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Heat shock protects neuronal cells from programmed cell death by apoptosis
0306-4522/93$6.00+ 0.00
NeuroscienceVol. 55, No. 3, pp. 621427, 1993 Printed in Great Britain
PergamonPress Ltd Q 1993 IBRO
HEAT SHOCK PROTECTS NEURONAL CELLS FROM PROGRAMMED CELL DEATH BY APOPTOSIS C. MAILHOS, M. K. HOWARD Medical
and D. S.
LATCHMAN
Molecular
Biology Unit, Department of Biochemistry, Division of Molecular Pathology, University College London Medical School, The Windeyer Building, Cleveland Street, London WIP 6DB, U.K. Abstract-The programmed cell death (apoptosis) of a proportion of the neurons which form plays a critical role in the development of the nervous system and ensures that the correct number of mature neurons am ultimately present. We show that the prior exposure of neuronal cells to an elevated temperature sufficient to induce the heat-shock response partially protects the cells from apoptotic cell death following subsequent transfer to serum-free medium. The degree of protection observed in experiments using different heat-shock or recovery times correlates with the extent of heat-shock protein synthesis. Similarly activation of heat-shock protein synthesis by inducers other than elevated temperature also results in protection from apoptosis. The mechanism by which the heat-shock proteins may protect neuronal cells from apoptosis is discussed.
functioning in disease. However, whilst a number of factors such as nerve growth factor and cyclic nucleotides which can influence neuronal survival in viva’-“9” and in vitro ‘*,” have been identified, it has not yet proved possible to identify specific proteins which enhance or inhibit apoptosis in neuronal cells, as has been achieved for example in the immune system.lo,X Interestingly inhibitors of protein synthesis and gene transcription can protect neuronal cells from apoptosis as occurs in other cell types.*“~33 However, no significant activation of genes involved in cell death in other cell types (e.g. c-myc or TRPM-2) has been observed in neuronal cells undergoing apoptosis.i9 In a number of different cell systems, the enhanced synthesis of the heat shock or stress proteins (hsps) has been shown to exert a protective effect on cellular survival (for review, see Ref. 17). Thus whilst several of these proteins are present in normal cells they are synthesized at enhanced levels in response to a variety of stresses. Cells containing elevated levels of these proteins either following a previous stress or due to arti~cial over-expression of one or more of the hsps show an enhanced resistance to subsequent stresses ranging from ethanol to cardiac ischaemia.‘5A’ In common with other cell types, neuronal cells induce increased synthesis of hsps following a variety of stresses*‘,*’ and such synthesis has been shown, for example, to protect neuronal cells from glutamate toxicity.‘*,3o We have therefore investigated whether prior induction of the hsps can protect neuronal cells from death by apoptosis. To do this we have used the proliferating neuronai cell line ND7, one of a series of cell lines prepared by fusing non-divi~ng dorsal root ganglion cells with the N18 neuroblastoma
The normal development
of a number of mammalian organ systems involves the death of a larger number of individual cells scattered throughout the tissue (for
reviews, see Refs 10, 40). The death of these cells takes place by an active, energy-dependent process known as programmed cell death or apoptosism This death by apoptosis is distinct from death by necrosis which is an abnormal event in which groups of adjacent cells are passively killed by toxic or noxious agents and which does not occur in normal deveiopment,~ The process of programmed cell death plays a particularly critical role in the development of the nervous system where a large number of the neuronal cells which form die by apoptosis ensuring that the correct number of mature neurons ultimately remain (for review, see Ref. 25). Interestingly the enhanced survival of motorneurons during development (due to reduced cell death) results in the mouse disease muscular dysgenesis” whilst the human inherited disease familial dysautonomia and infantile spinal muscular atrophy involve a reduced level of specific neuronal cell types which has been suggested to be due to enhanced programmed cell death during development? Similarly the enhanced cell death which occurs in human neurodegenerative diseases such as Alzheimer’s or Parkinson’s diseases may occur by apoptosis induced by the absence of specific neurotrophic factors.25.34 Hence the mechanisms regulating programmed cell death are of importance both for the normal functioning of the nervous system and its malAbbreuiarions: BSA, bovine serum aibumin; EDTA, ethylen~ia~netetra acetate; hsp, heat-shuck protein; PBS, phosphate-buffe~d saline; SDS, sodium dodecyl sulphate; TBS, T&-buffered saline. 621
cell line and selecting for the drug resistance of the ganglion cells.“” The resulting cell lines proliferate indefinitely in culture whilst retaining many characteristics of the ganglion cells not found in the parental neuroblastoma.is~3s Upon transfer to defined serumfree medium, ND7 cells cease dividing and a proportion of the cells differentiate into a mature neuronal phenotype exhibiting numerous dendritic processes.” Following such transfer, however, a number of cells do not differentiate but instead undergo a process of programmed cell death involving all the characteristic features of apoptosis including cellular DNA degradation into oligonucleosomal fragments. dependence on protein synthesis and membrane blebbing accompanied by nuclear condensation followed by explosive fragmentation. l4 The extent of apoptosis can be increased by addition of retinoic acid and decreased by addition of cyclic AMP to the serum-free medium indicating that this process can be regulated. We have therefore determined the effect of a prior heat-shock and induction of hsp synthesis on the extent of apoptosis which occurs following subsequent transfer of ND7 cells to serum-free medium. EXPERIMENTAL
PROCEDURES
The ND (neuroblastoma/DRG) series of immortalized ganglionic neurons was preparedis by fusing the NISTGZ azaguanine resistant neuroblastoma (4 x 106 cells) with neonatal Sprague-Dawley rat dorsal root ganglion neurons (5 x lo5 cells) in serum free L-15 medium (Gibco) containing 50% polyethylene glycol 1500 (Kodak) at 37’C for 1.5 min. Cell lines were then selected in L-l.5 medium containing HAT (hypoxanthine amniopterin thymidine), 10% fetal calf serum and 10% bovine endothelial cell-conditioned me~um. Routinely ND cells were grown in RPM1 medium supplemented with 10% fetal calf serum. To induce the cells to cease dividing and undergo morphological differentiation or apoptosis they were transferred to serumfree medium consisting of a 1: 1 mix of Dulbecco’s minimum essential medium (Gibco) and Nutrient mix Ham’s F12 (Gibco) supplemented with human transferrin (S pg/ml), bovine insulin (250 ngjml) and sodium s&mite (30 nM). To analyse the effect of heat-shock, cells were routinely exposed to a temperature of 42°C for 30 min in serum-containing medium, allowed to recover for 2 h at 37°C in the same medium and then transferred to serum-free medium. In other experiments the duration of the heat-shock and recovery periods were varied as indicated. Treatment with propanol or ethanol was carried out by exposing the cells to a 1.5 M concentration in medium for 30 min followed by removal of the alcohol-containing medium and addition of fresh serum-free medium.
dodecylsulphate (SDS), 0.0625 M TrisiHC’I ptl 7V, Io’!~, glycerol, 5% beta-mercaptoethanol and Bromophenol Blue, After boiling for 2 min, aliquots of the samples from equai numbers of cells were split in two and run in duplicate on an SDS-polyacrylamide gel. One half of the gel was stained and destained with Coomassie Blue to allow quantitation of total protein whilst the other half was transferred to nttrocellulose and probed with the AC888 monoclonal antibody which recognizes the mammalian 90,000 mol. wt heat shock protein hsp90.” Prior to exposure to the antibody the blots were first blocked with 3% bovine serum albumin (BSA);lrisbuffered saline (TBS) 0.05% Tween-20 for I 11at room temperature. then washed three times in 0.3% BSA/TBS: 0.05% Tween-20 (Buffer A) and once in TBS for 5 min each. The blots were then incubated with ACKX (I?.! )lg,ml in Buffer A) for 2 h at room temperature and washed as above. They were then exposed to a l/300 dilution of rabbit antimouse immuno~ob~in conjugated to horseradish peroxidase (Dakopak) for 2 h at room temperature as above. Blots were developed using a chemiluminescence kit, with multiple autoradiographic exposures of 5~~30 s. Hsp90 was quantitated by scanning the autoradtographs using the VD620 densitometer (Bio-rad) within the linear range. The values obtained from different samples were equalized for any differences in protein content by cxpressing the result relative to the value obtained by scanning the actin band in the other half of the sample on the stained! destained half of the gel.‘-’ In each case the total protein content of each sample was also determined by the method of Bradford.’ Similar results were obtained by expressing
c
t-i
97--+
69---
Trypan Blue staining At intervals after transfer to serum-free medium the extent of cell death was quantitated by measuring the percentage of viable cells able to exclude Trypan Biue. Cells were aspirated and spun for IOmin at 4000r.p.m. at 4°C. The cell pellet was resuspended in 50 ~1 of medium and an equal volume of 0.4% Trypan Blue in phosphate-buffered saline (PBS) was added. The mixture was incubated at room temperature for 5 min and the proportion of cells able to exclude Trypan Blue counted in a counting chamber. Western blotting Protein samples for blotting were prepared sion of cell pellets in sample buffer containing
by resuspen2.3% sodium
46--+
Fig. I. Western blot with the monoclonal antibody ACXX which reacts with hsp90 using protein extracts from ND7 cells maintained at 37°C (C) or transferred to 42’C for thirty minutes and allowed to recover at 37’C for 2 h (H). The positions of protein molecular weigh& markers of the sizes shown (in mol. wt x IO --‘) are indicated.
Heat shock protects neuronal cells hsp90 levels relative to total protein indicating that the actin level did not alter relative to total protein. DNA isolation
5 x lO*cells were lysed in 0.5 ml DNA lysis buffer (1% SDS, 10 mM Tris/HCl, 100 mM NaCl, 0.1 mM EDTA, pH 8.0) and proteinase K added to 208 pg/ml. After overnight incubation at 37°C the mixture was extracted twice with phenol-chloroform (I: 1) for 1 h on a rotary wheel and then
extracted once with chloroform and ethanol precipitated. The pellet was redissolved in 50 ~1 of Tris-EDTA buffer at 37°C for 1 h and then digested with 10 ng of RNase A for 30 min. DNA samoles were electronhoresed on a 1.2% agarose gel at low Voltage overnight:
623 RESULTS
In initial experiments ND7 cells in serum ing medium were exposed to a temperature for 30 min and allowed to recover for 2 h This treatment induced synthesis of the measured by the increased levels of the ~,~
containof 42°C at 37°C. hsps as mol. wt hsp (hsp90) which was detectable by western blotting with the AC88 antibody (Fig. 1). To assess the effect of heat-shock on the rate of cell death by apoptosis, the cells were transferred following the recovery period to serum-free medium either
100 -
80 “a L 5*
60 -
3 48-
48
24
8)
full
Tlmct
(hours)
Time
(hours)
serum 100 -
80 z ‘5 ; IO
60 -
dr 40 -
20 -
o-c24
48
Fig. 2. (A) Survival (as measured by the percentage of cells able to exclude Trypan Blue) of ND7 cells at the indicated time-points after transfer to serum free medium with or without retinoic acid. Cells were either exposed to a prior heat-shock in full medium or maintained at 37°C throughout the experiment. Solid bars, heat-shocked cells transferred to medium without retinoic acid; hatched bars, control cells transferred to medium without retinoic acid; stippled bars, heat-shocked cells transferred to medium with retinoic acid; open bars, control cells transferred to medium with retinoic acid. Values are the mean of three separate experiments whose standard error is shown by the bars. (B) Survival of parallel cultures of ND7 cells maintained in full serum medium throughout the experiment. Bars are shaded as in A.
6’3
(
M
,411HOS c’/
of IO ” M retmoic acid. We have previously shownI that such transfer results in the death of a proportion of the cells by apoptosis and that the rate of apoptotic cell death is accelerated in the presence of retinoic acid. In agreement with this. following transfer to serum-free medium there was a progressive decrease in the proportion of viable cells in the culture without retinoic acid as judged by the ability to exclude Trypan Blue and this effect was enhanced in the retinoic acid treated culture (Fig. 2A). As expected, ceils which remained in serumcontaining medium did not show significant cell death and were not affected by the addition of retinoic acid (Fig. 2B). Interestingly, however, the cells which had been heat shocked displayed enhanced viability in serum free medium at all time-points after heat-shock both in the presence or absence of retinoic acid and this difference was consistently observed in three separate experiments (Fig. 2A). This suggests that prior heat-shock can partially protect neuronal cells from cell death induced by transfer to serum-free medium and particularly from the enhancement of this effect by retinoic acid treatment. In order to confirm that the cell death observed following transfer to serum free medium was occurring by apoptosis, we prepared DNA from cells exposed to the various treatments, 19 h after transfer to serum-free medium. In these experiments cells exposed to serum-free medium in the presence or absence of retinoic acid showed the characteristic ladder of small DNA fragments of oligonucleosomal size (Fig. 3) indicating that the DNA degradation characteristic of apoptosis”’ was occurring in these cells. In contrast DNA degradation was significantly reduced in the samples exposed to heat-shock prior to transfer. This experiment indicates therefore that heat shock exerts a protective effect by reducing the extent of apoptosis in ND7 cells. In order to determine the relationship between the protective effect of heat-shock and enhanced hsp synthesis, we investigated the effect of varying the duration of exposure to 42°C with a constant recovery period (2.5 h) and subsequent transfer to serum-free medium as before. In these experiments (Figs 4, 5) a brief IO-min heat-shock resulted in only low level synthesis of hsp90 and did not result in enhanced survival following transfer to serum free medium. Indeed the degree of survival was reduced compared to control cells possibly due to a mild toxic effect of heat shock itself. As the period of heat-shock was increased to 20 or 30 minutes, however. there was a progressive increase in hsp90 synthesis which was paralleled by enhanced cell survival. A similar association between the level of hsp synthesis and the degree of protection was also seen when the recovery time at 37’C prior to transfer to serum-free medium was varied following a constant-duration (20 min) heat-shock (data not shown). In order to confirm the involvement of enhanced hsp synthesis in the protective effects we observed, in the
presence
rd.
+
or absence
+
_
CCHH
IoocI-
517 396 344 -
Fig. 3. Agarose gel elctrophoresia of DNA prepared from ND7 cells 19 h after transfer to serum-free medium with (+) or without (-) retinoic acid. Cells were either exposed to heat-shock in full medium prior to transfer (tracks labelled H) or maintained at 37°C throughout the experiment (tracks labelled C). Arrows indicate the positions of DNA markers of the sizes shown (in base pairs)
ND7 cells were treated with either ethanol or propanol for 30 min. Both of these treatments are known to induce hsp synthesis”,‘6 and this was confirmed by measuring hsp90 levels in the treated cells with the AC88 antibody (data not shown). When the cells were transferred to serum-free medium, the treated cells displayed enhanced survival compared to untreated cells in the presence or absence of retinoic acid confirming that induction of the hsps by other means than elevated temperature can protect neuronal cells from apoptosis (Fig. 6). Similar results were also obtained using other concentrations of these alcohols to induce the hsps (data not shown). DISCUSSION
As in other cell types the induction of the heatshock response has been shown to protect neuronal cells from the damaging effects of toxic stimuli both in oh and in uitro. Thus prior exposure to elevated temperature in vim protects neuronal cells against subsequent damage induced by exposure to light2 or ischaemia6 whilst similar exposure in vitro results in increased resistance to treatment with the excitotoxin glutamate.‘s~30 Here we extend these findings to show that prior heat-shock can partially protect neuronal cells from
Heat shock protects neuronal cells 25 20
60
15 40
10 20
5
YT
0
2; 10 lime of heatshock(min)
3b
Fig. 5. Levels of hsp90 (as determined by densitometric scanning of the blots such as that shown in Fig. 4) and cell survival 24 h after transfer to serum-free medium following different periods of heat-shock and a constant recovery period of 2.5 h at 37°C in full medium. Figures are the mean of three experiments whose standard error is shown by the bars.
69-
46Fig. 4. Western blot with the AC88 monoclonal antibody using cells exposed to the indicated period of heat shock (in minutes). The positions of protein molecular weight markers of the sizes shown (in mol. wt x 10m3) are indicated.
apoptosis which unlike the toxic or necrotic agents used by others is a natural event in neuronal development and plays a critical role in ensuring that the correct number of neurons eventually form.25 Indeed it has been suggested** that many cells require continuous signals from other cell types in order to survive and that in the absence of such signals the cells cease to suppress a constitutively active death pathway. Hence developing neuronal cells which fail to receive appropriate trophic factors from the target field’ or ND7 cells when transferred to serum-free
100
medium lacking the survival factors present in serum undergo death by apoptosis. To our knowledge this is the first demonstration in any cell type of the ability of a prior heat-shock to partially protect cells from apoptosis. In many other situations, elevated levels of the hsps have been shown to be responsible for the protective effects observed.‘5s4’ It is likely therefore that the induction of hsp synthesis is also responsible for the protective effect of heat shock on apoptosis in ND7 cells. It was not possible to test the requirement for hsp synthesis in this situation directly by investigating the effects of heat shock in the presence of protein synthesis inhibitors since apoptosis itself is dependent upon de nova protein synthesis both in our cellsI and in other cell types40 In experiments varying heat-shock and recovery times, however, we did observe a clear correlation between degree of induction of the hsps
1
1
‘i L ? 2 ap
80 1 80
n q
40
24h 48h
20
1.5M
propanol
1.5M
ethanol
control
Fig. 6. Survival of ND7 cells at intervals after transfer to serum-free medium with (+) or without (-) retinoic acid following prior exposure to ethanol, propanol or without such exposure (control). Solid bars indicate the results after 24 h in serum free medium, hatched bars the results after 48 h. In this batch of cells. no survival was observed in control cells treated with retinoic acid.
as measured by the increased level of one of these proteins, hsp90 and the extent of the protective effect. Moreover, treatment of ND cells with other inducers of hsp synthesis also resulted in protection from apoptosis upon transfer to serum-free medium confirming that the protective effect of a prior heat shock is likely to be dependent upon enhanced hsp synthesis rather than on some other effect occurring upon exposure to elevated temperature. In general the hsps have been shown to play a critical role in the regulation of protein folding and unfolding in both normal and stressed cells (for reviews, see Refs 8, 27). Thus for example hsp90 is thought to maintain proteins such as the steroid hormone receptors or the Src oncogene product in an inactive form prior to exposure to the appropriate physiologicai signal for activation.5,32 Similarly the hsp70 proteins are believed to play a critical role in catalysing the correct folding and assembly of multi-protein complexes in both the cytoplasm and the endoplasmic reticulum.“-” A protective role of the hsps in apoptosis may therefore involve some aspect of their role in protein folding involving for example the maintenance in an inactive form of the nuclease which digests the cellular DNA during the onset of apoptosis leading to the characteristic breakdown of cellular DNA into oligonucleosome length fragments.” It should be noted that whilst we have measured hsp90 levels as indicative of the changes in hsp synthesis in heat-
shock, our data do not indicate whether all or one of the different hsps are required for the observed protective effect. Similarly it is possible that induction of the hsps is not responsible for the protective e&cc we observe following heat-shock which could involve, for example, inhibition of the synthesis of ;I suicide protein. We are currently taking advantage of the fact that proliferating ND7 cells unlike primary neurons are readily transfectable in order to artihcally ovcrexpress each of the hsps in these cells and thereby identify which, if any, of these proteins is responsible for the protective effect. CONCLUSION
Whatever the precise role of the hsps, however, it is clear that heat shock can partially protect neuronal cells both from damage by toxic agents and from apoptosis. It is possible therefore that the identification of specific non-abusive procedures which elevate hsp levels in neuronal cells in vicc~ may have considerable application in the treatment of neurological diseases involving neuronal damage or enhanced neuronal cell death. Acknowledgements-We are most grateful to David Toft for the supply of the AC88 antibody and to Mary Collins, Sean Thomas and John Wood for helpful discussion. MKH is supported a grant from by Sir Jules Thorn Trust. CM is supported by the Countess of Lisburne Scholarship from UCLMS and an Overseas Research Student Award.
REFERENCES
1. Angelidis C. E., Lazarides I. and Paguolatos G. N. (1991) Constitutive expression of heat-shock protein 70 in mammalian cells confers thermotolerance. Eur. J. Biochem. 99, 35-39. protects against light damage in the rat retina. 2 Barbe M., Tytell M., Gower D. and Welch W. (1988) Hyperthermia Science 241, I8 I771 820. 3 Barde Y. (1989) Trophic factors and neuronal survival. Neuron 2, 1525-1534. 4. Bradford M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Analyf. Biochem. 72, 248-254. 5. Brugge J. S., Yonomoto W. and Darrow D. (1983) Interactions between the Rous Sarcoma virus transforming protein and two cellular phosphoproteins. Molec. cell. Biol. 3, 9-19. 6. Chopp M.. Chen H., Ho K.-L., Dereski M. 0.. Brown E., Hetzel F. W. and Welch K. M. (1989) Transient hyperthermia protects against subsequent forebrain ischemic cell damage in the rat. Neurology 39, 1396 1398. 7. Dhillon V. B., McCallum S., Norton P. M., Twomey B. M., Erkeller-Yuksel F., Lydyard P., Isenberg D. A. and Latchman D. S. (1993) Differential heat shock protein over-expression and its clinical relevance in systemic lupus erythematosus. Ana. rheu. Db. (in press). 8. Ellis R. J. (1990) Molecular Chaperones. Seminurs CeN Biol. 1, 1~72. 9. Gamstrop I. and Sarnat H. B. (1984) Progressive spinal muscular atrophies. New York, Raven Press. 10. Golstein P., Ojcius D. M. and Young J. D.-E. (1991) Cell death mechanisms and the immune system. fmmun. Rec. 121, 30-65. Il. Greene L. A. (1977) Quantitative in rifro studies on the nerve growth factor (NGF) requirements of neurons. 1. Sympathetic neurons. Deul Biol. 58, 96-105. 12. Hahn G. M., Shiu E. C., West B., Goldstein L. and Li G. C. (1985) Mechanistic implications of the induction of thermotolerance in Chinese Hamster cells by organic solvents. Cancer Rex 45, 4138-4143. 13. Hamburger V., Bruno-Bechtold J. K. and Yip J. (1981) Neuronal death in the spinal ganglia of the chick embryo and its reduction by nerve growth factor. J. Neurosci. 1, 60-71. 14. Howard M. K., Burke L. C., Mailhos C., Pizzey A., Gilbert C. S., Lawson W. D., Collins M. K. L., Thomas N. S. B. and Latchman D. S. (1993) Cell cycle arrest of proliferating neuronal cells by serum deprivation can result in either apoptosis or differentiation. J. Neurochem. (in press). 15. Li G. C. (1983) Induction of thermotolerance and enhanced heat shock protein synthesis in Chinese hamster fibroblasts by sodium arsenite and by ethanol. J. Cell Physiol. 115, 116-122. 16. Li G. C. and Hahn G. M. (1978) Ethanol-induced thermotolerance to heat and to Adriamycin. Nafure 274, 699- 701, 17. Lindquist S. and Craig E. A. (1988) The heat shock proteins. A. Rev. Gen. 22, 631--677. 18. Lowenstein D. H., Chan P. H. and Miles M. F. (1991) The stress protein response in cultured neurons: characterization and evidence for a protective role in excitotoxicity. Neuron 7, 1053~1060.
Heat shock protects neuronal cells
621
19. Martin D. P., Ito A., Horigome K., Lampe P. A. and Johnson E. M. Jr (1992) Biochemical characterization of programmed cell death in NGF-deprived sympathetic neurons. J. Neurobiol. 23, 1205-1220. 20. Martin D. P., Schmidt R. E., Di Stefano P. S., Lowry 0. H., Carter J. G. and Johnson E. M. Jr (1988) Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation. J. Cell Biol. 186.829-844. 21. Miller E., Roese J. B. and Morrison-Bogorad M. (1991) Expression of heat shock protein 70 and heat shock cognate 70 messenger RNAs in rat cortex and cerebellum after heat shock or amphetamine treatment. J. Neurochem. 56, 2060-2071. 22. Norton P. M., Isenberg D. A. and Latchman D. S. (1988) Elevated levels of the 90kD heat shock protein in a proportion of SLE patients with active disease. J. Auroimmun. 2, 187-195. 23. Nowak T. S. J. (1985) Synthesis of a stress protein following transient ischaemia in the gerbil. J. Neurochem. 45, 1635-1641. 24. Gppenheim R. W., Haverkamp L. J., Prevette D., MacManaman J. L., Appel S. H. (1988) Reduction of naturally occurring motoneuron death in uiuo by a target derived neurotrophic factor. Science 240, 919-922. 25. Gppenheim R. W. (1991) Cell death during development of the nervous system. A. Rev. Neurosci. 14, 453-501. 26. Pelham H. R. B. (1986) Speculations on the functions of the major heat-shock and glucose regulated proteins. Cell 46, 959-961. 21. Pelham H. R. B. (1989) Heat shock and the sorting of luminal ER proteins. Eur. molec. Biol. Org. J. 8, 3171-3176. 28. Raff M. C. (1992) Social controls on cell survival and cell death. Nature 356, 397-400. 29. Riehl R. M., Sullivan W. P., Vroman B. T., Bauer V. J., Pearson G. R. and Toft D. 0. (1985) Immunological evidence that the nonhormone binding component of avian steroid receptors exists in a wide range of tissues and species. Biochemistry 24, 6586-6591. 30. Rordorf G., Koroshetx W. J. and Bonventne J. V. (1991) Heat shock protects cultured neurons from glutamate toxicity. Neuron 7, 1043-1051. 31. Rydell R. E. and Greene L. A. (1988) CAMP analogues promote survival and neurite outgrowth in cultures of rat sympathetic and sensory neurons independently of nerve growth factor. Proc. natn. Acad. Sci. U.S.A. 85, 1257-1261. 32. Sanchez E. R., Meshinchi S., Tienrungrog W., Schlesinger M. J., Toft D. 0. and Pratt W. B. (1987) Relationship of the 90kDa murine heat shock protein to the untransformed and transformed states of the L cell glucocorticoid receptors. J. biol. Chem. 262, 6986-6991. 33. Scott S. A. and Davies A. M. (1990) Inhibitors of protein synthesis prevent cell death in sensory and parasympathetic neurons deprived of neurotrophic factor in vitro. J. Neurobiol. 21, 630-638. 34. Snider W. D. and Johnson E. M. (1989) Neurotrophic molecules. Ann. Neural. 26, 489-506. 35. Suburo A. M., Wheatley S. C., Horn D. A., Gibson S. J., John R., Fischer-Colbrie R., Wood J. N., Latchman D. S. and Polak J. M. (1992) Intracellular redistribution of neuropeptides and secretory proteins during differentiation of neuronal cell lines. Neuroscience 46, 881-889. 36. Vaux D. L., Cory S. and Adams J. M. (1988) Bcl-2 gene promotes haemopoietic cell survival and co-operates with c-myc to immortalize pre-B cells. Naiure 335, 440-442. 31. Weill C. L. and Greene D. P. (1984) Prevention of natural motoneurons cell death by dibutyryl cyclic GMP. Nature 308, 452-454.
38. Wood J. N., Bevan S. J., Coote P., Darn P. M., Hogan P., Latchman D. S., Morrison C., Rougor G., Theveniau M. and Wheatley S. C. (1990) Novel cell lines display the properties of nociceptive sensory neurons. Proc. R. Sot. B 241, 187-194. 39. Wylie A. H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Narure 284, 555-556. 40. Wylie A. H., Kerr J. F. R. and Currie A. R. (1980) Cell death: the significance of apoptosis. Int. Rev. Cytol. 68,251-306. 41. Yellon D. M. and Latchman D. S. (1991) Stress proteins and myocardial protection. J. molec. cell. Cardiol. 24, 113-124. (Accepted 3 March 1993)
NeuroscienceVol. 55, No. 3, pp. 621427, 1993 Printed in Great Britain
PergamonPress Ltd Q 1993 IBRO
HEAT SHOCK PROTECTS NEURONAL CELLS FROM PROGRAMMED CELL DEATH BY APOPTOSIS C. MAILHOS, M. K. HOWARD Medical
and D. S.
LATCHMAN
Molecular
Biology Unit, Department of Biochemistry, Division of Molecular Pathology, University College London Medical School, The Windeyer Building, Cleveland Street, London WIP 6DB, U.K. Abstract-The programmed cell death (apoptosis) of a proportion of the neurons which form plays a critical role in the development of the nervous system and ensures that the correct number of mature neurons am ultimately present. We show that the prior exposure of neuronal cells to an elevated temperature sufficient to induce the heat-shock response partially protects the cells from apoptotic cell death following subsequent transfer to serum-free medium. The degree of protection observed in experiments using different heat-shock or recovery times correlates with the extent of heat-shock protein synthesis. Similarly activation of heat-shock protein synthesis by inducers other than elevated temperature also results in protection from apoptosis. The mechanism by which the heat-shock proteins may protect neuronal cells from apoptosis is discussed.
functioning in disease. However, whilst a number of factors such as nerve growth factor and cyclic nucleotides which can influence neuronal survival in viva’-“9” and in vitro ‘*,” have been identified, it has not yet proved possible to identify specific proteins which enhance or inhibit apoptosis in neuronal cells, as has been achieved for example in the immune system.lo,X Interestingly inhibitors of protein synthesis and gene transcription can protect neuronal cells from apoptosis as occurs in other cell types.*“~33 However, no significant activation of genes involved in cell death in other cell types (e.g. c-myc or TRPM-2) has been observed in neuronal cells undergoing apoptosis.i9 In a number of different cell systems, the enhanced synthesis of the heat shock or stress proteins (hsps) has been shown to exert a protective effect on cellular survival (for review, see Ref. 17). Thus whilst several of these proteins are present in normal cells they are synthesized at enhanced levels in response to a variety of stresses. Cells containing elevated levels of these proteins either following a previous stress or due to arti~cial over-expression of one or more of the hsps show an enhanced resistance to subsequent stresses ranging from ethanol to cardiac ischaemia.‘5A’ In common with other cell types, neuronal cells induce increased synthesis of hsps following a variety of stresses*‘,*’ and such synthesis has been shown, for example, to protect neuronal cells from glutamate toxicity.‘*,3o We have therefore investigated whether prior induction of the hsps can protect neuronal cells from death by apoptosis. To do this we have used the proliferating neuronai cell line ND7, one of a series of cell lines prepared by fusing non-divi~ng dorsal root ganglion cells with the N18 neuroblastoma
The normal development
of a number of mammalian organ systems involves the death of a larger number of individual cells scattered throughout the tissue (for
reviews, see Refs 10, 40). The death of these cells takes place by an active, energy-dependent process known as programmed cell death or apoptosism This death by apoptosis is distinct from death by necrosis which is an abnormal event in which groups of adjacent cells are passively killed by toxic or noxious agents and which does not occur in normal deveiopment,~ The process of programmed cell death plays a particularly critical role in the development of the nervous system where a large number of the neuronal cells which form die by apoptosis ensuring that the correct number of mature neurons ultimately remain (for review, see Ref. 25). Interestingly the enhanced survival of motorneurons during development (due to reduced cell death) results in the mouse disease muscular dysgenesis” whilst the human inherited disease familial dysautonomia and infantile spinal muscular atrophy involve a reduced level of specific neuronal cell types which has been suggested to be due to enhanced programmed cell death during development? Similarly the enhanced cell death which occurs in human neurodegenerative diseases such as Alzheimer’s or Parkinson’s diseases may occur by apoptosis induced by the absence of specific neurotrophic factors.25.34 Hence the mechanisms regulating programmed cell death are of importance both for the normal functioning of the nervous system and its malAbbreuiarions: BSA, bovine serum aibumin; EDTA, ethylen~ia~netetra acetate; hsp, heat-shuck protein; PBS, phosphate-buffe~d saline; SDS, sodium dodecyl sulphate; TBS, T&-buffered saline. 621
cell line and selecting for the drug resistance of the ganglion cells.“” The resulting cell lines proliferate indefinitely in culture whilst retaining many characteristics of the ganglion cells not found in the parental neuroblastoma.is~3s Upon transfer to defined serumfree medium, ND7 cells cease dividing and a proportion of the cells differentiate into a mature neuronal phenotype exhibiting numerous dendritic processes.” Following such transfer, however, a number of cells do not differentiate but instead undergo a process of programmed cell death involving all the characteristic features of apoptosis including cellular DNA degradation into oligonucleosomal fragments. dependence on protein synthesis and membrane blebbing accompanied by nuclear condensation followed by explosive fragmentation. l4 The extent of apoptosis can be increased by addition of retinoic acid and decreased by addition of cyclic AMP to the serum-free medium indicating that this process can be regulated. We have therefore determined the effect of a prior heat-shock and induction of hsp synthesis on the extent of apoptosis which occurs following subsequent transfer of ND7 cells to serum-free medium. EXPERIMENTAL
PROCEDURES
The ND (neuroblastoma/DRG) series of immortalized ganglionic neurons was preparedis by fusing the NISTGZ azaguanine resistant neuroblastoma (4 x 106 cells) with neonatal Sprague-Dawley rat dorsal root ganglion neurons (5 x lo5 cells) in serum free L-15 medium (Gibco) containing 50% polyethylene glycol 1500 (Kodak) at 37’C for 1.5 min. Cell lines were then selected in L-l.5 medium containing HAT (hypoxanthine amniopterin thymidine), 10% fetal calf serum and 10% bovine endothelial cell-conditioned me~um. Routinely ND cells were grown in RPM1 medium supplemented with 10% fetal calf serum. To induce the cells to cease dividing and undergo morphological differentiation or apoptosis they were transferred to serumfree medium consisting of a 1: 1 mix of Dulbecco’s minimum essential medium (Gibco) and Nutrient mix Ham’s F12 (Gibco) supplemented with human transferrin (S pg/ml), bovine insulin (250 ngjml) and sodium s&mite (30 nM). To analyse the effect of heat-shock, cells were routinely exposed to a temperature of 42°C for 30 min in serum-containing medium, allowed to recover for 2 h at 37°C in the same medium and then transferred to serum-free medium. In other experiments the duration of the heat-shock and recovery periods were varied as indicated. Treatment with propanol or ethanol was carried out by exposing the cells to a 1.5 M concentration in medium for 30 min followed by removal of the alcohol-containing medium and addition of fresh serum-free medium.
dodecylsulphate (SDS), 0.0625 M TrisiHC’I ptl 7V, Io’!~, glycerol, 5% beta-mercaptoethanol and Bromophenol Blue, After boiling for 2 min, aliquots of the samples from equai numbers of cells were split in two and run in duplicate on an SDS-polyacrylamide gel. One half of the gel was stained and destained with Coomassie Blue to allow quantitation of total protein whilst the other half was transferred to nttrocellulose and probed with the AC888 monoclonal antibody which recognizes the mammalian 90,000 mol. wt heat shock protein hsp90.” Prior to exposure to the antibody the blots were first blocked with 3% bovine serum albumin (BSA);lrisbuffered saline (TBS) 0.05% Tween-20 for I 11at room temperature. then washed three times in 0.3% BSA/TBS: 0.05% Tween-20 (Buffer A) and once in TBS for 5 min each. The blots were then incubated with ACKX (I?.! )lg,ml in Buffer A) for 2 h at room temperature and washed as above. They were then exposed to a l/300 dilution of rabbit antimouse immuno~ob~in conjugated to horseradish peroxidase (Dakopak) for 2 h at room temperature as above. Blots were developed using a chemiluminescence kit, with multiple autoradiographic exposures of 5~~30 s. Hsp90 was quantitated by scanning the autoradtographs using the VD620 densitometer (Bio-rad) within the linear range. The values obtained from different samples were equalized for any differences in protein content by cxpressing the result relative to the value obtained by scanning the actin band in the other half of the sample on the stained! destained half of the gel.‘-’ In each case the total protein content of each sample was also determined by the method of Bradford.’ Similar results were obtained by expressing
c
t-i
97--+
69---
Trypan Blue staining At intervals after transfer to serum-free medium the extent of cell death was quantitated by measuring the percentage of viable cells able to exclude Trypan Biue. Cells were aspirated and spun for IOmin at 4000r.p.m. at 4°C. The cell pellet was resuspended in 50 ~1 of medium and an equal volume of 0.4% Trypan Blue in phosphate-buffered saline (PBS) was added. The mixture was incubated at room temperature for 5 min and the proportion of cells able to exclude Trypan Blue counted in a counting chamber. Western blotting Protein samples for blotting were prepared sion of cell pellets in sample buffer containing
by resuspen2.3% sodium
46--+
Fig. I. Western blot with the monoclonal antibody ACXX which reacts with hsp90 using protein extracts from ND7 cells maintained at 37°C (C) or transferred to 42’C for thirty minutes and allowed to recover at 37’C for 2 h (H). The positions of protein molecular weigh& markers of the sizes shown (in mol. wt x IO --‘) are indicated.
Heat shock protects neuronal cells hsp90 levels relative to total protein indicating that the actin level did not alter relative to total protein. DNA isolation
5 x lO*cells were lysed in 0.5 ml DNA lysis buffer (1% SDS, 10 mM Tris/HCl, 100 mM NaCl, 0.1 mM EDTA, pH 8.0) and proteinase K added to 208 pg/ml. After overnight incubation at 37°C the mixture was extracted twice with phenol-chloroform (I: 1) for 1 h on a rotary wheel and then
extracted once with chloroform and ethanol precipitated. The pellet was redissolved in 50 ~1 of Tris-EDTA buffer at 37°C for 1 h and then digested with 10 ng of RNase A for 30 min. DNA samoles were electronhoresed on a 1.2% agarose gel at low Voltage overnight:
623 RESULTS
In initial experiments ND7 cells in serum ing medium were exposed to a temperature for 30 min and allowed to recover for 2 h This treatment induced synthesis of the measured by the increased levels of the ~,~
containof 42°C at 37°C. hsps as mol. wt hsp (hsp90) which was detectable by western blotting with the AC88 antibody (Fig. 1). To assess the effect of heat-shock on the rate of cell death by apoptosis, the cells were transferred following the recovery period to serum-free medium either
100 -
80 “a L 5*
60 -
3 48-
48
24
8)
full
Tlmct
(hours)
Time
(hours)
serum 100 -
80 z ‘5 ; IO
60 -
dr 40 -
20 -
o-c24
48
Fig. 2. (A) Survival (as measured by the percentage of cells able to exclude Trypan Blue) of ND7 cells at the indicated time-points after transfer to serum free medium with or without retinoic acid. Cells were either exposed to a prior heat-shock in full medium or maintained at 37°C throughout the experiment. Solid bars, heat-shocked cells transferred to medium without retinoic acid; hatched bars, control cells transferred to medium without retinoic acid; stippled bars, heat-shocked cells transferred to medium with retinoic acid; open bars, control cells transferred to medium with retinoic acid. Values are the mean of three separate experiments whose standard error is shown by the bars. (B) Survival of parallel cultures of ND7 cells maintained in full serum medium throughout the experiment. Bars are shaded as in A.
6’3
(
M
,411HOS c’/
of IO ” M retmoic acid. We have previously shownI that such transfer results in the death of a proportion of the cells by apoptosis and that the rate of apoptotic cell death is accelerated in the presence of retinoic acid. In agreement with this. following transfer to serum-free medium there was a progressive decrease in the proportion of viable cells in the culture without retinoic acid as judged by the ability to exclude Trypan Blue and this effect was enhanced in the retinoic acid treated culture (Fig. 2A). As expected, ceils which remained in serumcontaining medium did not show significant cell death and were not affected by the addition of retinoic acid (Fig. 2B). Interestingly, however, the cells which had been heat shocked displayed enhanced viability in serum free medium at all time-points after heat-shock both in the presence or absence of retinoic acid and this difference was consistently observed in three separate experiments (Fig. 2A). This suggests that prior heat-shock can partially protect neuronal cells from cell death induced by transfer to serum-free medium and particularly from the enhancement of this effect by retinoic acid treatment. In order to confirm that the cell death observed following transfer to serum free medium was occurring by apoptosis, we prepared DNA from cells exposed to the various treatments, 19 h after transfer to serum-free medium. In these experiments cells exposed to serum-free medium in the presence or absence of retinoic acid showed the characteristic ladder of small DNA fragments of oligonucleosomal size (Fig. 3) indicating that the DNA degradation characteristic of apoptosis”’ was occurring in these cells. In contrast DNA degradation was significantly reduced in the samples exposed to heat-shock prior to transfer. This experiment indicates therefore that heat shock exerts a protective effect by reducing the extent of apoptosis in ND7 cells. In order to determine the relationship between the protective effect of heat-shock and enhanced hsp synthesis, we investigated the effect of varying the duration of exposure to 42°C with a constant recovery period (2.5 h) and subsequent transfer to serum-free medium as before. In these experiments (Figs 4, 5) a brief IO-min heat-shock resulted in only low level synthesis of hsp90 and did not result in enhanced survival following transfer to serum free medium. Indeed the degree of survival was reduced compared to control cells possibly due to a mild toxic effect of heat shock itself. As the period of heat-shock was increased to 20 or 30 minutes, however. there was a progressive increase in hsp90 synthesis which was paralleled by enhanced cell survival. A similar association between the level of hsp synthesis and the degree of protection was also seen when the recovery time at 37’C prior to transfer to serum-free medium was varied following a constant-duration (20 min) heat-shock (data not shown). In order to confirm the involvement of enhanced hsp synthesis in the protective effects we observed, in the
presence
rd.
+
or absence
+
_
CCHH
IoocI-
517 396 344 -
Fig. 3. Agarose gel elctrophoresia of DNA prepared from ND7 cells 19 h after transfer to serum-free medium with (+) or without (-) retinoic acid. Cells were either exposed to heat-shock in full medium prior to transfer (tracks labelled H) or maintained at 37°C throughout the experiment (tracks labelled C). Arrows indicate the positions of DNA markers of the sizes shown (in base pairs)
ND7 cells were treated with either ethanol or propanol for 30 min. Both of these treatments are known to induce hsp synthesis”,‘6 and this was confirmed by measuring hsp90 levels in the treated cells with the AC88 antibody (data not shown). When the cells were transferred to serum-free medium, the treated cells displayed enhanced survival compared to untreated cells in the presence or absence of retinoic acid confirming that induction of the hsps by other means than elevated temperature can protect neuronal cells from apoptosis (Fig. 6). Similar results were also obtained using other concentrations of these alcohols to induce the hsps (data not shown). DISCUSSION
As in other cell types the induction of the heatshock response has been shown to protect neuronal cells from the damaging effects of toxic stimuli both in oh and in uitro. Thus prior exposure to elevated temperature in vim protects neuronal cells against subsequent damage induced by exposure to light2 or ischaemia6 whilst similar exposure in vitro results in increased resistance to treatment with the excitotoxin glutamate.‘s~30 Here we extend these findings to show that prior heat-shock can partially protect neuronal cells from
Heat shock protects neuronal cells 25 20
60
15 40
10 20
5
YT
0
2; 10 lime of heatshock(min)
3b
Fig. 5. Levels of hsp90 (as determined by densitometric scanning of the blots such as that shown in Fig. 4) and cell survival 24 h after transfer to serum-free medium following different periods of heat-shock and a constant recovery period of 2.5 h at 37°C in full medium. Figures are the mean of three experiments whose standard error is shown by the bars.
69-
46Fig. 4. Western blot with the AC88 monoclonal antibody using cells exposed to the indicated period of heat shock (in minutes). The positions of protein molecular weight markers of the sizes shown (in mol. wt x 10m3) are indicated.
apoptosis which unlike the toxic or necrotic agents used by others is a natural event in neuronal development and plays a critical role in ensuring that the correct number of neurons eventually form.25 Indeed it has been suggested** that many cells require continuous signals from other cell types in order to survive and that in the absence of such signals the cells cease to suppress a constitutively active death pathway. Hence developing neuronal cells which fail to receive appropriate trophic factors from the target field’ or ND7 cells when transferred to serum-free
100
medium lacking the survival factors present in serum undergo death by apoptosis. To our knowledge this is the first demonstration in any cell type of the ability of a prior heat-shock to partially protect cells from apoptosis. In many other situations, elevated levels of the hsps have been shown to be responsible for the protective effects observed.‘5s4’ It is likely therefore that the induction of hsp synthesis is also responsible for the protective effect of heat shock on apoptosis in ND7 cells. It was not possible to test the requirement for hsp synthesis in this situation directly by investigating the effects of heat shock in the presence of protein synthesis inhibitors since apoptosis itself is dependent upon de nova protein synthesis both in our cellsI and in other cell types40 In experiments varying heat-shock and recovery times, however, we did observe a clear correlation between degree of induction of the hsps
1
1
‘i L ? 2 ap
80 1 80
n q
40
24h 48h
20
1.5M
propanol
1.5M
ethanol
control
Fig. 6. Survival of ND7 cells at intervals after transfer to serum-free medium with (+) or without (-) retinoic acid following prior exposure to ethanol, propanol or without such exposure (control). Solid bars indicate the results after 24 h in serum free medium, hatched bars the results after 48 h. In this batch of cells. no survival was observed in control cells treated with retinoic acid.
as measured by the increased level of one of these proteins, hsp90 and the extent of the protective effect. Moreover, treatment of ND cells with other inducers of hsp synthesis also resulted in protection from apoptosis upon transfer to serum-free medium confirming that the protective effect of a prior heat shock is likely to be dependent upon enhanced hsp synthesis rather than on some other effect occurring upon exposure to elevated temperature. In general the hsps have been shown to play a critical role in the regulation of protein folding and unfolding in both normal and stressed cells (for reviews, see Refs 8, 27). Thus for example hsp90 is thought to maintain proteins such as the steroid hormone receptors or the Src oncogene product in an inactive form prior to exposure to the appropriate physiologicai signal for activation.5,32 Similarly the hsp70 proteins are believed to play a critical role in catalysing the correct folding and assembly of multi-protein complexes in both the cytoplasm and the endoplasmic reticulum.“-” A protective role of the hsps in apoptosis may therefore involve some aspect of their role in protein folding involving for example the maintenance in an inactive form of the nuclease which digests the cellular DNA during the onset of apoptosis leading to the characteristic breakdown of cellular DNA into oligonucleosome length fragments.” It should be noted that whilst we have measured hsp90 levels as indicative of the changes in hsp synthesis in heat-
shock, our data do not indicate whether all or one of the different hsps are required for the observed protective effect. Similarly it is possible that induction of the hsps is not responsible for the protective e&cc we observe following heat-shock which could involve, for example, inhibition of the synthesis of ;I suicide protein. We are currently taking advantage of the fact that proliferating ND7 cells unlike primary neurons are readily transfectable in order to artihcally ovcrexpress each of the hsps in these cells and thereby identify which, if any, of these proteins is responsible for the protective effect. CONCLUSION
Whatever the precise role of the hsps, however, it is clear that heat shock can partially protect neuronal cells both from damage by toxic agents and from apoptosis. It is possible therefore that the identification of specific non-abusive procedures which elevate hsp levels in neuronal cells in vicc~ may have considerable application in the treatment of neurological diseases involving neuronal damage or enhanced neuronal cell death. Acknowledgements-We are most grateful to David Toft for the supply of the AC88 antibody and to Mary Collins, Sean Thomas and John Wood for helpful discussion. MKH is supported a grant from by Sir Jules Thorn Trust. CM is supported by the Countess of Lisburne Scholarship from UCLMS and an Overseas Research Student Award.
REFERENCES
1. Angelidis C. E., Lazarides I. and Paguolatos G. N. (1991) Constitutive expression of heat-shock protein 70 in mammalian cells confers thermotolerance. Eur. J. Biochem. 99, 35-39. protects against light damage in the rat retina. 2 Barbe M., Tytell M., Gower D. and Welch W. (1988) Hyperthermia Science 241, I8 I771 820. 3 Barde Y. (1989) Trophic factors and neuronal survival. Neuron 2, 1525-1534. 4. Bradford M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Analyf. Biochem. 72, 248-254. 5. Brugge J. S., Yonomoto W. and Darrow D. (1983) Interactions between the Rous Sarcoma virus transforming protein and two cellular phosphoproteins. Molec. cell. Biol. 3, 9-19. 6. Chopp M.. Chen H., Ho K.-L., Dereski M. 0.. Brown E., Hetzel F. W. and Welch K. M. (1989) Transient hyperthermia protects against subsequent forebrain ischemic cell damage in the rat. Neurology 39, 1396 1398. 7. Dhillon V. B., McCallum S., Norton P. M., Twomey B. M., Erkeller-Yuksel F., Lydyard P., Isenberg D. A. and Latchman D. S. (1993) Differential heat shock protein over-expression and its clinical relevance in systemic lupus erythematosus. Ana. rheu. Db. (in press). 8. Ellis R. J. (1990) Molecular Chaperones. Seminurs CeN Biol. 1, 1~72. 9. Gamstrop I. and Sarnat H. B. (1984) Progressive spinal muscular atrophies. New York, Raven Press. 10. Golstein P., Ojcius D. M. and Young J. D.-E. (1991) Cell death mechanisms and the immune system. fmmun. Rec. 121, 30-65. Il. Greene L. A. (1977) Quantitative in rifro studies on the nerve growth factor (NGF) requirements of neurons. 1. Sympathetic neurons. Deul Biol. 58, 96-105. 12. Hahn G. M., Shiu E. C., West B., Goldstein L. and Li G. C. (1985) Mechanistic implications of the induction of thermotolerance in Chinese Hamster cells by organic solvents. Cancer Rex 45, 4138-4143. 13. Hamburger V., Bruno-Bechtold J. K. and Yip J. (1981) Neuronal death in the spinal ganglia of the chick embryo and its reduction by nerve growth factor. J. Neurosci. 1, 60-71. 14. Howard M. K., Burke L. C., Mailhos C., Pizzey A., Gilbert C. S., Lawson W. D., Collins M. K. L., Thomas N. S. B. and Latchman D. S. (1993) Cell cycle arrest of proliferating neuronal cells by serum deprivation can result in either apoptosis or differentiation. J. Neurochem. (in press). 15. Li G. C. (1983) Induction of thermotolerance and enhanced heat shock protein synthesis in Chinese hamster fibroblasts by sodium arsenite and by ethanol. J. Cell Physiol. 115, 116-122. 16. Li G. C. and Hahn G. M. (1978) Ethanol-induced thermotolerance to heat and to Adriamycin. Nafure 274, 699- 701, 17. Lindquist S. and Craig E. A. (1988) The heat shock proteins. A. Rev. Gen. 22, 631--677. 18. Lowenstein D. H., Chan P. H. and Miles M. F. (1991) The stress protein response in cultured neurons: characterization and evidence for a protective role in excitotoxicity. Neuron 7, 1053~1060.
Heat shock protects neuronal cells
621
19. Martin D. P., Ito A., Horigome K., Lampe P. A. and Johnson E. M. Jr (1992) Biochemical characterization of programmed cell death in NGF-deprived sympathetic neurons. J. Neurobiol. 23, 1205-1220. 20. Martin D. P., Schmidt R. E., Di Stefano P. S., Lowry 0. H., Carter J. G. and Johnson E. M. Jr (1988) Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation. J. Cell Biol. 186.829-844. 21. Miller E., Roese J. B. and Morrison-Bogorad M. (1991) Expression of heat shock protein 70 and heat shock cognate 70 messenger RNAs in rat cortex and cerebellum after heat shock or amphetamine treatment. J. Neurochem. 56, 2060-2071. 22. Norton P. M., Isenberg D. A. and Latchman D. S. (1988) Elevated levels of the 90kD heat shock protein in a proportion of SLE patients with active disease. J. Auroimmun. 2, 187-195. 23. Nowak T. S. J. (1985) Synthesis of a stress protein following transient ischaemia in the gerbil. J. Neurochem. 45, 1635-1641. 24. Gppenheim R. W., Haverkamp L. J., Prevette D., MacManaman J. L., Appel S. H. (1988) Reduction of naturally occurring motoneuron death in uiuo by a target derived neurotrophic factor. Science 240, 919-922. 25. Gppenheim R. W. (1991) Cell death during development of the nervous system. A. Rev. Neurosci. 14, 453-501. 26. Pelham H. R. B. (1986) Speculations on the functions of the major heat-shock and glucose regulated proteins. Cell 46, 959-961. 21. Pelham H. R. B. (1989) Heat shock and the sorting of luminal ER proteins. Eur. molec. Biol. Org. J. 8, 3171-3176. 28. Raff M. C. (1992) Social controls on cell survival and cell death. Nature 356, 397-400. 29. Riehl R. M., Sullivan W. P., Vroman B. T., Bauer V. J., Pearson G. R. and Toft D. 0. (1985) Immunological evidence that the nonhormone binding component of avian steroid receptors exists in a wide range of tissues and species. Biochemistry 24, 6586-6591. 30. Rordorf G., Koroshetx W. J. and Bonventne J. V. (1991) Heat shock protects cultured neurons from glutamate toxicity. Neuron 7, 1043-1051. 31. Rydell R. E. and Greene L. A. (1988) CAMP analogues promote survival and neurite outgrowth in cultures of rat sympathetic and sensory neurons independently of nerve growth factor. Proc. natn. Acad. Sci. U.S.A. 85, 1257-1261. 32. Sanchez E. R., Meshinchi S., Tienrungrog W., Schlesinger M. J., Toft D. 0. and Pratt W. B. (1987) Relationship of the 90kDa murine heat shock protein to the untransformed and transformed states of the L cell glucocorticoid receptors. J. biol. Chem. 262, 6986-6991. 33. Scott S. A. and Davies A. M. (1990) Inhibitors of protein synthesis prevent cell death in sensory and parasympathetic neurons deprived of neurotrophic factor in vitro. J. Neurobiol. 21, 630-638. 34. Snider W. D. and Johnson E. M. (1989) Neurotrophic molecules. Ann. Neural. 26, 489-506. 35. Suburo A. M., Wheatley S. C., Horn D. A., Gibson S. J., John R., Fischer-Colbrie R., Wood J. N., Latchman D. S. and Polak J. M. (1992) Intracellular redistribution of neuropeptides and secretory proteins during differentiation of neuronal cell lines. Neuroscience 46, 881-889. 36. Vaux D. L., Cory S. and Adams J. M. (1988) Bcl-2 gene promotes haemopoietic cell survival and co-operates with c-myc to immortalize pre-B cells. Naiure 335, 440-442. 31. Weill C. L. and Greene D. P. (1984) Prevention of natural motoneurons cell death by dibutyryl cyclic GMP. Nature 308, 452-454.
38. Wood J. N., Bevan S. J., Coote P., Darn P. M., Hogan P., Latchman D. S., Morrison C., Rougor G., Theveniau M. and Wheatley S. C. (1990) Novel cell lines display the properties of nociceptive sensory neurons. Proc. R. Sot. B 241, 187-194. 39. Wylie A. H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Narure 284, 555-556. 40. Wylie A. H., Kerr J. F. R. and Currie A. R. (1980) Cell death: the significance of apoptosis. Int. Rev. Cytol. 68,251-306. 41. Yellon D. M. and Latchman D. S. (1991) Stress proteins and myocardial protection. J. molec. cell. Cardiol. 24, 113-124. (Accepted 3 March 1993)