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RESEARCH COMMUNICATION |
a Université de CAEN, CNRS UMR 6551, Laboratoire de Neurosciences, bd H. Becquerel, BP 5229, 14074 CAEN Cedex, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: excitotoxicity • serpins • neuronal cell death • apoptosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several growth factors attenuate neuronal death when induced by a large variety of insults. For example, many growth factors can inhibit several forms of axotomy-induced death, an apoptotic death that most likely reflects a failure of target-supplied trophic factors to reach the cell body. It is now recognized that apoptosis, characterized by a decrease in cell volume, membrane blebbing, chromatin condensation, and DNA fragmentation (4), is implicated in the pathogenesis of many neurodegenerative diseases (5). Accordingly, the concept has emerged that the survival-promoting properties of growth factors are crucial in the determination of apoptotic injury. Consistent with these data, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 all attenuate apoptosis in neurons (6). In contrast, when applied to cerebellar neurons, TGF-ß1 does not seem to exert a neurotrophic effect, but rather surprisingly induces apoptosis in neurons (7).
As several studies have described elevated concentrations of TGF-ß1 in brain biopsies from patients with Parkinson's (8) or Alzheimer's (9) disease and stroke (10), this growth factor has been characterized as an injury-related peptide. However, the beneficial or deleterious effect of the increased concentration of TGF-ß1 in altered brain tissue remains controversial (11, 12).
TGF-ß1 activates the transcription of genes important in the progression of the cell cycle, such as cyclin dependent kinase inhibitors, (13) as well as some involved in the maintenance of the extracellular matrix such as 
2(1) procollagen, fibronectin, and plasminogen activator inhibitor type 1 (PAI-1) (14, 15). The extracellular matrix degradation–regulation effect of the TGF-ß isoforms has been described in many systems and seems to be mediated primarily by an overexpression of PAI-1 (15, 16). PAI-1 is an inhibitory peptide for tissue-type plasminogen activator (t-PA), a serine protease that converts inactive plasminogen to active protease plasmin (16). The activity of t-PA has been studied extensively in neural tissue and seems to be implicated not only in neurite outgrowth, cell migration (17), and synaptic plasticity (18) but also in the excitotoxic cascade after focal cerebral ischemia (19). Based on the above results, we postulate that the neuroprotective effect of TGF-ß1 may involve the modulation of t-PA activity through an increased PAI-1 synthesis in astrocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate, and (+)5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine maleate (MK-801) were from Tocris (Bristol, U.K.). Staurosporine and cycloheximide were purchased from Sigma Chemical Co. (Isle D'Abeau, France). Eagle's minimal essential medium (MEM), cytosine arabinoside, horse serum, and fetal bovine serum were obtained from Sigma; poly-D-lysine and laminin were from Life Technologies. Anti-MAP-2 and anti-GFAP antibodies were purchased from Sigma; anti-PAI-1 and anti-t-PA antibodies were from American Diagnostica (Greenwich, Conn.). All other chemicals used were obtained from Sigma.
Cell culture
Mixed cortical cultures containing both neuronal and glial cells were prepared from fetal mice at 15–16 days gestation (20). Cerebral cortices were dissected and incubated for 20–30 min in 0.025% trypsin in media stock (MS, Eagle's minimal essential medium augmented with 2 mM glutamine and 25 mM glucose) and transferred to MS supplemented with 5% fetal bovine and 5% horse serum for trituration. Dissociated cells were plated at a density of about 3 x 105 cells per well on an established bed of glia in MS supplemented with 10% fetal bovine serum and 10% horse serum. Cortical astrocyte cell cultures were prepared similarly from 1- to 3-day-old postnatal pups, using plating medium supplemented with epidermal growth factor (10 ng/ml) (20).
All cultures were kept at 37°C in a humidified 5% CO2-containing atmosphere. After 3–7 days in vitro, glial cell division was halted by exposure to 10 µM cytosine arabinoside. Cells were subsequently shifted into maintenance medium, which was identical to the plating medium but lacking fetal bovine serum. The medium was changed twice weekly. Experiments were performed on cortical cultures after 14–15 days in vitro.
Near-pure neuronal cell cultures containing less than 5% astrocytes were prepared as previously detailed (21). Dissociated cortical cells in MS supplemented with 5% fetal bovine and 5% horse serum were plated in multiwell vessels that had previously been coated with poly-D-lysine and laminin, using conditioned medium from astrocyte cultures with 3 µM cytosine arabinoside. There was no further exchange of the media. Experiments were performed on neuronal cultures after 14–15 days in vitro.
Apoptosis
Serum deprivation (SD) was initiated by transferring pure neuronal cultures (DIV 7) into growth medium lacking serum. Neuronal cell death was assessed by cell counts after staining with 0.4% trypan blue dye.
Staurosporine (STP) exposure was induced at 37°C in mixed cortical cell culture (DIV 14) by a 24 h exposure to 200 nM of STP in MS supplemented with glycine. MK-801 (10 µM) was always added concurrently with STP to block secondary NMDA receptor activation.
Excitotoxicity
Slowly triggered excitotoxicity was induced at 37°C by a 24 h exposure to 15 µM NMDA, 10 µM AMPA, or 35 µM kainate in MS supplemented with glycine. MK-801 (10 µM) was always added concurrently with AMPA or kainate to block secondary NMDA receptor activation. TGF-ß1 was coapplied with the excitotoxin and left in the bathing media for 24 h.
Assessment of neuronal cell death
Neuronal death was estimated by examination of the cultures under phase-contrast microscopy, and quantitated by measurement of lactate dehydrogenase (LDH) release by damaged cells into the bathing medium 1 day after the onset of excitotoxic exposure (22). The LDH level corresponding to near-complete neuronal death (without glial death) was determined in sister cultures exposed to 200 µM NMDA for 24 h in MS supplemented with glycine. Background LDH levels were determined in sister cultures subjected to sham wash and subtracted from experimental values to yield the signal specific to experimentally induced injury.
Reverse transcription and polymerase chain reaction
Total RNAs were transcribed into cDNA using poly-dT oligonucleotides. An aliquot of the cDNAs so produced was amplified using sense and antisense primers for ß-actin [539 bp polymerase chain reaction (PCR) product], PAI-1 (331 bp PCR product), and t-PA (252 bp PCR product), respectively (30 cycles of PCR were chosen corresponding to 50% of the saturating curve of the PCR products). ß-Actin sense oligonucleotide: GTG GGC CGC TCT AGG CAC AA, and antisense oligonucleotide: CTC TTT GAT GTC ACGCAC GAT TTC; PAI-1 sense oligonucleotide: ATG AGA TCA GTA CTG CGG ATG CCA, and antisense oligonucleotide: GCA CAG AGA CGG TGC TGC. t-PA sense oligonucleotide: GAC GAT ACT TAT GAC AAC GAC, and antisense oligonucleotide: TAT TAA ACA GAT GCT GTG AGG. The specificity of both PAI-1 and t-PA PCR products were confirmed by sequencing.
Transfection and luciferase assays
Cells were transiently transfected with the indicated constructs using the lipofection method (Lipofectamine, Life Technologies) and stimulated with 1 ng/ml of human recombinant TGF-ß1 (R&D). Luciferase activities were quantified 24 h later by using a commercial kit (Promega). Values were normalized with respect of ß-galactosidase activity as expressed by the SV40 ß-galactosidase vector (Promega).
PAI-1 expression
Cells were incubated in methionine/cysteine-free minimal essential medium containing 50 µCi/ml of 35S-methionine/35S-cysteine (Trans-label, NEN, 1100 Ci/mmol.) for 6 h at 37°C in the presence of TGF-ß1 (1 ng/ml). Cells were removed by washing three times in a solution containing: 10 mM Tris-HCl (pH 8.0), 0.5% sodium deoxycholate with 1 mM phenylmethylsulfonyl fluoride. Cell matrix proteins were extracted and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in the presence of dithiothreitol (DTT) and ß-mercaptoethanol.
Immunoprecipitation
Metabolically labeled cells were solubilized in 1 ml of lytic buffer in the presence of protease inhibitors at 4°C for 30 min. By using specific antibodies, immunoprecipitations were performed from the supernatant of the lysis solution for 2 h at 4°C and followed by adsorption to protein G-Sepharose (Pharmacia Biotech). Bound proteins were eluted in DTT and ß-mercaptoethanol containing a loading sample buffer. Then an SDS-PAGE was performed.
Growth inhibition assay
After 20 h of treatment in the presence of 1 ng/ml of TGF-ß1 in serum free medium, cells were labeled with 0.5 µCi/ml of 3H-thymidine (specific activity 2200 Ci/mmol, NEN) for an additional 4 h. Cells were precipitated and washed three times in 5% trichloroacetic acid before extraction at 4°C in 0.1 N NaOH. The extracts were counted in a beta counter.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To induce apoptosis, we transferred cortical pure neuronal cultures after 7 days in vitro (DIV 7) to a serum-deficient medium (24), which provoked the death of approximately 50% of neurons over 24 h. This paradigm evidenced three features typical of apoptosis: 1) the neurons exhibited a gradual shrinkage of the cell body; 2) death was almost completely obviated by the addition of cycloheximide, a protein synthesis inhibitor; and 3) death was accompanied by the appearance of DNA fragmentation (data not shown). Addition of TGF-ß1 to the bathing medium (at 1 ng/ml) failed to modify the progression and extent of neuronal degeneration.
We also induced neuronal apoptosis by exposing mixed neuron–glia cultures (DIV 14) to staurosporine, a nonspecific protein kinase inhibitor, which caused a neuronal degeneration evolving over 24 h (25). Whereas cycloheximide totally blocked neuronal death, the addition of TGF-ß1 (at 1 ng/ml) was without effect (
Fig. 1A).
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Our next step was to determine the influence of TGF-ß1 on necrosis, a process morphologically distinct from apoptosis and characterized by a prominent and early cell swelling. To induce necrosis, we used the following glutamatergic agonists: NMDA, AMPA, and kainate (26). The exposure of mixed cortical neuron–glia cultures or pure cortical neuronal cultures (DIV 14) to these agonists produced acute swelling of neuronal bodies, followed 24 h later by widespread neuronal degeneration; the glia remained intact. Coincubation with increasing concentrations of TGF-ß1 reduced neuronal death only in the instance of NMDA-induced necrosis in mixed neuron–glia cultures (
Fig. 1B, C). In contrast, TGF-ß1 failed to produce any neuroprotective effect in pure cortical cultures of neurons ( Fig. 1D).
Expression of TGF-ß dominant negative receptor in astrocytes abolished TGF-ß1 neuroprotective effect against NMDA-induced necrosis
We demonstrate here that TGF-ß1 exerts its beneficial effects against excitotoxic necrosis only in mixed cortical neuron–glia cultures, results that would suggest an obligatory involvement of glia in the mechanisms responsible for the neuroprotective effect of TGF-ß1 in the necrotic-type of cell death.
To test the hypothesis as to whether glia truly mediate this neuroprotective effect, we attempted to prevent the TGF-ß1 response of astrocytes in mixed neuron–glia cultures. By using lipofection, we transfected the mixed neuron–glia cultures with ß-galactosidase cDNA: these experiments revealed that only astrocytes expressed ß-galactosidase ( Fig. 2A).
We estimated, by cell counting, that approximately 70% of the astrocytes were transfected. In mixed cortical cultures subjected to transfection, LDH levels were not modified (up to 48 h), which demonstrates that the procedure did not affect neuronal survival (data not shown). Then we selectively transfected astrocytes contained in mixed neuron–glia cultures with cDNA encoding for a TGF-ß dominant negative receptor (TßR-IIEI). This chimeric receptor was generated by fusion of the extracellular domain of TßR-II with the transmembrane and the intracellular domain of TßR-I, preventing TGF-ß1 responses (27). The expression of the transfected TßR-IIEI receptor was determined in glia cultures by metabolic labeling of the TGF-ß receptors, followed by immunoprecipitation with antibodies raised against the TßR-II extracellular domain. Complexes formed of TßRI and both endogenous TßRII or transfected TßRIIEI were expressed by astrocytes 24 h after the transfection protocol ( Fig. 2B). As it was important to determine whether the TßR-IIEI dominant negative receptor was able to prevent the TGF-ß1-induced biological response, we studied the inhibitory role of TGF-ß1 on astrocytic proliferation (28). Transfection of astrocytes with the cDNA encoding for TßR-IIEI markedly reduced (-75%) the TGF-ß1-induced inhibition of glial proliferation ( Fig. 2C). These data show that transfection of the cDNA encoding for TßR-IIEI rendered astrocytes less responsive to TGF-ß1. When the mixed neuron–glia cultures, transfected with TßR-IIEI, were exposed to NMDA, the neuroprotective effect of TGF-ß1 was abolished in sister cultures ( Fig. 2D). This result underlines the fact that TGF-ß1 protects neurons against NMDA-induced cell death when, and only when, TGF-ß1-responsive astrocytes are present in the mixed cortical cultures.
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PAI-1 and t-PA expression pattern in astrocytes and neurons: modulation by TGF-ß1
First, to quantify PAI-1 transcriptional activity that depends on TGF-ß1, we developed a luciferase reporter gene driven by the PAI-1 promotor (PAI-luciferase). The luciferase activity directly reflects the PAI-1 transcriptional level. TGF-ß1 enhanced luciferase activity up to 60% and cotransfection with the TßR-IIEI dominant negative receptor totally prevented this response ( Fig. 3A).
Next, we studied the cell type that expressed t-PA or PAI-1 in both glial cultures and pure cultures of cortical neurons. By using reverse transcriptase PCR (RT-PCR), we showed that t-PA mRNA was expressed in both neurons and glia whereas PAI-1 mRNA expression was restricted to glia ( Fig. 3B). Moreover, enhanced expression of PAI-1 mRNA after TGF-ß1 treatment was characteristic of the astrocytes. Expression of the t-PA mRNA was not influenced by TGF-ß1 treatment in neuronal and astrocytic cultures. Furthermore, the increased PAI-1 expression in astrocytes subsequent to treatment with TGF-ß1 was visualized, at the protein level, by SDS-PAGE of extracellular matrix-associated protein. As shown in Fig. 3C, TGF-ß1 induced the overexpression of a protein with an approximate molecular mass of 50 kDa specifically immunoprecipitated by an antibody raised against PAI-1. The light microscope distribution of the specific antibodies for the two proteins studied matched the distribution of t-PA and PAI-1 mRNAs ( Fig. 4).
To study the possibility of an interaction between PAI-1 and t-PA, immunoprecipitation with antibodies raised against PAI-1 and t-PA was performed both in mixed and in pure neuronal cultures. SDS-PAGE electrophoresis revealed the presence of complexes formed from PAI-1 and t-PA in mixed cultures of neurons and astrocytes but not in pure cultures of neurons ( Fig. 3D).
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Blockade of PAI-1 activity prevents TGF-ß1-induced neuroprotection
To test whether the neuroprotective effect of TGF-ß1 against NMDA-mediated necrosis involved the overexpression of PAI-1 in astrocytes, NMDA-induced toxicity in mixed cultures of neurons and astrocytes was performed in the presence of an antibody raised against PAI-1 that neutralizes the t-PA binding capacity of PAI-1 (29, 30). Although coincubation of cultures with 100 µg/ml of this antibody did not modify NMDA-induced cell death, this treatment abolished the neuroprotective effect of TGF-ß1 ( Fig. 5A, B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We also demonstrate that application of TGF-ß1 reduces excitotoxicity in cortical cell cultures. These results are in accordance with prior observations by Prehn et al. (23), which described a broad spectrum of neuroprotection induced by TGF-ß1. In our study, the neuroprotective activity of TGF-ß1 was limited to NMDA receptor-mediated neuronal cell death. AMPA- and kainate-induced cell death (associated with MK 801) were not affected by a coincubation with TGF-ß1, which would indicate that activation of TGF-ß receptors specifically modulates NMDA-induced cell death. Additional investigations are needed to determine the reason of this selective neuroprotective effect of TGF-ß1 against NMDA-induced excitotoxicity. Even though several reports (23, 31) have described the neuroprotective potential of this cytokine against necrosis, the mechanism of this action remains unclear.
We document here that this growth factor exerts its beneficial effects against excitotoxic necrosis only in mixed cortical cultures. This result underlines the fact that TGF-ß1 protects neurons against NMDA-induced cell death when, and only when, TGF-ß1-responsive astrocytes are present in the mixed cortical cultures. We confirmed and extended this observation by demonstrating that TGF-ß1 fails to exert any neuroprotective activity against NMDA-mediated excitotoxicity in pure neuronal cultures (containing less than 5% of astrocytes). These results would infer an involvement of glia in the mechanisms responsible for the neuroprotective effect of TGF-ß1 in necrosis. Moreover, our results clearly show that PAI-1, a serine protease inhibitor, expressed only in astrocytes and overexpressed after TGF-ß1 treatment, mediates the neuroprotective effect of TGF-ß1. These observations suggest the involvement of extracellular matrix components in growth factor-induced neurons/glia communication.
Increasing evidence suggests that members of the serine protease family, such as the tissue-type plasminogen activator (t-PA) or thrombin, may play a role in normal and diseased nervous system. Serine proteases increase in the neural parenchyma and cerebrospinal fluid after brain injury (17, 18). Recent data have demonstrated that t-PA increases neuronal damage after focal cerebral ischemia (32), suggesting that t-PA may promote neuronal injury through its plasminogen-activating activity. Thus, proteases may dramatically influence the outcomes of various neuropathological insults. An equilibrium between serine proteases and their specific inhibitors (serpins) exists in many tissues where these agents are involved in physiological processes associated with inflammation, connective tissue turnover; nonetheless, no clear involvement of serpins was evident in either physiological or pathological processes in the central nervous system. We have shown that PAI-1, a selective inhibitor of tPA exclusively expressed in astrocytes, is up-regulated by TGF-ß1 treatment. By blocking the ability of PAI-1 to inhibit tPA, we abolished the neuroprotective activity of TGF-ß1 against NMDA neurotoxicity. These results underline the potential neuroprotective effect that would be obtained by a modulation of the t-PA/PAI-1 axis.
Regardless of the underlying mechanisms of the involvement of t-PA in excitotoxic injury, our observations might caution against the indiscriminate application of t-PA for the treatment of hypoxic–ischemic injury in brain tissue. Even when net beneficial effects are observed, the possibility that the overall neuroprotection is limited by concomitant potentiation of excitotoxic necrosis should be explored in appropriate animal models.
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FOOTNOTES |
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1 Correspondence: Université de CAEN-CNRS UMR 6551, Laboratoire de Neurosciences, BP 5229, 14074 CAEN Cedex, France. E-mail: neurolab{at}criuc.unicaen.fr
3 Abbreviations: DTT, dithiothreitol; TGF-ß1, transforming growth factor ß1; NMDA, N-methyl-D-aspartate; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionate; MK-801, (+)5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine maleate; LDH, lactate dehydrogenase; MS, Eagle's minimal essential medium augmented with 2 mM glutamine and 25 mM glucose; MEM, Eagle's minimal essential medium; PAI-1, plasminogen activator inhibitor type 1; RT-PCR, reverse transcriptase-polymerase chain reaction; t-PA, tissue type plasminogen activator; SD, serum deprivation; STP, staurosporine.
Received for publication May 11, 1998.
Revision received July 6, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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