Neurotoxicity Research

, Volume 32, Issue 2, pp 163–171 | Cite as

4′-Chlorodiazepam Protects Mitochondria in T98G Astrocyte Cell Line from Glucose Deprivation

  • Eliana Baez
  • Gina Paola Guio-Vega
  • Valentina Echeverria
  • Daniel Andres Sandoval-Rueda
  • George E. Barreto


The translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor (PBR), is considered an important regulator of steroidogenesis and a potential therapeutic target in neurological disorders. Previous evidence suggests that TSPO ligands can protect cells during injury and prevent apoptosis in central nervous system (CNS) cells. However, its actions on astrocytic cells under metabolic injury are not well understood. In this study, we explored whether 4′-chlorodiazepam (Ro5–4864), a TSPO ligand, might protect astrocyte mitochondria under glucose deprivation. Our results showed that 4′-chlorodiazepam preserved cell viability and reduced nuclear fragmentation in glucose-deprived cells. These effects were accompanied by a reduced production of free radicals and maintenance of mitochondrial functions in cells treated with 4′-chlorodiazepam. Finally, our findings suggest that TSPO might be involved in reducing oxidative stress by preserving mitochondrial functions in astrocytic cells exposed to glucose withdrawal.


TSPO Glucose deprivation Mitochondria 4′-chlorodiazepam Astrocytes 


  1. Acaz-Fonseca E, Avila-Rodriguez M, Garcia-Segura LM, Barreto GE (2016) Regulation of astroglia by gonadal steroid hormones under physiological and pathological conditions. Prog Neurobiol 144:5–26. doi: 10.1016/j.pneurobio.2016.06.002 CrossRefPubMedGoogle Scholar
  2. Alho H, Varga V, Krueger KE (1994) Expression of mitochondrial benzodiazepine receptor and its putative endogenous ligand diazepam binding inhibitor in cultured primary astrocytes and C-6 cells: relation to cell growth. Cell Growth Differ 5:1005–1014PubMedGoogle Scholar
  3. Arbo BD, Benetti F, Garcia-Segura LM, Ribeiro MF (2015) Therapeutic actions of translocator protein (18 kDa) ligands in experimental models of psychiatric disorders and neurodegenerative diseases. J Steroid Biochem Mol Biol 154:68–74. doi: 10.1016/j.jsbmb.2015.07.007 CrossRefPubMedGoogle Scholar
  4. Arbo BD et al (2016) 4′-Chlorodiazepam is neuroprotective against amyloid-beta through the modulation of survivin and bax protein expression in vitro. Brain Res 1632:91–97. doi: 10.1016/j.brainres.2015.12.018 CrossRefPubMedGoogle Scholar
  5. Arbo BD, Vieira-Marques C, Ruiz-Palmero I, Ortiz-Rodriguez A, Arevalo MA, Garcia-Segura LM, Ribeiro MF (2017) 4′-Chlorodiazepam modulates the development of primary hippocampal neurons in a sex-dependent manner. Neurosci Lett 639:98–102. doi: 10.1016/j.neulet.2016.12.067 CrossRefPubMedGoogle Scholar
  6. Austin CJ, Kahlert J, Kassiou M, Rendina LM (2013) The translocator protein (TSPO): a novel target for cancer chemotherapy. Int J Biochem Cell Biol 45:1212–1216. doi: 10.1016/j.biocel.2013.03.004 CrossRefPubMedGoogle Scholar
  7. Avila-Rodriguez M, Garcia-Segura LM, Hidalgo-Lanussa O, Baez E, Gonzalez J, Barreto GE (2016) Tibolone protects astrocytic cells from glucose deprivation through a mechanism involving estrogen receptor beta and the upregulation of neuroglobin expression. Mol Cell Endocrinol 433:35–46. doi: 10.1016/j.mce.2016.05.024 CrossRefPubMedGoogle Scholar
  8. Avila Rodriguez M, Garcia-Segura LM, Cabezas R, Torrente D, Capani F, Gonzalez J, Barreto GE (2014) Tibolone protects T98G cells from glucose deprivation. J Steroid Biochem Mol Biol 144(Pt B):294–303. doi: 10.1016/j.jsbmb.2014.07.009 CrossRefPubMedGoogle Scholar
  9. Barreto G, Veiga S, Azcoitia I, Garcia-Segura LM, Garcia-Ovejero D (2007) Testosterone decreases reactive astroglia and reactive microglia after brain injury in male rats: role of its metabolites, oestradiol and dihydrotestosterone. Eur J Neurosci 25:3039–3046. doi: 10.1111/j.1460-9568.2007.05563.x CrossRefPubMedGoogle Scholar
  10. Barreto G, White RE, Ouyang Y, Xu L, Giffard RG (2011) Astrocytes: targets for neuroprotection in stroke. Cent Nerv Syst Agents Med Chem 11:164–173CrossRefPubMedPubMedCentralGoogle Scholar
  11. Barron AM, Garcia-Segura LM, Caruso D, Jayaraman A, Lee JW, Melcangi RC, Pike CJ (2013) Ligand for translocator protein reverses pathology in a mouse model of Alzheimer’s disease. J Neurosci 33:8891–8897. doi: 10.1523/JNEUROSCI.1350-13.2013 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Black KL, Ikezaki K, Santori E, Becker DP, Vinters HV (1990) Specific high-affinity binding of peripheral benzodiazepine receptor ligands to brain tumors in rat and man. Cancer 65:93–97CrossRefPubMedGoogle Scholar
  13. Cabezas R, Avila MF, Gonzalez J, El-Bacha RS, Barreto GE (2015) PDGF-BB protects mitochondria from rotenone in T98G cells. Neurotox Res 27:355–367. doi: 10.1007/s12640-014-9509-5 CrossRefPubMedGoogle Scholar
  14. Carre E, Ogier M, Boret H, Montcriol A, Bourdon L, Jean-Jacques R (2013) Metabolic crisis in severely head-injured patients: is ischemia just the tip of the iceberg? Front Neurol 4:146. doi: 10.3389/fneur.2013.00146 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Casellas P, Galiegue S, Basile AS (2002) Peripheral benzodiazepine receptors and mitochondrial function. Neurochem Int 40:475–486CrossRefPubMedGoogle Scholar
  16. Cosenza-Nashat M, Zhao ML, Suh HS, Morgan J, Natividad R, Morgello S, Lee SC (2009) Expression of the translocator protein of 18 kDa by microglia, macrophages and astrocytes based on immunohistochemical localization in abnormal human brain. Neuropathol Appl Neurobiol 35:306–328. doi: 10.1111/j.1365-2990.2008.01006.x CrossRefPubMedGoogle Scholar
  17. Costa B, Da Pozzo E, Giacomelli C, Barresi E, Taliani S, Da Settimo F, Martini C (2016) TSPO ligand residence time: a new parameter to predict compound neurosteroidogenic efficacy. Sci Rep 6:18164. doi: 10.1038/srep18164 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Costa B et al (2015) TSPO ligand residence time influences human glioblastoma multiforme cell death/life balance. Apoptosis 20:383–398. doi: 10.1007/s10495-014-1063-3 CrossRefPubMedGoogle Scholar
  19. Cunnane S et al (2011) Brain fuel metabolism, aging, and Alzheimer’s disease. Nutrition 27:3–20. doi: 10.1016/j.nut.2010.07.021 CrossRefPubMedGoogle Scholar
  20. Chen MK, Guilarte TR (2008) Translocator protein 18 kDa (TSPO): molecular sensor of brain injury and repair. Pharmacol Ther 118:1–17. doi: 10.1016/j.pharmthera.2007.12.004 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Choi J, Ifuku M, Noda M, Guilarte TR (2011) Translocator protein (18 kDa)/peripheral benzodiazepine receptor specific ligands induce microglia functions consistent with an activated state. Glia 59:219–230. doi: 10.1002/glia.21091 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Chung JY, Chen H, Midzak A, Burnett AL, Papadopoulos V, Zirkin BR (2013) Drug ligand-induced activation of translocator protein (TSPO) stimulates steroid production by aged brown Norway rat Leydig cells. Endocrinology 154:2156–2165. doi: 10.1210/en.2012-2226 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Dixon SJ, Stockwell BR (2014) The role of iron and reactive oxygen species in cell death. Nat Chem Biol 10:9–17. doi: 10.1038/nchembio.1416 CrossRefPubMedGoogle Scholar
  24. Falchi AM et al (2007) Intracellular cholesterol changes induced by translocator protein (18 kDa) TSPO/PBR ligands. Neuropharmacology 53:318–329. doi: 10.1016/j.neuropharm.2007.05.016 CrossRefPubMedGoogle Scholar
  25. Giatti S et al (2009) Neuroprotective effects of a ligand of translocator protein-18 kDa (Ro5-4864) in experimental diabetic neuropathy. Neuroscience 164:520–529. doi: 10.1016/j.neuroscience.2009.08.005 CrossRefPubMedGoogle Scholar
  26. Gulyas B et al (2009) A comparative autoradiography study in post mortem whole hemisphere human brain slices taken from Alzheimer patients and age-matched controls using two radiolabelled DAA1106 analogues with high affinity to the peripheral benzodiazepine receptor (PBR) system. Neurochem Int 54:28–36. doi: 10.1016/j.neuint.2008.10.001 CrossRefPubMedGoogle Scholar
  27. Gut P et al (2013) Whole-organism screening for gluconeogenesis identifies activators of fasting metabolism. Nat Chem Biol 9:97–104. doi: 10.1038/nchembio.1136 CrossRefPubMedGoogle Scholar
  28. Hannestad J et al (2013) The neuroinflammation marker translocator protein is not elevated in individuals with mild-to-moderate depression: a [(1)(1)C]PBR28 PET study. Brain Behav Immun 33:131–138. doi: 10.1016/j.bbi.2013.06.010 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Jack CR Jr et al (2010) Hypothetical model of dynamic biomarkers of the Alzheimer’s pathological cascade. Lancet Neurol 9:119–128. doi: 10.1016/S1474-4422(09)70299-6 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Kalk NJ et al (2017) Decreased hippocampal translocator protein (18 kDa) expression in alcohol dependence: a [11C]PBR28 PET study. Transl Psychiatry 7:e996. doi: 10.1038/tp.2016.264 CrossRefPubMedGoogle Scholar
  31. King LS (1975) Viewpoints in the teaching of medical history. I Introductory comments Clio Med 10:129–132PubMedGoogle Scholar
  32. Kugler W, Veenman L, Shandalov Y, Leschiner S, Spanier I, Lakomek M, Gavish M (2008) Ligands of the mitochondrial 18 kDa translocator protein attenuate apoptosis of human glioblastoma cells exposed to erucylphosphohomocholine. Cell Oncol 30:435–450PubMedPubMedCentralGoogle Scholar
  33. Lacapere JJ, Delavoie F, Li H, Peranzi G, Maccario J, Papadopoulos V, Vidic B (2001) Structural and functional study of reconstituted peripheral benzodiazepine receptor. Biochem Biophys Res Commun 284:536–541. doi: 10.1006/bbrc.2001.4975 CrossRefPubMedGoogle Scholar
  34. Lavisse S et al (2012) Reactive astrocytes overexpress TSPO and are detected by TSPO positron emission tomography imaging. J Neurosci 32:10809–10818. doi: 10.1523/JNEUROSCI.1487-12.2012 CrossRefPubMedGoogle Scholar
  35. Leonelli E et al (2007) Progesterone and its derivatives are neuroprotective agents in experimental diabetic neuropathy: a multimodal analysis. Neuroscience 144:1293–1304. doi: 10.1016/j.neuroscience.2006.11.014 CrossRefPubMedGoogle Scholar
  36. Leonelli E et al (2005) Ro5-4864, a synthetic ligand of peripheral benzodiazepine receptor, reduces aging-associated myelin degeneration in the sciatic nerve of male rats. Mech Ageing Dev 126:1159–1163. doi: 10.1016/j.mad.2005.06.001 CrossRefPubMedGoogle Scholar
  37. Li J, Smith JA, Dawson ES, Fu A, Nickels ML, Schulte ML, Manning HC (2017) Optimized translocator protein ligand for optical molecular imaging and screening. Bioconjug Chem. doi: 10.1021/acs.bioconjchem.6b00711 Google Scholar
  38. Liu F et al (2009) Reduced O-GlcNAcylation links lower brain glucose metabolism and tau pathology in Alzheimer’s disease. Brain 132:1820–1832. doi: 10.1093/brain/awp099 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Liu Y, Song XD, Liu W, Zhang TY, Zuo J (2003) Glucose deprivation induces mitochondrial dysfunction and oxidative stress in PC12 cell line. J Cell Mol Med 7:49–56CrossRefPubMedGoogle Scholar
  40. Mazzeo AT, Beat A, Singh A, Bullock MR (2009) The role of mitochondrial transition pore, and its modulation, in traumatic brain injury and delayed neurodegeneration after TBI. Exp Neurol 218:363–370. doi: 10.1016/j.expneurol.2009.05.026 CrossRefPubMedGoogle Scholar
  41. Midzak A, Zirkin B, Papadopoulos V (2015) Translocator protein: pharmacology and steroidogenesis. Biochem Soc Trans 43:572–578. doi: 10.1042/BST20150061 CrossRefPubMedGoogle Scholar
  42. Mitro N et al (2012) LXR and TSPO as new therapeutic targets to increase the levels of neuroactive steroids in the central nervous system of diabetic animals. Neurochem Int 60:616–621. doi: 10.1016/j.neuint.2012.02.025 CrossRefPubMedGoogle Scholar
  43. Morohaku K, Pelton SH, Daugherty DJ, Butler WR, Deng W, Selvaraj V (2014) Translocator protein/peripheral benzodiazepine receptor is not required for steroid hormone biosynthesis. Endocrinology 155:89–97. doi: 10.1210/en.2013-1556 CrossRefPubMedGoogle Scholar
  44. Notter T et al (2017) Translational evaluation of translocator protein as a marker of neuroinflammation in schizophrenia. Mol Psychiatry. doi: 10.1038/mp.2016.248 Google Scholar
  45. Oda T, Ueda A, Shimizu N, Handa H, Kasahara T (2002) Suppression of monocyte chemoattractant protein 1, but not IL-8, by alprazolam: effect of alprazolam on c-Rel/p65 and c-Rel/p50 binding to the monocyte chemoattractant protein 1 promoter region. J Immunol 169:3329–3335CrossRefPubMedGoogle Scholar
  46. Ortore G, Tuccinardi T, Martinelli A (2012) Computational studies on translocator protein (TSPO) and its ligands. Curr Top Med Chem 12:352–359CrossRefPubMedGoogle Scholar
  47. Panieri E, Gogvadze V, Norberg E, Venkatesh R, Orrenius S, Zhivotovsky B (2013) Reactive oxygen species generated in different compartments induce cell death, survival, or senescence. Free Radic Biol Med 57:176–187. doi: 10.1016/j.freeradbiomed.2012.12.024 CrossRefPubMedGoogle Scholar
  48. Papadopoulos V, Lecanu L (2009) Translocator protein (18 kDa) TSPO: an emerging therapeutic target in neurotrauma. Exp Neurol 219:53–57. doi: 10.1016/j.expneurol.2009.04.016 CrossRefPubMedPubMedCentralGoogle Scholar
  49. Papadopoulos V, Lecanu L, Brown RC, Han Z, Yao ZX (2006) Peripheral-type benzodiazepine receptor in neurosteroid biosynthesis, neuropathology and neurological disorders. Neuroscience 138:749–756. doi: 10.1016/j.neuroscience.2005.05.063 CrossRefPubMedGoogle Scholar
  50. Peters A et al (2004) The selfish brain: competition for energy resources. Neurosci Biobehav Rev 28:143–180. doi: 10.1016/j.neubiorev.2004.03.002 CrossRefPubMedGoogle Scholar
  51. Rechichi M et al (2008) TSPO over-expression increases motility, transmigration and proliferation properties of C6 rat glioma cells. Biochim Biophys Acta 1782:118–125. doi: 10.1016/j.bbadis.2007.12.001 CrossRefPubMedGoogle Scholar
  52. Repalli J (2014) Translocator protein (TSPO) role in aging and Alzheime’s disease. Curr Aging Sci 7:168–175CrossRefPubMedPubMedCentralGoogle Scholar
  53. Ricchelli F, Sileikyte J, Bernardi P (2011) Shedding light on the mitochondrial permeability transition. Biochim Biophys Acta 1807:482–490. doi: 10.1016/j.bbabio.2011.02.012 CrossRefPubMedGoogle Scholar
  54. Ritta MN, Campos MB, Calandra RS (1987) Effect of GABA and benzodiazepines on testicular androgen production. Life Sci 40:791–798CrossRefPubMedGoogle Scholar
  55. Rupprecht R et al (2010) Translocator protein (18 kDa) (TSPO) as a therapeutic target for neurological and psychiatric disorders. Nat Rev Drug Discov 9:971–988. doi: 10.1038/nrd3295 CrossRefPubMedGoogle Scholar
  56. Samarghandian S, Shabestari MM (2013) DNA fragmentation and apoptosis induced by safranal in human prostate cancer cell line. Indian J Urol 29:177–183. doi: 10.4103/0970-1591.117278 CrossRefPubMedPubMedCentralGoogle Scholar
  57. Samplaski MK, Dimitromanolakis A, Lo KC, Grober ED, Mullen B, Garbens A, Jarvi KA (2015) The relationship between sperm viability and DNA fragmentation rates. Reprod Biol Endocrinol 13:42. doi: 10.1186/s12958-015-0035-y CrossRefPubMedPubMedCentralGoogle Scholar
  58. Sena LA, Chandel NS (2012) Physiological roles of mitochondrial reactive oxygen species. Mol Cell 48:158–167. doi: 10.1016/j.molcel.2012.09.025 CrossRefPubMedPubMedCentralGoogle Scholar
  59. Shah K, Desilva S, Abbruscato T (2012) The role of glucose transporters in brain disease: diabetes and Alzheimer’s disease. Int J Mol Sci 13:12629–12655. doi: 10.3390/ijms131012629 CrossRefPubMedPubMedCentralGoogle Scholar
  60. Skaper SD (2007) The brain as a target for inflammatory processes and neuroprotective strategies. Ann N Y Acad Sci 1122:23–34. doi: 10.1196/annals.1403.002 CrossRefPubMedGoogle Scholar
  61. Soustiel JF, Vlodavsky E, Milman F, Gavish M, Zaaroor M (2011) Improvement of cerebral metabolism mediated by Ro5-4864 is associated with relief of intracranial pressure and mitochondrial protective effect in experimental brain injury. Pharm Res 28:2945–2953. doi: 10.1007/s11095-011-0463-0 CrossRefPubMedGoogle Scholar
  62. Soustiel JF, Zaaroor M, Vlodavsky E, Veenman L, Weizman A, Gavish M (2008) Neuroprotective effect of Ro5-4864 following brain injury. Exp Neurol 214:201–208. doi: 10.1016/j.expneurol.2008.08.008 CrossRefPubMedGoogle Scholar
  63. Strohmeier R, Roller M, Sanger N, Knecht R, Kuhl H (2002) Modulation of tamoxifen-induced apoptosis by peripheral benzodiazepine receptor ligands in breast cancer cells. Biochem Pharmacol 64:99–107CrossRefPubMedGoogle Scholar
  64. Toro-Urrego N, Garcia-Segura LM, Echeverria V, Barreto GE (2016) Testosterone protects mitochondrial function and regulates neuroglobin expression in astrocytic cells exposed to glucose deprivation. Front Aging Neurosci 8:152. doi: 10.3389/fnagi.2016.00152 CrossRefPubMedPubMedCentralGoogle Scholar
  65. Tu LN, Morohaku K, Manna PR, Pelton SH, Butler WR, Stocco DM, Selvaraj V (2014) Peripheral benzodiazepine receptor/translocator protein global knock-out mice are viable with no effects on steroid hormone biosynthesis. J Biol Chem 289:27444–27454. doi: 10.1074/jbc.M114.578286 CrossRefPubMedPubMedCentralGoogle Scholar
  66. Turkheimer FE et al (2007) Reference and target region modeling of [11C]-(R)-PK11195 brain studies. J Nucl Med 48:158–167PubMedGoogle Scholar
  67. Veenman L et al (2004) Peripheral-type benzodiazepine receptor density and in vitro tumorigenicity of glioma cell lines. Biochem Pharmacol 68:689–698. doi: 10.1016/j.bcp.2004.05.011 CrossRefPubMedGoogle Scholar
  68. Veenman L, Papadopoulos V, Gavish M (2007) Channel-like functions of the 18-kDa translocator protein (TSPO): regulation of apoptosis and steroidogenesis as part of the host-defense response. Curr Pharm Des 13:2385–2405CrossRefPubMedGoogle Scholar
  69. Veiga S, Azcoitia I, Garcia-Segura LM (2005) Ro5-4864, a peripheral benzodiazepine receptor ligand, reduces reactive gliosis and protects hippocampal hilar neurons from kainic acid excitotoxicity. J Neurosci Res 80:129–137. doi: 10.1002/jnr.20430 CrossRefPubMedGoogle Scholar
  70. Voloboueva LA, Suh SW, Swanson RA, Giffard RG (2007) Inhibition of mitochondrial function in astrocytes: implications for neuroprotection. J Neurochem 102:1383–1394. doi: 10.1111/j.1471-4159.2007.4634.x CrossRefPubMedPubMedCentralGoogle Scholar
  71. Werry EL, King VA, Barron ML, Banister SD, Sokias R, Kassiou M (2017) Derivatives of the pyrazolo[1,5-a]pyrimidine acetamide DPA-713 as translocator protein (TSPO) ligands and pro-apoptotic agents in human glioblastoma. Eur J Pharm Sci 96:186–192. doi: 10.1016/j.ejps.2016.09.026 CrossRefPubMedGoogle Scholar
  72. Winkeler A et al (2012) The translocator protein ligand [(1)(8)F]DPA-714 images glioma and activated microglia in vivo. Eur J Nucl Med Mol Imaging 39:811–823. doi: 10.1007/s00259-011-2041-4 CrossRefPubMedPubMedCentralGoogle Scholar
  73. Yousefi OS et al (2013) The 1,4-benzodiazepine Ro5-4864 (4-chlorodiazepam) suppresses multiple pro-inflammatory mast cell effector functions. Cell Commun Signal 11:13. doi: 10.1186/1478-811X-11-13 CrossRefPubMedPubMedCentralGoogle Scholar
  74. Zinnhardt B et al (2017) Combined PET imaging of the inflammatory tumor microenvironment identifies margins of unique radiotracer uptake. Cancer Res. doi: 10.1158/0008-5472.CAN-16-2628 PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Eliana Baez
    • 1
  • Gina Paola Guio-Vega
    • 1
  • Valentina Echeverria
    • 2
  • Daniel Andres Sandoval-Rueda
    • 1
  • George E. Barreto
    • 1
    • 3
  1. 1.Departamento de Nutrición y Bioquímica, Facultad de CienciasPontificia Universidad JaverianaBogotá D.C.Colombia
  2. 2.Universidad San SebastiánConcepciónChile
  3. 3.Instituto de Ciencias BiomédicasUniversidad Autónoma de ChileSantiagoChile

Personalised recommendations