Neuroprotection by Glial Metabotropic Glutamate Receptors Is Mediated by Transforming Growth Factor-␤

The Journal of Neuroscience, December 1, 1998, 18(23):9594–9600 Neuroprotection by Glial Metabotropic Glutamate Receptors Is Mediated by Transforming Growth Factor-b V. Bruno,1 G. Battaglia,1 G. Casabona,1 A. Copani,2 F. Caciagli,3 and F. Nicoletti1,2 1Istituto Neurologico Mediterraneo Neuromed, 86077 Pozzilli, Italy, 2Institute of Pharmacology, School of Pharmacy, University of Catania, 95125 Catania, Italy, and 3Department of Pharmacological Sciences, University of Chieti, 66013 Chieti, Italy The medium collected from cultured astrocytes transiently ex- protective activity of DCG-IV or 4C3HPG, as well as the activity posed to the group-II metabotropic glutamate (mGlu) receptor of GM/DCG-IV or GM/4C3HPG; and (3) a transient exposure of 9 9 9 agonists (2S ,1 R ,2 R ,3 R )-2-(2,3-dicarboxycyclopropyl)glycine cultured astrocytes to either DCG-IV or 4C3HPG led to a de- (DCG-IV) or (S)-4-carboxy-3-hydroxyphenylglycine (4C3HPG) layed increase in both intracellular and extracellular levels of is neuroprotective when transferred to mixed cortical cultures TGFb. We therefore conclude that a transient activation of challenged with NMDA (Bruno et al., 1997). The following data group-II mGlu receptors (presumably mGlu3 receptors) in as- indicate that this particular form of neuroprotection is mediated trocytes leads to an increased formation and release of TGFb, by transforming growth factor-b (TGFb). (1) TGFb1 and -b2 which in turn protects neighbor neurons against excitotoxic were highly neuroprotective against NMDA toxicity, and their death. These results offer a new strategy for increasing the local action was less than additive with that produced by the medium production of neuroprotective factors in the CNS. collected from astrocytes treated with DCG-IV or 4C3HPG Key words: metabotropic glutamate receptors; glial cells; (GM/DCG-IV or GM/4C3HPG); (2) antibodies that specifically transforming growth factor-b; neuroprotection; excitotoxic neu- neutralized the actions of TGFb1or-b2 prevented the neuro- ronal death; astrocyte; cortical cultures Metabotropic glutamate (mGlu) receptors form a family of eight inhibition of glutamate release may contribute to, but is not subtypes (mGlu1–8), which have been subdivided into three sufficient to explain, neuroprotection mediated by group-II mGlu groups. Group-I includes mGlu1 and -5, which are coupled to receptors. Accordingly, mGlu2 or -3 receptor agonists protect not polyphosphoinositide (PI) hydrolysis as well as to various classes only against excitotoxic death but also against apoptosis induced 1 b of K channels via a Go or Gq GTP binding protein (for review, by -amyloid peptide or hypoxia combined with glucose depriva- see Pin and Duvoisin, 1995). Group-II (mGlu2 and -3) and tion in the presence of a mixture of ionotropic receptor antago- group-III (mGlu4, -6, -7, -8) receptors are coupled to a Gi- nists, i.e., under conditions in which neurodegeneration develops protein, and their activation inhibits adenylyl cyclase activity in in the absence of any excitotoxic component (Buisson and Choi, heterologous expression systems (Tanabe et al., 1992, 1993). 1995; Copani et al., 1995). We recently showed that neuroprotec- However, native group-II or group-III mGlu receptors in the tion mediated by group-II mGlu receptors involves a novel form CNS are coupled to multiple transduction pathways, including of glial–neuronal interaction, which is promoted by the activation 1 inhibition of voltage-sensitive Ca 2 channels, stimulation or in- of mGlu3 receptors present in astrocytes (Bruno et al., 1997). The hibition of cAMP formation, stimulation of PI hydrolysis, and medium collected from cultured astrocytes 2–20 hr after a brief activation of the mitogen-activated protein (MAP) kinase path- exposure to mGlu3 receptor agonists is highly neuroprotective way (Winder and Conn, 1992; Genazzani et al., 1994; for review, against NMDA toxicity (Bruno et al., 1997, 1998). Neuroprotec- see Pin and Duvoisin, 1995). tion is attenuated after treating the astrocytes with cycloheximide Recently, mGlu receptors have been considered a potential or after heating the medium, suggesting that astrocytes produce target for neuroprotective drugs. In particular, activation of and release a proteic neuroprotective factor in response to mGlu3 group-II or group-III mGlu receptors protects neurons against receptor activation (Bruno et al., 1997). This novel mechanism excitotoxic death or other forms of degeneration (for review, see may offer a new strategy to increase the local production of Nicoletti et al., 1996). However, the mechanism responsible for neurotrophic factors in the CNS. We now demonstrate that neuroprotection differs between these two classes of mGlu recep- neuroprotection by glial mGlu3 receptors is mediated by trans- tor subtypes. mGlu4, -7, and -8 receptors are exclusively localized forming growth factor-b1 (TGFb1) and TGFb2, which are re- in neurons, and their activation inhibits glutamate release (Shige- leased from astrocytes and exert a potent neuroprotective activity moto et al., 1997). Hence, group-III mGlu receptor agonists are in in vitro and in vivo models of excitotoxic death. of potential value in the experimental therapy of epilepsy or neurodegenerative disorders of excitotoxic origin. In contrast, MATERIALS AND METHODS Mixed cortical cultures. Mixed cortical cultures containing both neurons Received July 6, 1998; revised Sept. 8, 1998; accepted Sept. 10, 1998. and astrocytes were prepared from fetal mice at 14–16 d of gestation, as Correspondence should be addressed to Dr. Ferdinando Nicoletti, Institute of described by Rose et al. (1992). In brief, dissociated cortical cells were Pharmacology, School of Pharmacy, University of Catania, Viale AA Doria 6, 95125 plated in 15 mm multiwell vessels (Falcon Primaria, Lincoln Park, NY) Catania, Italy. on a layer of confluent astrocytes [prepared as described by Rose et al. Copyright © 1998 Society for Neuroscience 0270-6474/98/189594-07$05.00/0 (1992)], using a plating medium of MEM-Eagle’s salts (supplied Bruno et al. • Glial mGlu3 Receptors and Neuroprotection J. Neurosci., December 1, 1998, 18(23):9594–9600 9595 glutamine-free) supplemented with 5% heat-inactivated horse serum, Table 1. Neuroprotection by the medium collected from glial cultures 2 5% fetal bovine serum, glutamine (2 mM), glucose (21 mM), and or 20 hr after a transient exposure to group-II mGlu receptor agonists NaHCO3 (25 mM). After 3–5 d in vitro (DIV), non-neuronal cell division m was halted by a 1–3 d exposure to 10 M cytosine arabinoside, and NMDA toxicity cultures were shifted to a maintenance medium identical to plating medium but lacking fetal bovine serum. Subsequent partial medium LDH release replacement was performed twice a week. Cultures at 13–14 DIV were Number of dead cells (mO.D./min) used. 6 6 Glial cultures. Glial cell cultures were prepared from postnatal mice Basal 7 0.7 15 7 (1–3 d after birth), as described previously (Rose et al., 1992). Dissoci- NMDA, 100 mM 155 6 9 119 6 8 ated cortical cells were grown in 15 mm multiwell vessels using a plating 1 GM/control, 2 hr 168 6 11 116 6 22 medium of MEM-Eagle’s salts supplemented with 10% of heat- 1 GM/control, 20 hr 183 6 24 104 6 17 inactivated horse serum, 10% fetal bovine serum, 2 mM glutamine, 1 GM/DCG-IV, 2 hr 67 6 7* 59 6 9* sodium bicarbonate (25 mM), and glucose (21 mM). Cultures were kept at 1 GM/DCG-IV, 20 hr 74 6 15* 57 6 6* 37°C in a humidified CO2 atmosphere until they reached confluency (7–14 DIV). Confluent cultures were then used for the experiments or as 1 GM/4C3HPG, 2 hr 71 6 9* 63 6 11* a support for mixed cultures. 1 GM/4C3HPG, 20 hr 56 6 5* 60 6 8* Assessment of neuronal death in culture. For induction of excitotoxic death, mixed cultures were exposed to NMDA for 10 min at room Values are means 6 SEM of 12–16 determinations. *p , 0.01 (one-way ANOVA 1 temperature in a HEPES-buffered salt solution containing (in mM): 120 Fisher PLSD) versus NMDA alone. GM/control, Glial medium (GM) collected 2 or 20 hr after a 10 min exposure of pure cultures of astrocytes to the buffer alone; NaCl, 5.4 KCl, 0.8 MgCl2 , 1.8 CaCl2 , 20 HEPES, and 15 glucose. Afterward, the cultures were extensively washed and incubated in MEM- GM/DCG-IV and GM/4C3HPG, medium collected after a 10 min exposure of cultured astrocytes to 1 mM DCG-IV or 100 mM 4C3HPG. Collected medium was Eagle’s (supplemented with 25 mM NaHCO and 21 mM glucose) (MS) 3 transferred to mixed cultures immediately after the toxic pulse with NMDA and at 37°C. In some experiments, glial conditioned medium (GM) was added maintained for the following 20 hr. The astrocyte medium did not affect neuronal to the cultures immediately after the NMDA pulse and maintained viability in the absence of NMDA (data not shown). The number of dead cells was during the following 24 hr of incubation. GM was prepared as follows. determined by counting neurons stained with trypan blue in three random micro- Glial cortical cultures were exposed for 10 min to group-II mGlu receptor scopic fields. See Bruno et al. (1997) for a more detailed characterization. mO.D., agonists, and then drugs were washed out and cultures were kept in MS Milli-Optical density. (which does not contain serum) at 37°C for the following 20 hr. At the end of this incubation, the medium was collected and immediately transferred to mixed cultures. Neuronal injury was estimated by examining the cultures with phase- contrast microscopy 24 hr after the insult, when the process of cell death was largely complete. Neuronal damage was quantitatively assessed by trypan blue staining. Stained neurons were counted from three random fields per well.

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