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Opposing effects of transforming growth factor-β1 on glutamate neurotoxicity
Neuroscience
Pergamon
0306-4522(94)EOO82-F
Vol. 60, No. I, pp. 7-10, 1994 Elsevier Science Ltd 1994 IBRO Printed in Great Britain 0306-4522/94 36.00 + 0.00
Letter to neuroscience OPPOSING EFFECTS OF TRANSFORMING GROWTH FACTOR-P 1 ON GLUTAMATE NEUROTOXICITY J. H. M. Institut
F’REHN*
and J.
KRIEGLSTEINT
und Toxikologie, FB Pharmazie Philipps-Universitlt, 35032 Marburg/Lahn,
fiir Phamakologie
und Lebensmittelchemie, Germany
Activation of microglia has been emphasized as a responsible for the injury-induced expression of Of particular interest, however, are the critical step in the pathophysiology of degenerative and TGF-p 1.‘6*20 inflammatory processes of the CNS. Activated micro- effects of this microglia-derived cytokine on neurons glia release low molecular weight compounds, such as that undergo selective degeneration in many types of excitatory amino acids, that are directly toxic to brain pathology. In this regard, previous studies have shown that TGF-fi 1 is able to decrease hypoxic neurons. Here we demonstrate that a microgliaderived cytokine, transforming growth factor-/l 1, directly alters and excitotoxic neuronal injury in oitro,24-26and also the susceptibility of neurons to glutamate-induced reduces ischemic brain injury in uivo.8,24 Beside potential neuronotrophic/neuroprotective cell damage. Transforming growth factor-j1 acts as a neuroprotectant following short-term exposure to factors, activated microglia also release compounds glutamate, whereas, following chronic exposure to that are neurotoxic.’ A large portion of their neuroglutamate, similar concentrations of transforming toxic potency seems to be due to the release of low growth factor-/l 1 actually potentiate excitotoxic cell molecular weight molecules that activate excitatory death. This complex interaction may play an important amino acid receptorqz3 either glutamate itself22 or a role in determining the extent of local tissue damage. closely related molecule.9 In view of the increasing The transforming growth factor+s (TGF-Bs) are evidence for the involvement of microglia-derived disulphide-linked polypeptide dimers that are synfactors in degenerative and inflammatory processes of thesized in most mammalian cell types. Their role as the CNS,’ we have investigated how the presence of important mediators in the regulation of cell growth, both glutamate and TGF-/I1 influences the survival differentiation, inflammation and tissue repair is of cultured central neurons. well established.” Three highly conserved isoforms, Non-neuronal cells synthesize substantial amounts TGF-fis 1, 2 and 3, are found in humans and other of growth factors and cytokines that could mask the mammalian species. 27In the adult CNS, TGF-/? iso- direct interaction between TGF-/3 1 and glutamate in forms 2 and 3 have been identified in many neuronal a neuronal survival assay. We therefore used primary populations as well as in astrocytes, whereas TGF-/I 1 neuronal cultures from the telencephalon of sevenis virtually absent in both neuronal and glial cell~.‘~~~’ day-old chick embryos that can be cultured in the In contrast, strong TGF-/3 1 expression is detectable absence of glial cells. 2’126 Cultured chick telencephalic in the rat brain after a brain lesion, administration of neurons become sensitive to glutamate neurotoxicity excitotoxins, or a hypoxic-ischemic insult.‘0~‘3~‘4,‘6.‘9,20 after four to five days in vitro, a process mediated In human postmortem brains, TGF-Bl immunovia both N-methyl-D-aspartate(NMDA) and nonreactivity has been demonstrated at the site of neuroNMDA receptors.25 Cells were exposed to the pathological changes associated with Alzheimer’s excitatory amino acid L-glutamate under serum-free disease, Down’s syndrome and AIDS neuropathconditions after six days in vitro. For this purpose, ology.30*3’Activated microglia appear to be initially the cultures were rinsed twice with 5 ml of serum free Dulbecco’s modified Eagles medium (DMEM) and were then exposed to serum-free DMEM’ supple*Present address: Department of Pharmacological and Physiological Sciences, The University of Chicago, mented with 1 mM sodium L-glutamate for periods 947 E. 58th Street, Chicago, IL 60637, U.S.A. of either 60 min or 18 h. Neuronal injury was detertTo whom correspondence should be addressed. mined 18 h after the onset of the glutamate exposure Abbreviations: DMEM, Dulbecco’s modified Eagles medium; with a calorimetric assay using the metabolic dye MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium zolium bromide; NMDA, N-methyl-D-aspartate; TGF-,V, transforming growth factor-/l. bromide (MTT).‘*
8
J. H. M. PREHNand J. KRIEGLSTEIN
Table 1. Transforming growth factor-p I protects against excitotoxic injury following short-term exposure to glutamate Exposure to glutamate (h)
TGF-p 1 (ngiml)
0
0
0
3
1
0
I
I
0.1 0.3
4 4 5 4
I I 1
I 3
n
Optical density (% of control)
4 4
100.0 + 4.2 102.3 k 3.9 76.6 + 82.6 f 87.1 k 93.1 * 81.4 k
3.0 3.9 2.4* 3.0** 5.4
Glutamate exposure was performed on primary cultures of chick telencephalic neurons after six days in vitro. Cultures were exposed to serum-free DMEM culture medium supplemented with 1 mM L-glutamate at 37°C for a period of 60min. Afterwards, the cultures were washed twice and maintained in DMEM culture medium for a further 17 h. TGF-fi 1 was present in the cultures both during the exposure to glutamate and throughout the recovery period. Eighteen hours after the onset of the glutamate exposure, the cultures were incubated with DMEM culture medium supplemented with I mg/ml MTT for exactly 4 h. After this period, the MTTcontaining medium was aspirated and the blue formazan was extracted by adding 5 ml of acidified (0.04 N HCl) 2-propanol to the cultures. The optical density of the extract was determined spectrophotometrically at a wavelength of 570 nm using a reference wavelength of 690nm. To compare the results from different experiments, the values are expressed relative to the mean value of sham-washed control sister cultures that had not been exposed to glutamate. Data given are means + S.E.M. Experiment was performed in triplicate with similar results. Different from glutamate-exposed controls: *P < 0.05; **P < 0.01 (one-way analysis of variance and Duncan’s post hoc test).
Exposure to glutamate for 60 min led to a delayed neuronal degeneration that did not affect the entire neuronal population. Biochemically, this was reflected by a 23.4 + 3.0% decrease in the optical density Table 2. Transforming growth factor-p 1 potentiates excitotoxic injury resulting from chronic exposure to glutamate Exposure to glutamate (h)
TGF-P 1 (ngiml)
n
Optical density (% of control) 100.0 & 2.5 99.9 & 0.4
0
0
0
10
5 4
0 0.3 1 3 10
6 5 5 5 4
18 18 18 18 18
49. I f 44.2 + 35.3 + 39.3 7 37.1 +
2.5 4.8 1.1** 1.6* 1.1**
Glutamate exposure was performed on primary cultures of chick telencephalic neurons after six days in vitro. Cultures were exposed to serum-free DMEM culture medium supplemented with 1mM L-glutamate for a period of 18 h. TGF-fi 1 was present in the cultures during the entire period of the glutamate exposure. Eighteen hours after the onset of the glutamate exposure, neuronal injury was quantified as described in the legend of Table 1. Data given are means rt S.E.M. Experiment was performed in thplicate with similar results. Different from glutamate-exposed controls: *P c 0.05; **P i 0.01 (oneway analysis of variance and Duncan’s post hoc test).
compared with sham-washed control sister cultures. Morphologically, the portion of the neurons damaged by glutamate exhibited shrunken, dark cell bodies, fragmentation of neurites, loss of phasebrightness and vacuolization.26 In order to investigate the effects of TGF-/l 1 on glutamate-induced neuronal injury, the cultures were treated with increasing concentrations of TGF-fi I (R&D Systems, Minneapolis, MN) both during the glutamate exposure and throughout the recovery period. Treatment with TGFpl led to a statistically significant neuroprotection, indicated by an increase in the optical density of the cultures (Table 1). Chronic exposure to glutamate for 18 h led to a 50.9 f 2.5% decrease in the optical density compared with sham-washed controls. However, treatment of the cultures with TGF$l during the entire period of the glutamate exposure did not protect the cultures from excitotoxic injury. On the contrary, TGF-/ll significantly potentiated glutamate-induced neuronal injury (Table 2). In many biological systems, the TGF-/Js mediate their effects in a complex interaction with other growth factors and cytokines2’ a mode of action that has also been observed in neurons.3s’1 Thus, we were interested in determining whether both effects of TGF-b 1, i.e. protection and potentiation of excitotoxic neuronal damage, could also be observed in an environment where other growth factors and cytokines were present. To this end, identical treatment schedules were applied with the exception that both the glutamate-containing exposure medium and the recovery culture medium contained 8% (V/V) fetal calf serum. Interestingly, treatment of glutamate-exposed cultures with TGF-fi 1 in the presence of serum showed effects generally comparable to those observed under serum-free conditions: protection from excitotoxic injury after short-term (60 min) exposure to glutamate (l-10 ng/ml TGF-b l), and potentiation of excitotoxic injury after chronic (18 h) exposure to glutamate (l-3 ng/ml TGF-/l 1; data not shown). Among the wide range of endogenous and exogenous compounds able to modulate glutamate-induced neuronal injury, TGF-11, an injury-related cytokine and signalling peptide, shows a unique bimodal effect in that it is able to both inhibit and potentiate glutamate-induced neuronal damage. The expression of TGF-81 in the injured brain has been shown to originate from activated brain macrophages.‘6,20 However, the injury-related production and release of TGF-/I 1 is not necessarily confined to these cell types. Astrocytes in culture produce and secrete increasing amounts of TGF-Pl upon stimulation with cytokines, including interleukin-1 and TGF-/? 1 itself.6,‘7 In vivo, TGF-/3 1 immunoreactivity has been detected in glial cells after a stab wound injury in the rat brain,14 as well as in astrocytes surrounding neuropathological sites in human brains.‘x3’ In the latter
case, it has been demonstrated that TGF-b 1 immuno-
TGF-p 1 and glutamate neurotoxicity
reactivity is frequently accompanied by interleukin-1 immunoreactivity.5 It is therefore conceivable that TGF-fi 1 serves as an important signalling link between the immune system and the central nervous system in response to a brain injury. Moreover, it has recently been demonstrated that neurons are able to secrete TGF-/3 1 following a lesion performed in vitro or in vivo, and may also be able to transport TGF-81 anterogradely and release it at the site of injury.28 Assuming the involvement of glutamate receptormediated processes in many aspects of neuronal degeneration and neuronal plasticity: the interaction between glutamate and TGF-pl could play an important role in determining the extent of tissue injury and the degree of functional recovery. Although it has recently been demonstrated that the TGF-/?s activate membrane-bound serine/ threonine kinases,7x’2the precise molecular mechanism of TGF-fl l-induced modulation of excitotoxic injury remains to be elucidated. Growth factors that are linked to tyrosine kinase activity, such as fibroblast growth factors, have also been shown to prevent excitotoxic injury of cultured neurons.” However,
9
experiments with these growth factors require a longer pre-treatment of the cultures in order to produce an anti-excitotoxic effectI This was not the case for TGF-/?l in the present study. We thus suggest TGF-/I’s mode of action is distinct from that of growth factors linked to tyrosine kinases and is one which does not depend on the presence of other growth factors or cytokines in the environment. In view of the increasing evidence for TGF-fi 1 as a control switch in many biological processes,2g27it is conceivable that, in the injured CNS, TGF-b 1 has a similar function: TGF-PI may have the capacity to improve the survival of neurons undergoing a toxic environment insult. On the other hand, this signalling peptide may also be able to accelerate the removal of severely damaged neurons by supporting the neurotoxic activity of excitatory amino acids released from macrophages. From a teleological point of view, both processes may be similarily important to protect or maintain brain function. Acknowledgements-The
authors are grateful to Sandra Engel for technical assistance. Supported by the Deutsche Forschungsgemeinschaft (Kr 354/15-l).
REFERENCES
1. Banati R. B., Gehrmann J., Schubert P. and Kreutzberg G. W. (1993) Cytotoxicity of microgha. Glia 7, 111-118. 2. Battegay E. J., Raines E. W., Seifert R. A., Bowen-Pope D. F. and Ross R. (1990) TGF-8 induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop. Cell 63, 515-524. 3. Chalazonitis A., Kalberg J., Twardzik D. R., Morrison R. S. and Kessler J. A. (1992) Transforming growth factor B has neurotrophic actions on sensory neurons in vitro and is synergistic with nerve growth factor. Deul Biol. 152,121-132. 4. Choi D. W. (1988) Glutamate neurotoxicity and diseases of the nervous system. Neuron 1, 623-634. 5. da Cunha A., Jefferson J. A., Jackson R. W. and Vitkovic L. (1993) Glial cell-specific mechanisms of TGF-jll induction by IL-1 in cerebral cortex. J. Neuroimmunol. 42, 71-86. 6. da Cunha A. and Vitkovic L. (1992) Transforming growth factor-/?1 (TGF-81) expression and regulation in rat neocortical astrocytes. J. Neuroimmunol. 36, 157-169. 7. Ebner R., Chen R.-H., Shum L., Lawler S., Zioncheck T. F., Lee A., Lopez A. R. and Derynck R. (1993) Cloning of a type I TGF-/l receptor and its effect on TGF-/l binding to the type II receptor. Science 260, 13441348. 8. Gross C. E., Bednar M. M., Howard D. B. and Sporn M. B. (1993) Transforming growth factor-81 reduces infarct size after experimental cerebral ischemia in a rabbit model. Stroke 24, 558-562. 9. Giulian D., Vaca K. and Corpuz M. (1993) Brain glia release factors with opposing actions upon neuronal survival. J. Neurosci. 13, 29-37. 10. Klempt N. D., Sirimanne
11. 12. 13. 14. 15. 16. 17. 18. 19.
E., Gunn A. J., Klempt M., Singh K., Williams C. and Gluckman P. D. (1992) Hypoxia-ischemia induces transforming growth factor /? 1 mRNA in the infant rat brain. Molec. Brain. Res. 13,93-101. Lefebvre P. P., van de Water T. R., Weber T., Rogister B. and Moonen G. (1991) Growth factor interactions in cultures of dissociated adult acoustic ganglia: neuronotrophic effects. Bruin Res. 567, 306-312. Lin H. Y., Wang X.-F., Ng-Eaton E., Weinberg R. A. and Lodish H. F. (1992) Expression cloning of the TGF-fi type II receptor, a functional transmembrane serine/threonine kinase. Cell 68, 775-785. Lindholm D., Hengerer B., Zafra F. and Thoenen H. (1992) Transforming growth factor-/I 1 in the rat brain: Increase after injury and inhibition of astrocyte proliferation. J. Cell Biol. 117, 395-400. Logan A., Frautschy S. A., Gonzalez A.-M., Spom M. B. and Baird A. (1992) Enhanced expression of transforming growth factor b 1 in the rat brain after a localized brain injury. Brain Res. 587, 216-225. Mattson M. P., Murrain M., Guthrie P. B. and Kater S. B. (1989) Fibroblast growth factor and glutamate: opposing roles in the generation and degeneration of hippocampal neuroarchitecture. J. Neurosci. 9, 3728-3740. Morgan T. E., Nichols N. R., Pasinetti G. M. and Finch C. E. (1993) TGF-/ll mRNA increases in macrophagei microglial cells of the hippocampus in response to deafferentation and kainic acid-induced neurodegeneration. Expl Neural. 120, 291-301. Morganti-Kossmann M. C., Kossmann T., Brandes M. E., Mergenhagen S. E. and Wahl S. M. (1992) Autocrine and paracrine regulation of astrocyte function by transforming growth factor-p. J. Neuroimmunol. 39, 163-174. Mosmann T. (1983) Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Meth. 65, 55-63. Nichols N. R., Laping N. J., Day J. R. and Finch C. E. (1991) Increases in transforming growth factor-b mRNA in
hippocampus during response to entorhinal cortex lesions in intact and adrenaleetomized rats. J. Neurosci. Res. 28, 134-139.
20.
Pasinetti G. M., Nichols N. R., TOCCO G., Morgan T., Laping N. and Finch C. E. (1993) Transforming growth factor 81 and gbronectin messenger RNA in rat brain: Responses to injury and cell-type localization. Neuroscience ~4, 893-907.
10
J.H.M.
PREHN andJ.
KRIEGLSTEIN
21. Pettmann B., Louis J. C. and Sensenbrenner M. (1979) Morphological and biochemical maturation of neurones cultured in the absence of glial cells. Nature 281, 378-380. 22. Piani D., Frei K., Do K., Cuenod M. and Fontana A. (1991) Murine brain macrophages induce NMDA receptor mediated neurotoxicity in vitro by secreting glutamate. Neurosci. L&t. 133, 1599162. 23. Piani D., Spranger M., Frei K., Schaffner A. and Fontana A. (1992) Macrophage-induced cytotoxicty of N-methylo-aspartate receptor positive neurons involves excitatory amino acids rather than reactive oxygen intermediates and cytokines. Eur. J. Immunol. 22, 242992436. 24. Prehn J. H. M., Backhaul C. and Krieglstein J. (1993) Transforming growth factor-b 1 prevents glutamate neurotoxicity in rat neocortical cultures and protects mouse neocortex from ischemic injury in uiuo. J. Cereh. Blood Flow Metab. 13, 521-525. 25. Prehn J. H. M. and Krieglstein J. (1991) Primary neuronal cultures from chick embryo cerebral hemispheres-a model for studying toxic and trophic effects of excitatory amino acids. J. Cereb. Blood Flow Metab. 11, S317. 26. Prehn J. H. M., Peruche B., Unsicker K. and Krieglstein J. (1993) Isoform-specific effects of transforming growth factors-8 on degeneration of primary neuronal cultures induced by cytotoxic hypoxia or glutamate. J. Neurochem. 60, 166551672. 27. Roberts A. B. and Sporn M. B. (1990) The transforming growth factors-D. In Handbook of Experimental Pharmacology, Peptide Growth Factors and Their Receptors I (eds Sporn M. B. and Roberts A. B.), Vol. 95, pp. 419-472, Springer, Heidelberg. 28. Rogister B., Delree P., Leprince P., Martin D., Sadzot C., Malgrange B., Munaut C., Rigo J. M., Lcfebvre P. P., Octave J.-N., Schoenen J. and Moonen G. (1993) Transforming growth factor p as a neuronoglial signal during peripheral nervous system response to injury. J. Neurosci. Res. 34, 32-43. 29. Unsicker K., Flanders K. C., Cissel D. S., Lafyatis R. and Sporn M. B. (1991) Transforming growth factor beta isoforms in the adult rat central and peripheral nervous system. Neuroscience 44, 613-625. 30. van der Wal E. A., Gomez-Pinilla F. and Cotman C. W. (1993) Transforming growth factor-b1 is in plaques in Alzheimer and Down pathologies. NeuroReport 4, 69-72. 31. Wahl S. M., Allen J. B., McCartney-Francis N., Morganti-Kossmann M. C., Kossmann T., Ellingsworth L.. Mai U. E. H., Mergenhagen S. E. and Orenstein J. M. (1991) Macrophageand astrocyte-derived transforming growth factor /J as a mediator of central nervous system dysfunction in acquired immune deficiency syndrome. J. exp. Med. 173, 981-991. (Accepted 27 December 1993)
Pergamon
0306-4522(94)EOO82-F
Vol. 60, No. I, pp. 7-10, 1994 Elsevier Science Ltd 1994 IBRO Printed in Great Britain 0306-4522/94 36.00 + 0.00
Letter to neuroscience OPPOSING EFFECTS OF TRANSFORMING GROWTH FACTOR-P 1 ON GLUTAMATE NEUROTOXICITY J. H. M. Institut
F’REHN*
and J.
KRIEGLSTEINT
und Toxikologie, FB Pharmazie Philipps-Universitlt, 35032 Marburg/Lahn,
fiir Phamakologie
und Lebensmittelchemie, Germany
Activation of microglia has been emphasized as a responsible for the injury-induced expression of Of particular interest, however, are the critical step in the pathophysiology of degenerative and TGF-p 1.‘6*20 inflammatory processes of the CNS. Activated micro- effects of this microglia-derived cytokine on neurons glia release low molecular weight compounds, such as that undergo selective degeneration in many types of excitatory amino acids, that are directly toxic to brain pathology. In this regard, previous studies have shown that TGF-fi 1 is able to decrease hypoxic neurons. Here we demonstrate that a microgliaderived cytokine, transforming growth factor-/l 1, directly alters and excitotoxic neuronal injury in oitro,24-26and also the susceptibility of neurons to glutamate-induced reduces ischemic brain injury in uivo.8,24 Beside potential neuronotrophic/neuroprotective cell damage. Transforming growth factor-j1 acts as a neuroprotectant following short-term exposure to factors, activated microglia also release compounds glutamate, whereas, following chronic exposure to that are neurotoxic.’ A large portion of their neuroglutamate, similar concentrations of transforming toxic potency seems to be due to the release of low growth factor-/l 1 actually potentiate excitotoxic cell molecular weight molecules that activate excitatory death. This complex interaction may play an important amino acid receptorqz3 either glutamate itself22 or a role in determining the extent of local tissue damage. closely related molecule.9 In view of the increasing The transforming growth factor+s (TGF-Bs) are evidence for the involvement of microglia-derived disulphide-linked polypeptide dimers that are synfactors in degenerative and inflammatory processes of thesized in most mammalian cell types. Their role as the CNS,’ we have investigated how the presence of important mediators in the regulation of cell growth, both glutamate and TGF-/I1 influences the survival differentiation, inflammation and tissue repair is of cultured central neurons. well established.” Three highly conserved isoforms, Non-neuronal cells synthesize substantial amounts TGF-fis 1, 2 and 3, are found in humans and other of growth factors and cytokines that could mask the mammalian species. 27In the adult CNS, TGF-/? iso- direct interaction between TGF-/3 1 and glutamate in forms 2 and 3 have been identified in many neuronal a neuronal survival assay. We therefore used primary populations as well as in astrocytes, whereas TGF-/I 1 neuronal cultures from the telencephalon of sevenis virtually absent in both neuronal and glial cell~.‘~~~’ day-old chick embryos that can be cultured in the In contrast, strong TGF-/3 1 expression is detectable absence of glial cells. 2’126 Cultured chick telencephalic in the rat brain after a brain lesion, administration of neurons become sensitive to glutamate neurotoxicity excitotoxins, or a hypoxic-ischemic insult.‘0~‘3~‘4,‘6.‘9,20 after four to five days in vitro, a process mediated In human postmortem brains, TGF-Bl immunovia both N-methyl-D-aspartate(NMDA) and nonreactivity has been demonstrated at the site of neuroNMDA receptors.25 Cells were exposed to the pathological changes associated with Alzheimer’s excitatory amino acid L-glutamate under serum-free disease, Down’s syndrome and AIDS neuropathconditions after six days in vitro. For this purpose, ology.30*3’Activated microglia appear to be initially the cultures were rinsed twice with 5 ml of serum free Dulbecco’s modified Eagles medium (DMEM) and were then exposed to serum-free DMEM’ supple*Present address: Department of Pharmacological and Physiological Sciences, The University of Chicago, mented with 1 mM sodium L-glutamate for periods 947 E. 58th Street, Chicago, IL 60637, U.S.A. of either 60 min or 18 h. Neuronal injury was detertTo whom correspondence should be addressed. mined 18 h after the onset of the glutamate exposure Abbreviations: DMEM, Dulbecco’s modified Eagles medium; with a calorimetric assay using the metabolic dye MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium zolium bromide; NMDA, N-methyl-D-aspartate; TGF-,V, transforming growth factor-/l. bromide (MTT).‘*
8
J. H. M. PREHNand J. KRIEGLSTEIN
Table 1. Transforming growth factor-p I protects against excitotoxic injury following short-term exposure to glutamate Exposure to glutamate (h)
TGF-p 1 (ngiml)
0
0
0
3
1
0
I
I
0.1 0.3
4 4 5 4
I I 1
I 3
n
Optical density (% of control)
4 4
100.0 + 4.2 102.3 k 3.9 76.6 + 82.6 f 87.1 k 93.1 * 81.4 k
3.0 3.9 2.4* 3.0** 5.4
Glutamate exposure was performed on primary cultures of chick telencephalic neurons after six days in vitro. Cultures were exposed to serum-free DMEM culture medium supplemented with 1 mM L-glutamate at 37°C for a period of 60min. Afterwards, the cultures were washed twice and maintained in DMEM culture medium for a further 17 h. TGF-fi 1 was present in the cultures both during the exposure to glutamate and throughout the recovery period. Eighteen hours after the onset of the glutamate exposure, the cultures were incubated with DMEM culture medium supplemented with I mg/ml MTT for exactly 4 h. After this period, the MTTcontaining medium was aspirated and the blue formazan was extracted by adding 5 ml of acidified (0.04 N HCl) 2-propanol to the cultures. The optical density of the extract was determined spectrophotometrically at a wavelength of 570 nm using a reference wavelength of 690nm. To compare the results from different experiments, the values are expressed relative to the mean value of sham-washed control sister cultures that had not been exposed to glutamate. Data given are means + S.E.M. Experiment was performed in triplicate with similar results. Different from glutamate-exposed controls: *P < 0.05; **P < 0.01 (one-way analysis of variance and Duncan’s post hoc test).
Exposure to glutamate for 60 min led to a delayed neuronal degeneration that did not affect the entire neuronal population. Biochemically, this was reflected by a 23.4 + 3.0% decrease in the optical density Table 2. Transforming growth factor-p 1 potentiates excitotoxic injury resulting from chronic exposure to glutamate Exposure to glutamate (h)
TGF-P 1 (ngiml)
n
Optical density (% of control) 100.0 & 2.5 99.9 & 0.4
0
0
0
10
5 4
0 0.3 1 3 10
6 5 5 5 4
18 18 18 18 18
49. I f 44.2 + 35.3 + 39.3 7 37.1 +
2.5 4.8 1.1** 1.6* 1.1**
Glutamate exposure was performed on primary cultures of chick telencephalic neurons after six days in vitro. Cultures were exposed to serum-free DMEM culture medium supplemented with 1mM L-glutamate for a period of 18 h. TGF-fi 1 was present in the cultures during the entire period of the glutamate exposure. Eighteen hours after the onset of the glutamate exposure, neuronal injury was quantified as described in the legend of Table 1. Data given are means rt S.E.M. Experiment was performed in thplicate with similar results. Different from glutamate-exposed controls: *P c 0.05; **P i 0.01 (oneway analysis of variance and Duncan’s post hoc test).
compared with sham-washed control sister cultures. Morphologically, the portion of the neurons damaged by glutamate exhibited shrunken, dark cell bodies, fragmentation of neurites, loss of phasebrightness and vacuolization.26 In order to investigate the effects of TGF-/l 1 on glutamate-induced neuronal injury, the cultures were treated with increasing concentrations of TGF-fi I (R&D Systems, Minneapolis, MN) both during the glutamate exposure and throughout the recovery period. Treatment with TGFpl led to a statistically significant neuroprotection, indicated by an increase in the optical density of the cultures (Table 1). Chronic exposure to glutamate for 18 h led to a 50.9 f 2.5% decrease in the optical density compared with sham-washed controls. However, treatment of the cultures with TGF$l during the entire period of the glutamate exposure did not protect the cultures from excitotoxic injury. On the contrary, TGF-/ll significantly potentiated glutamate-induced neuronal injury (Table 2). In many biological systems, the TGF-/Js mediate their effects in a complex interaction with other growth factors and cytokines2’ a mode of action that has also been observed in neurons.3s’1 Thus, we were interested in determining whether both effects of TGF-b 1, i.e. protection and potentiation of excitotoxic neuronal damage, could also be observed in an environment where other growth factors and cytokines were present. To this end, identical treatment schedules were applied with the exception that both the glutamate-containing exposure medium and the recovery culture medium contained 8% (V/V) fetal calf serum. Interestingly, treatment of glutamate-exposed cultures with TGF-fi 1 in the presence of serum showed effects generally comparable to those observed under serum-free conditions: protection from excitotoxic injury after short-term (60 min) exposure to glutamate (l-10 ng/ml TGF-b l), and potentiation of excitotoxic injury after chronic (18 h) exposure to glutamate (l-3 ng/ml TGF-/l 1; data not shown). Among the wide range of endogenous and exogenous compounds able to modulate glutamate-induced neuronal injury, TGF-11, an injury-related cytokine and signalling peptide, shows a unique bimodal effect in that it is able to both inhibit and potentiate glutamate-induced neuronal damage. The expression of TGF-81 in the injured brain has been shown to originate from activated brain macrophages.‘6,20 However, the injury-related production and release of TGF-/I 1 is not necessarily confined to these cell types. Astrocytes in culture produce and secrete increasing amounts of TGF-Pl upon stimulation with cytokines, including interleukin-1 and TGF-/? 1 itself.6,‘7 In vivo, TGF-/3 1 immunoreactivity has been detected in glial cells after a stab wound injury in the rat brain,14 as well as in astrocytes surrounding neuropathological sites in human brains.‘x3’ In the latter
case, it has been demonstrated that TGF-b 1 immuno-
TGF-p 1 and glutamate neurotoxicity
reactivity is frequently accompanied by interleukin-1 immunoreactivity.5 It is therefore conceivable that TGF-fi 1 serves as an important signalling link between the immune system and the central nervous system in response to a brain injury. Moreover, it has recently been demonstrated that neurons are able to secrete TGF-/3 1 following a lesion performed in vitro or in vivo, and may also be able to transport TGF-81 anterogradely and release it at the site of injury.28 Assuming the involvement of glutamate receptormediated processes in many aspects of neuronal degeneration and neuronal plasticity: the interaction between glutamate and TGF-pl could play an important role in determining the extent of tissue injury and the degree of functional recovery. Although it has recently been demonstrated that the TGF-/?s activate membrane-bound serine/ threonine kinases,7x’2the precise molecular mechanism of TGF-fl l-induced modulation of excitotoxic injury remains to be elucidated. Growth factors that are linked to tyrosine kinase activity, such as fibroblast growth factors, have also been shown to prevent excitotoxic injury of cultured neurons.” However,
9
experiments with these growth factors require a longer pre-treatment of the cultures in order to produce an anti-excitotoxic effectI This was not the case for TGF-/?l in the present study. We thus suggest TGF-/I’s mode of action is distinct from that of growth factors linked to tyrosine kinases and is one which does not depend on the presence of other growth factors or cytokines in the environment. In view of the increasing evidence for TGF-fi 1 as a control switch in many biological processes,2g27it is conceivable that, in the injured CNS, TGF-b 1 has a similar function: TGF-PI may have the capacity to improve the survival of neurons undergoing a toxic environment insult. On the other hand, this signalling peptide may also be able to accelerate the removal of severely damaged neurons by supporting the neurotoxic activity of excitatory amino acids released from macrophages. From a teleological point of view, both processes may be similarily important to protect or maintain brain function. Acknowledgements-The
authors are grateful to Sandra Engel for technical assistance. Supported by the Deutsche Forschungsgemeinschaft (Kr 354/15-l).
REFERENCES
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