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Keywords:

  • Alzheimer's disease;
  • apoptosis;
  • chromogranin A;
  • metabotropic glutamate receptor;
  • microglia;
  • neurotoxicity

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

Regulation of microglial reactivity and neurotoxicity is critical for neuroprotection in neurodegenerative diseases. Here we report that microglia possess functional group II metabotropic glutamate receptors, expressing mRNA and receptor protein for mGlu2 and mGlu3, negatively coupled to adenylate cyclase. Two different agonists of these receptors were able to induce a neurotoxic microglial phenotype which was attenuated by a specific antagonist. Chromogranin A, a secretory peptide expressed in amyloid plaques in Alzheimer's disease, activates microglia to a reactive neurotoxic phenotype. Chromogranin A-induced microglial activation and subsequent neurotoxicity may also involve an underlying stimulation of group II metabotropic glutamate receptors since their inhibition reduced chromogranin A-induced microglial reactivity and neurotoxicity. These results show that selective inhibition of microglial group II metabotropic glutamate receptors has a positive impact on neuronal survival, and may prove a therapeutic target in Alzheimer's disease.

Abbreviations used
Aβ25–35

amyloid β peptide 25–35

CGA

chromogranin A

DAPI

4,5-diamidino-2-phenylindole

DCS

distinct cell sorter grade

DCGIV

2S,2′R,3′R-2-(2′,3′-dicarboxy-cyclopropyl)glycine

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

iNOS

inducible nitric oxide synthase

l-CCG-I

l-carboxycyclopropylglycine-I

MCCG

2S,3S,4S-2-methyl-2-(carboxycyclopropyl)glycine

mGlu

metabotropic glutamate

NGS

normal goat serum

NO

nitric oxide

PBS

phosphate-buffered saline

SDS–PAGE

sodium dodecyl sulphate–polyacrylamide gel electrophoresis

TTBS

Tris-HCl buffer containing Tween 20.

Activated microglia accumulate at α-amyloid plaques (Itagaki et al. 1989) in Alzheimer's diseased brain. The secretory peptide chromogranin A (CGA) shows enhanced expression in neurodegenerative diseases such as Alzheimer's disease (Munoz et al. 1990; Yasuhara et al. 1994) and is present in the dystrophic neurones of amyloid plaques (Munoz 1991). CGA activates microglia to a reactive phenotype and stimulates the release of microglial cytotoxins (Taupenot et al. 1996; Ciesielski-Treska et al. 1998; Kingham et al. 1999; Kingham and Pocock 2000), suggesting that this peptide may contribute to the continued and neurotoxic activation of microglia in Alzheimer's disease. Previously we have shown that CGA-induced activation of microglia induces nitric oxide (NO) production, mitochondrial depolarization and release of potential neurotoxins including glutamate and the cysteine protease, cathepsin B (Kingham et al. 1999; Kingham and Pocock 2000, 2001). Amyloid beta peptides, the major components of amyloid plaques in Alzheimer's disease, also induce an inflammatory neurotoxic activation of microglia (Combs et al. 1999).

Glutamate is a major determinant of microglial evoked neurotoxicity (Piani et al. 1991, 1992) and can cause NMDA receptor-mediated toxicity to neurones in vitro (Piani et al. 1991; Piani and Fontana 1994; Klegeris and McGeer 1997; Klegeris et al. 1997; Kingham et al. 1999). Glutamate release from CGA-stimulated microglia occurs via the cystine–glutamate antiporter as well as by a bafilomycin A-sensitive mechanism (Kingham et al. 1999). Overstimulation of microglia with CGA or lipopolysaccharide leads to microglial apoptosis which may have consequences for the ability of the brain to recover from insult (Kingham et al. 1999; Kingham and Pocock 2000; Liu et al. 2001). Glutamate release is also triggered from microglia following exposure to amyloid beta peptides (Barger and Basile 2001). Glutamate may be involved in an autologous feedback loop to damage microglia, triggering microglial reactivity by stimulation of metabotropic glutamate (mGlu) receptors (Kingham et al. 1999). Inhibition of CGA-induced microglial apoptosis by (R,S)-α-methyl-4-sulphonophenylglycine (MSPG), a general antagonist of mGlu receptors, indicates the involvement of mGlu receptors in microglial apoptosis (Kingham et al. 1999).

The mGlu receptor family includes at least eight subtypes classified into three groups on the basis of signal transduction mechanisms, pharmacological properties and gene sequence homology; group I includes mGlu1 and mGlu5, group II includes mGlu2 and mGlu3, and group III includes mGlu4, 6, 7 and 8 (Pin and Duvoisin 1995; Conn and Pin 1997; Schoepp et al. 1999). Several studies have demonstrated that mGlu3 and mGlu5 are the predominant mGlu receptors to be expressed in glial cells in vivo and in vitro (Ohishi et al. 1993; Romano et al. 1995; Petralia et al. 1996; Schools and Kimelberg 1999) although mRNA, but no receptor expression, was found for mGlu8 in astrocytes under certain culture conditions (Janssens and Lesage 2001). Although cultured microglia have been shown to express mGlu5 (Biber et al. 1999), little evidence exists for the expression of other subtypes or for the function of mGlu receptors on microglia.

In this paper we show that microglia possess functional group II mGlu receptors (mGlu2 and mGlu3). Furthermore, we have shown that activation of these receptors promotes a neurotoxic microglial phenotype which appears to underlie neurotoxicity. Inhibition of microglial group II mGlu receptors can reduce CGA-stimulated microglial reactivity, providing neuroprotection. Moreover, the apoptosis induced in microglia following exposure to amyloid beta peptide 25–35 (Aβ25–35) also displays an underlying group II mGlu receptor stimulation because 2S,3S,4S-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), an antagonist of these receptors, attenuated the microglial apoptosis. These data suggest that modulation of microglial reactivity by the selective inhibition of specific mGlu receptors could provide therapeutic benefit in Alzheimer's disease and other neurodegenerative diseases in which the activation of mGlu receptors has been implicated (Nicoletti et al. 1996; Bruno et al. 1998), and where excessive glutamate release may result in stimulation of microglial mGlu receptors.

Materials

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

Fetal calf serum and minimum essential medium with Earle's salts were obtained from Life Technologies Ltd (Paisley, UK). Tissue culture plastic ware was obtained from Scientific Laboratory Supplies Ltd (Nottingham, UK). CGA was purchased from Scientific Marketing Associates (Barnet, Hertfordshire, UK). Aβ25–35 peptide was purchased from Bachem UK Ltd (St Helens, Merseyside, UK). All mGlu receptor agonists and antagonists were from Tocris Cookson Ltd (Bristol, UK). 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazole carboncyanine iodide (JC-1) was from Molecular Probes Europe (Leiden, the Netherlands). Oligo d(T) primer and M-MLV reverse transcriptase were from Life Technologies Ltd. Taq DNA polymerase and the 100-bp DNA ladder were from Promega (Southampton, UK). PCR primers were from Operon Europe (Cologne, Germany). Cyclic AMP kit was from Affiniti Research Products, Exeter, UK. Rabbit anti-mGlu2/3 IgG and rabbit anti-mGlu2 IgG were from Upstate Biotechnology (NY, USA). Vectastain antirabbit IgG biotinylated antibody and rhodamine avidin-distinct, cell sorter grade (DCS) were from Vector Laboratories Inc (Burlingame, CA, USA). Inducible nitric oxide synthase (iNOS)/NOS type II antibody (NP32030) was supplied by Transduction Laboratories (Affiniti Research Products) and mouse anti-rat ED1 was from Serotec, Oxford, UK. ImmunoFluore mounting medium was from ICN Pharmaceuticals (Oxford, UK). All other chemicals and reagents were purchased from Sigma (Poole, UK) unless specified otherwise.

Preparation of microglial cultures

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

Microglial cells were isolated from 3–6 day-old Wistar rat pups as previously described (Kingham and Pocock 2000). Briefly, animals were killed by cervical dislocation and decapitation in accordance with the Scientific Procedures Act 1986, UK. Brains were removed into ice-cold phosphate-buffered saline (PBS; 140 mm NaCl, 5 mm KCl, 25 mm Na2HPO4, 2.9 mm NaH2PO4.2H2O, 11 mm glucose, pH 7.4), and homogenized. The homogenate was centrifuged at 500 g for 10 min and the pellet resuspended in 70% isotonic Percoll diluted with PBS. This was overlaid with 35% Percoll and then PBS. The Percoll gradient was centrifuged at 1250 g for 45 min and cells were collected from the 35–70% interface. After washing, the cells were plated at 6 × 104 per 13-mm coverslip. Cultures were maintained at 37°C in 5% CO2 in minimum essential medium with Earle's salts supplemented with 25 mm KCl, 30 mm glucose, 25 mm NaHCO3, 1 mm glutamine, 10% fetal calf serum, 50 U/mL penicillin and 50 µg/mL streptomycin. Cells were routinely used 1 day after plating. We confirmed our previous findings that after 1 day in vitro microglial cells exhibit a resting, ramified morphology with only a small number of cells staining positive for ED-1 (a marker for activated microglia) (Table 1a, Kingham et al. 1999) and that cultures were enriched with microglia with few contaminating cells, as shown by positive immunoreactivity to the microglial marker OX-42 (data not shown) (Kingham et al. 1999) or the lectin marker isolectin B4 (Table 1b, Fig. 2).

Table 1.  Percentage of total number of cells staining with ED1, isolectin B4 and antibodies to mGlu2 and mGlu2/3 receptors
 Percentage of total number of cells
  1. (a) Percentage of cells staining with ED1 the microglial marker for activation. Primary cultured microglia were treated for 24 h with 500 nm DCGIV, 100 µm l-CCG-I, 50 nm CGA or solvent only (controls). Cells were fixed and stained with ED-1 (1 : 500), and counterstained with haemotoxylin. Cells were counted in three separate fields on four coverslips per condition and the number of ED1-positive cells expressed as a percentage of the total cell number (haemotoxylin-positive cells) ± SEM. (b) Primary cultured microglia were fixed after 1 day in vitro (DIV) and stained with G. simplicifolia isolectin B4 (1 : 20) and either mGlu2 (1 : 250) or mGlu2/3 (1 : 250) antibodies and counterstained with DAPI (1 : 1000) for total cell number. Cells were counted on a minimum of three separate fields on four coverslips per condition and expressed as a percentage of the total cell number (DAPI-stained cells) ± SEM.

(a) Staining with ED1
 Control2.93 ± 0.69
 DCGIV53.03 ± 5.10
 l-CCG-I59.25 ± 4.98
 CGA77.80 ± 4.70
(b) Staining with marker
 Isolectin B499.05 ± 0.95
 mGlu298.43 ± 1.27
 mGlu2/397.72 ± 1.98
image

Figure 2. Microglia express group II mGlu2 and mGlu3 mRNA. Ethidium bromide-stained gel of RT-PCR analysis of mGlu2 and mGlu3 receptor mRNA in extracts from the equivalent of 100 ng of total RNA extracted from rat microglial preparations. Equivalent amounts of each PCR product were size fractionated on a 1% agarose gel. A 100-bp DNA ladder marker was also run (left lane) with the brightest band equal to 500 bp. PCR product sizes were 444 bp (mGlu2), 396 bp (mGlu3) (a) and 559 bp (GAPDH) (b).

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Preparation of neuronal cultures

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

Cerebellar granule neurones were isolated from 3–6-day-old rat pups and prepared as previously described (Evans and Pocock 1999). Cells were plated on 13-mm poly-d-lysine-coated glass coverslips at a density of 0.65 × 106 per coverslip and maintained in minimum essential medium (as above). After 24 h in vitro, cytosine furanoarabinoside (10 µm) was added to prevent proliferation of non-neuronal cells. The cultures were maintained at 37°C in 5% CO2, and used following 7 days in vitro.

Treatment of microglial cultures

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

Microglial cells were exposed to either mGlu receptor agonists and/or antagonists for 1 h before activation by the addition of CGA (50 nm) or Aβ25–35 (50 µm) to the culture medium, to mGlu receptor agonists/antagonists alone, or to CGA or Aβ25–35 alone. The concentrations of specific mGlu agonists and antagonists used were: 500 nm 2S,2′R,3′R-2-(2′,3′-dicarboxy-cyclopropyl)glycine (DCGIV), 100 µm l-carboxycyclopropylglycine-I (l-CCG-I) and 500 µm 2S,3S,4S-2-methyl-2-(carboxycyclopropyl)glycine(MCCG). Where used, inhibitors of the NMDA receptor such as 5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801; 10 µm) or l-(+)-2-amino-5-phophonopentanoic acid (AP5; 50 µm) were added to the cells 1 h before treatment with mGlu receptor modulators, or CGA. Microglia were incubated for 24 h with CGA before tissue culture medium was collected and the cells evaluated for mitochondrial depolarization, apoptosis and protein expression by western blotting.

Measurement of cyclic AMP

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

Microglial cultures were treated with 1 mm 3-isobutyl-1-methylxanthine, an inhibitor of cyclic AMP degradation, for 10 min before the addition of forskolin (100 µm). The cells were incubated for 10 min before exposure to DCGIV (500 nm) or l-CCG-I (100 µm) for a further 10 min. Cyclic AMP production in these cells was compared with that in cells treated with forskolin alone and in untreated control cultures. Cells were lysed with 0.1 m HCl at room temperature for 15 min and cyclic AMP was measured in cell supernatants using an enzyme immunoassay kit (Biomol) according to the manufacturers' instructions for the acetylated method. Cyclic AMP concentrations were calculated by comparison with a standard curve constructed with known concentrations of cyclic AMP.

Co-cultures

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

Microglial cells (two coverslips) were placed in a 35-mm Petri dish in 2 mL medium and mGlu receptor agonists/antagonists and/or CGA were added as described above. After 24 h, neuronal cells (two coverslips) were added to each dish and the preparation was maintained at 37°C in 5% CO2 for a further 24 h. Cells were then fixed and apoptosis was assessed with Hoechst 33342 staining. Control cultures consisted of microglia alone, neurones alone or microglia with neurones.

Measurement of microglial mitochondrial membrane polarization

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

Microglial mitochondrial membrane polarization was assessed with the fluorescent probe JC-1. At 490 nm cells with depolarized mitochondria contained JC-1 predominantly in the monomeric form and fluoresced green. Cells with polarized mitochondria predominantly contain JC-1 in aggregate form, and the mitochondria fluoresce red/orange (Salvioli et al. 1997). We have previously shown that the state of mitochondrial depolarization determined with JC-1 staining is the same that obtained following staining with tetramethylrhodamine ethyl ester, another mitochondrial membrane potential-sensitive fluorescence dye, and that the fluorescence associated with either stain subsequently decreases from microglial mitochondria following addition of the mitochondrial uncoupler carbonyl cyanide p-(trifluoro methoxy)phenylhydrazone (FCCP) (Kingham and Pocock 2000). Microglia were incubated with JC-1 (5 µm) in basic medium (153 mm NaCl, 3.5 mm KCl, 0.4 mm KH2PO4, 20 mmN-tris(hydroxymethyl)methyl-2-aminoethanesulphonic acid, 5 mm NaHCO3, 5 mm glucose, 1.2 mm Na2SO4, 1.3 mm CaCl2, pH 7.4) at 37°C for 10 min, washed and placed on a thermostatted stage at 37°C. Coverslips were viewedusing an Olympus IX70 inverted fluorescence microscope (Olympus Optical Co. Ltd, London, UK) with excitation at 490 nm and emission at > 520 nm. Microglia with polarized mitochondriacontained distinct mitochondria fluorescing red/orange; in microglia with depolarized mitochondria, the cell cytoplasm and mitochondria appeared green (Kingham and Pocock 2000). The numbers of cells with mitochondria stained red/orange or green were counted, and the degree of mitochondrial depolarization was expressed as the percentage of green cells per field. A minimum of six fields per coverslip were counted, and between six and 10 coverslips from at least four independent experiments were assessed for each variable.

Assessment of apoptosis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

Following measurement of mitochondrial polarization, the same cells were then fixed with 4% formaldehyde in PBS (4°C) for 10 min and then incubated with Hoechst 33342 (5 µg/mL) for10 min (Yan et al. 1994; Kingham et al. 1999). Nuclear morphology was viewed using a fluorescence microscope with excitation at 360 nm and emission > 490 nm. Apoptotic cells were identified as those cells containing brightly stained pyknotic nuclei compared with healthy cells in which the nuclei appeared less bright and less condensed. A minimum of six fields per coverslip were counted, and between six and 10 coverslips were assessed for each variable from at least four independent experiments.

Glutamate receptor mRNA expression by RT-PCR

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

Equivalent amounts of total RNA (2 µg) from microglial preparations were reverse transcribed into single-stranded cDNA in a reaction mixture containing 10 mm dithiothreitol, 40 units RNase inhibitor, 10 mm Tris-HCl, pH 8.3, 15 mm KCl, 0.6 mm MgCl2, 0.5 mm dNTPs, 250 ng oligo d(T) primer and 600 units M-MLV reverse transcriptase at 37°C for 80 min. Incubation for 10 min at 70°C terminated the reverse transcription reaction. All samples from individual experiments were reverse transcribed simultaneously. Negative controls were prepared by incubation of samples without reverse transcriptase.

PCR was carried out on the equivalent of 100 ng of reverse transcribed total RNA from each sample as previously described (Copelman et al. 2000). A Perkin Elmer thermocycler (Perkin Elmer, Warrington, UK) was used with a reaction mixture containing 50 mm KCl, 10 mm Tris-HCl, pH 9, 0.1% Triton X-100, 0.5 MgCl2, 200 µm dNTPs, 1 µm each of upstream and downstream primer and 2.5 units of Taq DNA polymerase. Complementary DNA sequences were obtained from a nucleotide sequence database GenBank (National Centre of Biotechnology Information, Bethesda, MD, USA) and resulting PCR primer sequences (Operon Europe), accession numbers, MgCl2 concentrations and optimal annealing temperatures were as follows.

  • mGlu2: 0.5 MgCl2, optimal annealing temperature 63°C, accession no. M92075, forward primer 5′-GCCCCCTTTCGCCCAGCAGATAC-3′, reverse primer 5′-CAGGCAGGCGATGGTGACAGGT-3′.

  • mGlu3: 1 mm MgCl2, optimal annealing temperature 59°C, accession no. M92076, forward primer 5′-GCTCCAACATCCGCAAGTCCTA-3′, reverse primer 5′-TGTCCATGGCCAGGTGCTTGTC-3′.

  • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 2 mm MgCl2, optimal annealing temperature 60°C, accession no. M17701, forward primer 5′-TGGTGCCAAAAGGGTCATCATCTCC-3′, reverse primer 5′-GCCAGCCCCAGCATCAAAGGTG-3′.

PCR carried out on non-reverse transcribed total RNA samples did not produce any detectable products. The thermal cycle profile for each set of primers included a primary denaturation cycle at 94°C for 5 min and a final extension period at 72°C for 10 min. The intervening PCR cycle consisted of 1-min segments of primer denaturation, annealing and extension, and was repeated for 40 cycles for each primer pair.

Equivalent quantities (20 µL) of each PCR product were size fractionated on a 1% agarose gel and the product size was verified by running the samples against a 100-bp DNA ladder. The PCR products were visualized by staining the agarose gels with 0.5 µg/mL ethidium bromide and viewing under ultraviolet light.

Immunolocalization

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

Microglia were fixed in 4% formaldehyde and incubated at 37°C for 30 min with 4% normal goat serum (NGS) in PBS. Following washing in PBS, the cells were incubated overnight at 4°C with 1 : 250 mGlu2/3 or mGlu2 antibody in PBS plus 4% NGS. After washing, cells were exposed to 1 : 200 goat anti-rabbit IgG biotinylated antibody for 1 h at room temperature, washed in PBS and then exposed to rhodamine avidin DCS 1 : 100 for 1 h at room temperature in the dark. At the same time, cells were incubated with isolectin B4 FITC-labelled lectin (Griffonia simplicifolia) 1 : 100. As a control for lectin binding, lectin was bound with 500 mm d-galactose in HEPES with 0.1 mm CaCl2 for 30 min at room temperature before exposure to the cells at the same concentration. Cells were washed and briefly exposed to 1 : 1000 4,5-diamidino-2-phenylindole (DAPI) to stain the nuclei. For ED1 staining, cells were fixed in methanol for 10 min at − 20°C and then preincubated for 30 min at room temperature with PBS containing 2.5% normal horse serum. Cells were incubated overnight at 4°C with mouse anti-rat ED1 (1 : 200 in PBS plus 2.5% serum). After washing, cells were exposed to biotinylated anti-mouse IgG antibody (1 : 200) for 1 h at room temperature. Following 1 h incubation with avidin–biotin–horseradish peroxidase complex, staining was developed by exposure to diaminobenzidine tetrahydrochloride. In all cases, negative controls were treated in the same way but the primary antibody was omitted. Following washing, cells were mounted in ImmunoFluore mounting medium and observed by fluorescence microscopy.

Western blot analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

Cells on coverslips were lysed by the addition of 20 µm lysis buffer (20 mm Tris-acetate, 1 mm EDTA, 1 mm EGTA, 10 mm sodium α-glycerophosphate, 1 mm sodium orthovandate, 5% glycerol, 1% Triton X-100, 0.27 mm sucrose, 1 mm benzamidine, 4 µg/mL leupeptin, 0.1% α-mercaptoethanol, pH 7.4) (Evans and Pocock 1999). Protein (45 µg) was resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) on a 10% gel (Bio-Rad Laboratories Ltd, Hemel Hempstead, Hertfordshire, UK) and transferred to Immobilon P polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). After blocking (5% milk powder in 10 mm Tris-HCl containing 150 mm NaCl and 0.1% Tween 20 (T-TBS), pH 7.4) the membrane was incubated with the primary antibody for 2 h (iNOS, 1 : 500; α-actin, 1 : 1000) at room temperature. For the mGlu antibodies, the membrane was incubated with primary antibody for 4 h at room temperature (mGlu2, 1 : 1000; mGlu2/3, 1 : 1000). In all cases the membrane was then repeatedly washed in T-TBS before exposing to a horseradish peroxidase-conjugated secondary antibody (1 : 500) for 2 h at room temperature. Following washing, blots were developed by enhanced chemiluminescence detection. The resulting blots were scanned by densitometry and optical density related to actin expression.

Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

Since CGA induces microglial apoptosis mediated by an upstream mitochondrial depolarization (Kingham and Pocock 2000), we determined the effects of modulation of group II mGlu receptors on this mitochondrial depolarization and apoptosis. We chose a timepoint following exposure to CGA, Aβ25–35 or group II mGlu receptor agonists ± antagonist of 24 h since we have shown that at this time after CGA exposure approximately 50% of the microglia are apoptotic (Kingham et al. 1999). In naïve, non-stimulated microglial cultures, the majority of cells exhibited JC-1 staining which was punctate red/orange indicative of polarized mitochondria. Following CGA stimulation, the staining became green and diffuse, indicative of mitochondrial depolarization (Kingham and Pocock 2000).

Exposure of microglia to the group II agonist DCGIV (500 nm) without CGA resulted in a significant increase in the number of cells displaying depolarized mitochondria as well as pyknotic nuclei compared with control, non-activated microglia (Fig. 1a). Thus at 24 h, there was approximately a 40% increase above controls of microglia displaying depolarized mitochondria and pyknotic nuclei, which increased to mean ± SEM 80 ± 2.4% at 48 h (data not shown). The group II antagonist MCCG (500 µm) alone did not cause mitochondrial depolarization or apoptosis above control values (data not shown). Addition of MCCG together with DCGIV reduced both the changes in membrane polarisation and apoptosis to control levels (Fig. 1a). Similar findings were observed with another agonist of group II mGlu receptors, l-CCG-I (100 µm); mitochondrial depolarization and apoptosis following exposure to l-CCG-I exhibited a similar trend to that with DCGIV, and the increases could be abrogated by MCCG.

image

Figure 1. Activation of microglial mGlu receptors can induce microglial mitochondrial depolarization and cellular apoptosis. Primary microglia were exposed to the specific mGlu receptor agonists (DCGIV, 500 nm or l-CCG-I, 100 µm) and antagonist (MCCG, 500 µm) (a) alone (b) together with 50 nm CGA or (c) together with 50 µm Aβ25–35). After 24 h the number of cells with depolarized mitochondria (determined by JC-1 staining) and those exhibiting apoptotic nuclei (as indicated by Hoechst 33342 staining) were counted. Activation of group II mGlu receptors without CGA significantly increased microglial depolarization and apoptosis (a). CGA- or Aβ25–35-induced apoptosis was not reduced or enhanced by DCGIV or l-CCG-I, but was decreased with MCCG (b, c). Data are the mean ± SEM of at least four determinations. Significance values are shown for mGlu receptor agonist compared with agonist + antagonist (a, b), or Aβ25–35 alone compared with Aβ25–35 + agonist or antagonist (c). ***p < 0.0005, **p < 0.005 (Welch's two-sided t-test).

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Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

Exposing microglia to 50 nm CGA alone for 24 h resulted in a marked increase in the number of cells with depolarized mitochondria, from a mean ± SEM basal level of 4.5 ± 1.0 to 40.7 ± 3.4% following exposure to CGA. Apoptosis was also significantly increased from a basal level of 3.8 ± 0.6% to 40.0 ± 2.6% at 24 h (Fig. 1b). Activation of group II mGlu receptors with DCGIV + CGA or l-CCG-I + CGA did not significantly increase mitochondrial depolarization or apoptosis above that induced by CGA alone (Fig. 1b). The group II mGluR antagonist MCCG significantly reduced CGA-induced mitochondrial depolarization and apoptosis (data not shown). When the group II agonists and MCCG were added together with CGA, mitochondrial depolarization and apoptosis were reduced to control levels. These data suggest that group II mGlu receptor activation may underlie CGA-induced microglial activation and that the activation of these mGlu receptors is intimately coupled to the apoptotic cascade induced by CGA. The effects of DCGIV or l-CCG-I on microglial mitochondrial membrane potential and apoptosis could not be attenuated with specific antagonists of group I mGlu receptors [(R,S)-1-aminoindan-1,5-dicarboxylic acid (AIDA) 250 µm or 6-methyl-2-(phenylazo)-3-pyridinol (SIB 1757) (100 µm)], or by the specific group III mGlu receptor antagonist (S)-2-amino-2-methyl-4-phosphobutanoic acid (MAP4) (500 µm), indicating that the observed effects were not due to the activation of other groups of mGlu receptors (data not shown). Microglia exposed to DCGIV or l-CCG-I became immunoreactive for ED1 (Table 1a) as did microglia exposed to CGA (Table 1a), as shown previously (Kingham et al. 1999). Microglia exposed to 50 µm Aβ25–35 for 24 h also underwent apoptosis (Fig. 1c). The level of apoptosis induced by Aβ25–35 could not be further enhanced by concurrent exposure to the group II mGlu receptor agonists DCGIV (500 nm) or l-CCG-I (100 µm). However, the level of apoptosis induced by Aβ25–35 could be significantly attenuated by the group II mGlu receptor antagonist MCCG (500 µm), suggesting that, as with CGA, stimulation of microglia with Aβ25–35 leads to an underlying activation of group II mGlu receptors.

Expression and functional activation of group II mGlu receptors

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

The above data suggest that microglia may express functional group II mGlu receptors so we sought to characterize this expression. RT-PCR analysis revealed that primary cultured rat microglia express single-band amplification products of the expected sizes for mGlu2 (444 bp) and mGlu3 (396 bp) (Fig. 2a). Figure 2b indicates the RT-PCR product for the housekeeping gene GAPDH as an internal standard. Immunolocalization using commercially available antibodies to mGlu2 (Fig. 3ai) and mGlu2/3 receptors (Fig. 3bi) indicated that these receptor subtypes are expressed on primary cultured microglia. Positive staining with G. simplicifolia isolectin B4 confirmed that these cells are microglia (Streit 1990; Hailer et al. 1996; Takeuchi et al. 2001) (Figs 3aii and 3bii). Receptor expression for mGlu3 was not assessed because of a lack of an antibody to this subtype. Controls in which primary antibody was omitted did not show any fluorescence labelling (Fig. 3ci) despite the presence of microglia (Fig. 3cii). Analysis of the cells stained with the microglial marker isolectin B4 revealed that a large number of the cells in the cultures were microglia. That the majority of these cells showed staining with antibodies to mGlu2 and mGlu2/3 receptors suggested there was not heterogeneity in the microglial population for these two receptors and that the PCR results were not due to contamination (Table 1b). We also carried out immunolocalization of freshly plated microglia (1 h after plating) and found that the microglia stained positively for mGlu2 and mGlu2/3 receptor protein, negating suggestions that the expression may be an artifact of culture conditions (data not shown). Western blotting of microglia revealed the presence of prominant bands at around 105 kDa (for mGlu2) and 100–110 kDa (for mGlu2/3) (Fig. 3d).

image

Figure 3. Microglia express group II mGlu2 and mGlu3 receptor protein. Immunolocalization of mGlu2 (ai) and mGlu2/3 receptor protein (bi) in primary cultured non-stimulated microglia. The negative control without primary antibody is shown in (ci). Panels (aii) (bii) and (cii) represent staining with G. simplicifolia isolectin B4, a specific marker for microglia for the respective fields above. (d) Western blot of cell lysates from N9 microglia resolved by 10% SDS–PAGE and immunoblotted with anti-mGlu2 or anti-mGlu2/3 antibody (both 1 : 1000). RBM is a rat brain microsomal preparation used as a positive control. The sizes of molecular weight markers are given on the right of each blot.

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Since group II mGlu receptors have been shown in other cell types to signal intracellularly by inhibiting adenylate cyclase and inducing a decrease in cyclic AMP, we determined whether this was also the case in microglia (Fig. 4a). Forskolin (100 µm) induced a fourfold increase in cyclic AMP production in the microglia. This was significantly attenuated by DCGIV or l-CCG-I. Similar findings were also observed with the N9 microglial cell line (data not shown).

image

Figure 4. Stimulation of microglia with group II mGlu receptor agonists inhibits forskolin-induced cyclic AMP accumulation but does not act via NMDA receptors. (a) Inhibition of forskolin-induced cyclic AMP accumulation in microglia by group II mGlu receptor agonists. Microglia were treated with 100 µm forskolin for 10 min followed by DCGIV (500 nm) or l-CCG-I (100 µm) for a further 10 min. Control cells were not forskolin treated or exposed to group II mGlu receptor agonists. Data are the mean ± SEM of at least three determinations. Significance values are shown versus forskolin-treated cells. (b) Activation of group II mGlu receptors induces microglial apoptosis independently of an effect on NMDA receptors. Microglial apoptosis following DCGIV (500 nm) stimulation was not due to NMDA receptor activation since 10 µm MK-801 or 50 µm AP5 did not significantly reduce DCGIV-induced apoptosis. Data are the mean ± SEM of at least four determinations. Unless indicated, significance values are shown compared with DCGIV alone. (c) CGA-induced microglial apoptosis was not prevented by MK-801 or AP5 suggesting that there is no underlying activation of NMDA receptors. Data are the mean ± SEM of at least four determinations. Significance values are shown compared with CGA alone. ns, Not significant. ***p < 0.0005, **p < 0.005, *p < 0.05 (Welch's two-sided t-test).

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Since DCGIV may also interact with NMDA receptors (Ishida et al. 1993; Brabet et al. 1998), we determined whether the apoptosis induced by DCGIV was mediated by activation of these receptors. Neither MK-801, a non-competitive antagonist at NMDA receptors, nor AP5, a competitive antagonist of these receptors, significantly attenuated DCGIV-induced microglial apoptosis (Fig. 4b) indicating that the effects of DCGIV are due to its action at group II mGlu receptors rather than at ionotropic glutamate receptors. Furthermore, the microglial apoptosis induced by CGA was not due to an activation of NMDA receptors (Fig. 4c).

Activated microglia induce neuronal death; modulation by mGlu receptors on microglia

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

To determine how modulation of microglial group II mGlu receptors affects the neurotoxicity of microglia, we exposed cultured cerebellar granule neurones to conditioned medium from microglia treated as above. Conditioned medium from CGA-stimulated microglia induces death of cultured cerebellar granule cells by apoptosis (Kingham et al. 1999). At 24 h, neuronal apoptosis was approximately 50% of the total cells (Fig. 5b), rising to 95% at 48 h (data not shown). Owing to the loss of cell adhesion as the number of apoptotic cells increased, we measured apoptosis at 24 h as cell numbers were more consistent than those observed at longer timepoints. Neither CGA alone nor conditioned medium from non-stimulated microglia had any significant deleterious effect on neuronal survival indicating that the presence of microglia and CGA together were required for neurotoxicity (Kingham et al. 1999). To determine how prior exposure of microglia to agonists or antagonists of group II mGlu receptors modulated subsequent neurotoxicity following CGA stimulation, neuronal cultures were incubated with conditioned medium from microglia exposed to mGlu receptor agonists and/or antagonist before CGA activation, and the level of apoptosis induced in neurones exposed to microglial conditioned medium for 24 h was assessed by Hoechst 33342 staining.

image

Figure 5. Activation of microglial group II mGlu receptors with CGA stimulation is neurotoxic. Cerebellar granule neurones were incubated with medium from control microglia or microglia exposed to CGA and mGlu receptor agonists and antagonists. Apoptosis was measured by Hoechst 33342 staining 24 h later. (a) Neuronal death was observed with conditioned medium from DCGIV- or l-CCG-I-stimulated microglia without CGA and this neurotoxicity was reduced by the antagonist MCCG. (b) Medium from CGA-activated microglia induced neuronal death, as did CGA + DCGIV medium and CGA + l-CCG-I medium. Inhibition of group II mGlu receptors with MCCG reduced microglial-mediated neurotoxicity. Data are the mean ± SEM of at least four determinations. Significance values, unless indicated, are shown for agonist compared with agonist + antagonist. (c) Medium from Aβ25–35-activated microglia induced neuronal death as did Aβ25–35 + DCGIV medium and Aβ25–35 + LCCG-I. Inhibition of microglial group II mGlu receptors with MCCG did not reduce microglial-mediated neurotoxicity. Data are the mean ± SEM of at least four determinations. Significance values are shown for Aα25–35 alone compared with Aα25–35 + mGlu receptor agonist/antagonist. (d) Neuronal apoptosis was not induced following 24 h exposure to conditioned medium from unstimulated microglia together with direct application to the neuronal cultures of the mGlu receptor agonists or antagonists. However, direct application of 50 µm Aβ25–35 was neurotoxic. Significance values are shown compared with control conditioned medium (control CM). (e) Co-cultures of microglia activated with CGA, DCGIV or l-CCG-I are toxic to neurones. Neuronal apoptosis was measured with Hoechst 33342 staining following 24 h microglial–neuronal co-culture. Microglia without stimulation in co-culture with neurones (control) did not induce apoptosis above that in primary cultures of cerebellar granule neurones cultured alone (CGC alone). Addition of CGA, DCGIV or l-CCG-I produced comparable levels of neuronal apoptosis, all significantly raised above control. CGA + DCGIV or CGA + l-CCG-I produced comparable levels of apoptosis above control. MCCG was not neurotoxic when added in co-culture. While apoptosis with CGA and agonists + antagonist was still above control levels, apoptosis was significantly reduced by MCCG compared with CGA + agonists. ns, Not significant. ***p < 0.0005, **p < 0.005, *p < 0.05 (Welch's two-sided t-test).

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Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

To determine whether direct stimulation of microglial mGlu receptors without CGA exposure was also neurotoxic, neurones were incubated with medium from microglia that had been exposed to mGlu receptor agonists and/or antagonist only. Conditioned medium from microglia incubated for 24 h with DCGIV or l-CCG-I induced significant increases in apoptosis compared with that in control cultures exposed to conditioned medium from non-activated microglia (Fig. 5a). Interestingly, medium from microglia exposed to l-CCG-I with or without CGA was more toxic to neurones than that from microglia exposed to DCGIV with or without CGA. Exposure of microglia to the group II antagonist MCCG did not induce neuronal apoptosis (data not shown). MCCG attenuated the DCGIV- or l-CCG-I-induced microglial neurotoxicity to basal levels (Fig. 5a). This suggests that stimulation of microglial group II mGlu receptors can lead to a neurotoxic microglial phenotype and that subsequent inhibition of these receptors can attenuate this toxicity.

Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

Medium from microglia incubated with the group II agonist DCGIV during exposure to 50 nm CGA resulted in a similar level of neuronal apoptosis as seen with conditioned medium from microglia stimulated with CGA alone (mean ± SEM 44 ± 3% and 46 ± 4.1% respectively) (Fig. 5b). Medium from microglia exposed to l-CCG-I and CGA was slightly more toxic (Fig. 5b). Neuronal apoptosis was significantly decreased in neurones exposed to medium from microglia incubated with MCCG during CGA activation (data not shown). Furthermore, conditioned medium from microglia stimulated with CGA in the presence of both MCCG and DCGIV or MCCG and l-CCG-I resulted in significantly attenuated levels of neuronal apoptosis. Conditioned medium from microglia exposed to Aβ25–35 induced neuronal apoptosis (Fig. 5c) as did medium from microglia exposed to DCGIV + Aβ25–35 and l-CCG-I + Aβ25–35. However, in this case the neuronal apoptosis could not be abrogated if the microglia were also exposed to the group II mGlu receptor antagonist MCCG.

The observed changes in neuronal apoptosis were due to the different microglial conditioned media as direct application of either CGA or mGlu receptor agonists and antagonists to neuronal cultures did not increase neuronal apoptosis above control levels (data not shown). When conditioned medium from control microglia was added to neuronal cultures together with direct application of the mGlu agonists/antagonists, there was no significant modulation of neuronal apoptosis (either increased or decreased) above basal levels (Fig. 5d). In particular, this suggests that neither DCGIV nor l-CCG-I exert a neurotoxic effect by a direct activation of neuronal mGlu receptors or by activation of NMDA receptors on the neurones, but rather that they trigger the production of neurotoxins from microglia. However, Aβ25–35 together with conditioned medium from control microglia was neurotoxic, suggesting that the amyloid peptide is directly neurotoxic.

Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

Since the addition of microglial conditioned medium to neurones allows the assessment of released stable components of neurotoxicity, to determine whether similar results might be obtained by allowing the exposure of neurones to non-stable microglial-released toxins, microglia were co-cultured with neurones in the presence of mGlu receptor agonists/antagonists and CGA. Similar results for microglial and neuronal apoptosis following CGA activation and mGlu receptor modulation were obtained when cells were grown in co-culture (Fig. 5e). This indicates that soluble, stable toxins are released from microglia and that the presence of neurones does not modulate the microglial response to CGA stimulation in the presence or absence of mGlu receptor agonists/antagonists. These findings demonstrate that medium from CGA-activated microglia is neurotoxic and that modulation of different microglial mGlu receptors can attenuate or exacerbate neuronal death induced by stimulation of microglia with CGA or by sole stimulation of microglial group II mGlu receptors.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References

Our results show for the first time that primary cultured microglia from rat brain express mGlu receptors of the group II subtype. This is supported by our findings that RT-PCR products for mGlu2 and mGlu3 receptors were present in rat microglia and that immunolocalization using commercially available antibodies to mGlu2 and mGlu2/3 receptors revealed the presence of receptor protein in microglia. These receptors appear to be negatively coupled to adenylate cyclase since forskolin-induced cyclic AMP generation could be inhibited by either DCGIV or l-CCG-I. The coupling of these receptors to an inhibition of cyclic AMP formation is in line with previous findings for group II mGlu receptors in other cell types (Conn and Pin 1997).

Interestingly, we were able to detect both mGlu3 and mGlu2 receptor mRNA in microglia as well as receptor protein expression of the two subtypes on isolectin B4-positive microglia. This differs from the results of Biber et al. (1999) who found only significant expression of mGlu5 in cultured microglia. It should be noted, however, that their method of microglial isolation and culture is rather different from the method used in the present study, the microglia being in culture for much longer than in our method of isolation and culture. Culturing microglia has been shown to change the pattern of a number of receptors and ion channels (Kettenmann et al. 1993; Eder et al. 1997). Previously, astrocytes have been shown to express mGlu3 but not mGlu2 receptor mRNA or protein (Schools and Kimelberg 1999; Janssens and Lesage 2001). While this suggests a fundamental difference in receptor expression between microglia and astrocytes, it may also be due to the age of the animals used and the method of cell isolation, since both these variables can affect expression (Catania et al. 1994; Schools and Kimelberg 1999).

Microglia exposed to the selective group II mGlu receptor agonists DCGIV or l-CCG-I (Brabet et al. 1998) with or without CGA showed enhanced mitochondrial depolarization and apoptosis. We have previously shown that mitochondrial depolarization precedes apoptosis following CGA stimulation of microglia (Kingham and Pocock 2000) and it is thus likely that the inhibition of mitochondrial depolarization by MCCG modulates downstream apoptotic cascades. These findings also suggest that stimulation of group II mGlu receptors on microglia, independently of any co-stimulus, can activate microglia to a reactive and neurotoxic phenotype. This was reflected in the increased staining with ED1 following exposure to either DCGIV or l-CCG-I (Table 1a).

Activation of group II mGlu receptors underlies CGA-induced mitochondrial depolarization and subsequent apoptosis as well as Aβ25–35-induced apoptosis, since DCGIV or l-CCG-I in conjunction with CGA or Aβ25–35 stimulation did not appear to significantly potentiate mitochondrial depolarization or apoptosis induced by CGA or apoptosis induced by Aβ25–35, while MCCG, a selective group II mGlu receptor antagonist (Knöpfel et al. 1995), inhibited CGA-induced mitochondrial depolarization and apoptosis and Aβ25–35-induced apoptosis. It has been reported that DCGIV may act as an agonist of NMDA receptors at high concentrations (Ishida et al. 1993; Brabet et al. 1998). Our findings that MK-801 or AP5 did not attenuate DCGIV-induced microglial apoptosis suggests that our responses with DCGIV do not involve activation of an NMDA receptor-mediated pathway. Furthermore, the concentrations of DCGIV and l-CCG-I used in this study are similar to those used to assess the functions of group II mGlu receptors in mixed glial cultures (Bruno et al. 1997). We found that the effects of l-CCG-I on microglial apoptosis at concentrations between 10 and 500 µm were saturable and completely reversed by the group II mGlu receptor antagonist MCCG. In addition, microglial apoptosis induced by either DCGIV or l-CCG-I could not be attenuated by antagonists of group I or group III mGlu receptors, suggesting that the two mGlu receptor agonists used were activating specific receptors.

Conditioned medium from microglia exposed to DCGIV or l-CCG-I alone induced neuronal apoptosis. This neurotoxicity was due to the downstream production of microglia-derived toxins or triggers to subsequent neurotoxicity because direct addition of DCGIV or l-CCG-I to neurones together with conditioned medium from non-activated microglia was not toxic.

In contrast to the effects on microglia, direct addition of DCGIV to neurones has been shown to be neuroprotective against a number of different insults including NMDA and kainate (Bruno et al. 1995; Buisson and Choi 1996). In neurones, stimulation of group II mGlu receptors exerts anticonvulsant effects and attenuates traumatic neuronal injury (Attwell et al. 1998; Allen et al. 1999). Under the current conditions, carry over of DCGIV or l-CCG-I to neuronal cultures did not appear to be neuroprotective following either addition of microglial conditioned medium or in co-culture. Neuroprotection mediated by stimulation of group II mGlu receptors with DCGIV or l-CCG-I in mixed cortical neuronal–glial cultures requires new protein synthesis (Bruno et al. 1997). In our experiments, it is possible that there was insufficient time for protein synthesis triggered by any carry over of DCGIV or LCCG-I to be protective. Other neuroprotective effects may be due to the acute regulation (depression) of glutamatergic neurotransmission (Dietrich et al. 1997). While we observed no neurotoxicity or modulation of neuronal survival compared with controls when the agonists of group II mGlu receptors were added directly to the neuronal cultures, it is possible that carry over of these compounds during addition of microglial conditioned medium or during co-culture may, if anything, reduce the neuronal death observed. Although activation of group II mGlu receptors on astrocytes protects neurones against excitotoxic death and other forms of degeneration (Nicoletti et al. 1996; Bruno et al. 1998), our results suggest that activation of group II mGlu receptors on microglia may not be beneficial for neuronal survival.

Whilst CGA triggers the production of NO in microglia mediated by iNOS (Taupenot et al. 1996; Kingham et al. 1999), conditioned medium from CGA-stimulated microglia does not mediate NO or peroxynitrite-dependent neuronal death (Ciesielski-Treska et al. 1998; Kingham et al. 1999). In support of this, similar results were obtained with microglial conditioned medium (in which NO would have degraded) as with microglia and neurones grown in co-culture (in which NO produced by the microglia would have been able to induce neurotoxicity). These findings suggest therefore that NO is not likely to be involved in the neurotoxicity observed here as a consequence of activation of microglial group II mGlu receptors. However, the current findings do not exclude the possibility that NO generated in the neuronal cultures triggered by some factor produced by the microglia could contribute to the subsequent neurotoxicity. Aβ25–35 induced microglial reactivity which could be inhibited by MCCG, suggesting an underlying group II mGlu receptor activation, similar to that observed with CGA. However, in contrast to CGA, inhibition of group II mGlu receptors on microglia exposed to Aβ25–35 did not reduce their neurotoxicity. We found that conditioned medium from non-activated microglia together with Aβ25–35 induced neuronal apoptosis. Thus the likelihood is that carry over of Aβ25–35 in the microglial conditioned medium is directly neurotoxic. This is not unlikely since Aβ25–35 has been shown to be toxic to cultured cerebellar granule cells (Schorzielli et al. 1996; Allen et al. 2001).

Our findings suggest that stimulation of group II mGlu receptors on microglia can induce neuronal death via the release of soluble, non-labile toxins. Activated microglia can release a variety of potentially neurotoxic substances (Banati et al. 1993), including reactive oxygen species, arachidonic acid, prostaglandins (Klegeris and McGeer 1997; Minghetti and Levi 1998), glutamate (Piani et al. 1991; Kingham et al. 1999) and proteases (Kingham and Pocock 2001) which may be implicated in current pathways. While activation of group II mGlu receptors alone without any other stimulus is sufficient to induce microglial reactivity and neurotoxicity, it also appears that a concurrent stimulation of group II mGlu receptors may underlie an activating stimulus of microglia. We are currently assessing whether the same neurotoxins are released following direct group II mGlu receptor stimulation and CGA stimulation. The normal functional role of group II mGlu receptors on microglia is currently unclear. However, ithas been suggested that the activation of mGlu3 on astrocytes may relieve inhibition of iNOS induction by reducing cyclic AMP levels, influence receptor/transporter expression or reduce proliferation (Ciccarelli et al. 1997; Porter and McCarthy 1997; Schools and Kimelberg 1999). Furthermore, as a consequence of synaptic activity, brain injury or neurodegenerative disease, extracellular glutamate may increase as a result of exocytosis, transporter reversal or cell lysis, leading to activation of mGlu receptors on surrounding microglia as has been shown for astrocytes (Porter and McCarthy 1996). Whether any of these mechanisms is relevant to the activation of microglial group II mGlu receptors remains to be determined.

In conclusion, the present results provide evidence that group II mGlu receptors expressed by microglia are involved in the signalling pathways leading to microglial activation and subsequent neurotoxicity following exposure to CGA. Suppression of microglial activation by selective modulation of group II microglial mGlu receptors may be a putative therapeutic strategy in Alzheimer's disease and other neurodegenerative diseases.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Preparation of microglial cultures
  6. Preparation of neuronal cultures
  7. Treatment of microglial cultures
  8. Measurement of cyclic AMP
  9. Treatment of neuronal cultures
  10. Co-cultures
  11. Measurement of microglial mitochondrial membrane polarization
  12. Assessment of apoptosis
  13. Isolation of total RNA
  14. Glutamate receptor mRNA expression by RT-PCR
  15. Immunolocalization
  16. Western blot analysis
  17. Statistical analysis
  18. Results
  19. Group II mGlu receptor activation can induce microglial mitochondrial depolarization and apoptosis
  20. Group II mGlu receptor activation underlies CGA- or Aβ25–35 induced microglial activation
  21. Expression and functional activation of group II mGlu receptors
  22. Activated microglia induce neuronal death; modulation by mGlu receptors on microglia
  23. Stimulation of group II mGlu receptors on microglia without co-exposure to CGA produces a neurotoxic phenotype
  24. Activation of microglial group II mGlu receptors with CGA or Aβ25–35 stimulation is neurotoxic
  25. Microglial mGlu receptor stimulation in co-culture indicates neurotoxicity is mediated by microglia-derived stable toxins
  26. Discussion
  27. Acknowledgement
  28. References
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