- Email: [email protected]
Opposing regulation of tau protein levels by ionotropic and metabotropic glutamate receptors in human NT2 neurons
Neuroscience Letters 243 (1998) 77–80
Opposing regulation of tau protein levels by ionotropic and metabotropic glutamate receptors in human NT2 neurons Marta Paterlini*, Alessandra Valerio, Francesca Baruzzi, Maurizio Memo, PierFranco Spano Division of Pharmacology, Department of Biomedical Sciences and Biotechnologies, Brescia University Medical School, Via Valsabbina 19, 25123 Brescia, Italy Received 22 September 1997; received in revised form 9 January 1998; accepted 16 January 1998
Abstract Human NT2-N neurons derived from retinoic acid treatment of the NTera 2 cell line were used to determine the consequences of ionotropic glutamate receptor (iGluR) hyperstimulation and possible modulatory role(s) exerted by metabotropic glutamate receptor (mGluR) activation. We found that NT2-N neurons express the NR1 subunit of N-methyl-D-aspartate (NMDA) iGluRs and mRNA encoding the 1a isoform of mGluRs. A 15 min pulse with 100 mM NMDA induced an increase in the levels of tau proteins in NT2-N cells. This effect was prevented by incubating NT2-N neurons in the presence of the mGluR agonist (1S,3R)-1 aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD). This phenomenon was related, in terms of doses and time, with the observed 1S,3R-ACPD-mediated protection against NMDA-induced NT2-N cell death. Our findings suggest that iGluRs and mGluRs might participate in the control of human neuron viability by differentially affecting the expression of tau proteins. 1998 Elsevier Science Ireland Ltd.
Keywords: NT2-N neurons; Human; Glutamate receptors; Tau protein; Neurodegeneration
There is mounting evidence that excitotoxicity associated with an excessive stimulation of glutamate receptors contributes to the neuronal loss in chronic neurodegenerative diseases such as amyotrophic lateral sclerosis and Huntington’s and Alzheimer’s diseases (AD) [5]. Studies of molecular biology and pharmacology of glutamate receptors have identified two broad classes of receptor proteins in the mammalian central nervous system. It is well known that the overstimulation of ionotropic glutamate receptors (iGluRs) results in neurotoxic effects, whereas the impact of metabotropic glutamate receptor (mGluR) stimulation on neuron viability is undergoing active investigation [8]. Stimulation of mGluRs has been found to attenuate excitotoxicity [8–10,13]. This finding led to the hypothesis of possible ‘cross-talking’ between receptors belonging to the different glutamate receptors classes, suggesting that mGluRs may act as modulators of the iGluR activity. To date, eight mGluRs have been cloned and characterized in three sub* Corresponding author. Tel.: +39 30 3715291; fax: +39 30 3701157; e-mail: [email protected]
groups. Among these, group I mGluRs (mGluR1a-d and mGluR5a-b) are coupled to the phospholipase C signal transduction pathway while group II and III mGluRs are negatively coupled to adenylate cyclase activity [7]. We have recently reported that the activation of mGluRs belonging to group I reduces glutamate-mediated rise in cytosolic calcium concentration ([Ca2+]i) in rat cerebellar granule cells through a mechanism involving protein kinase C (PKC) activation and resulting in neuroprotection [10]. We have also previously demonstrated that exposure of rat cerebellar granule cells to glutamate induces an increase in the expression of the cytoskeleton-associated tau protein [11]. Tau proteins are the major constituents of paired helical filaments, whose accumulation is associated with disorganization of the normal cytoskeleton in AD neurons [3]. Blocking the enhancement of tau expression with specific antisense oligonucleotides prevents the glutamate-mediated intracellular program leading to neuronal death [6,11]. There are, however, certain limitations associated with the use of rodent primary cultured neurons to explore human neurodegeneration. A clonal line of human neurons
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00087- 1
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that exhibits excitotoxicity would be ideal for these investigations. Unlike various neuroblastomas considered and investigated, the cell line NTera 2 (NT2) seems to represent a useful tool for studying the cellular and molecular mechanisms with relevance to human neurodegenerative diseases [12]. NT2 cells are a teratocarcinoma cell line, which differentiates in response to retinoic acid, yielding a pure population of postmitotic human neurons (NT2-N) which express tau proteins [12] and receptors responsive to the iGluR agonist N-methyl-D-aspartate (NMDA) [17]. In fact, both NMDA and non-NMDA iGluR channels have been identified electrophysiologically and mRNAs for several subunits of iGluRs have been detected [2,17]. To our knowledge, however, no attempt has been made to assess the presence and function of mGluRs in NT2-N cells. In the present study, we used NT2-N neurons to: (1) investigate on the effects of NMDA on tau protein levels; (2) verify the expression of mGluRs; (3) evaluate the possible regulatory effects of mGluR agonists on NMDA-induced modifications in tau levels and neuron viability. NT2 cells (Stratagene, La Jolla, CA, USA) were grown as described by Pleasure et al. [12]. For differentiation, NT2 cells were treated with 1 mM retinoic acid (RA) twice weekly for 5 weeks. NT2-N neurons were purified by non-neuronal cells as described [12] and allowed to differentiate up to 28 days in vitro (DIV). The response of NT2-N cells to excitotoxicity was examined as previously described [10]. Neurons were exposed to a 15 min pulse with GluR
Fig. 1. Representative immunoblot analysis of the NR1 subunit of NMDA iGluR in NT2 and NT2-N cells using anti-NR1 antibody. Protein extracts (20 mg) were loaded on each well of an 8% sodium dodecyl sulfate-polyacrylamide gel (lane 1, undifferentiated NT2 cells; lane 2, differentiated NT2-N at DIV 3). The size of the MW marker is indicated on the left. MW of NR1 band is roughly 120 kDa.
Fig. 2. RT-PCR assay of the expression of mGluR1, mGluR5 and bactin mRNAs by NT2-N cells (lanes 1 and 3), rat cerebellar granule cells (lanes 2 and 4) and rat striatum (lane 5). ‘St’ refers to the MW standard. Sizes of the fragment are: 517, 396, 344, 298 and 220 bp.
agonists. After 3 h washout, cells were harvested and proteins extracted for immunoblotting. A parallel set of cells was treated with the GluR agonists in the same way and cultured for 24 h before performing intravital staining with a mixture of fluorescein diacetate and propidium iodide. The percentage of surviving neurons was computed by calculating the ratio between the viable, fluorescein diacetate-positive cells and total cell counts in photomicrographs [10]. Total RNA from NT2-N and rat cerebellar granule cells was isolated and reverse-transcribed as previously described [16]. The polymerase-chain reaction (PCR) for detection of the mGluR1 and mGluR5 receptor cDNAs and co-amplification of b-actin cDNA was performed according with the scheme and primers described in Valerio et al. [16]. Immunoblotting was conducted as previously described [15], with minor modifications. Filters were incubated at room temperature (RT) for 90 min with anti-tau polyclonal antibody (Sigma, St. Louis, MO, USA) diluted 1:100. For immunodetection, a goat anti-rabbit alkaline phosphatase conjugate antibody (Promega, Madison, WI, USA), 1:7500 dilution, was used. For studying NR1 subunit of NMDA receptor, filters were incubated at RT for 90 min with anti NR1 antibody (UBI, Lake Placid, NY, USA) diluted 1:20. Immunodetection was performed using a HRP-labelled anti-rabbit antibody, 1:1000 dilution, and the enhanced chemiluminescence kit from Amersham (Buckingamshire, UK). To verify the expression of NMDA receptor protein by NT2-N cells, we performed an immunoblot analysis using an antibody specifically recognizing the NR1 subunit of NMDA receptors. As shown in Fig. 1, protein extracts from undifferentiated NT2 cells did not react with antiNR1 antibody; on the contrary, protein extracts from NT2-N neurons at 3 DIV showed an immunoreactive band of the apparent molecular weight (MW) of 120 kDa, corresponding to the NR1 subunit. Moreover, we investigated the expression of the mRNAs encoding for mGluR1 and mGluR5 receptors. The pattern of group I mGluR mRNA expression in NT2-N was compared to that observed in rat cerebellar granule cells. Reverse
M. Paterlini et al. / Neuroscience Letters 243 (1998) 77–80
Fig. 3. Representative immunoblot analysis of tau proteins in NT2–N (DIV4) cells using polyclonal anti-tau antibody. Protein extracts (25 mg) were loaded on each well of an 10% SDS-polyacrylamide gel (lane 1, control; lane 2, 100 mM 1S,3R-ACPD; lane 3, 100 mM NMDA; lane 4, 100 mM NMDA + 100 mM 1S,3R-ACPD). The size of MW marker is indicated on the left. NT2-N cells express two anti-tauimmunoreactive bands of the apparent MW of 68 and 54 kDa.
transcription PCR (RT-PCR) for mGluR1 mRNA assay was conducted using a pair of primers flanking the alternatively spliced exon, so that the amplification of template cDNA could generate two PCR products of 293 and 378 bp, corresponding to the mGluR1a and 1b isoforms, respectively. RT-PCR revealed that both NT2-N and granule cells express mGluR1a and 1b isoforms. In particular, a fragment of 293 bp, corresponding to mGluR1a, was predominant in NT2-N cells (Fig. 2, lane 1), while two fragments of 293 and 378 bp corresponding to mGluR1a and mGluR1b, respectively, were expressed in cerebellar granule cells (Fig. 2, lane 2). RT-PCR for mGluR5 assay was performed using a pair of primers flanking the alternatively spliced portion of mGluR5. We found that mGluR5 was expressed neither in NT2-N or in granule cells (Fig. 2, lanes 3 and 4). Amplification of corpus striatum cDNA (Fig. 2, lane 5) was performed as a positive control of mGluR5 mRNA expression, resulting in a fragment of the expected length of 432 bp, corresponding to the predominantly expressed 5b subtype. As a control of the amount of starting cDNA template in the different samples, b-actin cDNA was co-amplified in each tube, resulting in similar amounts of a fragment of 241 bp. We then investigated the possible changes in tau protein levels induced by iGluR and mGluR agonist application. A representative immunoblot analysis of protein extracts from NT2-N cells using anti-tau antibody is illustrated in Fig. 3.
79
As expected [12], this polyclonal antibody clearly recognized a set of proteins migrating in the range of 68 to 54 kDa in control NT2-N cells (lane 1). After NMDA treatment, the total amount of tau protein increased. In fact, an increase in the immunostaining of the bands was appreciable 3 h after a pulse with 100 mM NMDA (lane 3). If cells were simultaneously treated with the mGluR-selective agonist 1S,3R-ACPD (100 mM), the increase in tau protein content induced by NMDA was attenuated (lane 4). 1S,3R-ACPD per se did not significantly modify basal tau protein levels (lane 2). Neurotoxicity assays were performed in parallel experiments on each cell preparation to correlate the iGluR and mGluR agonist-induced effects on tau content with their modulatory effects on cell viability. A dose-dependent decrease in cell survival was observed 24 h after the pulse with 100 or 200 mM NMDA. Moreover, the NMDA-induced cell death was counteracted by a simultaneous treatment with 100 mM 1S,3R-ACPD (Fig. 4). There is an emerging consensus that glutamate, through the interaction with specific receptor subtypes, activates a complex transcriptional program which regulates the expression of various proteins, including tau [6]. In this context, we have recently demonstrated that preventing the glutamate-induced increase in tau expression counteract the excitotoxic death of rat cerebellar granule neurons. We designed the present study to evaluate the glutamatergic control of tau protein levels in human neurons. We show that differentiated NT2-N neurons express functional NR1 subunits that contribute to form iGluR channels possibly involved in mediating neurotoxicity. In fact, hyperstimulation of iGluRs with NMDA triggers NT2-N cell death. We also show that NMDA administration, at the same doses which provokes cell injury, causes tau pro-
Fig. 4. Effect of the selective mGluR agonist 1S,3R ACPD on the NMDA-induced NT2-N cell death. Cells were exposed for 15 min to increasing doses of NMDA in the absence or in the presence of 100 mM 1S, 3R-ACPD. *P , 0.05 versus control.
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tein levels to rise. This latter phenomenon appears during the lag time with precedes neuronal death. It is therefore conceivable that, as we have already shown in rat granule cells [11], also in human neurons the increase in the expression of tau proteins participates in the cascade of events promoted by iGluR agonists and leading to neurodegeneration. Our results also show that NT2-N neurons express a group I mGluR pattern superimposable to that found in granule cells. In fact, we observed that both cell types express mGluR1 but not mGluR5. In particular, mGluR1a isoform appears to be predominantly expressed by NT2-N, while both mGluR1a and 1b isoforms are present in granule cells. The lack of mGluR5 in granule neurons suggests that the group I mGluR-mediated blockade of glutamateinduced [Ca2+]i rise and neurotoxicity, which was previously observed in such cell preparation [10], are indeed mediated by mGluR1.The role of group I mGluRs on excitotoxicity is at present under debate, since both facilitatory [8] and inhibitory [10,13] actions on neurodegeneration have been reported. These apparent discrepancies could be in part explained by the involvement of different group I mGluR subtypes and their regional heterogeneity [1] and also by a different subunit composition of native NMDA receptors in the examined models [14], which may influence their capability to be modulated by PKC and mGluRs. In line with our suggestions, use of knock out mice that lack mGluR1 has shown evidences against a permissive role of this receptor in excitotoxicity [1]. Exposure of NT2-N neurons to the mGluR agonist 1S,3RACPD prevents the NMDA-induced rise in tau protein levels and neuronal death. Further studies using subtypeselective mGluR ligands will be necessary to dissect the contribution of individual mGluRs, including mGluR1a, to NT2-N neuroprotection. The expression and function of group II and III mGluRs should also be investigated in NT2-N cells, since activation of adenylate cyclase-inhibiting mGluRs has been found to be neuroprotective [8]. Activation of mGluR1a increases the release of soluble forms of amyloid precursor protein [4], which reduce b-amyloid production. NT2-N neurons may be a promising tool to further elucidate the capability of mGluRs to regulate the expression of tau protein and interfere with other molecular events underlying AD. The authors are grateful to Dr. Marina Pizzi for helpful discussions and to Ms. Mery Biofava for technical assistance. [1] Ferraguti, F., Pietra, C., Valerio, E., Corti, C., Chiamulera, C. and Conquet, F., Evidence against a permissive role of the metabotropic glutamate receptor 1 in acute excitotoxicity, Neuroscience, 79 (1997) 1–5.
[2] Hardy, M., Younkin, D., Tang, C.-M., Pleasure, J., Shi, Q.-Y., Williams, M. and Pleasure, D., Expression of non-NMDA glutamate receptor channel genes by clonal human neurons, J. Neurochem., 63 (1994) 482–489. [3] Kosik, K.S., Joachim, C.L. and Selkoe, D.J., Microtubule-associated protein tau is the major antigenic component of paired helical filaments in Alzheimer’s disease, Proc. Natl. Acad. Sci. USA, 83 (1986) 4044–4048. [4] Lee, R.K., Wurtman, R.J., Cox, A.J. and Nitsch, R.M., Amyloid precursor protein processing is stimulated by metabotropic glutamate receptors, Proc. Natl. Acad. Sci. USA, 92 (1995) 8083– 8087. [5] Lipton, S.A. and Rosemberg, P.A., Excitatory amino acids as a final common pathway for neurologic disorders, N. Eng. J. Med., 330 (9 ) (1994) 613–622. [6] Memo, M., Pizzi, M., Valerio, A., Grilli, M. and Spano, P.F., Molecular mechanisms of glutamate-induced neurodegeneration, Int. Rev. Psychiatry, 7 (1995) 339–348. [7] Nakanishi, S., Metabotropic glutamate receptors: synaptic transmission, modulation, and plasticity, Neuron, 13 (1994) 1031–1037. [8] Nicoletti, F., Bruno, V., Copani, A., Casabona, G. and Kno¨pfel, T., Metabotropic glutamate receptors: a new target for the therapy of neurodegenerative disorders?, Trends Neurosci., 19 (1996) 267–271. [9] Pizzi, M., Consolandi, O., Memo, M. and Spano, P.F., Activation of multiple metabotropic glutamate receptor subtypes prevents NMDA-induced excitotoxicity in rat hippocampal slices, Eur. J. Neurosci., 8 (1995) 1516–1521. [10] Pizzi, M., Galli, P., Consolandi, O., Arrighi, V., Memo, M. and Spano, P.F., Metabotropic and ionotropic transducers of glutamate signal inversely control cytoplasmic Ca2+ concentration and excitotoxicity in cultured cerebellar granule cells: pivotal role of protein kinase C, Mol. Pharmacol., 49 (1996) 586–594. [11] Pizzi, M., Valerio, A., Arrighi, V., Galli, P., Belloni, M., Ribola, M., Alberici, A., Spano, P.F. and Memo, M., Inhibition of glutamate-induced neurotoxicity by a tau antisense oligonucleotide in primary culture of rat cerebellar granule cells, Eur. J. Neurosci., 7 (1995) 1603–1613. [12] Pleasure, S.J., Page, C.D. and Lee, W.M., Pure, postmitotic, polarized human neurons derived from NTera 2 cells provide a system for expressing exogenous proteins in terminally differentiated neurons, J. Neurosci., 12 (1992) 1802–1815. [13] Reymann, K.G., Schroder, U.H., Ja¨ger, T., Opitz, T., Sabelhaus, C.F. and Breder, J., Protein kinase C is involved in the protective effect of class I metabotropic glutamate receptor agonists against hypoxic/hypoglycemic injury in hippocampal slices, Neuropharmacology, 35 (1996) A25. [14] Sucher, N.J., Awobului, M., Choi, Y.-B. and Lipton, S., NMDA receptors: from genes to channels, Trends Pharmacol. Sci., 17 (1996) 348–355. [15] Valerio, A., Alberici, A., Paterlini, M., Grilli, M., Galli, P., Memo, M. and Spano, P.F., Opposing regulation of amyloid precursor protein by ionotropic and metabotropic glutamate receptors, NeuroReport, 6 (1995) 1317–1321. [16] Valerio, A., Paterlini, M., Boifava, M., Memo, M. and Spano, P.F., Metabotropic glutamate receptor mRNA expression in rat spinal cord, NeuroReport, 8 (1997) 2695–2699. [17] Younkin, D.P., Tang, C., Hardy, M., Reddy, U.R., Shi, Q.-Y., Pleasure, S.J., Lee, V.M.-Y. and Pleasure, D., Inducible expression of neuronal glutamate receptor channels in the NT2 human cell line, Proc. Natl. Acad. Sci. USA, 90 (1993) 2174–2178.
Opposing regulation of tau protein levels by ionotropic and metabotropic glutamate receptors in human NT2 neurons Marta Paterlini*, Alessandra Valerio, Francesca Baruzzi, Maurizio Memo, PierFranco Spano Division of Pharmacology, Department of Biomedical Sciences and Biotechnologies, Brescia University Medical School, Via Valsabbina 19, 25123 Brescia, Italy Received 22 September 1997; received in revised form 9 January 1998; accepted 16 January 1998
Abstract Human NT2-N neurons derived from retinoic acid treatment of the NTera 2 cell line were used to determine the consequences of ionotropic glutamate receptor (iGluR) hyperstimulation and possible modulatory role(s) exerted by metabotropic glutamate receptor (mGluR) activation. We found that NT2-N neurons express the NR1 subunit of N-methyl-D-aspartate (NMDA) iGluRs and mRNA encoding the 1a isoform of mGluRs. A 15 min pulse with 100 mM NMDA induced an increase in the levels of tau proteins in NT2-N cells. This effect was prevented by incubating NT2-N neurons in the presence of the mGluR agonist (1S,3R)-1 aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD). This phenomenon was related, in terms of doses and time, with the observed 1S,3R-ACPD-mediated protection against NMDA-induced NT2-N cell death. Our findings suggest that iGluRs and mGluRs might participate in the control of human neuron viability by differentially affecting the expression of tau proteins. 1998 Elsevier Science Ireland Ltd.
Keywords: NT2-N neurons; Human; Glutamate receptors; Tau protein; Neurodegeneration
There is mounting evidence that excitotoxicity associated with an excessive stimulation of glutamate receptors contributes to the neuronal loss in chronic neurodegenerative diseases such as amyotrophic lateral sclerosis and Huntington’s and Alzheimer’s diseases (AD) [5]. Studies of molecular biology and pharmacology of glutamate receptors have identified two broad classes of receptor proteins in the mammalian central nervous system. It is well known that the overstimulation of ionotropic glutamate receptors (iGluRs) results in neurotoxic effects, whereas the impact of metabotropic glutamate receptor (mGluR) stimulation on neuron viability is undergoing active investigation [8]. Stimulation of mGluRs has been found to attenuate excitotoxicity [8–10,13]. This finding led to the hypothesis of possible ‘cross-talking’ between receptors belonging to the different glutamate receptors classes, suggesting that mGluRs may act as modulators of the iGluR activity. To date, eight mGluRs have been cloned and characterized in three sub* Corresponding author. Tel.: +39 30 3715291; fax: +39 30 3701157; e-mail: [email protected]
groups. Among these, group I mGluRs (mGluR1a-d and mGluR5a-b) are coupled to the phospholipase C signal transduction pathway while group II and III mGluRs are negatively coupled to adenylate cyclase activity [7]. We have recently reported that the activation of mGluRs belonging to group I reduces glutamate-mediated rise in cytosolic calcium concentration ([Ca2+]i) in rat cerebellar granule cells through a mechanism involving protein kinase C (PKC) activation and resulting in neuroprotection [10]. We have also previously demonstrated that exposure of rat cerebellar granule cells to glutamate induces an increase in the expression of the cytoskeleton-associated tau protein [11]. Tau proteins are the major constituents of paired helical filaments, whose accumulation is associated with disorganization of the normal cytoskeleton in AD neurons [3]. Blocking the enhancement of tau expression with specific antisense oligonucleotides prevents the glutamate-mediated intracellular program leading to neuronal death [6,11]. There are, however, certain limitations associated with the use of rodent primary cultured neurons to explore human neurodegeneration. A clonal line of human neurons
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00087- 1
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that exhibits excitotoxicity would be ideal for these investigations. Unlike various neuroblastomas considered and investigated, the cell line NTera 2 (NT2) seems to represent a useful tool for studying the cellular and molecular mechanisms with relevance to human neurodegenerative diseases [12]. NT2 cells are a teratocarcinoma cell line, which differentiates in response to retinoic acid, yielding a pure population of postmitotic human neurons (NT2-N) which express tau proteins [12] and receptors responsive to the iGluR agonist N-methyl-D-aspartate (NMDA) [17]. In fact, both NMDA and non-NMDA iGluR channels have been identified electrophysiologically and mRNAs for several subunits of iGluRs have been detected [2,17]. To our knowledge, however, no attempt has been made to assess the presence and function of mGluRs in NT2-N cells. In the present study, we used NT2-N neurons to: (1) investigate on the effects of NMDA on tau protein levels; (2) verify the expression of mGluRs; (3) evaluate the possible regulatory effects of mGluR agonists on NMDA-induced modifications in tau levels and neuron viability. NT2 cells (Stratagene, La Jolla, CA, USA) were grown as described by Pleasure et al. [12]. For differentiation, NT2 cells were treated with 1 mM retinoic acid (RA) twice weekly for 5 weeks. NT2-N neurons were purified by non-neuronal cells as described [12] and allowed to differentiate up to 28 days in vitro (DIV). The response of NT2-N cells to excitotoxicity was examined as previously described [10]. Neurons were exposed to a 15 min pulse with GluR
Fig. 1. Representative immunoblot analysis of the NR1 subunit of NMDA iGluR in NT2 and NT2-N cells using anti-NR1 antibody. Protein extracts (20 mg) were loaded on each well of an 8% sodium dodecyl sulfate-polyacrylamide gel (lane 1, undifferentiated NT2 cells; lane 2, differentiated NT2-N at DIV 3). The size of the MW marker is indicated on the left. MW of NR1 band is roughly 120 kDa.
Fig. 2. RT-PCR assay of the expression of mGluR1, mGluR5 and bactin mRNAs by NT2-N cells (lanes 1 and 3), rat cerebellar granule cells (lanes 2 and 4) and rat striatum (lane 5). ‘St’ refers to the MW standard. Sizes of the fragment are: 517, 396, 344, 298 and 220 bp.
agonists. After 3 h washout, cells were harvested and proteins extracted for immunoblotting. A parallel set of cells was treated with the GluR agonists in the same way and cultured for 24 h before performing intravital staining with a mixture of fluorescein diacetate and propidium iodide. The percentage of surviving neurons was computed by calculating the ratio between the viable, fluorescein diacetate-positive cells and total cell counts in photomicrographs [10]. Total RNA from NT2-N and rat cerebellar granule cells was isolated and reverse-transcribed as previously described [16]. The polymerase-chain reaction (PCR) for detection of the mGluR1 and mGluR5 receptor cDNAs and co-amplification of b-actin cDNA was performed according with the scheme and primers described in Valerio et al. [16]. Immunoblotting was conducted as previously described [15], with minor modifications. Filters were incubated at room temperature (RT) for 90 min with anti-tau polyclonal antibody (Sigma, St. Louis, MO, USA) diluted 1:100. For immunodetection, a goat anti-rabbit alkaline phosphatase conjugate antibody (Promega, Madison, WI, USA), 1:7500 dilution, was used. For studying NR1 subunit of NMDA receptor, filters were incubated at RT for 90 min with anti NR1 antibody (UBI, Lake Placid, NY, USA) diluted 1:20. Immunodetection was performed using a HRP-labelled anti-rabbit antibody, 1:1000 dilution, and the enhanced chemiluminescence kit from Amersham (Buckingamshire, UK). To verify the expression of NMDA receptor protein by NT2-N cells, we performed an immunoblot analysis using an antibody specifically recognizing the NR1 subunit of NMDA receptors. As shown in Fig. 1, protein extracts from undifferentiated NT2 cells did not react with antiNR1 antibody; on the contrary, protein extracts from NT2-N neurons at 3 DIV showed an immunoreactive band of the apparent molecular weight (MW) of 120 kDa, corresponding to the NR1 subunit. Moreover, we investigated the expression of the mRNAs encoding for mGluR1 and mGluR5 receptors. The pattern of group I mGluR mRNA expression in NT2-N was compared to that observed in rat cerebellar granule cells. Reverse
M. Paterlini et al. / Neuroscience Letters 243 (1998) 77–80
Fig. 3. Representative immunoblot analysis of tau proteins in NT2–N (DIV4) cells using polyclonal anti-tau antibody. Protein extracts (25 mg) were loaded on each well of an 10% SDS-polyacrylamide gel (lane 1, control; lane 2, 100 mM 1S,3R-ACPD; lane 3, 100 mM NMDA; lane 4, 100 mM NMDA + 100 mM 1S,3R-ACPD). The size of MW marker is indicated on the left. NT2-N cells express two anti-tauimmunoreactive bands of the apparent MW of 68 and 54 kDa.
transcription PCR (RT-PCR) for mGluR1 mRNA assay was conducted using a pair of primers flanking the alternatively spliced exon, so that the amplification of template cDNA could generate two PCR products of 293 and 378 bp, corresponding to the mGluR1a and 1b isoforms, respectively. RT-PCR revealed that both NT2-N and granule cells express mGluR1a and 1b isoforms. In particular, a fragment of 293 bp, corresponding to mGluR1a, was predominant in NT2-N cells (Fig. 2, lane 1), while two fragments of 293 and 378 bp corresponding to mGluR1a and mGluR1b, respectively, were expressed in cerebellar granule cells (Fig. 2, lane 2). RT-PCR for mGluR5 assay was performed using a pair of primers flanking the alternatively spliced portion of mGluR5. We found that mGluR5 was expressed neither in NT2-N or in granule cells (Fig. 2, lanes 3 and 4). Amplification of corpus striatum cDNA (Fig. 2, lane 5) was performed as a positive control of mGluR5 mRNA expression, resulting in a fragment of the expected length of 432 bp, corresponding to the predominantly expressed 5b subtype. As a control of the amount of starting cDNA template in the different samples, b-actin cDNA was co-amplified in each tube, resulting in similar amounts of a fragment of 241 bp. We then investigated the possible changes in tau protein levels induced by iGluR and mGluR agonist application. A representative immunoblot analysis of protein extracts from NT2-N cells using anti-tau antibody is illustrated in Fig. 3.
79
As expected [12], this polyclonal antibody clearly recognized a set of proteins migrating in the range of 68 to 54 kDa in control NT2-N cells (lane 1). After NMDA treatment, the total amount of tau protein increased. In fact, an increase in the immunostaining of the bands was appreciable 3 h after a pulse with 100 mM NMDA (lane 3). If cells were simultaneously treated with the mGluR-selective agonist 1S,3R-ACPD (100 mM), the increase in tau protein content induced by NMDA was attenuated (lane 4). 1S,3R-ACPD per se did not significantly modify basal tau protein levels (lane 2). Neurotoxicity assays were performed in parallel experiments on each cell preparation to correlate the iGluR and mGluR agonist-induced effects on tau content with their modulatory effects on cell viability. A dose-dependent decrease in cell survival was observed 24 h after the pulse with 100 or 200 mM NMDA. Moreover, the NMDA-induced cell death was counteracted by a simultaneous treatment with 100 mM 1S,3R-ACPD (Fig. 4). There is an emerging consensus that glutamate, through the interaction with specific receptor subtypes, activates a complex transcriptional program which regulates the expression of various proteins, including tau [6]. In this context, we have recently demonstrated that preventing the glutamate-induced increase in tau expression counteract the excitotoxic death of rat cerebellar granule neurons. We designed the present study to evaluate the glutamatergic control of tau protein levels in human neurons. We show that differentiated NT2-N neurons express functional NR1 subunits that contribute to form iGluR channels possibly involved in mediating neurotoxicity. In fact, hyperstimulation of iGluRs with NMDA triggers NT2-N cell death. We also show that NMDA administration, at the same doses which provokes cell injury, causes tau pro-
Fig. 4. Effect of the selective mGluR agonist 1S,3R ACPD on the NMDA-induced NT2-N cell death. Cells were exposed for 15 min to increasing doses of NMDA in the absence or in the presence of 100 mM 1S, 3R-ACPD. *P , 0.05 versus control.
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tein levels to rise. This latter phenomenon appears during the lag time with precedes neuronal death. It is therefore conceivable that, as we have already shown in rat granule cells [11], also in human neurons the increase in the expression of tau proteins participates in the cascade of events promoted by iGluR agonists and leading to neurodegeneration. Our results also show that NT2-N neurons express a group I mGluR pattern superimposable to that found in granule cells. In fact, we observed that both cell types express mGluR1 but not mGluR5. In particular, mGluR1a isoform appears to be predominantly expressed by NT2-N, while both mGluR1a and 1b isoforms are present in granule cells. The lack of mGluR5 in granule neurons suggests that the group I mGluR-mediated blockade of glutamateinduced [Ca2+]i rise and neurotoxicity, which was previously observed in such cell preparation [10], are indeed mediated by mGluR1.The role of group I mGluRs on excitotoxicity is at present under debate, since both facilitatory [8] and inhibitory [10,13] actions on neurodegeneration have been reported. These apparent discrepancies could be in part explained by the involvement of different group I mGluR subtypes and their regional heterogeneity [1] and also by a different subunit composition of native NMDA receptors in the examined models [14], which may influence their capability to be modulated by PKC and mGluRs. In line with our suggestions, use of knock out mice that lack mGluR1 has shown evidences against a permissive role of this receptor in excitotoxicity [1]. Exposure of NT2-N neurons to the mGluR agonist 1S,3RACPD prevents the NMDA-induced rise in tau protein levels and neuronal death. Further studies using subtypeselective mGluR ligands will be necessary to dissect the contribution of individual mGluRs, including mGluR1a, to NT2-N neuroprotection. The expression and function of group II and III mGluRs should also be investigated in NT2-N cells, since activation of adenylate cyclase-inhibiting mGluRs has been found to be neuroprotective [8]. Activation of mGluR1a increases the release of soluble forms of amyloid precursor protein [4], which reduce b-amyloid production. NT2-N neurons may be a promising tool to further elucidate the capability of mGluRs to regulate the expression of tau protein and interfere with other molecular events underlying AD. The authors are grateful to Dr. Marina Pizzi for helpful discussions and to Ms. Mery Biofava for technical assistance. [1] Ferraguti, F., Pietra, C., Valerio, E., Corti, C., Chiamulera, C. and Conquet, F., Evidence against a permissive role of the metabotropic glutamate receptor 1 in acute excitotoxicity, Neuroscience, 79 (1997) 1–5.
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