Neurochemical Research

, Volume 32, Issue 9, pp 1476–1482 | Cite as

Beta-Amyloid Toxicity in Embryonic Rat Astrocytes

  • Poincyane Assis-Nascimento
  • Karen M. Jarvis
  • Jeremy R. Montague
  • Laura M. Mudd
Original Paper

Abstract

The senile plaques of Alzheimer’s disease contain a high concentration of beta-amyloid (βA) protein, which may affect the glial population in the septal nucleus, an area of increased risk in AD. βA toxicity was measured in septal glia, via a dose-response experiment, by quantifying the effects of three different doses (0.1, 1, and 10 μM) of βA on cell survival. Astrocytes from embryonic day-16 rats were grown in serum-free media in a single layer culture. Cells were treated on day in vitro (DIV)1 and survival was determined on DIV3 to ascertain which concentration was most toxic. In a separate set of experiments, an attempt was made to protect glial cells from the degenerative effects of βA, with treatments of growth factors and estrogen. βA (10 μM) treatment was administered on DIV1, on DIV2 the cells were treated with estrogen (EST, 10 nM), insulin-like growth factors (IGF1 and IGF2, each 10 ng/ml), basic fibroblast growth factor (bFGF, 5 ng/ml) or nerve growth factor (NGF, 100 ng/ml), and on DIV3 the cells were visualized and quantified by fluorescence microscopy with DAPI (4,6-diamidino-2-phenylindole). In addition to dose-response and glial protection, experiments were also conducted to determine whether toxic effects were due to apoptosis. Our results suggest that the survival of glial populations is significantly affected in all three concentrations (0.1, 1.0, and 10 μM) of βA. Glial protection was evident in the presence of NGF, for it showed the significantly highest survival rate relative to the βA treatment alone. Furthermore, toxic effects of βA appear to be due primarily to apoptosis. Significant reversal of βA-induced apoptosis was seen with bFGF and IGF1.

Keywords

Beta-amyloid Rat Neurotoxicity Apoptosis 

Notes

Acknowledgments

The authors thank Dr. Christophe Hengartner for his generous technical assistance. Poincyane Assis was supported by NIH-NIGMS MARCU*STAR Grant, T34GM08082, NIH-NIGMS RISE Grant, R25 GM9244, and NIH MBRS SCORE Grant S06 GM45455. Laura Mudd and Jeremy Montague were supported by NIH MBRS SCORE Grant S06 GM45455. The photographer, Louie Jarvis, is acknowledged for the fluorescence microscopy images.

References

  1. 1.
    Parihar MS, Hemnani T (2004) Alzheimer’s disease pathogenesis and therapeutic interventions. J Clin Neurosci 11(5):456–467PubMedCrossRefGoogle Scholar
  2. 2.
    Ribaut-Barassin C, Dupont JL, Haeberle AM et al (2003) Alzheimer’s disease proteins in cerebellar and hippocampal synapses during postnatal development and aging of the rat. Neuroscience 120(2):405–423PubMedCrossRefGoogle Scholar
  3. 3.
    Sanchez-Alavez M, Gallegos RA, Kalafut MA et al (2002) Loss of medial septal modulation of dentate gyrus physiology in young mice overexpressing human beta-amyloid precursor protein. Neurosci Lett 330(1):45–48PubMedCrossRefGoogle Scholar
  4. 4.
    LeBlanc AC, Papadopoulos M, Belair C et al (1997) Processing of amyloid precursor protein in human primary neuron and astrocyte cultures. J Neurochem 68(3):1183–1190PubMedCrossRefGoogle Scholar
  5. 5.
    Pike CJ, Walencewicz-Wasserman AJ, Kosmoski J et al (1995) Structure-activity analyses of beta-amyloid peptides: contributions of the beta 25–35 region to aggregation and neurotoxicity. J Neurochem 64(1):253–265PubMedCrossRefGoogle Scholar
  6. 6.
    Parihar MS, Hemnani T (2004) Alzheimer’s disease pathogenesis and therapeutic interventions. J Clin Neurosci 11(5):456–467PubMedCrossRefGoogle Scholar
  7. 7.
    Kobayashi K, Hayashi M, Nakano H et al (2004) Correlation between astrocyte apoptosis and Alzheimer changes in gray matter lesions in Alzheimer’s disease. J Alzheim Dis 6(6):623–632; discussion 673–681Google Scholar
  8. 8.
    Takuma K, Baba A, Matsuda T (2004) Astrocyte apoptosis: implications for neuroprotection. Prog Neurobiol 72(2):111–127PubMedCrossRefGoogle Scholar
  9. 9.
    Nagele RG, Wegiel J, Venkataraman V et al (2004) Contribution of glial cells to the development of amyloid plaques in Alzheimer’s disease. Neurobiol Aging 25(5):663–674PubMedCrossRefGoogle Scholar
  10. 10.
    Cotter DR, Pariante CM, Everall IP (2001) Glial cell abnormalities in major psychiatric disorders: the evidence and implications. Brain Res Bull 55(5):585–595PubMedCrossRefGoogle Scholar
  11. 11.
    Gee JR, Keller JN (2005) Astrocytes: regulation of brain homeostasis via apolipoprotein E. Int J Biochem Cell Biol 37(6):1145–1150PubMedCrossRefGoogle Scholar
  12. 12.
    Marchetti B (1997) Cross-talk signals in the CNS: role of neurotrophic and hormonal factors, adhesion molecules and intercellular signaling agents in luteinizing hormone-releasing hormone (LHRH)-astroglial interactive network. Frontline Biosci (online) 2:D88–D125Google Scholar
  13. 13.
    Zaheer A, Zhong W, Lim R (1995) Expression of mRNAs of multiple growth factors and receptors by neuronal cell lines: detection with RT-PCR. Neurochem Res 20(12):1457–1463PubMedCrossRefGoogle Scholar
  14. 14.
    Chiang YH, Silani V, Zhou FC (1996) Morphological differentiation of astroglial progenitor cells from EGF-responsive neurospheres in response to fetal calf serum, basic fibroblast growth factor, and retinol. Cell Transplant 5(2):179–189PubMedCrossRefGoogle Scholar
  15. 15.
    Yokoyama M, Black IB, Dreyfus CF (1993) NGF increases brain astrocyte number in culture. Exp Neurol 124(3):377–380PubMedCrossRefGoogle Scholar
  16. 16.
    Heese K, Hock C, Otten U (1998) Inflammatory signals induce neurotrophin expression in human microglial cells. J Neurochem 70(2):699–707PubMedCrossRefGoogle Scholar
  17. 17.
    Jousimaa J MJ, Rauvala H (1984) Neurite outgrowth of neuroblastoma cells induced by proteins covalently coupled to glass coverslips. Eur J Cell Biol 35(1):55–61PubMedGoogle Scholar
  18. 18.
    Kapuscinski J (1995) DAPI: a DNA-specific fluorescent probe. Biotech Histochem 70(5):220–233PubMedGoogle Scholar
  19. 19.
    Zar JH (1996) Biostatistical analysis, 3rd edn. Prentice Hall, Upper Saddle River, NJ, pp 662 + appGoogle Scholar
  20. 20.
    Sokal RR, Rohlf SJ (1995) Biometry, 3rd edn. W.H. Freeman and Company, New York, pp 887Google Scholar
  21. 21.
    Meda L, Baron P, Scarlato G (2001) Glial activation in Alzheimer’s Disease: the role of AB and its associated proteins. Neurobiol Aging 22(2001):885–893PubMedCrossRefGoogle Scholar
  22. 22.
    Klegeris A, Walker DG, McGeer PL (1997) Interaction of Alzheimer beta-amyloid peptide with the human monocytic cell line THP-1 results in a protein kinase C-dependent secretion of tumor necrosis factor-alpha. Brain Res 747(1):114–121PubMedCrossRefGoogle Scholar
  23. 23.
    Zheng WH, Bastianetto S, Mennicken F et al (2002) Amyloid beta peptide induces tau phosphorylation and loss of cholinergic neurons in rat primary septal cultures. Neurosci 115(1):201–211CrossRefGoogle Scholar
  24. 24.
    Law A, Gauthier S, Quirion R (2001) Neuroprotective and neurorescuing effects of isoform-specific nitric oxide synthase inhibitors, nitric oxide scavenger, and antioxidant against beta-amyloid toxicity. Br J Pharmacol 133(7):1114–1124PubMedCrossRefGoogle Scholar
  25. 25.
    Yakovlev AG, Faden AI (2004) Mechanisms of neural cell death: implications for development of neuroprotective treatment strategies. NeuroRx 1(1):5–16PubMedCrossRefGoogle Scholar
  26. 26.
    Kim H, Li Q, Hempstead BL et al (2004) Paracrine and autocrine functions of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in brain-derived endothelial cells. J Biol Chem 279(32):33538–33546PubMedCrossRefGoogle Scholar
  27. 27.
    Wie MB, Koh JY, Won MH et al (2001) BAPTA/AM, an intracellular calcium chelator, induces delayed necrosis by lipoxygenase-mediated free radicals in mouse cortical cultures. Prog Neuropsychopharmacol Biol Psychiatry 25(8):1641–1659PubMedCrossRefGoogle Scholar
  28. 28.
    Lobner D, Ali C (2002) Mechanisms of bFGF and NT-4 potentiation of necrotic neuronal death. Brain Res 954(1):42–50PubMedCrossRefGoogle Scholar
  29. 29.
    Ryu BR, Ko HW, Jou I et al (1999) Phosphatidylinositol 3-kinase-mediated regulation of neuronal apoptosis and necrosis by insulin and IGF-I. J Neurobiol 39(4):536–546PubMedCrossRefGoogle Scholar
  30. 30.
    Pekny M, Nilsson M (2005) Astrocyte activation and reactive gliosis. Glia 50(4):427–434PubMedCrossRefGoogle Scholar
  31. 31.
    Lee TH, Kato H, Pan LH et al (1998) Localization of nerve growth factor, trkA and P75 immunoreactivity in the hippocampal formation and basal forebrain of adult rats. Neurosci 83(2):335–349CrossRefGoogle Scholar
  32. 32.
    Soltys Z, Janeczko K, Orzylowska-Sliwinska O et al (2003) Morphological transformations of cells immunopositive for GFAP, TrkA or p75 in the CA1 hippocampal area following transient global ischemia in the rat. A quantitative study. Brain Res 987(2):186–193PubMedCrossRefGoogle Scholar
  33. 33.
    Amantea D, Russo R, Bagetta G et al (2005) From clinical evidence to molecular mechanisms underlying neuroprotection afforded by estrogens. Pharmacol Res 52(2):119–132PubMedCrossRefGoogle Scholar
  34. 34.
    Nakagawara A (2001) Trk receptor tyrosine kinases: a bridge between cancer and neural development. Cancer Lett 169(2):107–114PubMedCrossRefGoogle Scholar
  35. 35.
    Tuszynski MH, Thal L, Pay M et al (2005) A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease. Nature Med 11(6):551–555PubMedGoogle Scholar
  36. 36.
    Ajo R, Cacicedo L, Navarro C et al (2003) Growth hormone action on proliferation and differentiation of cerebral cortical cells from fetal rat. Endocrinol 144(3):1086–1097CrossRefGoogle Scholar
  37. 37.
    Auletta M, Nielsen FC, Gammeltoft S (1992) Receptor-mediated endocytosis and degradation of insulin-like growth factor I and II in neonatal rat astrocytes. J Neurosci Res 31(1):14–20PubMedCrossRefGoogle Scholar
  38. 38.
    Walter HJ, Berry M, Hill DJ et al (1999) Distinct sites of insulin-like growth factor (IGF)-II expression and localization in lesioned rat brain: possible roles of IGF binding proteins (IGFBPs) in the mediation of IGF-II activity. Endocrinol 140(1):520–532CrossRefGoogle Scholar
  39. 39.
    Chrysis D, Calikoglu AS, Ye P et al (2001) Insulin-like growth factor-I overexpression attenuates cerebellar apoptosis by altering the expression of Bcl family proteins in a developmentally specific manner. J Neurosci 21(5):1481–1489PubMedGoogle Scholar
  40. 40.
    Cardona-Gomez GP, Mendez P, DonCarlos LL et al (2001) Interactions of estrogens and insulin-like growth factor-I in the brain: implications for neuroprotection. Brain Res Brain Res Rev 37(1–3):320–334PubMedCrossRefGoogle Scholar
  41. 41.
    Chaban VV, Lakhter AJ, Micevych P (2004) A membrane estrogen receptor mediates intracellular calcium release in astrocytes. Endocrinol 145(8):3788–3795CrossRefGoogle Scholar
  42. 42.
    Garcia-Segura LM, Naftolin F, Hutchison JB et al (1999) Role of astroglia in estrogen regulation of synaptic plasticity and brain repair. J Neurobiol 40(4):574–584PubMedCrossRefGoogle Scholar
  43. 43.
    Leadbeater WE, Gonzalez AM, Logaras N et al (2006) Intracellular trafficking in neurones and glia of fibroblast growth factor-2, fibroblast growth factor receptor 1 and heparan sulphate proteoglycans in the injured adult rat cerebral cortex. J Neurochem 96(4):1189–1200PubMedCrossRefGoogle Scholar
  44. 44.
    Neary JT, Kang Y, Shi YF (2004) Signaling from nucleotide receptors to protein kinase cascades in astrocytes. Neurochem Res 29(11):2037–2042PubMedCrossRefGoogle Scholar
  45. 45.
    Lenhard T, Schober A, Suter-Crazzolara C et al (2002) Fibroblast growth factor-2 requires glial-cell-line-derived neurotrophic factor for exerting its neuroprotective actions on glutamate-lesioned hippocampal neurons. Mol Cell Neurosci 20(2):181–197PubMedCrossRefGoogle Scholar
  46. 46.
    de la Monte SM, Wands JR (2005) Review of insulin and insulin-like growth factor expression, signaling, and malfunction in the central nervous system: relevance to Alzheimer’s disease. J Alzheim Dis 7(1):45–61Google Scholar
  47. 47.
    Trendelenburg G, Dirnagl U (2005) Neuroprotective role of astrocytes in cerebral ischemia: focus on ischemic preconditioning. Glia 50(4):307–320PubMedCrossRefGoogle Scholar
  48. 48.
    Nedergaard M, Dirnagl U (2005) Role of glial cells in cerebral ischemia. Glia 50(4):281–286PubMedCrossRefGoogle Scholar
  49. 49.
    Feng Z, Zhang JT (2004) Protective effect of melatonin on beta-amyloid-induced apoptosis in rat astroglioma C6 cells and its mechanism. Free Radic Biol Med 37(11):1790–1801PubMedCrossRefGoogle Scholar
  50. 50.
    Emsley JG, Mitchell BD, Kempermann G et al (2005) Adult neurogenesis and repair of the adult CNS with neural progenitors, precursors, and stem cells. Prog Neurobiol 75(5):321–341PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Poincyane Assis-Nascimento
    • 1
  • Karen M. Jarvis
    • 1
  • Jeremy R. Montague
    • 1
  • Laura M. Mudd
    • 1
  1. 1.School of Natural and Health SciencesBarry UniversityMiami ShoresUSA

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