular mechanisms of glutamate-dependent neurodegeneration in ischemia and traumatic brain injury. Cell Mol Life Sci. 117 necrosis. Cell. 2008;135(7):1161–1163.
- Grohm, " Molecular regulation of mitochondrial dynamics by dynamin-related protein 1 (Drp1) and Bid in model systems of neuronal cell death " , dissertation 10 Publications 119
- Wu W, Liu P, Li J. Necroptosis: An emerging form of programmed cell death. Crit. Rev. Oncol. Hematol. 2012;82(3):249–258.
- Christofferson DE, Yuan J. Necroptosis as an alternative form of programmed cell death. Curr Opin Cell Biol. 2010;22(2):263–268.
- Vanden Berghe T, Vanlangenakker N, Parthoens E, Deckers W, Devos M, Festjens N, et al. Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features. Cell Death Differ. 2009;17(6):922–930.
- Xu X, Chua CC, Kong J, Kostrzewa RM, Kumaraguru U, Hamdy RC, et al. Necrostatin-1 protects against glutamate-induced glutathione depletion and caspase-independent cell death in HT-22 cells. J Neurochem. 2007;103(5):2004–2014.
- S Diemert, S Krieg, S W Kim, N Plesnila, C Culmsee; Neuroprotection through targeted deletion of CYLD, Society for Neuroscience, Chicago, USA 17.10.2009-21.10.2009
- Kaltschmidt B, Kaltschmidt C. NF-kappaB in the nervous system. Cold Spring Harb Perspect Biol. 2009;1(3):a001271.
- Zhu X, Yu Q-S, Cutler RG, Culmsee CW, Holloway HW, Lahiri DK, et al. Novel p53 inactivators with neuroprotective action: syntheses and pharmacological evaluation of 2-imino-2,3,4,5,6,7-hexahydrobenzothiazole and 2-imino-2,3,4,5,6,7-hexahydrobenzoxazole derivatives. J. Med. Chem. 2002;45(23):5090–5097.
- S Diemert, J Grohm, S Tobaben, C Culmsee; Online-measurement of neuronal cell death, 51. Jahrestagung der Deutschen Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie Mainz, 23.3.2010-25.3.2010
- Tan S, Schubert D, Maher P. Oxytosis: a novel form of programmed cell death. Current topics in medicinal chemistry. 2001;1(6):497–506.
- S Diemert, J Grohm, S Tobaben, A Dolga, C Culmsee; Real time detection of neuronal cell death by the xCELLigence system, Jahrestagung der deutschen pharmazeutischen Gesellschaft, Jena, 28.9.2009-1.10.2009
- Yazdanpanah B, Wiegmann K, Tchikov V, Krut O, Pongratz C, Schramm M, et al. Riboflavin kinase couples TNF receptor 1 to NADPH oxidase. Nature. 2009;460(7259):1159–1163.
- Lu J, Bai L, Sun H, Nikolovska-Coleska Z, Mceachern D, Qiu S, et al. SM- 164: A Novel, Bivalent Smac Mimetic That Induces Apoptosis and Tumor Regression by Concurrent Removal of the Blockade of cIAP-1/2 and XIAP. Cancer Research. 2008;68(22):9384–9393.
- Kasof G, Prosser J, Liu D, Lorenzi M, Gomes B. The RIP-like kinase, RIP3, induces apoptosis and NF-kappa B nuclear translocation and localizes to mitochondria. Febs Lett. 2000;473(3):285–291.
- Tobaben. The role of 12/15-lipoxygenases in ROS-mediated neuronal cell death " , dissertation 2011
- Komander D, Lord CJ, Scheel H, Swift S, Hofmann K, Ashworth A, et al. The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module. Molecular Cell. 2008;29(4):451– 464.
- Kovalenko A, Chable-Bessia C, Cantarella G, Israël A, Wallach D, Courtois G. The tumour suppressor CYLD negatively regulates NF-kappaB signalling by deubiquitination. Nature. 2003;424(6950):801–805.
- Kalai M, Van Loo G, Vanden Berghe T, Meeus A, Burm W, Saelens X, et al. Tipping the balance between necrosis and apoptosis in human and murine cells treated with interferon and dsRNA. Cell Death Differ. 2002;9(9):981– 994.
- Morgan MJ, Kim Y-S, Liu Z-G. TNFalpha and reactive oxygen species in necrotic cell death. Cell Res. 2008;18(3):343–349.
- Kim Y-S, Morgan MJ, Choksi S, Liu Z-G. TNF-Induced Activation of the Nox1 NADPH Oxidase and Its Role in the Induction of Necrotic Cell Death. Molecular Cell. 2007;26(5):675–687.
- Wang L, Du F, Wang X. TNF-α Induces Two Distinct Caspase-8 Activation Pathways. Cell. 2008;133(4):693–703.
- Culmsee C, Zhu X, Yu QS, Chan SL, Camandola S, Guo Z, et al. A synthetic inhibitor of p53 protects neurons against death induced by ischemic and excitotoxic insults, and amyloid beta-peptide. J Neurochem. 2001;77(1):220– 228.
- Festjens N, Kalai M, Smet J, Meeus A, Van Coster R, Saelens X, et al. Butylated hydroxyanisole is more than a reactive oxygen species scavenger. Cell Death Differ. 2006;13(1):166–169.
- Northington FJ, Chavez-Valdez R, Graham EM, Razdan S, Gauda EB, Martin LJ. Necrostatin decreases oxidative damage, inflammation, and injury after neonatal HI. Journal of Cerebral Blood Flow & Metabolism. 2011;31(1):178– 189.
- Billen LP, Shamas-Din A, Andrews DW. Bid: a Bax-like BH3 protein. Oncogene. 2008;27 Suppl 1S93–104.
- Tan S, Sagara Y, Liu Y, Maher P, Schubert D. The regulation of reactive oxygen species production during programmed cell death. The Journal of Cell Biology. 1998;141(6):1423–1432.
- Murphy T, Myamoto M, Sastre A, Schnaar RL, Coyle JT. Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress. Neuron. 1989;2(6):1547–1558.
- Tamura Y,Chiba T, Tanioka T, Shimizu N. NO donor induces Nec-1-inhibitable, but RIP1-independent, necrotic cell death in pancreatic β-cells. 2011;585(19):3058–3064.
- He S, Wang L, Miao L, Wang T, Du F, Zhao L, et al. Receptor Interacting Protein Kinase-3 Determines Cellular Necrotic Response to TNF-α. Cell. 2009;137(6):1100–1111.
- Cho Y, Challa S, Moquin D, Genga R, Ray TD, Guildford M, et al. Phosphorylation-Driven Assembly of the RIP1-RIP3 Complex Regulates Programmed Necrosis and Virus-Induced Inflammation. Cell. 2009;137(6):1112–1123.
- S Diemert, A Dolga, S Tobaben, J Grohm, S Pfeifer, E Oexler, C Culmsee; Impedance measurement for real time detection of neuronal cell death. Neuroscience methods, 2012 Jan 15;203(1):69-77.
- Grohm J, Plesnila N, Culmsee C. Bid mediates fission, membrane permeabilization and peri-nuclear accumulation of mitochondria as a prerequisite for oxidative neuronal cell death. Brain, Behavior, and Immunity. 2010;24(5):831–838.
- Bertrand MJM, Milutinovic S, Dickson KM, Ho WC, Boudreault A, Durkin J, et al. cIAP1 and cIAP2 Facilitate Cancer Cell Survival by Functioning as E3 Ligases that Promote RIP1 Ubiquitination. Molecular Cell. 2008;30(6):689– 700.
- Chan FK-M, Shisler J, Bixby JG, Felices M, Zheng L, Appel M, et al. A role for tumor necrosis factor receptor-2 and receptor-interacting protein in programmed necrosis and antiviral responses. J Biol Chem. 2003;278(51):51613–51621.