, Volume 231, Issue 17, pp 3293–3312 | Cite as

Neuroprotection by the synthetic neurosteroid enantiomers ent-PREGS and ent-DHEAS against Aβ25–35 peptide-induced toxicity in vitro and in vivo in mice

  • Fadia El Bitar
  • Johann Meunier
  • Vanessa Villard
  • Marion Alméras
  • Kathiresan Krishnan
  • Douglas F. Covey
  • Tangui Maurice
  • Yvette Akwa
Original Investigation



Pregnenolone sulfate (PREGS) and dehydroepiandrosterone sulphate (DHEAS) are pro-amnesic, anti-amnesic and neuroprotective steroids in rodents. In Alzheimer’s disease (AD) patient’s brains, their low concentrations are correlated with high levels of Aβ and tau proteins. The unnatural enantiomer ent-PREGS enhanced memory in rodents. We investigated here whether ent-PREGS and ent-DHEAS could be neuroprotective in AD models.


The effects of PREGS, ent-PREGS, DHEAS and ent-DHEAS against Aβ25–35 peptide-induced toxicity were examined in vitro on B104 neuroblastoma cells and in vivo in mice.


B104 cells pretreated with the steroids before Aβ25–35 were analysed by flow cytometry measuring cell viability and death processes. Mice injected intracerebroventricularly with Aβ25–35 and the steroids were analysed for their memory abilities. Additionally, lipid peroxidation levels in the hippocampus were measured.


ent-PREGS and PREGS significantly attenuated the Aβ25–35-induced decrease in cell viability. Both steroids prevented the Aβ25–35-induced increase in late apoptotic cells. PREGS further attenuated the ratio of necrotic cells. ent-DHEAS and DHEAS significantly reduced the Aβ25–35-induced toxicity and prevented the cells from entering late apoptosis and necrosis. All steroids stimulated neurite outgrowth per se and prevented the Aβ25–35-induced decrease. In vivo, ent-PREGS and ent-DHEAS significantly attenuated the Aβ25–35-induced decrease in memory (spontaneous alternation and passive avoidance) and an increase in lipid peroxidation levels. In contrast to the natural steroids, both enantiomers prevented amnesia when injected 6 h before Aβ25–35 in contrast to the natural steroids.


The unnatural steroids ent-PREGS and ent-DHEAS are potent neuroprotective agents and could be effective therapeutical tools in AD.


Alzheimer’s disease Neurosteroid Enantiomer β-amyloid toxicity Learning and memory Oxidative stress Pregnenolone sulphate Dehydroepiandrosterone sulphate Neuroprotection Memory 



We thank Dr A. Meiniel for generously providing us with B104 neuroblastoma cells. We thank the Flow Cytometry Core Facility at King Faisal Specialist Hospital & Research Center (Riyadh) for help in cytometry experiments. This work is supported in part by external resources of the Institut National de la Santé et de la Recherche Médicale (INSERM, Paris) and the University of Montpellier 2 (Montpellier), and by the United States National Institutes of Health grant GM 47969 (DFC).

Conflict of interest

JM and VV are now employees of Amylgen (Montpellier). TM is the scientific director of Amylgen and scientific board adviser of Anavex Life Sciences (Hoboken, NJ, USA). DFC holds equity in Sage Therapeutics Inc. The companies were not involved, scientifically or financially, in the present experiments. The authors declare that they have no other conflict of interest.


  1. Akan P, Kizildag S, Ormen M, Genc S, Oktem MA, Fadiloglu M (2009) Pregnenolone protects the PC-12 cell line against amyloid β peptide toxicity but its sulfate ester does not. Chem Biol Interact 177:65–70PubMedCrossRefGoogle Scholar
  2. Akwa Y, Ladurelle N, Covey DF, Baulieu EE (2001) The synthetic enantiomer of pregnenolone sulfate is very active on memory in rats and mice, even more so than its physiological neurosteroid counterpart: distinct mechanisms? Proc Natl Acad Sci U S A 98:14033–14037PubMedCentralPubMedCrossRefGoogle Scholar
  3. Behl C, Davis JB, Klier FG, Schubert D (1994) Amyloid β peptide induces necrosis rather than apoptosis. Brain Res 645:253–264PubMedGoogle Scholar
  4. Behl C (1997) Amyloid β-protein toxicity and oxidative stress in Alzheimer’s disease. Cell Tissue Res 290:471–480PubMedCrossRefGoogle Scholar
  5. Blennow K, de Leon MJ, Zetterberg H (2006) Alzheimer’s disease. Lancet 368:387–403PubMedCrossRefGoogle Scholar
  6. Butterfield DA, Drake J, Pocernich C, Castegna A (2001) Evidence of oxidative damage in Alzheimer’s disease brain: central role for amyloid β-peptide. Trends Mol Med 7:548–554PubMedCrossRefGoogle Scholar
  7. Butterfield DA, Castegna A, Lauderback CM, Drake J (2002a) Evidence that amyloid β-peptide-induced lipid peroxidation and its sequelae in Alzheimer’s disease brain contribute to neuronal death. Neurobiol Aging 23:655–664PubMedCrossRefGoogle Scholar
  8. Butterfield DA, Griffin S, Munch G, Pasinetti GM (2002b) Amyloid β-peptide and amyloid pathology are central to the oxidative stress and inflammatory cascades under which Alzheimer’s disease brain exists. J Alzheimers Dis 4:193–201PubMedGoogle Scholar
  9. Compagnone NA, Mellon SH (1998) Dehydroepiandrosterone: a potential signalling molecule for neocortical organization during development. Proc Natl Acad Sci U S A 95:4678–4683PubMedCentralPubMedCrossRefGoogle Scholar
  10. Compagnone NA, Mellon SH (2000) Neurosteroids: biosynthesis and function of these novel neuromodulators. Front Neuroendocrinol 21:1–56PubMedCrossRefGoogle Scholar
  11. Covey DF (2009) ent-Steroids: novel tools for studies of signaling pathways. Steroids 74:577–585PubMedCentralPubMedCrossRefGoogle Scholar
  12. Covey DF, Evers AS, Mennerick S, Zorumski CF, Purdy RH (2001) Recent developments in structure-activity relationships for steroid modulators of GABAA receptors. Brain Res Brain Res Rev 37:91–97PubMedCrossRefGoogle Scholar
  13. Deshpande A, Mina E, Glabe C, Busciglio J (2006) Different conformations of amyloid beta induce neurotoxicity by distinct mechanisms in human cortical neurons. J Neurosci 26:6011–6018PubMedCrossRefGoogle Scholar
  14. Ekinci FJ, Linsley MD, Shea TB (2000) β-amyloid-induced calcium influx induces apoptosis in culture by oxidative stress rather than tau phosphorylation. Brain Res Mol Brain Res 76:389–395PubMedGoogle Scholar
  15. El Bitar F, Dastugue B, Meiniel A (1999) Neuroblastoma B104 cell line as a model for analysis of neurite outgrowth and neuronal aggregation induced by Reissner’s fiber material. Cell Tissue Res 298:233–242PubMedCrossRefGoogle Scholar
  16. Farr SA, Banks WA, Uezu K, Gaskin FS, Morley JE (2004) DHEAS improves learning and memory in aged SAMP8 mice but not in diabetic mice. Life Sci 75:2775–2785PubMedCrossRefGoogle Scholar
  17. Flood JF, Smith GE, Roberts E (1988) Dehydroepiandrosterone and its sulfate enhance memory retention in mice. Brain Res 447:269–278PubMedGoogle Scholar
  18. Forloni G, Bugiani O, Tagliavini F, Salmona M (1996) Apoptosis-mediated neurotoxicity induced by β-amyloid and PrP fragments. Mol Chem Neuropathol 28:163–171PubMedCrossRefGoogle Scholar
  19. Gridley KE, Green PS, Simpkins JW (1997) Low concentrations of estradiol reduce β-amyloid25-35-induced toxicity, lipid peroxidation and glucose utilization in human SK-N-SH neuroblastoma cells. Brain Res 778:158–165PubMedGoogle Scholar
  20. Gursoy E, Cardounel A, Kalimi M (2001) Pregnenolone protects mouse hippocampal (HT-22) cells against glutamate and amyloid-β protein toxicity. Neurochem Res 26:15–21PubMedCrossRefGoogle Scholar
  21. Hales TG, Tyndale RF (1994) Few cell lines with GABAA mRNAs have functional receptors. J Neurosci 14:5429–5436PubMedGoogle Scholar
  22. Hardy J, Gwinn-Hardy K (1998) Genetic classification of primary neurodegenerative disease. Science 282:1075–1079PubMedCrossRefGoogle Scholar
  23. Harkany T, Hortobagyi T, Sasvari M, Konya C, Penke B, Luiten PG, Nyakas C (1999) Neuroprotective approaches in experimental models of β-amyloid neurotoxicity: relevance to Alzheimer’s disease. Prog Neuropsychopharmacol Biol Psychiatry 23:963–1008PubMedCrossRefGoogle Scholar
  24. Hermes-Lima M, Willmore WG, Storey KB (1995) Quantification of lipid peroxidation in tissue extracts based on Fe(III)xylenol orange complex formation. Free Radic Biol Med 19:271–280PubMedCrossRefGoogle Scholar
  25. Holscher C, Gengler S, Gault VA, Harriott P, Mallot HA (2007) Soluble β-amyloid[25–35] reversibly impairs hippocampal synaptic plasticity and spatial learning. Eur J Pharmacol 561:85–90PubMedCrossRefGoogle Scholar
  26. Kaminsky YG, Marlatt MW, Smith MA, Kosenko EA (2010) Subcellular and metabolic examination of amyloid-β peptides in Alzheimer disease pathogenesis: evidence for Aβ25-35. Exp Neurol 221:26–37PubMedCrossRefGoogle Scholar
  27. Loo DT, Copani A, Pike CJ, Whittemore ER, Walencewicz AJ, Cotman CW (1993) Apoptosis is induced by β-amyloid in cultured central nervous system neurons. Proc Natl Acad Sci U S A 90:7951–7955PubMedCentralPubMedCrossRefGoogle Scholar
  28. Malouf AT (1992) Effect of β-amyloid peptides on neurons in hippocampal slice cultures. Neurobiol Aging 13:543–551PubMedCrossRefGoogle Scholar
  29. Mark RJ, Blanc EM, Mattson MP (1996) Amyloid β-peptide and oxidative cellular injury in Alzheimer’s disease. Mol Neurobiol 12:211–224PubMedCrossRefGoogle Scholar
  30. Markowski M, Ungeheuer M, Bitran D, Locurto C (2001) Memory-enhancing effects of DHEAS in aged mice on a win-shift water escape task. Physiol Behav 72:521–525PubMedCrossRefGoogle Scholar
  31. Mathis C, Vogel E, Cagniard B, Criscuolo F, Ungerer A (1996) The neurosteroid pregnenolone sulfate blocks deficits induced by a competitive NMDA antagonist in active avoidance and lever-press learning tasks in mice. Neuropharmacology 35:1057–1064PubMedCrossRefGoogle Scholar
  32. Mattson MP, Cheng B, Davis D, Bryant K, Lieberburg I, Rydel RE (1992) β-Amyloid peptides destabilize calcium homeostasis and render human cortical neurons vulnerable to excitotoxicity. J Neurosci 12:376–389PubMedGoogle Scholar
  33. Maurice T, Lockhart BP, Privat A (1996) Amnesia induced in mice by centrally administered β-amyloid peptides involves cholinergic dysfunction. Brain Res 706:181–193PubMedGoogle Scholar
  34. Maurice T, Gregoire C, Espallergues J (2006) Neuro(active)steroids actions at the neuromodulatory sigma1 (σ1) receptor: biochemical and physiological evidences, consequences in neuroprotection. Pharmacol Biochem Behav 84:581–597PubMedCrossRefGoogle Scholar
  35. Maurice T, Junien JL, Privat A (1997) Dehydroepiandrosterone sulfate attenuates dizocilpine-induced learning impairment in mice via sigma 1-receptors. Behav Brain Res 83:159–164PubMedCrossRefGoogle Scholar
  36. Maurice T, Su TP, Privat A (1998) Sigma1 (σ1) receptor agonists and neurosteroids attenuate β25–35-amyloid peptide-induced amnesia in mice through a common mechanism. Neuroscience 83:413–428PubMedCrossRefGoogle Scholar
  37. Meunier J, Ieni J, Maurice T (2006) The anti-amnesic and neuroprotective effects of donepezil against amyloid β25–35 peptide-induced toxicity in mice involve an interaction with the sigma1 receptor. Br J Pharmacol 149:998–1012PubMedCentralPubMedGoogle Scholar
  38. Miranda S, Opazo C, Larrondo LF, Munoz FJ, Ruiz F, Leighton F (2000) The role of oxidative stress in the toxicity induced by amyloid β-peptide in Alzheimer’s disease. Prog Neurobiol 62:633–648PubMedCrossRefGoogle Scholar
  39. Nilsson KR, Zorumski CF, Covey DF (1998) Neurosteroid analogues. 6. The synthesis and GABAA receptor pharmacology of enantiomers of dehydroepiandrosterone sulfate, pregnenolone sulfate, and (3α,5β)-3-hydroxypregnan-20-one sulfate. J Med Chem 41:2604–2613PubMedCrossRefGoogle Scholar
  40. Petit GH, Tobin C, Krishnan K, Moricard Y, Covey DF, Rondi-Reig L, Akwa Y (2011) Pregnenolone sulfate and its enantiomer: differential modulation of memory in a spatial discrimination task using forebrain NMDA receptor deficient mice. Eur Neuropsychopharmacol 21:211–215PubMedCentralPubMedCrossRefGoogle Scholar
  41. Pike CJ, Walencewicz-Wasserman AJ, Kosmoski J, Cribbs DH, Glabe CG, Cotman CW (1995) Structure-activity analyses of β-amyloid peptides: contributions of the β25–35 region to aggregation and neurotoxicity. J Neurochem 64:253–265PubMedCrossRefGoogle Scholar
  42. Sayre LM, Zelasko DA, Harris PL, Perry G, Salomon RG, Smith MA (1997) 4-Hydroxynonenal-derived advanced lipid peroxidation end products are increased in Alzheimer’s disease. J Neurochem 68:2092–2097PubMedCrossRefGoogle Scholar
  43. Schubert D, Brass B, Dumas JP (1986) Protein complexity of central nervous system cell lines. J Neurosci 6:2829–2836PubMedGoogle Scholar
  44. Schubert D, Heinemann S, Carlisle W, Tarikas H, Kimes B, Patrick J, Steinbach JH, Culp W, Brandt BL (1974) Clonal cell lines from the rat central nervous system. Nature 249:224–227PubMedCrossRefGoogle Scholar
  45. Selkoe DJ (1997) Alzheimer’s disease: genotypes, phenotypes, and treatments. Science 275:630–631PubMedCrossRefGoogle Scholar
  46. Solovyan V, Bezvenyuk Z, Huotari V, Tapiola T, Suuronen T, Salminen A (1998) Distinct mode of apoptosis induced by genotoxic agent etoposide and serum withdrawal in neuroblastoma cells. Mol Brain Res 62:43–55PubMedCrossRefGoogle Scholar
  47. Stepanichev MY, Moiseeva YV, Lazareva NA, Onufriev MV, Gulyaeva NV (2003) Single intracerebroventricular administration of amyloid-β25–35 peptide induces impairment in short-term rather than long-term memory in rats. Brain Res Bull 61:197–205PubMedCrossRefGoogle Scholar
  48. Tyndale RF, Hales TG, Olsen RW, Tobin AJ (1994) Distinctive patterns of GABAA receptor subunit mRNAs in 13 cell lines. J Neurosci 14:5417–5428PubMedGoogle Scholar
  49. Vallée M, Mayo W, Le Moal M (2001a) Role of pregnenolone, dehydroepiandrosterone and their sulfate esters on learning and memory in cognitive aging. Brain Res Brain Res Rev 37:301–312PubMedCrossRefGoogle Scholar
  50. Vallée M, Shen W, Heinrichs SC, Zorumski CF, Covey DF, Koob GF, Purdy RH (2001b) Steroid structure and pharmacological properties determine the anti- amnesic effects of pregnenolone sulphate in the passive avoidance task in rats. Eur J Neurosci 14:2003–2010PubMedCrossRefGoogle Scholar
  51. Villard V, Espallergues J, Keller E, Alkam T, Nitta A, Yamada K, Nabeshima T, Vamvakides A, Maurice T (2009) Antiamnesic and neuroprotective effects of the aminotetrahydrofuran derivative ANAVEX1-41 against amyloid β25–35-induced toxicity in mice. Neuropsychopharmacology 34:1552–1566PubMedCrossRefGoogle Scholar
  52. Wang X, Dykens JA, Perez E, Liu R, Yang S, Covey DF, Simpkins JW (2006) Neuroprotective effects of 17β-estradiol and nonfeminizing estrogens against H2O2 toxicity in human neuroblastoma SK-N-SH cells. Mol Pharmacol 70:395–404PubMedCrossRefGoogle Scholar
  53. Weaver CE Jr, Wu FS, Gibbs TT, Farb DH (1998) Pregnenolone sulfate exacerbates NMDA-induced death of hippocampal neurons. Brain Res 803:129–136PubMedGoogle Scholar
  54. Weill-Engerer S, David JP, Sazdovitch V, Liere P, Eychenne B, Pianos A, Schumacher M, Delacourte A, Baulieu EE, Akwa Y (2002) Neurosteroid quantification in human brain regions: comparison between Alzheimer’s and nondemented patients. J Clin Endocrinol Metab 87:5138–5143PubMedCrossRefGoogle Scholar
  55. Xu B, Yang R, Chang F, Chen L, Xie G, Sokabe M (2012) Neurosteroid PREGS protects neurite growth and survival of newborn neurons in the hippocampal dentate gyrus of APPswe/PS1dE9 mice. Curr Alzheimer Res 9:361–372PubMedCrossRefGoogle Scholar
  56. Yang R, Chen L, Wang H, Xu B, Tomimoto H (2012) Anti-amnesic effect of neurosteroid PREGS in Aβ25–35-injected mice through σ1 receptor- and α7nAChR-mediated neuroprotection. Neuropharmacology 63:1042–1050PubMedCrossRefGoogle Scholar
  57. Yankner BA, Duffy LK, Kirschner DA (1990) Neurotrophic and neurotoxic effects of amyloid β protein: reversal by tachykinin neuropeptides. Science 250:279–282PubMedCrossRefGoogle Scholar
  58. Zussy C, Brureau A, Delair B, Marchal S, Keller E, Ixart G, Naert G, Meunier J, Chevallier N, Maurice T, Givalois L (2011) Time-course and regional analyses of the physiopathological changes induced after cerebral injection of an amyloid-β fragment in rats. Am J Pathol 179:315–334PubMedCentralPubMedCrossRefGoogle Scholar
  59. Zwain IH, Yen SS (1999) Neurosteroidogenesis in astrocytes, oligodendrocytes, and neurons of cerebral cortex of rat brain. Endocrinology 140:3843–3852PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Fadia El Bitar
    • 1
    • 2
  • Johann Meunier
    • 3
  • Vanessa Villard
    • 3
  • Marion Alméras
    • 3
  • Kathiresan Krishnan
    • 4
  • Douglas F. Covey
    • 4
  • Tangui Maurice
    • 3
  • Yvette Akwa
    • 1
  1. 1.INSERM U788 and Université Paris SUD, Faculté de Médecine, UMR-S788Le Kremlin-BicêtreFrance
  2. 2.Department of Genetics, Research CentreKing Faisal Specialist Hospital and Research CentreRiyadhSaudi Arabia
  3. 3.INSERM U710 and Université de Montpellier 2MontpellierFrance
  4. 4.School of Medicine, Department of Developmental BiologyWashington University in St. LouisSt. LouisUSA

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