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Keywords:

  • Astrocytes;
  • Dopaminergic neurones;
  • 1-Methyl-4-phenylpyridinium;
  • Nitric oxide;
  • 6-Hydroxydopamine;
  • Parkinson's disease

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Determination of the effects of altered glial function on the susceptibility of neurones to MPP+- and 6-OHDA-induced cytotoxicity in astrocytic/ventral mesencephalic co-cultures
  5. RESULTS
  6. Effects of altered glial function on neuronal susceptibility to MPP+- or 6-OHDA-induced toxicity
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

Abstract : Altered glial function in the substantia nigra in Parkinson's disease may lead to the release of toxic substances that cause dopaminergic cell death or increase neuronal vulnerability to neurotoxins. To investigate this concept, we examined the effects of subjecting astrocytes to lipopolysaccharide (LPS)-induced activation alone or combined with l-buthionine-[S,R]-sulfoximine-induced glutathione depletion or inhibition of complex I activity by 1-methyl-4-phenylpyridinium (MPP+) on the viability of primary ventral mesencephalic neurones or susceptibility to MPP+ and 6-hydroxydopamine (6-OHDA) in co-cultures. LPS-activated astrocytes caused neuronal death in a time-dependent manner, but glutathione-depleted or complex l-inhibited astrocytes had no effect on neuronal viability. The neurotoxicity of LPS-activated astrocytes was inhibited by the inducible nitric oxide synthase inhibitor aminoguanidine, by the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and by reduced glutathione (GSH). MPP+-induced neuronal death was greater in ventral mesencephalic cultures previously cultured with LPS-activated, glutathione-depleted, or complex l-inhibited astrocytes compared with co-cultures containing normal astrocytes. The increased neuronal susceptibility to MPP+ caused by LPS-activated or complex l-inhibited astrocytes and glutathione-depleted astrocytes was inhibited by the NMDA/glutamate antagonist MK-801 and by GSH, respectively. Neuronal death caused by 6-OHDA was increased in ventral mesencephalic cultures previously cultured with LPS-activated and glutathione-depleted, but not complex l-inhibited astrocytes, compared with co-cultures containing normal astrocytes. Treatment of co-cultures with GSH prevented the increased neuronal susceptibility to OHDA. These findings suggest that glial dysfunction may cause neuronal death or render neurones susceptible to toxic insults via a mechanism involving the release of free radicals and glutamate. Such a mechanism may play role in the development or progression of nigrostriatal degeneration in Parkinson's disease.

The major pathological change in Parkinson's disease (PD) is degeneration of the dopamine-containing neurones of the substantia nigra pars compacta and the appearance of Lewy bodies (Forno, 1981). Nigral cell death in PD is also accompanied by glial proliferation and reactive microgliosis at the sites of neurodegeneration (McGeer and McGeer, 1997 ; Banati et al., 1998). The cause of dopaminergic cell death in PD is unknown, but there is evidence to suggest that altered glial function may contribute to the initiation or progression of the neurodegenerative process (Chao et al., 1996a ; Hirsch et al., 1998). Postmortem studies using homogenates of PD nigral tissue have identified changes in biochemical parameters indicative of oxidative stress, impaired mitochondrial function, and excitotoxicity (Jenner and Olanow, 1996). In particular, there is a 40% decrease in the levels of reduced glutathione (GSH) (Sian et al., 1994), an increase in iron levels (Dexter et al., 1991), and a 33% increase in mitochondrial manganese-dependent super-oxide dismutase activity (Saggu et al., 1989). Free radical-mediated damage is apparent from increased lipid peroxidation (Dexter et al., 1994), elevated protein carbonyls (Alam et al., 1997b), and DNA fragmentation (Sanchez-Ramos et al., 1994 ; Alam et al., 1997a) in substantia nigra. Mitochondrial dysfunction also occurs in PD as evident from an ~40% decrease of complex I activity (Schapira et al., 1990) and reduced immunostaining for the α-ketoglutarate dehydrogenase complex in substantia nigra (Mizuno et al., 1994). The neurodegenerative process may also involve nitric oxide (NO) and glutamate-related toxic processes, because inducible NO synthase (iNOS) mRNA is increased in substantia nigra in PD (Hunot et al., 1996) and inhibition of mitochondrial function by NO leads to glutamate release and excitotoxicity (Beal, 1998 ; McNaught and Brown, 1998). The magnitude of the nigral alterations, however, is too great to take place primarily in dopaminergic neurones, which make up only 1-2% of the total cell population in substantia nigra in normal brain, and up to 75% less in PD. This suggests that GSH depletion and complex I inhibition, for example, occur predominantly in glial cells. Indeed, most GSH in brain is present in glial cells (Sagara et al., 1993), and only glial cells stain positive for elevated nonhaem iron in substantia nigra in PD (Morris and Edwardson, 1994). Similarly, superoxide dismutase and complex I activities are significantly higher in astrocytes than in neurones (Savolainen, 1978 ; Stewart et al., 1998), and iNOS is located almost exclusively in astrocytes and microglial in normal and PD brain (Hunot et al., 1996). These observations suggest that the occurrence of oxidative stress or mitochondrial dysfunction in normal or activated glial cells may contribute to nigral dopaminergic cell death in PD.

Altered glial function may contribute to neuronal death by the release of free radicals and glutamate that cause neuronal injury. Indeed, previous investigations have shown that activation of glial cells in culture or in vivo by cytokines leads to the expression of iNOS mRNA and the production of NO (Chao et al., 1996b). Furthermore, we showed recently that lipopolysaccharide (LPS)-induced activation of cultured primary astrocytes also causes extracellular accumulation of hydrogen peroxide (H2O2) and glutamate, as well as NO (McNaught and Jenner, 1999). In addition, inhibition of complex I activity led to extracellular accumulation of H2O2 and glutamate (McNaught and Jenner, 1999). Therefore, glial activation, GSH depletion, or complex I inhibition may contribute to neuronal death in substantia nigra in PD. Indeed, the levels of inflammatory cytokines such as interleukin-1β and tumor necrosis factor-α are elevated in brain in PD (Boka et al., 1994 ; Mogi et al., 1994, and previous studies have shown that cytokine-induced activation of glial cells causes neuronal death in cortical cultures via a mechanism involving NO production (Dawson et al., 1994 ; Bolaños et al., 1996 ; Chao et al., 1996b). However, it is know if the release of NO, reactive oxygen species, or glutamate from LPS/cytokine-activated or complex I-inhibited glial cells induces dopaminergic cell death. Also, it is not known if such alterations of glial function increase the susceptibility of dopaminergic neurones to the actions of neurotoxins relevant to the aetiology of PD.

To determine the effect of altered glial function on neuronal survival, we have examined the effects of glial cell activation alone or in combination with glutathione depletion and/or complex I inhibition on dopaminergic cell viability and susceptibility to 1-methyl-4-phenylpyridinium (MPP+)- or 6-hydroxydopamine (6-OHDA)-induced toxicity in primary astrocytic/ventral mesencephalic co-cultures.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Determination of the effects of altered glial function on the susceptibility of neurones to MPP+- and 6-OHDA-induced cytotoxicity in astrocytic/ventral mesencephalic co-cultures
  5. RESULTS
  6. Effects of altered glial function on neuronal susceptibility to MPP+- or 6-OHDA-induced toxicity
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

Materials

Hanks' balanced salt solution (HBSS), high-glucose Dulbecco's modified Eagle's medium (DMEM), bovine serum albumin (fraction V), foetal calf serum, gentamicin solution, antibiotic and antimycotic solution (AAS ; 10,000 units of penicillin, 10 mg of streptomycin, and 25 μg of amphotericin B per milliliter of 0.9% NaCl), l-valine, poly-l-lysine, Triton X-100, normal goat serum, 3,3′-diaminobenzidine tetrahydrochloride, 0.25% trypsin-EDTA solution, bovine pancreatic trypsin (type III), soybean trypsin inhibitor, LPS (E. coli, 026 : B6), l-buthionine-[S,R]-sulfoximine (l-BSO), GSH, and 6-OHDA hydrochloride were obtained from Sigma Chemical Co. (Poole, U.K.). Primary tyrosine hydroxylase (TH) rabbit antibody was obtained from Pel-Freez Biologicals (Rogers, AR, U.S.A.). Biotinylated goat anti-rabbit IgG, avidin, and biotin were components of the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, U.S.A.). Bovine pancreatic DNase I was obtained from Boehringer Mannheim (East Sussex, U.K.). D-Valine-containing minimum essential medium (D-Val MEM) was obtained from GibcoBRL (Paisley, U.K.). MPP+ iodide was obtained from Research Biochemicals (Natick, MA, U.S.A.). Aminoguanidine hydrochloride and (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine [dizocilpine ; (+)-MK-801 maleate] were obtained from Tocris (Bristol, U.K.). 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (carboxy PTIO) was obtained from Alexis Corp. (Nottingham, U.K.). Cell culture plastics were obtained from Sigma and GibcoBRL. All other reagents and materials were of analytical or cell culture grade and obtained from commercial sources. Reagents and cell culture materials were sterilized before use.

Astrocytic culture

Primary astrocytes were prepared from the forebrain of postnatal (1-2 days old) Wistar rats (Bantin & Kingman, Hull, U.K.) and plated in poly-l-lysine-coated (5 μg/ml) 162-cm2 vented cell culture flasks containing 20 ml d/l-Val MEM (MEM containing 0.1 mg/ml l-valine, 10% foetal calf serum, and 2.5% AAS) as described elsewhere (Marriott et al., 1995). The cells were grown in a hamidified atmosphere of 5% CO2/95% air at 37°C. The culture medium was replaced with d/l-Val MEM after 1 and 3 days in culture. After 7 days in culture, and every 3 days thereafter, the culture medium was replaced with D-Val MEM (MEM containing 10% foetal calf serum and 2.5% ASS) to retard the growth of fibroblasts and meningeal cells. When the astrocytes reached confluence (after ~ 14 days in culture), the cells were removed from the flasks by trypsinization, then plated in removable 10-mm Nunc tissue culture inserts (polycarbonate membrane, 0.4 μm) in 24-well culture plates at a density of 105 cells/cm2 containing 1.0 ml of D-Val MEM, and grown as described above. One day later, the astrocytes were used for experimentation. Immunocytochemical analyses have shown that this method produces cultures comprising >95% glial fibrillary acidic protein-positive astrocytes (Marriott et al., 1995).

Neuronal culture

Primary neuronal cultures were prepared from embryonic 15-16 days of gestation) ventral mesencephalon as previously described (Barker and Johnson, 1995). Pregnant Sprague-Dawley rats (Charles River, U.K.) were killed by decapitation, and the foetuses (10-15) removed, cleared of membranes, and placed into a petri dish containing HBSS. The foetal ventral mesencephalon was removed under a dissecting stereomicroscope, placed into 10-ml tubes containing HBSS, and then centrifuged at 100 g for 1 min. The supernatant was discarded and 2 ml of 0.1% trypsin solution was added and allowed to incubated at 37°C for 6 min. Subsequently, 2 ml of 10 μg/ml DNase I solution was added and the suspension centrifuged at 100 g for 2 min. The supernatant was discarded, 2 ml of triturating solution (1 mg/ml bovine serum albumin, 10 μg/ml DNase I, and 0.5 mg/ml soybean trypsin inhibitor) was added, and the tissue was triturated 20 times using a Pasteur pipette. Cells were suspended in 85% DMEM (DMEM containing 15% foetal calf serum, 0.6 mg/ml glutamine, and 0.25% gentamicin), plated at a density of 104 cells/cm2 on 10-mm 10 μg/ml poly-l-lysine-coated coverslips in 24-well culture plates, and grown in a humidified atmosphere of 5% CO2/95% air at 37°C. Three days later, the culture medium was changed to 85% DMEM supplemented with 10 μM cytosine arabinoside to control glial proliferation (Bolaños et al., 1996). Neuronal cultures were used for experimentation 7 days after preparation.

Determination of neuronal death

Dopamine-containing neurones in primary ventral mesencephalic cultures were visualized by TH immunoreactivity and counted as previously described (McNaught et al., 1996). Coverslips containing primary ventral mesencephalic neurones were washed with 0.1 M phosphate-buffered saline (PBS ; pH 7.4) and then fixed in 4% paraformaldehyde for at least 1 h. Cells were permeabilized in 0.1% Triton X-100 for 30 min, washed in PBS, and then incubated in 10% normal goat serum for 1 h. After washing in PBS, cells were incubated in primary rabbit anti-TH antibody (1 : 200 dilution in 1% normal goat serum) overnight. Coverslips were washed with PBS and then incubated in secondary antibody (biotinylated goat anti-rabbit IgG, 1 : 200 dilution) for 1 h. After washing with PBS, cells were incubated in ABC reagent (1 : 1 of 1 : 200 avidin and 1 : 200 biotin) for 1 h. Subsequently, coverslips were washed with PBS and 50 mM Tris-HCl (pH 7.4) and then incubated with 0.05% (wt/vol) diaminobenzidine in Tris-HCl buffer for 10 min. H2O2 was added at a final concentration of 0.01% and incubated until the brown TH-positive cells were visible (~5 min). The reaction was terminated by replacing the reaction mixture with Tris-HCl. Coverslips were washed with PBS, dehydrated through ethanol, and then mounted onto slides. Slides were examined under a Zeiss Axioskop microscope equipped with a grid-containing eyepiece, the number of TH-positive neurones were counted manually in several grids, and then the average was taken. In some studies, we also examined the release of lactate dehydrogenase as a biochemical indicator of cell death and found a good correlation with the extent of cell death as determined by TH immunocytochemistry/cell counting.

Determination of the effects of altered glial function on neuronal viability in astrocytic/ventral mesencephalic co-cultures

The culture medium of primary astrocytes in inserts in 24-well culture plates was replaced with D-Val MEM containing 30 μg/ml LPS, 300 μMl-BSO, and/or 30 μM MPP+ for 1 day. These parameters were used in this study, because our previous investigation showed that LPS stimulated NO release to near-maximal levels, l-BSO depleted total glutathione by up to 90%, and MPP+ inhibited complex I activity by up to 43% (McNaught and Jenner, 1999). The inserts containing astrocytes were removed, washed with 85% DMEM, and placed in 24-well culture plates above an established (7 days old) bed of primary ventral mesencephalic neurones. Cells were co-cultured for up to 3 days in 85% DMEM with or without 0.5 mM aminoguanidine, 0.1 mM carboxy PTIO, 10 μM MK-801, or 1 mM GSH. The inserts containing astrocytes were removed and neuronal death determined as described above.

Determination of the effects of altered glial function on the susceptibility of neurones to MPP+- and 6-OHDA-induced cytotoxicity in astrocytic/ventral mesencephalic co-cultures

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Determination of the effects of altered glial function on the susceptibility of neurones to MPP+- and 6-OHDA-induced cytotoxicity in astrocytic/ventral mesencephalic co-cultures
  5. RESULTS
  6. Effects of altered glial function on neuronal susceptibility to MPP+- or 6-OHDA-induced toxicity
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

The culture medium of primary astrocytes in inserts in 24-well culture plates was replaced with D-Val MEM containing 30 μg/ml LPS, 300 μMl-BSO, or 30 μM MPP+. One day later, the inserts containing astrocytes were removed, washed with DMEM, and placed in 24-well culture plates above an established (7 days old) bed of primary ventral mesencephalic neurones. Cells were co-cultured for 3 days in 85% DMEM with or without 0.5 mM aminoguanidine, 0.1 mM carboxy PTIO, 10 μM MK-801, or 1 mM GSH. The inserts containing astrocytes and cultured medium were removed, 85% DMEM containing 30 μM MPP+ or 6-OHDA was added, and cells were grown for 3 days. Neuronal death was determined as described above.

Statistical analysis

Results were analysed statistically using Student's t test or Mann-Whitney U test. Data presented are from four to six separate cultures with n = 3-6.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Determination of the effects of altered glial function on the susceptibility of neurones to MPP+- and 6-OHDA-induced cytotoxicity in astrocytic/ventral mesencephalic co-cultures
  5. RESULTS
  6. Effects of altered glial function on neuronal susceptibility to MPP+- or 6-OHDA-induced toxicity
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

Effects of altered glial function on neuronal viability

Primary ventral mesencephalic neurones were co-cultured with normal or abnormal (activated, glutathione-depleted, or complex I-inhibited) primary astrocytes for up to 3 days, after which neuronal death was determined. In co-cultures with normal astrocytes, mean neuronal death after 1 and 3 days was 8 and 14% of the TH-positive neuronal population, respectively (Fig. 1).

image

Figure 1.  Effects of LPS-activated astrocytes on neuronal viability in primary astrocytic/ventral mesencephalic co-cultures. Primary astrocytes in porous inserts in 24-well culture plates (105 cells/cm2) were treated with 30 μg/ml LPS in D-Val MEM. One day later, the inserts containing astrocytes were removed, washed with 85% DMEM, and placed in 24-well culture plates above an established bed of primary ventral mesencephalic neurones (104 cells/cm2). Cells were co-cultured for 1 (□) or 3 (▪) days in 85% DMEM with or without 0.5 mM aminoguanidine, 0.1 mM carboxy PTIO, 10 μM MK-801, or 1 mM GSH. Neuronal death was determined as described in Materials and Methods. Results were analysed statistically using Student's t test and are presented as means ± SEM (n = 6). *p < 0.01, compared with Normal Astrocyte. #p < 0.01, compared with LPS-activated Astrocyte.

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LPS activation. Culturing ventral mesencephalic neurones with LPS-activated astrocytes resulted in neuronal death, which increased with time. Thus, after 1 and 3 days in co-culture, neuronal death increased to 12 and 23%, respectively (Fig. 1). Treatment of LPS-activated astrocytic/ventral mesencephalic co-cultures with the iNOS inhibitor aminoguanidine (0.5 mM) reduced neuronal death to 10 and 18% after 1 and 3 days, respectively (Fig. 1). Similarly, the NO scavenger carboxy PTIO (0.1 mM) reduced neuronal death to 9 and 18% after 1 and 3 days, respectively (Fig. 1). Treatment of co-cultures with GSH decreased neuronal death. Thus, treatment of LPS-activated astrocytic/ventral mesencephalic co-cultures with 1 mM GSH decreased neuronal death to 8 and 14% after 1 and 3 days, respectively (Fig. 1). However, treatment of LPS-activated astrocytic/ventral mesencephalic co-cultures with the NMDA/glutamate antagonist MK-801 had no effect on neuronal death caused by LPS-activated astrocytes (Fig. 1). Co-culturing ventral mesencephalic neurones with glutathione-depleted or complex I-inhibited astrocytes for up to 3 days did not cause neuronal death (data not shown).

Combined treatment.

Neuronal death was higher in co-cultures with LPS-activated/glutathione-depleted astrocytes than in co-cultures with LPS-activated astrocytes alone. Thus, LPS-activated/glutathione-depleted astrocytes caused 18 and 27% neuronal death after 1 and 3 days, respectively (Fig. 2). Neuronal death was inhibited by both 0.5 mM aminoguanidine and 1 mM GSH, resulting in 14 and 12% neuronal death after 3 days, respectively (Fig. 2). Neuronal death in co-cultures with LPS-activated/complex I-inhibited astrocytes was higher than neuronal death in co-cultures with LPS-activated astrocytes alone. Thus, LPS-activated/complex I-inhibited astrocytes caused 17 and 25% neuronal death after 1 and 3 days, respectively (Fig. 2). Neuronal death was inhibited by both 0.5 mM aminoguanidine and 1 mM GSH, resulting in 16 and 15% neuronal death after 3 days, respectively (Fig. 2). In co-cultures with glutathione-depleted/complex I-inhibited astrocytes, neuronal death was 10 and 17% after 1 and 3 days, respectively (Fig. 2.

image

Figure 2.  Effects of altered astrocytic function on neuronal viability in primary astrocytic/ventral mesencephalic co-cultures. Primary astrocytes in porous inserts in 24-well culture plates (105 cells/cm2) were treated with 30 μg/ml LPS + 300 μMl-BSO, 30 μg/ml LPS + 30 μM MPP+, or 300 μMl-BSO + 30 μM MPP+ in D-Val MEM. One day later, the inserts containing astrocytes were removed, washed with 85% DMEM, and placed in 24-well culture plates above an established bed of primary ventral mesencephalic neurones (104 cells/cm2). Cells were co-cultured mesencephalic neurones (104 cells/cm2). Cells were co-cultured for 1 (□) or 3 (▪) days in 85% DMEM with or without 0.5 mM aminoguanidine or 1 mM GSH. Neuronal death was determined as described in Materials and Methods. Results were analysed statistically using Student's t test and are presented as means ± SEM (n = 6). *p < 0.01, compared with Normal Astrocyte. #p < 0.01, compared with LPS-activated/GSH-depleted Astrocyte or LPS-activated/Complex I-inhibited Astrocyte.

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Effects of altered glial function on neuronal susceptibility to MPP+- or 6-OHDA-induced toxicity

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Determination of the effects of altered glial function on the susceptibility of neurones to MPP+- and 6-OHDA-induced cytotoxicity in astrocytic/ventral mesencephalic co-cultures
  5. RESULTS
  6. Effects of altered glial function on neuronal susceptibility to MPP+- or 6-OHDA-induced toxicity
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

Primary ventral mesencephalic neurones were co-cultured with normal or abnormal (activated, glutathione-depleted, or complex I-inhibited) primary astrocytes for 3 days, then astrocytes were removed, and the neurones were treated with 30 μM MPP+ or 6-OHDA. Neuronal death was examined after 3 days. In ventral mesencephalic cultures previously co-cultured with normal astrocytes, the mean neuronal death caused by 30 μM MPP+ and 6-OHDA was 17 and 37% of the TH-positive neuronal population, respectively (Figs. 3 and 4).

image

Figure 3.  Effects of LPS-activated astrocytes on neuronal susceptibility to MPP+ - or 6-OHDA-induced toxicity. Primary astrocytes in porous inserts in 24-well culture plates (105 cells/cm2) were treated with 30 μg/ml LPS in d-Val MEM. One day later, the inserts containing astrocytes were removed, washed with 85% DMEM, and placed in 24-well culture plates above an established bed of primary ventral mesencephalic neurones (104 cells/cm2). Cells were co-cultured for 1 day in 85% DMEM with or without 0.5 mM aminoguanidine, 0.1 mM carboxy PTIO, 10 μM MK-801, or 1 mM GSH. The inserts and medium were removed, the primary ventral mesencephalic neurones treated with 30 μM MPP+ (□) or 6-OHDA (▪) in 85% DMEM, and cells grown for 3 days. Neuronal death was determined as described in Materials and Methods. Results were analysed statistically using Mann-Whitney U test and are presented as means ± SEM (n = 3). *p <0.05, compared with Normal Astrocyte. #p <0.05, compared with LPS-activated Astrocyte.

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image

Figure 4.  Effects of GSH-depleted and complex I-inhibited astrocytes on neuronal susceptibility to MPP+ - or 6-OHDA-induced toxicity. Primary astrocytes in porous inserts in 24-well culture plates (105 cells/cm2) were treated with 300 μMl-BSO or 30 μM MPP+. One day later, the inserts containing astrocytes were removed, washed with 85% DMEM, and placed in 24-well culture plates above an established bed of primary ventral mesencephalic neurones (104 cells/cm2). Cells were co-cultured for 1 day in 85% DMEM with or without 1 mM GSH or 10 μM MK-801. The inserts and medium were removed, the primary ventral mesencephalic neurones treated with 30 μM MPP+ (□) or 6-OHDA (▪) in 85% DMEM, and cells grown for 3 days. Neuronal death was determined as described in Materials and Methods. Results were analysed statistically using Mann-Whitney U test and are presented as means ± SEM (n = 3). *p <0.05, compared with Normal Astrocyte. #p <0.05, compared with GSH-depleted Astrocyte or Complex I-inhibited Astrocyte.

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LPS activation. Co-culturing ventral mesencephalic neurones with LPS-activated astrocytes increased MPP+ -induced neuronal death to 26% (Fig. 3). Treatment of ventral mesencephalic cultures with 10 μM MK-801, but not 0.5 mM aminoguanidine, 0.1 mM carboxy PTIO, or 1 mM GSH, attenuated the increased susceptibility of neurones to MPP+ caused by LPS-activated astrocytes, resulting in only 17% neuronal death (Fig. 3). Co-culturing ventral mesencephalic neurones with LPS-activated astrocytes increased 6-OHDA-induced neuronal death to 61% (Fig. 3). Treatment of co-cultures with 1 mM GSH, but not 0.5 mM aminoguanidine, 0.1 mM carboxy PTIO, or 10 μM MK-801, attenuated the increased susceptibility of neurones to 6-OHDA caused by LPS-activated astrocytes, resulting in only 46% neuronal death (Fig. 3).

Glutathione depletion.

Co-culturing ventral mesencephalic neurones with glutathione-depleted astrocytes increased neuronal death caused by MPP+ to 26% (Fig. 4). Treatment of co-cultures with 1 mM GSH attenuated the increased susceptibility of neurones to MPP+ caused by GSH-depleted astrocytes, resulting in 20% neuronal death (Fig. 4). Co-culturing ventral mesencephalic neurones with glutathione-depleted astrocytes increased neuronal death caused by 6-OHDA to 49% (Fig. 4). Treatment of co-cultures with 1 mM GSH attenuated the increased susceptibility of neurones to 6-OHDA caused by glutatione-depleted astrocytes, resulting in 37% neuronal death (Fig. 4).

Complex I inhibition.

Co-culturing ventral mesencephalic neurones with complex I-inhibited astrocytes increased MPP+ -induced neuronal death to 28% (Fig. 4). Treatment of co-cultures with 10 μM MK-801 attenuated the increased susceptibility of neurones to MPP+ caused by complex I-inhibited astrocytes, resulting in 21% neuronal death (Fig. 4). Co-culturing ventral mesencephalic neurones with complex I-inhibited astrocytes did not alter neuronal death caused by 6-OHDA (Fig. 4).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Determination of the effects of altered glial function on the susceptibility of neurones to MPP+- and 6-OHDA-induced cytotoxicity in astrocytic/ventral mesencephalic co-cultures
  5. RESULTS
  6. Effects of altered glial function on neuronal susceptibility to MPP+- or 6-OHDA-induced toxicity
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES

Glial cells normally provide trophic support to neurones in the developing and adult brain. However, we and others have shown that alteration of glial function in cell cultures, in particular LPS-induced activation, depletion of glutathione levels, or inhibition of complex I activity, causes the extracellular accumulation of NO, H2O2, and glutamate, which are toxic to dopaminergic neurones (Bolaños et al., 1996, 1996 ; McNaught and Jenner, 1999), As these biochemical changes appear to occur in glial cells in substantia nigra in PD, it is postulated that abnormal glial function leads to the release of free radicals and glutamate, and these toxic substances cause dopaminergic cell death or render neurones vulnerable to other toxic insults. Accordingly, in the present investigation, we have shown that glial activation causes neuronal death in primary astrocytic/ventral mesencephalic co-cultures. Although glial glutathione depletion or complex I inhibition did not cause neuronal death, these alterations along with glial activation rendered neurones more susceptible to the toxic effects of MPP+ and 6-OHDA.

Previous studies have shown that activation of astrocytes or microglia by LPS or cytokines causes neuronal death in mixed glial/cortical neuronal co-cultures by a mechanism involving glial iNOS mRNA expression and production and release of NO from glial cells (Dawson et al., 1994 ; Bolaños et al., 1996 ; Chao et al., 1996b). These findings are consistent with our observations in this study showing that the specific iNOS inhibitor aminoguanidine (Griffith et al., 1993) and the NO scavenger carboxy PTIO (Akaike et al., 1993) attenuated the time-dependent neuronal death caused by LPS-activated astrocytes. In addition, we found that treatment of co-cultures with GSH attenuated neuronal death caused by LPS-activated astrocytes. This suggests that the generation of reactive oxygen species by LPS-activated astrocytes may also contribute to neuronal death, because the primary role of GSH is to scavenge superoxide radicals and convert H2O2 to water (Halliwell, 1996). Indeed, we have shown previously that activation of astrocytes in culture by LPS leads to the extracellular accumulation of H2O2 as well as NO in a time- and concentration-dependent manner (McNaught and Jenner, 1999). Recently, however, GSH has been suggested to react with NO, and so this mechanism may also contribute to the ability of GSH to protect neurones from toxicity in this investigation (Bolaños et al., 1996). The NMDA antagonist MK-801 did not protect neurones from LPS-activated astrocytes even though we previously showed that activation of astrocytes in culture results in the extracellular accumulation of glutamate (McNaught and Jenner, 1999). This observation may be explained by our previous finding that extracellular glutamate accumulation caused by LPS was rapid and complete after 1 day of treatment, unlike the continuous accumulation of NO and H2O2. Consequently, there would be no significant glutamate release from LPS-activated astrocytes leading to glutamate-mediated neurotoxicity, because in this study astrocytic/ventral mesencephalic co-cultures were established 1 day after astrocytes were treated with LPS. Nevertheless, activation of glial cells in vivo may cause neuronal death by a process involving the release of glutamate from glial cells (Beal, 1998). Indeed, intranigral injection of LPS in rats resulted in glial activation and nigrostriatal degeneration, but this was not attributed to NO production or a direct effect of LPS on neurones, because iNOS inhibitors did not prevent dopaminergic cell death, and LPS is not toxic to cultured mesencephalic neurones (Bronstein et al., 1995 ; Castaño et al., 1998).

Culturing ventral mesencephalic neurones with glutathione-depleted astrocytes did not cause neuronal death in this study. This observation is consistent with previous findings that depletion of glutathione levels in astrocytes did not cause the release of NO, H2O2, or glutamate, or cause glial cell death (Bolaños et al., 1996 ; McNaught and Jenner, 1999). Furthermore, glial cells and neurones are able to maintain antioxidant defence mechanisms in the presence of low concentrations of glutathione, but normal levels of catalase (Desagher et al., 1996 ; Dringen and Hamprecht, 1997). However, inhibition of complex I activity leads to the release of glutamate, but this was rapid and complete before the establishment of co-cultures, and so may not cause neuronal death as discussed previously.

As glial activation, GSH depletion, and complex I inhibition may occur simultaneously in substantia nigra in PD, we examined the effects of combined glial alterations on neuronal viability. It was found that glial activation combined with glutathione depletion or complex I inhibition resulted in increased neuronal death compared with neuronal death caused by either treatment alone. The biochemical basis of this is not known, but one possibility is that increased neuronal death results from the summated release of toxic species from glial cells caused by both treatments. However, this seems unlikely, because we previously showed that LPS-induced activation of glutathione-depleted or complex I-inhibited astrocytes resulted in a marked reduction in the accumulation of NO and H2O2 levels compared with LPS activation alone (McNaught and Jenner, 1999). An alternative explanation is that following combined treatment, NO reacts rapidly with superoxide radicals (before conversion to H2O2) to produce peroxynitrite and hydroxyl radicals, which are markedly more cytotoxic than NO or H2O2 (Halliwell, 1996).

LPS-activated or glutathione-depleted astrocytes potentiated the ability of MPP+ and 6-OHDA to cause neuronal toxicity, possibly by a mechanism involving the release of glutamate and free radicals, respectively. This observation is consistent with the mechanism by which both neurotoxins are thought to be toxic to dopamine-containing cells. MPP+ causes neuronal death in vivo and in cell cultures by virtue of its ability to inhibit complex I activity, resulting in ATP depletion, glutamate release, and excitotoxicity (Irwin and Langston, 1993). So, it is expected that factors increasing glutamate levels, such as LPS-induced release from astrocytes or inhibition of uptake, would cause neuronal supersensitivity to excitotoxicity or summate to increase the apparent toxicity of MPP+. Indeed, in this study, complex I-inhibited astrocytes also potentiated the toxicity of MPP+, but not 6-OHDA, and this effect was inhibited by MK-801. It is interesting that previous studies have shown that the NMDA antagonist MK-801 and other glutamate-channel blockers prevent the toxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/MPP+ following intracerebral administration in mice and primates (Zuddas et al., 1992 ; Beal, 1998). 6-OHDA induces cell death by the generation of reactive oxygen species (Lotharius et al., 1999). Consequently, the release of reactive oxygen species from LPS-activated astrocytes may exhaust neuronal defences before 6-OHDA treatment, or act synergistically with 6-OHDA to induce oxidative stress in neurones. This concept is supported by observations that depletion of brain GSH levels with L-BSO is itself not toxic to neurones, but increases the dopaminergic toxicity of MPTP, MPP+, or 6-OHDA following intracerebral administration to rats and mice (Seaton et al., 1996 ; Wüllner et al., 1996).

In conclusion, glial activation causes neuronal death via a mechanism that appears to involve the release of reactive oxygen/nitrogen species and glutamate from astrocytes. Glial glutathione depletion or complex I inhibition did not cause neuronal death, but potentiated the toxicity of MPP+ and 6-OHDA, possibly via the release of glutamate and reactive oxygen species from astrocytes. It is not clear if altered glial function is a primary event causing neuronal death in substantia nigra in PD, or if it is secondary to other factors, including neuronal degeneration. Either way, the findings in this investigation suggest that altered glial function may contribute to the development or progression of the neurodegenerative process in substantia nigra in PD.

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  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Determination of the effects of altered glial function on the susceptibility of neurones to MPP+- and 6-OHDA-induced cytotoxicity in astrocytic/ventral mesencephalic co-cultures
  5. RESULTS
  6. Effects of altered glial function on neuronal susceptibility to MPP+- or 6-OHDA-induced toxicity
  7. DISCUSSION
  8. Acknowledgements
  9. REFERENCES
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