Glia Increase Degeneration of Hippocampal Neurons through Release of Tumor Necrosis Factor-α

TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.

150, 271–276 (1998)

TO988406

Glia Increase Degeneration of Hippocampal Neurons through Release of Tumor Necrosis Factor-a Barbara Viviani, Emanuela Corsini, Corrado L. Galli, and Marina Marinovich Institute of Pharmacological Sciences, University of Milan, 20133 Milan, Italy Received September 29, 1997; accepted February 5, 1998

Glia Increase Degeneration of Hippocampal Neurons through Release of Tumor Necrosis Factor-a. Viviani, B., Corsini, E., Galli, C. L., and Marinovich, M. (1998). Toxicol. Appl. Pharmacol. 150, 271–276. This study characterizes the role of glial cells in chemically induced neurodegeneration. We evaluated the effect of trimethyltin, a trisubstituted organotin compound that elicits distinct lesions in the central nervous system in vivo, on a sandwich coculture of neurons and glia. Exposure of a 98% pure culture of rat hippocampal neurons to 0.1–1 mM trimethyltin for 24 h caused neural cell death and nuclear changes typical of apoptosis; at these doses glial cells viability was not affected but the cells released significant amounts of tumor necrosis factor-alpha (TNF-a). Neuronal apoptosis and TNF-a release from glial cells both increased when the two cell types were exposed together to trimethyltin, which indicates synergy. Treatment of a neuron– glia co-culture with TNF-a antibody prevented the increase in neuronal apoptosis, and TNF-a administration induced apoptosis in hippocampal cells. We conclude that glial cells and TNF-a both modulate trimethyltin-induced neurodegeneration. © 1998 Academic Press Key Words: cytokines; trimethyltin; cell death.

In the processes of development and maintenance of the nervous system there exists a complex interdependency between neurons and glial cells. Glial cells maintain normal functioning of the nervous system both by controlling the extracellular environment and by supplying metabolites and growth factors. After damage to the CNS, glia are thought to support neural growth and metabolism and to scavenge agents toxic to neurons (Giulian and Lachman, 1985; Hefti, 1986; Mattson and Rychlik, 1990). However, the idea that glia could participate in actually damaging neurons has also recently started to emerge. Nitric oxide, reactive oxygen species, and cytokines are released from glial cells in response to focal cerebral ischemia (Lees, 1993; Rothwell and Relton, 1993). Higher levels of cytokines synthesized within the CNS have been reported in patients suffering various infections including HIV (Stanley et al., 1991) and in several neurodegenerative disorders such as Alzheimer disease and Down syndrome (Griffin et al., 1989)

and multiple sclerosis (Hofman, 1989). The presence of such products in the brain after damage does not prove them either beneficial or detrimental, but nitric oxide concentrations can reach neurotoxic levels (Nowicki et al., 1991), and endogenous IL-1 directly mediates acute neuronal death during ischemia (Relton and Rothwell, 1992). Furthermore, Giulian et al. (1993) reported that activated microglia secrete neuron-killing factors while Dugan et al. (1995) observed that glia enhance the neurotoxicity of (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Although glial responses have been examined in experimental models of neurodegenerative diseases, direct observation of the interactions between glial cells and neurons in the development of neurodegeneration is new. Trimethyltin is a neurotoxic organotin compound, which induces neural degeneration in specific areas of the central nervous system such as hippocampus, pyriform cortex, neocortex, amygdala nucleus, and brain stem (Rehul et al., 1983). In vitro, trimethyltin has been shown to cause selective neural degeneration (Thompson et al., 1996), and this suggests a direct effect of this compound on neurons. Both in vivo and in vitro trimethyltin induces a glial response with increased levels of glial fibrillary acidic protein, an increased number of astrocytic processes (Monnet-Tschudi et al., 1995), and activation of microglia (McCann et al., 1996). Thus, neurons may also be affected indirectly, through the perturbation of glial homeostasis. Although the ability of trimethyltin to affect both neurons and glial cells has been recognized, the communication between these two cell populations in trimethyltin-induced neurodegeneration has never been studied. We have investigated in primary cell cultures the possible interaction between glial cells and hippocampal neurons in trimethyltin-induced neural degeneration. EXPERIMENTAL PROCEDURES Materials. Trimethyltin chloride was purchased from Societa’ Italiana Chimici (Rome, Italy). The ELISA system to detect cell death was from Boehringer Mannheim (Germany). Murine TNF-a and polyclonal rabbit antimurine TNF-a were from Genzyme (Cambridge, MA). Isolectin B4, labeled fluorescein isothiocyanate (FITC) from Griffonia simplicifolia and L-NAME (Nv-nitro-D-arginine methyl ester) were from Sigma Chemical Co. (St. Louis,

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0041-008X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

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MO) as well as all other chemicals and cell culture materials. Eicosatetraynoic acid (ETYA) was from Cayman (Ann Arbor, MI). Sandwich co-cultures of hippocampal neurons and glial cells (Goslin and Banker, 1991). Primary cultures of glial cells were prepared from 1- to 2-day-old newborn rats (Sprague–Dawley). Cerebral hemispheres were freed of the meninges and were mechanically disrupted. Cells were dissociated in a solution of trypsin 2.5% and DNAse 1%, filtered through a 100-mm nylon mesh, and plated in Petri dishes (140,000 cells per 35-mm dish) in minimum essential Eagle’s medium (MEM) supplemented with 10% fetal calf serum, 0.6% glucose, 0.1 mg/ml streptomycin, and 100 IU/ml penicillin. Glial cultures were fed twice a week and grown at 37°C in a humidified incubator with 5% CO2. Culture medium was replaced 24 h before the study with neuronal maintenance medium (Dulbecco’s modified Eagle’s medium and Ham’s nutrient mix F-12 supplemented with 5 mg/ml insulin, 100 mg/ml transferrin, 100 mg/ml putrescin, 30 nM Na selenite, 20 nM progesterone, and 100 IU/ml penicillin. Neuronal cultures were established from the hippocampus of 18-day rat fetuses. Brains were removed and freed of meninges and the hippocampus was isolated. Cells were dissociated by incubation for 15–20 min at 37°C in a 2.5% trypsin solution followed by trituration. Cell suspension was diluted in the medium used for glial cells and plated onto polyornithine-coated coverslips at a density of 160,000 cells per coverslip. The day after plating, coverslips were transferred to dishes containing a glial monolayer in neuronal maintenance medium supplemented with 5 mM cytosine arabinoside. Coverslips were inverted so that the hippocampal neurons faced the glia monolayer. Paraffin dots attached to the coverslips supported them above the glia, creating a narrow gap that precluded contact between the two cell types but allowed the mutual diffusion of soluble substances. The same size of paraffin dots applied on the coverslips allowed a constant distance between neurons and glia to be kept. These culture conditions allowed us to grow differentiated neuronal cultures with .98% homogeneity, as assessed by immunocytochemistry of microtubule-associated protein 2 and glial fibrillary acidic protein. Astrocytes and microglia cell cultures. A layer of astrocytic cells was obtained by vigorous shaking of a confluent 10-day-old monolayer of mixed glial cells, as described by McCarthy and De Vellis (1980). Cultures of enriched astroglia were treated further with 5 mM L-leucine methyl ester to eliminate microglia (97% homogeneity). Isolated astroglial preparations were then seeded in 24-well plates (700,000 cells/well) in MEM with supplements as above. Microglia were isolated by shaking glial cultures at 260 rpm for 2 h. Microglia dislodged into the medium were purified by plating for 30 min in 24-well plates (100,000 cells/well). Contaminating cells were removed with the supernatant. These conditions allowed us to obtain highly enriched microglial cultures with 98% homogeneity, as assessed by immunocytochemistry with Griffonia simplicifolia isolectin B4. Both astrocytes and microglia were fed nutrient twice a week and maintained at 37°C in a humidified incubator with 5% CO2. Experimental protocol. All experiments were performed on 4- to 6-dayold hippocampal neuron cultures and 2- to 3-week-old glial cell cultures. Astrocytes and microglia, obtained from 10-day-old glial cultures were generally used within 2 days after purification. Hippocampal neurons and glial cells, alone or in co-culture, were exposed to trimethyltin. To treat neurons alone with TMT, differentiated neurons on coverslips were divided by the glia monolayer and exposed to TMT dissolved in the growing medium. Assessment of cell survival. The viability of hippocampal neurons and glial cells was measured by the 3-(4,5-dimethyl-thiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Denizot and Lang, 1986). Nuclear morphology and DNA fragmentation. Treated hippocampal neurons on coverslips were washed and fixed with 80% (v/v) methanol/phosphate buffer for 15 min at 4°C. After fixation cells were rinsed, stained for 5 min with 5 mg/ml propidium iodide, and examined under a Zeiss Axioskop microscope with a 403 objective. Trimethyltin-induced apoptosis was also assessed by measuring with an

FIG. 1. Percent of cell death in primary hippocampal neurons and glial cells after trimethyltin exposure. ■, Neurons alone; E, Neurons exposed together with glial cells. Each value represents the means 6 SD of two experiments in triplicate. All data are significant p , 0.01 vs control, *p , 0.05 and **p , 0.01 vs neurons exposed alone; ANOVA followed by Tukey test.

ELISA detection kit the formation of immunoreactive oligonucleosomal fragments. This assay allows the specific determination of mono- and oligonucleosomes in the 20,000g supernatant fraction of cell lysates. Data are expressed as enrichment of oligonucleosomes 5 100 3 (sample absorbance/control absorbance) 2 100. Assay for TNF-a. TNF-a content was assayed by determining cytotoxicity against sensitive L929 cells, as previously described (Rosenthal and Corsini, 1995). Briefly, L929 cells were seeded into 96-well microtiter plates (25,000 cells/well) in RPMI-1640 culture medium supplemented with 10% fetal calf serum and incubated for 24 h at 37°C in a humidified atmosphere 5% in CO2. After removal of the medium, actinomycin D (0.6 mg/100 ml) was added to each well for 1 h at 37°C. Recombinant murine TNF-a and test samples were added and plates were incubated at 37°C for 18 h. Cells were stained and fixed with 0.2% crystal violet in 2% ethanol for 10 min at room temperature, washed, and then lysed with 1% SDS. Absorbance at 620 nm was read on a Multiscan reader. TNF-a concentration was calculated from a standard curve with known amounts of recombinant murine TNF-a. Results are expressed in pg/ml. Statistical analysis. The statistical significance of differences was determined by two-way analysis of variance (ANOVA) followed by a Tukey HSD multiple comparison test.

RESULTS

Trimethyltin-Induced Cell Death (Apoptosis) in Hippocampal Neurons but not in Primary Glial Cells Exposure of primary hippocampal neurons alone to concentrations of trimethyltin ranging from 0.1 to 1 mM for 24 h was followed by cell death, as assessed by the MTT test (Fig. 1). The effect is dose-dependent and was already significant at 0.1 mM trimethyltin (Fig. 1). By contrast, no decrease in cell viability was detectable after treatment of glial cells with up to 2.5 mM trimethyltin (Fig. 1, inset). The exposure of hippocampal neurons together with glial cells to 0.5–1 mM trimethyltin

GLIA EXACERBATES TRIMETHYLTIN NEUROTOXICITY

significantly increased the percentage of neuronal death (Fig. 1). Hippocampal cell death was associated with chromatin condensation (Fig. 2) and DNA fragmentation, typical features of apoptosis. DNA fragmentation was evident at 0.1 mM trimethyltin (35 6 3.1 enrichment in oligonucleosomes) and increased significantly with increasing dose (130 6 6.8 at 0.5 mM) and when neurons were co-exposed with glial cells (224 6 8 enrichment at 0.5 mM). Trimethyltin-Induced TNF-a Release From Primary Glial Cells: Involvement of Astrocytes and Microglia It has been reported (Maier et al., 1995) that trimethyltin treatment in vivo increases the levels of proinflammatory cytokines in rat hippocampus. Exposure of hippocampal neurons to trimethyltin up to 1 mM released no TNF-a (Fig. 3), but glial cells similarly exposed produced TNF-a; the combination of glial cells with neurons produced TNF-a even at 0.5 mM trimethyltin and far more TNF-a than with glial cells alone at 1 mM trimethyltin (Fig. 3). Exposure of primary cultures of astrocytes as well as of enriched cultures of microglia to 1 mM trimethyltin was followed by a drastic change in cell morphology (data not shown), which for microglial cells was already evident 6 h after exposure and lasted up to 24 h. Both astrocytes and microglial cultures were stimulated to release TNF-a some seven-fold by 1 mM trimethyltin (Table 1). TNF-a Exacerbates the Trimethyltin-Induced Neuronal Apoptosis We exposed hippocampal neurons plus glial cells to 0.5 mM trimethyltin in the presence of TNF-a antibody and assessed DNA fragmentation in the hippocampal neurons (Table 2). The antibody opposed the DNA-fragmenting effect, though it did not prevent it; the residual enrichment of oligonucleosomes in its presence was similar to that obtained when neurons alone were exposed to 0.5 mM trimethyltin or 0.25 ng/ml TNF-a (Table 2). TNF-a Release was not Blocked by Inhibitors of Nitric Oxide (NO) Synthase or Prostaglandin (PG) Synthesis Endeavors were made to identify the factor(s) released by damaged neurons that stimulated TNF-a release from glial cells. Since in the sandwich culture neurons and glia are in apposition but do not enter into direct contact, we focused on the most common soluble mediators of cell damage, namely NO and arachidonic acid derivatives. Incubation of the cell system with 10 mM ETYA, an inhibitor of prostaglandin and leukotriene synthesis, 30 min before the exposure to trimethyltin did not modify the release of TNF-a (126 6 3 vs 121 6 1.3 pg/ml). Similarly, addition of an inhibitor of NO synthase (50 mM L-NAME) had no effect (424 6 11 vs 399 6 14 pg/ml).

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DISCUSSION

The role of glial cells in neurophysiology extends well beyond passive structural support. In fact, early in development, glial cells and neurons establish a highly dynamic relationship that influences neural cell growth, morphology, behavior, and repair. The present study was undertaken to characterize biochemical communication between glial cells and neurons in chemically induced neurodegeneration. The use of a sandwich co-culture of neurons and glial cells helped us to address the problem. Freshly prepared primary hippocampal neurons were apposed to a glial monolayer but separated from it by a narrow gap. Although the two cell types were not in contact, soluble mediators could diffuse between them. We chose to study trimethyltin because it induces a specific pattern of neuronal degeneration and glial activation (MonnetTschudi et al., 1995; Richter-Landsberg and Besser, 1994) and represents a good model of selective neurotoxicity. Several theories have been put forward to account for the neurotoxic action of trimethyltin. It has been observed that trimethyltin in vitro alters the active and passive membrane properties of CA1 pyramidal neurons (Harkins and Armstrong, 1992) and the uptake and efflux of glutamate and D-aspartate (Naalsund and Fonnum, 1986) and inhibits mitochondrial ATP synthesis (Aldridge, 1977) and Na1/K1 ATPases (Stine et al., 1988). Trimethyltin brings about some of these effects by direct action on neurons, but some are due to its action on glia. Other authors have observed the inhibition of astrocytic Na1/K1 ATPases with consequent swelling of and glutamate release from astrocytes (Aschner and Aschner, 1992), the increase in number and/or clustering of GSI-B4 lectin-positive microglial cells (Monnet-Tschudi et al., 1995; McCann et al., 1996), and increased expression of glial–fibrillary acidic protein in astrocytes (Monnet-Tschudi et al., 1995; Richter-Landsberg and Besser, 1994). This activation of astrocytes and microglial cells could promote the synthesis of proinflammatory cytokines (Maier et al. 1995), some of which are neurotoxic. Hippocampal neurons represent the main target of trimethyltin in vivo (Chang and Dyer, 1983). We observed degeneration of primary hippocampal neurons in vitro at doses of trimethyltin (Fig. 1) similar to its concentration in brain after in vivo exposure (Lopachin and Aschner, 1993). Chromatin condensation and DNA fragmentation in trimethyltin-treated neurons (Fig. 2) suggest that trimethyltin triggers apoptosis. This effect was highly selective for neurons: at the same doses the viability of glial cells was not affected. Nevertheless, the exposure of glial cells to 1 mM trimethyltin was followed by morphological modification, suggesting glial activation. These modifications were evident both in astrocytes and microglia. Since in vivo exposure to trimethyltin increases the levels of cytokine (IL-1a, IL-6, and TNF-a) mRNAs in rat hippocampus (Maier et al., 1995), we determined whether trimethyltin-induced glial activation results in cytokine release. We focused on TNF-a because of its role in

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FIG. 2. Morphological evaluation of apoptosis in primary hippocampal neurons. The nuclei of hippocampal cells were stained with propidium iodide and visualized under a fluorescent microscope at 403 magnification. Arrows indicate apoptotic nuclei. (A) Control cells. (B) Hippocampal neurons exposed to 0.5 mM trimethyltin for 24 h.

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GLIA EXACERBATES TRIMETHYLTIN NEUROTOXICITY

TABLE 2 TNF-a and DNA Fragmentation in Neuron and Neuron 1 Glia Cultures Enrichment in oligonucleosomes in neuronal extracts Neurons Trimethyltin (0.5 mM) TNF-a (25 ng/ml) Neurons 1 glia Trimethyltin (0.5 mM) Trimethyltin 1 AbTNF-a

230 6 12 150 6 1 324 6 11** 173 6 5

** p , 0.01 vs neurons 1 trimethyltin, Student’s t-test. FIG. 3. TNF-a release from glia and neurons induced by different concentrations of trimethyltin. h Neurons; } glia; and E glia and neurons together. TNF-a was measured in the culture medium 24 h after the treatment. Each value represents the means 6 SD of two experiments in triplicate. °°p , 0.01 vs control, **p , 0.01 vs glia, ANOVA followed by Tukey test.

modulating neurodegeneration/neurorepair. Exposure of glial cells to trimethyltin did result in TNF-a release. Our data suggest that both astrocytes and microglia could release TNF-a (Table 1). Nevertheless, since it is possible to obtain only enriched astrocytic and microglial cultures (homogeneity 97%), this hypothesis should be furtherly confirmed by in situ hybridization or immunocytochemistry. Glial cells seem to be the only source of TNF-a, since under our experimental conditions TNF-a was not released from neurons. However, trimethyltin-exposed neurons significantly enhanced the TNF-a release from glial cells (Fig. 3). This effect is evident only at doses of trimethyltin (0.5 mM) that induce hippocampal cell death, which suggests that only damaged neurons modulate glial activity. The neuronal factor(s) involved in increasing TNF-a release from glia is still unknown: soluble mediators like nitric oxide and prostaglandins do not seem to be involved. TNF-a is not the only factor released by trimethyltin from glial cells that is able to affect neural function. As cited above, other cytokines like IL-1 and IL-6 could be also involved in

TABLE 1 TNF-a Release From Astrocytes and Microglia After Treatment with Trimethyltin Astrocytes

Microglia TNF-a (pg/ml)

Control Trimethyltin (1 mM)

42 6 3 254 6 10**

49 6 15 291 6 46**

Note. Data are means 6 SEM of three independent samples. **p , 0.01 vs the relative control, Student’s t-test.

trimethyltin-induced damage and their role is under investigation. Trimethyltin induces release of glutamate from hippocampal slices (Patel et al. 1990) and reduces its high-affinity uptake (Naalsund and Fonnum, 1986), thereby rendering neurons highly vulnerable to glutamate-mediated cytotoxicity. Astrocytes can play a part in this: trimethyltin treatment of primary astrocyte cultures increases the rate and amount of glutamate efflux (Aschner and Aschner, 1992). However, no direct evidence has been provided of a link between trimethyltin-induced glutamate release and trimethyltin-induced neural degeneration, whereas in our experimental system the increased apoptosis due to the presence of glia during trimethyltin exposure seems to be highly dependent on TNF-a (Table 2). It has been suggested (Chao and Hu, 1994) that neuronal excitotoxicity is potentiated by TNF-a through enhancement of postsynaptic glutamate binding and the inhibition of astrocytic glutamine synthetase. Thus, TNF-a could be the ultimate resultant of a complex cascade involving several mediators. In conclusion, the exposure of hippocampal neurons to trimethyltin together with the glial monolayer resulted in a higher rate of neural death and apoptosis than exposure of neurons alone. This was observed at doses at which a significant release of TNF-a was detectable and was very much reduced by a TNF-a antibody. At doses of trimethyltin that do not release TNF-a, is rather observed a reduction of neural cell death in the presence of glia. This could be an unspecific effect due to the higher number of cells present in the co-culture condition than in neurons alone. These results, together with the observation that TNF-a alone is able to induce apoptosis in primary hippocampal neurons, suggest a direct involvement of this cytokine in the mechanism underlying neurodegenerative phenomena. Trimethyltin, being able to affect both neuronal and glial cells, created a degenerative loop in which glial cells precipitate neuronal death and damaged neurons stimulate the production of their own killing factor.

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ACKNOWLEDGMENTS This work was supported by CEE grant EV5V-CT94-0508 (DG 12 SOLS) and by CNR grant 97.04658.CT13. We thank Dr G. E. Rovati for the very precious support for the statistical analysis.

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