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Proc Natl Acad Sci U S A. Mar 19, 1996; 93(6): 2312–2316.
PMCID: PMC39792

beta-Amyloid toxicity in organotypic hippocampal cultures: protection by EUK-8, a synthetic catalytic free radical scavenger.

Abstract

Oxygen free radicals have been proposed to mediate amyloid peptide (beta-AP)-induced neurotoxicity. To test this hypothesis, we evaluated the effects of EUK-8, a synthetic catalytic superoxide and hydrogen peroxide scavenger, on neuronal injury produced by beta-AP in organotypic hippocampal slice cultures. Cultures of equivalent postnatal day 35 (defined as mature) and 14 (defined as immature) were exposed to various concentrations of beta-AP (1-42 or 1-40) in the absence or presence of 25 microM EUK-8 for up to 72 hours. Neuronal injury was assessed by lactate dehydrogenase release and semiquantitative analysis of propidium iodide uptake at various times after the initiation of beta-AP exposure. Free radical production was inferred from the relative increase in dichlorofluorescein fluorescence, and the degree of lipid peroxidation was determined by assaying thiobarbituric acid-reactive substances. Treatment of mature cultures with beta-AP (50-250 microg/ml) in serum-free conditions resulted in a reproducible pattern of damage, causing a time-dependent increase in neuronal injury accompanied with formation of reactive oxygen species. However, immature cultures were entirely resistant to beta-AP-induced neurotoxicity and also demonstrated no dichlorofluorescein fluorescence or increased lipid peroxidation after beta-AP treatment. Moreover, mature slices exposed to beta-AP in the presence of 25 microM EUK-8 were significantly protected from beta-AP-induced neurotoxicity. EUK-8 also completely blocked beta-AP-induced free radical accumulation and lipid peroxidation. These results not only support a role for oxygen free radicals in beta-AP toxicity but also highlight the therapeutic potential of synthetic radical scavengers in Alzheimer disease.

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  • Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL, Beyreuther K. Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4245–4249. [PMC free article] [PubMed]
  • Selkoe DJ. The molecular pathology of Alzheimer's disease. Neuron. 1991 Apr;6(4):487–498. [PubMed]
  • Chartier-Harlin MC, Crawford F, Houlden H, Warren A, Hughes D, Fidani L, Goate A, Rossor M, Roques P, Hardy J, et al. Early-onset Alzheimer's disease caused by mutations at codon 717 of the beta-amyloid precursor protein gene. Nature. 1991 Oct 31;353(6347):844–846. [PubMed]
  • Itzhaki RF. Possible factors in the etiology of Alzheimer's disease. Mol Neurobiol. 1994 Aug-Dec;9(1-3):1–13. [PubMed]
  • Van Broeckhoven CL. Molecular genetics of Alzheimer disease: identification of genes and gene mutations. Eur Neurol. 1995;35(1):8–19. [PubMed]
  • Games D, Adams D, Alessandrini R, Barbour R, Berthelette P, Blackwell C, Carr T, Clemens J, Donaldson T, Gillespie F, et al. Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein. Nature. 1995 Feb 9;373(6514):523–527. [PubMed]
  • Pike CJ, Burdick D, Walencewicz AJ, Glabe CG, Cotman CW. Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state. J Neurosci. 1993 Apr;13(4):1676–1687. [PubMed]
  • Kowall NW, Beal MF, Busciglio J, Duffy LK, Yankner BA. An in vivo model for the neurodegenerative effects of beta amyloid and protection by substance P. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7247–7251. [PMC free article] [PubMed]
  • Forloni G, Chiesa R, Smiroldo S, Verga L, Salmona M, Tagliavini F, Angeretti N. Apoptosis mediated neurotoxicity induced by chronic application of beta amyloid fragment 25-35. Neuroreport. 1993 May;4(5):523–526. [PubMed]
  • Loo DT, Copani A, Pike CJ, Whittemore ER, Walencewicz AJ, Cotman CW. Apoptosis is induced by beta-amyloid in cultured central nervous system neurons. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):7951–7955. [PMC free article] [PubMed]
  • Sribanditmongkol P, Sheu MJ, Tejwani GA. Inhibition of morphine tolerance and dependence by diazepam and its relation to the CNS Met-enkephalin levels. Brain Res. 1994 May 9;645(1-2):1–12. [PubMed]
  • Behl C, Davis JB, Lesley R, Schubert D. Hydrogen peroxide mediates amyloid beta protein toxicity. Cell. 1994 Jun 17;77(6):817–827. [PubMed]
  • Lockhart BP, Benicourt C, Junien JL, Privat A. Inhibitors of free radical formation fail to attenuate direct beta-amyloid25-35 peptide-mediated neurotoxicity in rat hippocampal cultures. J Neurosci Res. 1994 Nov 1;39(4):494–505. [PubMed]
  • Podlisny MB, Stephenson DT, Frosch MP, Tolan DR, Lieberburg I, Clemens JA, Selkoe DJ. Microinjection of synthetic amyloid beta-protein in monkey cerebral cortex fails to produce acute neurotoxicity. Am J Pathol. 1993 Jan;142(1):17–24. [PMC free article] [PubMed]
  • Rimvall K, Keller F, Waser PG. Selective kainic acid lesions in cultured explants of rat hippocampus. Acta Neuropathol. 1987;74(2):183–190. [PubMed]
  • Vornov JJ, Tasker RC, Coyle JT. Direct observation of the agonist-specific regional vulnerability to glutamate, NMDA, and kainate neurotoxicity in organotypic hippocampal cultures. Exp Neurol. 1991 Oct;114(1):11–22. [PubMed]
  • Bruce AJ, Sakhi S, Schreiber SS, Baudry M. Development of kainic acid and N-methyl-D-aspartic acid toxicity in organotypic hippocampal cultures. Exp Neurol. 1995 Apr;132(2):209–219. [PubMed]
  • Tasker RC, Coyle JT, Vornov JJ. The regional vulnerability to hypoglycemia-induced neurotoxicity in organotypic hippocampal culture: protection by early tetrodotoxin or delayed MK-801. J Neurosci. 1992 Nov;12(11):4298–4308. [PubMed]
  • Newell DW, Malouf AT, Franck JE. Glutamate-mediated selective vulnerability to ischemia is present in organotypic cultures of hippocampus. Neurosci Lett. 1990 Aug 24;116(3):325–330. [PubMed]
  • Stoppini L, Buchs PA, Muller D. Lesion-induced neurite sprouting and synapse formation in hippocampal organotypic cultures. Neuroscience. 1993 Dec;57(4):985–994. [PubMed]
  • Butterfield DA, Hensley K, Harris M, Mattson M, Carney J. beta-Amyloid peptide free radical fragments initiate synaptosomal lipoperoxidation in a sequence-specific fashion: implications to Alzheimer's disease. Biochem Biophys Res Commun. 1994 Apr 29;200(2):710–715. [PubMed]
  • Friedlich AL, Butcher LL. Involvement of free oxygen radicals in beta-amyloidosis: an hypothesis. Neurobiol Aging. 1994 Jul-Aug;15(4):443–455. [PubMed]
  • Behl C, Davis J, Cole GM, Schubert D. Vitamin E protects nerve cells from amyloid beta protein toxicity. Biochem Biophys Res Commun. 1992 Jul 31;186(2):944–950. [PubMed]
  • Goodman Y, Steiner MR, Steiner SM, Mattson MP. Nordihydroguaiaretic acid protects hippocampal neurons against amyloid beta-peptide toxicity, and attenuates free radical and calcium accumulation. Brain Res. 1994 Aug 15;654(1):171–176. [PubMed]
  • Harris ME, Hensley K, Butterfield DA, Leedle RA, Carney JM. Direct evidence of oxidative injury produced by the Alzheimer's beta-amyloid peptide (1-40) in cultured hippocampal neurons. Exp Neurol. 1995 Feb;131(2):193–202. [PubMed]
  • Musleh W, Bruce A, Malfroy B, Baudry M. Effects of EUK-8, a synthetic catalytic superoxide scavenger, on hypoxia- and acidosis-induced damage in hippocampal slices. Neuropharmacology. 1994 Jul;33(7):929–934. [PubMed]
  • Yankner BA, Duffy LK, Kirschner DA. Neurotrophic and neurotoxic effects of amyloid beta protein: reversal by tachykinin neuropeptides. Science. 1990 Oct 12;250(4978):279–282. [PubMed]
  • Burdick D, Soreghan B, Kwon M, Kosmoski J, Knauer M, Henschen A, Yates J, Cotman C, Glabe C. Assembly and aggregation properties of synthetic Alzheimer's A4/beta amyloid peptide analogs. J Biol Chem. 1992 Jan 5;267(1):546–554. [PubMed]
  • Stoppini L, Buchs PA, Muller D. A simple method for organotypic cultures of nervous tissue. J Neurosci Methods. 1991 Apr;37(2):173–182. [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Koh JY, Choi DW. Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. J Neurosci Methods. 1987 May;20(1):83–90. [PubMed]
  • Kovachich GB, Mishra OP. Lipid peroxidation in rat brain cortical slices as measured by the thiobarbituric acid test. J Neurochem. 1980 Dec;35(6):1449–1452. [PubMed]
  • Pozzo Miller LD, Mahanty NK, Connor JA, Landis DM. Spontaneous pyramidal cell death in organotypic slice cultures from rat hippocampus is prevented by glutamate receptor antagonists. Neuroscience. 1994 Nov;63(2):471–487. [PubMed]
  • Games D, Khan KM, Soriano FG, Keim PS, Davis DL, Bryant K, Lieberburg I. Lack of Alzheimer pathology after beta-amyloid protein injections in rat brain. Neurobiol Aging. 1992 Sep-Oct;13(5):569–576. [PubMed]
  • Stein-Behrens B, Adams K, Yeh M, Sapolsky R. Failure of beta-amyloid protein fragment 25-35 to cause hippocampal damage in the rat. Neurobiol Aging. 1992 Sep-Oct;13(5):577–579. [PubMed]
  • Smyth MD, Kesslak JP, Cummings BJ, Cotman CW. Analysis of brain injury following intrahippocampal administration of beta-amyloid in streptozotocin-treated rats. Neurobiol Aging. 1994 Mar-Apr;15(2):153–159. [PubMed]
  • Malouf AT. Effect of beta amyloid peptides on neurons in hippocampal slice cultures. Neurobiol Aging. 1992 Sep-Oct;13(5):543–551. [PubMed]
  • Koh JY, Yang LL, Cotman CW. Beta-amyloid protein increases the vulnerability of cultured cortical neurons to excitotoxic damage. Brain Res. 1990 Nov 19;533(2):315–320. [PubMed]
  • Hensley K, Carney JM, Mattson MP, Aksenova M, Harris M, Wu JF, Floyd RA, Butterfield DA. A model for beta-amyloid aggregation and neurotoxicity based on free radical generation by the peptide: relevance to Alzheimer disease. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3270–3274. [PMC free article] [PubMed]
  • Sakamoto A, Ohnishi ST, Ohnishi T, Ogawa R. Relationship between free radical production and lipid peroxidation during ischemia-reperfusion injury in the rat brain. Brain Res. 1991 Jul 19;554(1-2):186–192. [PubMed]
  • Bose R, Schnell CL, Pinsky C, Zitko V. Effects of excitotoxins on free radical indices in mouse brain. Toxicol Lett. 1992 Apr;60(2):211–219. [PubMed]
  • Goodman Y, Steiner MR, Steiner SM, Mattson MP. Nordihydroguaiaretic acid protects hippocampal neurons against amyloid beta-peptide toxicity, and attenuates free radical and calcium accumulation. Brain Res. 1994 Aug 15;654(1):171–176. [PubMed]
  • Baudry M, Etienne S, Bruce A, Palucki M, Jacobsen E, Malfroy B. Salen-manganese complexes are superoxide dismutase-mimics. Biochem Biophys Res Commun. 1993 Apr 30;192(2):964–968. [PubMed]
  • Schubert D, Behl C, Lesley R, Brack A, Dargusch R, Sagara Y, Kimura H. Amyloid peptides are toxic via a common oxidative mechanism. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):1989–1993. [PMC free article] [PubMed]

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