Abstract
In this study the effects of 30 min heat-shock, ranging from 42°C to 46°C, on survival, protein synthesis and HSP72 expression were investigated in primary rat neuronal cultures. Heat-shock of 44°C resulted in a complete, but transient inhibition of protein synthesis which recovered within 24 h. 46°C heat-shock resulted in an irreversible inhibition of protein synthesis and complete neuronal loss within 24 h. Cycloheximide treatment of neuronal cultures resulted in aggravation of neuronal cell damage after heatshock of 44°C, indicating that the capacity for recovery of the overall protein synthesis is an important survival factor. In addition, the reduction of neuronal cell damage mediated by heat conditioning was abolished by cycloheximide treatment, indicating that the function of new proteins is important for induced thermotolerance. Induction of the strictly inducible member of the heat-shock protein 70kDa family, HSP72, was found in those few astrocytes which were contaminating the neuronal cell cultures, but not in neurons. These results indicate that newly synthesised proteins other than HSP72 are likely to mediate neuronal protection following heat shock in our experiments. These findings raise the possibility that induced tolerance may not necessarily be mediated by HSP72.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- CHX:
-
cycloheximide
- HSP:
-
heat shock protein
- DIV:
-
daysin vitro
- NF:
-
neurofilament
- GFAP:
-
glial fibrillary acidic protein
- PBS:
-
phosphate buffered saline
- TCA:
-
trichloroacetic acid
- SDS:
-
sodium dodecyl sulphate
- PAGE:
-
polyacrylamide gel electrophoresis
- NBT:
-
4-nitrobluetetrazolium chloride
- BCIP:
-
5-bromo-4-chloro-3-indolyl-phosphate 4-toluidine salt
- BSA:
-
bovine serum albumin
- SSC:
-
standard sodium citrate
- ABC:
-
avidin-biotin-complex
References
Abe H., and Nowak Jr. T.S. (1994). Stress protein induction is not required to express ischemic tolerance in the gerbil.Soc. Neurosci. Abstr. 20:1038.
Banker G.A. and Cowan W.M. (1977). Rat hippocampal neurons in dispersed cell culture.Brain Res. 126: 397–425.
Chopp M., Li Y., Dereski M.O., Levine S.R., Yoshida Y. and Garcia J.H. (1991). Neuronal injury and expression of 72-kDa heat-shock protein after forebrain ischemia in the rat.Acta Neuropathol. 83: 66–71.
Dienel G.A., Pulsinelli W.A., and Duffy T.E. (1980). Regional protein synthesis in rat brain following acute hemispheric ischemia.J. Neurochem. 35:1216–1226.
Duchen L.W. (1984). General pathology of the neuron. In (J.H. Adams, J.A.N. Corsellis, and L.W. Duchen, eds.),Greenfield's Neuropathology, Edward Arnold Ltd., London, 1, pp. 8–23.
Ferrini U., Falconi R., Delpino A., Cavaliere R., Zupi G. and Natali P.G. (1984). Heat-shock proteins produced by two human melanoma cell lines: Absence of correlation with thermosensitivity.Int. J. Cancer 34:651–655.
Gething M.-J. and Sambrook J. (1992). Protein folding in the cell.Nature 355:33–45.
Horn M. and Schlote W. (1992). Delayed neuronal death and delayed neuronal recovery in the human brain following global ischemia.Acta Neuropathol. 85:79–87.
Hossmann K.-A. (1993). Disturbances of cerebral protein synthesis and ischemic cell death.Prog. Brain Res. 96:161–177.
Johnston R.N. and Kucey B.L. (1988). Competitive inhibition of HSP-70 gene expression causes thermosensitivity.Science 242:1551–1554.
Kirino T. (1982). Delayed neuronal death in the gerbil hippocampus.Brain Res. 239:58–69.
Kirino T., Tsujita Y. and Tamura A. (1991). Induced tolerance to ischemia in gerbil hippocampal neurons.J. Cereb. Blood Flow Metab. 11:299–307.
Kitagawa K., Matsumoto M., Tagaya M., Kuwabara K., Hata R., Handa N.et al. (1991). Hyperthermia-induced neuronal protection against ischemic injury in gerbils.J. Cereb. Blood Flow Metab. 11:449–452.
Kleihues P. and Hossmann K.-A. (1971). Protein synthesis in the cat brain after prolonged cerebral ischemia.Brain Res. 35:409–418.
Kumar K. (1992). Heat shock proteins in brain ischemia: Role undefined as yet.Metab. Brain Dis. 7:115–123.
Landry J., Chretien P., Lambert H., Hickey E. and Weber L.A. (1989). Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells.J. Cell Biol. 109:7–15.
Lindquist S. (1986). The heat shock response.Ann. Rev. Biochem. 55:1151–1191.
Lowenstein D.H. Characterization and evidence for a protective role in excitotoxicity.Neuron 7:1053–1060.
Lowry O.H., Rosebrough N.J., Farr A.L., and Randall R.J. (1951). Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193:265–275.
Mailhos C., Howard M.K. and Latchman D. S. (1994). Heat shock proteins hsp90 and hsp70 protect neuronal cells from thermal stress but not from programmed cell death.J. Neurochem. 63:1787–1795.
Maniatis T., Fritsch E.F. and Sambrook J. (1982).Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York.
Manzerra P., Rush S.J. and Brown I.R. (1993). Temporal and spatial distribution of heat shock mRNA and protein (hsp70) in the rabbit cerebellum in response to hyperthermia.J. Neurosci. Res. 36:480–490.
Marini A.M., Kozuka M., Lipsky R.H. and Nowak Jr. T.S. (1990). 70-Kilodalton heat shock protein induction in cerebellar astrocytes and cerebellar granule cellsin vitro: comparison with immunocytochemical localization after hyperthermiain vivo.J. Neurochem. 54:1509–1516.
Mima T., Halaby I., Petito C. and Pulsinelli W. (1991). Induction of heat-shock proteins fail to attenuate ischemic damage to CA1 hippocampus.J. Cereb. Blood Flow Metab. 11 (Suppl. 2):S216.
Nakata N., Kato H. and Kogure K. (1993). Inhibition of ischemic tolerance in the gerbil hippocampus by quercetin and anti-heat shock protein-70 antibody.Neuroreport 4:695–698.
Nishi S., Taki W., Uemura Y., Higashi T., Kukuchi H., Kudoh H.et al. (1993). Ischemic tolerance due to the induction of HSP70 in a rat ischemic recirculation model.Brain Res. 615:281–288.
Nishimura R.N., Dwyer B.E., Cole R. and de Vellis J. (1988). Induction of the 68/72 kDa heat-shock protein during hydrogen peroxide toxicity.Neural Development and Regeneration H 22:227–231.
Nishimura R.N., Dwyer B.E., Cole R., de Vellis J. and Picard K. (1989). Induction of the major inducible 68-kDa heat-shock protein after rapid changes of extracellular pH in cultured rat astrocytes.Exp. Cell Res. 180:276–280.
Nowak Jr. T.S. (1990). Protein synthesis and the heat shock/stress response after ischemiaCerebrovasc. Brain Metab. Rev. 2:345–366.
Nowak Jr. T.S. (1993). Synthesis of heat shock/stress proteins during cellular injury.Ann. N.Y. Acad. Sci. 679:142–156.
Nowak Jr. T.S., Bond U. and Schlesinger M.J. (1990). Heat shock RNA levels in brain and other tissues after hyperthermia and transient ischemia.J. Neurochem. 54:451–458.
Ovelgonne J.H., Souren J.E.M., Wiegant F.A.C. and Vanwijk R. (1995). Relationship between cadmium-induced expression of heatshock genes, inhibition of protein synthesis and cell death.Toxicology 99:19–30.
Riabowol K.T., Mizzen L.A. and Welch W.J. (1988). Heat shock is lethal to fibroblasts microinjected with antibodies against HSP70.Science 242:433–436.
Tomioka C., Nishioka K. and Kogure K. (1993). A comparison of induced heat-shock protein in neurons destined to survive and those destined to die after transient ischemia in rats.Brain Res. 612:216–220.
Uto A., Dux E. and Hossmann K.-A. (1994). Effect of serum on intracellular calcium homeostasis and survival of primary cortical an hippocampal CA1 neurons following brief glutamate treatment.Metab. Brain Dis. 9:333–345.
Uto A., Dux E., Kusumoto M. and Hossmann K.-A. (1995). Delayed neuronal death after brief histotoxic hypoxiain vitro.J. Neurochem. 64:2185–2192.
Welch W.J. (1992). Mammalian stress response: Cell physiology, structure/function of stress proteins, and implications for medicine and disease.Physiol. Rev. 72:1063–1081.
Widelitz R.B., Magun B.E. and Gerner E.W. (1986). Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.Mol. Cell. Biol. 6:1088–1094.
Wiessner C., Neumann-Haefelin T., Vogel P., Back T. and Hossmann K.-A. (1995). Transient forebrain ischemia induces an immediate-early gene encoding the mitogen-activated protein kinase phosphatase 3CH134 in the adult rat brain.Neurosci. 64:959–966.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Vogel, P., Dux, E. & Wiessner, C. Effect of heat shock on neuronal cultures: Importance of protein synthesis and HSP72 induction for induced tolerance and survival. Metab Brain Dis 12, 203–217 (1997). https://doi.org/10.1007/BF02674613
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02674613