Activation of microglial NADPH oxidase is synergistic with glial iNOS expression in inducing neuronal death: a dual-key mechanism of inflammatory neurodegeneration
© Mander and Brown; licensee BioMed Central Ltd. 2005
Received: 25 July 2005
Accepted: 12 September 2005
Published: 12 September 2005
Inflammation-activated glia are seen in many CNS pathologies and may kill neurons through the release of cytotoxic mediators, such as nitric oxide from inducible NO synthase (iNOS), and possibly superoxide from NADPH oxidase (NOX). We set out to determine the relative role of these species in inducing neuronal death, and to test the dual-key hypothesis that the production of both species simultaneously is required for significant neuronal death.
Primary co-cultures of cerebellar granule neurons and glia from rats were used to investigate the effect of NO (from iNOS, following lipopolysaccharide (LPS) and/or cytokine addition) or superoxide/hydrogen peroxide (from NOX, following phorbol 12-myristate 13-acetate (PMA), ATP analogue (BzATP), interleukin-1β (IL-1β) or arachidonic acid (AA) addition) on neuronal survival.
Induction of glial iNOS caused little neuronal death. Similarly, activation of NOX alone resulted in little or no neuronal death. However, if NOX was activated (by PMA or BzATP) in the presence of iNOS (induced by LPS and interferon-γ) then substantial delayed neuronal death occurred over 48 hours, which was prevented by inhibitors of iNOS (1400W), NOX (apocynin) or a peroxynitrite decomposer (FeTPPS). Neurons and glia were also found to stain positive for nitrotyrosine (a putative marker of peroxynitrite) only when both iNOS and NOX were simultaneously active. If NOX was activated by weak stimulators (IL-1β, AA or the fibrillogenic prion peptide PrP106-126) in the presence of iNOS, it caused microglial proliferation and delayed neurodegeneration over 6 days, which was prevented by iNOS or NOX inhibitors, a peroxynitrite decomposer or a NMDA-receptor antagonist (MK-801).
These results suggest a dual-key mechanism, whereby glial iNOS or microglial NOX activation alone is relatively benign, but if activated simultaneously are synergistic in killing neurons, through generating peroxynitrite. This mechanism may mediate inflammatory neurodegeneration in response to cytokines, bacteria, ATP, arachidonate and pathological prions, in which case neurons may be protected by iNOS or NOX inhibitors, or scavengers of NO, superoxide or peroxynitrite.
Keywordsmicroglia peroxynitrite nitric oxide prion protein inflammation cytokines
Glia (microglia and astrocytes) can become inflammation activated in many CNS pathologies, including infectious, ischaemic, inflammatory and neurodegenerative disorders [1, 2]. Glial activation may be protective to the host, as it can lead to the removal of cell debris and killing of pathogens . However excessive or chronic glial activation can kill nearby neurons [4, 5]. Thus inflammation may contribute to many CNS pathologies including Alzheimer's, Parkinson's and motor neuron diseases, multiple sclerosis, meningitis, AIDS dementia, strokes, trauma and normal brain ageing [6, 7]. It is therefore important to understand the mechanisms by which inflammatory-activated glia kill neurons.
Astrocytes and microglia can become activated by a range of factors, including pathogens and pro-inflammatory cytokines, and can lead to the subsequent death of co-cultured neurons [8, 9]. Activated astrocytes and/or microglia produce a variety of factors which can mediate neuronal death, including reactive oxygen species (ROS) [10, 11], nitric oxide [8, 9, 12] and glutamate [8, 13], as well as pro-inflammatory cytokines that perpetuate glial activation, such as interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α) .
The neuroprotective effects of anti-oxidants have been established  and are thought to be due to the removal of ROS (such as superoxide) and as well as more toxic molecules (such as peroxynitrite) . There is evidence that NADPH oxidase is activated in Alzheimer's disease and AIDS dementia [17–19]. The major source of ROS during inflammation is NADPH oxidase [20, 21], although other sources may also contribute [22, 23]. NADPH oxidase is expressed mainly by microglia in the brain [21, 24], and produces superoxide (O2-) extracellularly or within phagocytic vesicles, in order to kill pathogens. The oxidase can be acutely activated by PMA, ATP, arachidonic acid, some chemokines and cytokines [25–28]. Superoxide is then broken down mainly by extracellular and intracellular superoxide dismutase to give hydrogen peroxide (H2O2).
iNOS is not normally expressed in the brain, but is induced in astrocytes and microglia by proinflammatory cytokines and pathogen components, such as lipopolysaccharide (LPS)/endotoxin of Gram-negative bacteria . Once expressed iNOS produces high, sustained levels of NO which can, in certain conditions, kill nearby neurons, by mechanisms including inhibition of mitochondrial respiration and the release of glutamate from neurons and glia, resulting in excitotoxicity . However, such mechanisms may require a relatively high level of NO and/or a relatively low level of oxygen [30, 31]. An alternative mechanism would be for NO to react with superoxide (e.g. from the NADPH oxidase) to produce peroxynitrite (ONOO-), which is potentially more neurotoxic to neurons than NO or superoxide [32, 33].
This suggests a dual-key hypothesis of inflammatory neurodegeneration whereby iNOS expression or NADPH oxidase activation alone is relatively benign, but when combined together at the same time causes neurodegeneration via peroxynitrite. We have previously shown that acute activation of the NADPH oxidase in isolated microglia expressing iNOS results in the rapid disappearance of NO and produces ONOO- . In this paper we report that activation of the microglial NADPH oxidase to produce superoxide is synergistic with NO from iNOS in inducing death of co-cultured neurons, whereas activation of either alone causes little or no death of co-cultured neurons.
Materials & methods
The following materials were purchased from the indicated sources: 1400W.dihydrochloride from Alexis (Nottingham, UK); MK-801 maleate, apocynin and FeTPPS (5,10,15,20-Tetrakis(4-sulfonatophenyl)porphyrinato Iron (III) chloride) from Calbiochem (Nottingham, UK). All other reagents were ordered from Sigma (Poole, UK).
Cerebellar granule cell (CGC) cultures were prepared from 7-day-old Wistar rats, as described in Bal-Price & Brown, 2001. Briefly, the pups were anaesthetised using 5% halothane in oxygen, followed by decapitation. Brains were removed under sterile conditions and the cerebellum dissected. Meninges were removed and the cerebella dissociated in Versene solution (1:5000, Gibco BRL) and plated at 0.25 × 106 cells/cm2 in 24-well plates (in 500 μl DMEM) coated with 0.001% poly-L-lysine. Cultures were maintained in DMEM (Gibco BRL) supplemented with 5% horse serum, 5% foetal calf serum, 38 mM glucose, 5 mM HEPES, 2 mM glutamine, 25 mM KCl and 10 μg/ml gentamicin. Cells were kept at 37°C in a humidified atmosphere of 5% CO2/95% air and used for experiments at 16–18 days in vitro (DIV). Cultures of CGC's contained 22 ± 4% astrocytes and 2 ± 1% microglia as assessed by immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP: a marker for astrocytes) and complement receptor-3 (a marker for microglia), CGC's were identified based on morphology and at 16–18 DIV 76 ± 5% of the cells in the culture were CGC's. All experiments were undertaken in accordance with the UK Animals (Scientific Procedures) Act 1986.
Activation of glia in neuronal-glial cultures
Lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria and interferon-γ (IFN-γ), a pro-inflammatory cytokine, are potent activators of glia when administered together. Neuronal-glial cultures were treated with 100 ng/ml LPS (from Salmonella typhimurium) and 10 ng/ml IFN-γ (rat recombinant, Sigma) for 48 hours (or longer where indicated). The proinflammatory cytokines tumour necrosis factor-α (TNF-α; 10 ng/ml, rat recombinant, Sigma) and interleukin-1β (IL-1β; 10 ng/ml, rat recombinant, Sigma) were also used in combination with IFN-γ to activate the glia in neuronal-glial cultures (48 hours). Where present, inhibitors were added at the same time as LPS/IFN-γ.
In some experiments IL-1β or arachidonic acid (AA, 30 μM) were added to the cultures as well as LPS/IFN-γ. In these experiments, IL-1β or AA were added 24 hours after LPS/IFN-γ addition, but inhibitors were added at the same time as LPS/IFN-γ. Activators, inhibitors and IL-1β or AA were added once only and neuronal death was assessed 144 hours after LPS/IFN-γ addition.
In some experiments, prion protein or a fragment of the human prion protein were used (kindly provided by David R. Brown, University of Bath). Recombinant mouse prion protein was expressed in bacteria and purified using a histidine-tagging method, as described previously . The prion peptide (PrP106-126) with sequence KTNMKHMAGAAAAGAVVGGLG was derived from amino acid residues 106–126 of the human prion protein sequence, and a scrambled sequence of the peptide was used as a control; sequence: NGAKALMGGHGATKVMVGAAA. Prion protein was used at 5 μg/ml and the prion protein peptides at 225 μg/ml.
To activate NADPH oxidase, phorbol 12-myristate 13-acetate (PMA, 50 ng/ml) or benzoyl(benzoyl)-ATP (BzATP, 1 mM) are used and are added to neuronal-glial cultures either alone or at the same time as LPS/IFN-γ.
Enrichment of microglia in neuronal-glial cultures
Primary rat microglia were obtained from mixed glial cultures (astrocytes and microglia). Glial cultures were prepared from the cerebral cortices of 7-day-old Wistar rats (the same brains that were used to isolate cerebellar granule neurons). Meninges were removed from the cerebral hemispheres and then dissociated using a solution of EBSS containing 0.3% BSA, 0.004% DNase I and 0.025% Trypsin. Cells were plated at 0.1 × 106 cells/cm2 in 75 cm2 cell culture flasks (Falcon) coated with 0.0005% poly-L-lysine. Cultures were maintained in DMEM supplemented with 10% foetal calf serum and 1% Penicillin-Streptomycin. Cells were kept at 37°C in a humidified atmosphere of 5% CO2/95% air.
At confluency, glial cultures were used to isolate microglial cells by gently shaking/tapping the mixed glial cultures to dislodge microglia loosely attached to astrocytes. Medium from the mixed glial cultures, containing microglia was removed and centrifuged (135 g for 5 minutes). Microglia were re-suspended in conditioned medium from CGC cultures and added to neuronal-glial cultures in some experiments (50, 000 microglia/cm2). Fifteen minutes after the addition of microglia to some neuronal-glial cultures, LPS/IFN-γ and inhibitors where appropriate were added together. Neuronal death was assessed 48 hours after LPS/IFN-γ addition.
Assessment of glial activation
Activation of glia in the neuronal-glial culture was assessed by NADPH diaphorase staining and measurements of nitrite in the medium. Nitric oxide synthase (NOS) is an NADPH diaphorase, using a chromogen (nitroblue tetrazolium, NBT), and NADPH as the reductant, diaphorase staining was used to detect cells with NOS activity. Following treatment (with cytokines or untreated for control staining) the neuronal glial cultures were fixed with 4% paraformaldehyde in phosphate buffer for 30 minutes at 4°C. After fixation, cells were incubated in 0.3% Triton X-100 (in phosphate buffer) for 5 minutes. Cells were then incubated for 2 hours at 37°C in 0.3% Triton X-100 containing 1 mg/ml NADPH and 0.2 mg/ml NBT. Cells were washed once with 0.3% Triton X-100 and then viewed using an inverted light microscope (Leica).
Nitrite levels in the medium were measured using the Griess reaction. Briefly, aliquots of medium following treatments were taken and centrifuged (8000 g for 5 minutes). 6 mM HCl was added to the supernatant and then 1 mM sulfanilamide and 1 mM N-1 (1-naphthyl)ethylenediamine (NEDA) were added. Absorbance at a wavelength of 548 nm was measured by plate reader (BMG, Fluostar Optima), before and after the addition of NEDA. Nitrite concentrations in samples were calculated from a standard curve of sodium nitrite in DMEM.
Assessment of cell viability
The viability of CGC's was assessed by propidium iodide (PI, 2 μg/ml) and Hoechst 33342 (6 μg/ml) staining, using a fluorescence microscope (Axiovert S-100) and filters for excitation at 365 nm and emission at 420 nm. The cell-impermeable nuclear dye, PI stains the nuclei of cells that have lost plasma membrane integrity and are considered to be necrotic. Using the cell-permeable DNA dye Hoechst 33342, the nuclear morphology of the CGC's was studied. Neuronal nuclei exhibiting irregular Hoechst staining, nuclear shrinkage, chromatin condensation and/or nuclear fragmentation but PI negative were classified as showing chromatin condensation (CC). Individual cells exhibiting both CC and PI staining were included in the PI data. Cells were counted in three microscopic fields in each well (3 wells per treatment) and expressed as a percentage of the total number of neurons. Each treatment was repeated at least three times.
Assessment of microglia proliferation
Microglia cells were identified using Isolectin IB4 (from Griffonia simplicifolia), which has strong affinity for microglia but not astrocytes. An Alexa Fluor 488 conjugate of isolectin IB4 (10 ng/ml) was added to cultures activated with LPS/IFN-γ and treated with IL-1β, AA or prion protein/peptide and incubated for 15 minutes at 37°C. Stained cells (microglia) were visualised and counted by viewing under a fluorescence microscope (excitation 488 nm, emission 530 nm).
Mixed neuronal-glial cultures were stained for the peroxynitrite marker, 3-nitrotyrosine (3-NT). Cultures were untreated (control) or treated with LPS/IFN-γ, PMA, LPS/IFN-γ/PMA or FeTPPS + LPS/IFN-γ/PMA. Cultures were fixed with 4% paraformaldehyde and then incubated with 10 μg/ml of anti-nitrotyrosine monoclonal antibody (Upstate). The primary antibody was detected using a Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories). 3-NT -positive cells were visualised using a fluorescence microscope (excitation 546 nm, emission 590 nm).
Data are expressed as mean ± SEM and were analysed for significance using ANOVA.
Inflammatory activation of glia in neuronal-glial cultures does not lead to substantial death of the co-cultured neurons
Effects of inflammatory activated-glia in mixed neuronal-glial cultures on neuronal death. Neuronal death was assessed by propidium iodide staining (PI, necrosis) or chromatin condensation of neuronal nuclei by Hoechst 33342 staining (CC, a marker of apoptosis) 48 hours after treatment. Nitrite (the primary breakdown product of NO) levels were measured in the culture medium 48 hours following treatments. Statistical differences were established using ANOVA at *p < 0.05 and ***p < 0.001. Data expressed is mean ± SEM, n = 3 or more.
0.9 ± 0.9
0.5 ± 0.6
2.7 ± 3.0
5.7 ± 3.4 *
3.6 ± 1.5 *
18.6 ± 8.4 ***
5.6 ± 0.4 ***
4.3 ± 2.9 *
4.2 ± 2.8
1.1 ± 1.1
0.6 ± 0.7
3.7 ± 2.1
6.3 ± 4.1 *
5.5 ± 3.2 *
4.5 ± 1.4
Relatively pure neuronal cultures (CGC cultures isolated as described in the methods section and then treated with 10 μM arabinoside cytosine at 18 hours to inhibit the proliferation of glia) consisting of 97 ± 4% neurons, 2 ± 1% astrocytes and 1 ± 1% microglia were not affected by the presence of cytokines alone (mean % of chromatin-condensed (CC) and propidium iodide-positive (PI) neurons ± SEM of 3 separate cultures; control: CC: 4 ± 3%, PI: 8 ± 4%; 10 ng/ml IL-1β: CC: 3 ± 2%, PI: 7 ± 3%; 10 ng/ml TNF-α: CC: 3 ± 2%, PI: 9 ± 4%). Additionally, no significant adverse effects were seen even if the concentrations of IL-1β or TNF-α were increased 10-fold (mean % of neurons ± SEM of 1 culture; control: CC: 4 ± 3%, PI: 8 ± 4%; 100 ng/ml IL-1β: CC: 4 ± 2%, PI: 5 ± 4%; 100 ng/ml TNF-α: CC: 4 ± 2%, PI: 6 ± 4%) or if combined with 10 ng/ml IFN-γ treatment (mean % of neurons ± SEM of 2 separate cultures; control: CC: 4 ± 3%, PI: 8 ± 4%; 10 ng/ml IL-1β + IFN-γ: CC: 3 ± 2%, PI: 5 ± 3%; 10 ng/ml TNF-α + IFN-γ: CC: 3 ± 3%, PI: 7 ± 2%).
These results suggest that the cytokines have no direct toxicity for neurons, and although nitric oxide (NO) is produced by iNOS expressed in glia following activation with LPS/IFN-γ, it is not able to kill the co-cultured neurons alone, or the quantities of NO produced are not sufficient to induce widespread death of these mature neuronal cultures.
Simultaneous activation of iNOS and NADPH oxidase results in massive neuronal death, mediated by peroxynitrite
As PMA activates the protein kinase C pathway, the effects of PMA might be due to reasons other than stimulating the microglial NADPH oxidase, such as increased iNOS expression leading to more NO production and neuronal death by NO and not peroxynitrite. However, the levels of nitrite and nitrate in the culture medium of neuronal-glial cultures treated with LPS/IFN-γ/PMA were not different to those found in the absence of PMA (Figure: 2b).
Activation of glia in microglia-enriched neuronal-glial cultures potently kills co-cultured neurons
Prion protein or PrP106-126 induce neuronal death in the presence of inflammatory activation mediated by microglia and NADPH oxidase activation
Prion protein or peptide (PrP106-126) does not affect neuronal survival. Neuronal-glial cultures treated once with either prion protein (5 μg/ml) or PrP106-126 (225 μg/ml) did not induce neuronal death over a period of 7 days (assessed by Hoechst 33342 to visualise chromatin condensation (CC) or propidium iodide (PI) to stain necrotic cells). However, prion protein or PrP106-126 did stimulate the proliferation of microglia in neuronal-glial cultures over the same period of time. Statistical differences were established using ANOVA at *p < 0.05, **p < 0.01 and ***p < 0.001 and are in comparison to untreated cultures (symbol *); data expressed is mean ± SEM, n = 3 or more.
Microglia per field
2.0 ± 1.6
0.6 ± 0.3
22 ± 5
2.9 ± 0.5
0.7 ± 0.6
53 ± 8 ***
1.0 ± 0.8
0.7 ± 0.4
51 ± 7 ***
We found that LPS/IFN-γ induced NOS activity within cultured glia, but induced relatively little death of co-cultured neurons. It has previously been reported that LPS/cytokine-induced iNOS expression in glia is sufficient [5, 8, 39, 40] or insufficient [41–43] to induce death of co-cultured neurons. Similarly, in vivo it has been reported that iNOS expression is sufficient [44, 45] or insufficient [46–48] to induce neuronal death. This suggests that either there is a threshold level for NO/iNOS induced neuronal death , or NO/iNOS-induced neuronal death is conditional upon some other factors. We have recently reported one such conditional factor (hypoxia) that synergises with NO/iNOS to induce neuronal death . In this report we have tested the hypothesis that NO/iNOS induced neuronal death is conditional upon microglial NADPH oxidase activation.
It has previously been shown that PMA stimulation of microglia results in superoxide production through stimulation of NADPH oxidase  and, in the presence of LPS/IFN-γ activated glia (producing NO from iNOS), the superoxide combines with NO to form peroxynitrite . We found that if the NADPH oxidase was stimulated by PMA in the presence of LPS/IFN-γ activated glia, it resulted in extensive death of the co-cultured neurons, while PMA alone induced very little neuronal death. In pathophysiological conditions, extracellular levels of ATP can increase , and ATP can activate purinergic receptors (more specifically P2X7 receptors), which can lead to the activation of NADPH oxidase . We used a specific P2X7 receptor agonist (BzATP) to activate the NADPH oxidase in the presence of iNOS expression (LPS/IFN-γ activated cultures) and we found extensive neuronal death, comparable to that induced by LPS/IFN-γ/PMA. Neuronal-glial cultures activated with LPS/IFN-γ/PMA or LPS/IFN-γ/BzATP induced delayed neuronal death that occurred over 2 days. This is partly due to the time taken for iNOS expression, but it also implies that once peroxynitrite is generated neuronal death is not immediate.
In both cases (LPS/IFN-γ/PMA or LPS/IFN-γ/BzATP), inhibitors of iNOS or NADPH oxidase or a scavenger of peroxynitrite prevented this neuronal death, implicating peroxynitrite as the potential mediator of neuronal death and the source of peroxynitrite as NO from iNOS and superoxide from NADPH oxidase. The putative peroxynitrite marker nitrotyrosine, was found in both neurons and some glia, implying that LPS/IFN-γ/PMA treatment does result in peroxynitrite production that reacts with neurons. Furthermore, the peroxynitrite decomposition catalyst prevents the occurrence of nitrotyrosine-positive neurons following LPS/IFN-γ/PMA treatment. FeTPPS has been shown to rapidly react and catalyse the decomposition of extracellular peroxynitrite  and inhibit tyrosine nitration . The presence of nitrotyrosine immunoreactivity in glia did not appear to induce glial death. It has been found that glia can up-regulate their antioxidant defences to become more resistant to oxidative stress , which may explain the lack of change in glial morphology.
The mechanism of peroxynitrite-induced neuronal death is still unclear but has been proposed to involve DNA-damage induced PARP activation , damage to the mitochondrial respiratory chain , and lipid peroxidation . It is still controversial whether peroxynitrite-induced neuronal death involves activation of the NMDA receptor [58, 59]. We found that a blocker of the NMDA-receptor did not prevent the relatively acute neuronal death induced by LPS/IFN-γ/PMA or LPS/IFN-γ/BzATP, but did prevent the relatively slow neuronal death induced by LPS/IFN-γ/IL-1β or LPS/IFN-γ/AA, although in both cases death was prevented by a peroxynitrite decomposer. It is possible that low, sustained levels of peroxynitrite induce neuronal death via the NMDA receptor, whereas high, acute levels induce death by other means, but we have not directly tested this. We found that IL-1β or AA activated NADPH oxidase hydrogen peroxide production to a lesser extent than PMA but, like PMA, either IL-1β or AA synergised with LPS/IFN-γ to induce neuronal death mediated by peroxynitrite following activation of iNOS and NADPH oxidase. However the neuronal death induced by LPS/IFN-γ/IL-1β or LPS/IFN-γ/AA occurred over 6 days, rather than 2 days as with LPS/IFN-γ/PMA or LPS/IFN-γ/BzATP. This relative delay might be due to the lower level of NADPH oxidase activation and thus peroxynitrite production. Additionally, Il-1β or AA caused microglial proliferation during the 6-day cultures, which may have contributed to the delayed neuronal death. Recently we found that IL-1β or AA stimulated microglial proliferation in microglia-astrocyte cultures via hydrogen peroxide production from NADPH oxidase (manuscript in preparation). Here we have shown that IL-1β or AA stimulate the proliferation of microglia in neuronal-glial cultures, even in the presence of LPS/IFN-γ (which itself inhibits microglial proliferation). In order to test whether an increase in microglia would potentiate LPS/IFN-γ induced neuronal death, we added extra isolated microglia to the neuronal-glial culture, increasing the microglial population from 2% to 15% of cells in the co-culture. In such microglia-enriched cultures, LPS/IFN-γ induced neuronal death was greatly increased. These observations suggest that microglia are essential for inflammatory activated glia-induced neuronal death, and one reason for this may be the expression of NADPH oxidase, which is predominantly localised to microglia .
Transmissible spongiform encephalopathies (Prion diseases) are lethal neurodegenerative disorders characterised by the progressive accumulation of a protease resistant isoform (PrPsc) of the normal host prion protein (PrPc), in amyloid plaques . An inflammatory response, predominantly mediated by microglia, is seen in post-mortem brain tissue, in transgenic models of the disease, and in culture . A peptide, consisting of residues 106–126 (PrP106-126) of the human prion protein, replicates many of the pathological mechanisms involved in prion diseases and provides a good in vitro model. Contrary to published data [38, 62], we observed no neurotoxicity following the addition of PrP106-126 alone to this mature neuronal-glial culture. We found that both prion protein and PrP106-126, but not a scrambled peptide, stimulated microglial proliferation when added to neuronal-glial cultures, and this proliferation was blocked by a NADPH oxidase inhibitor. Both prion protein and peptide were synergistic in killing neurons in the presence of glial iNOS, in a peroxynitrite and microglia-dependent mechanism. The addition of the cellular isoform of prion protein to cell cultures has previously been shown to have no toxic effects . However, in the presence of glial iNOS we found it to induce significant levels of neuronal death, although significantly less than that induced by PrP106-126.
We have shown that in a mature mixed culture of neurons and glia, activation of iNOS or NADPH oxidase alone does not result in substantial neuronal death, but that simultaneous activation of both is synergistic in killing co-cultured neurons. This neuronal death appears to be dependent on microglia, and microglial proliferation is itself stimulated by activating the NADPH oxidase. These results suggest a dual-key hypothesis for inflammatory neurodegeneration; i.e. that activation of glial iNOS or NADPH oxidase alone may be relatively benign, but when activated together they cause peroxynitrite-mediated neuronal death. The conditionality of NO/iNOS-induced neuronal death provides insight into the mechanisms of inflammatory neurodegeneration and suggests that microglial NADPH oxidase may be a key therapeutic target.
This work was supported by the BBSRC, MRC and Alzheimer's Research Trust.
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