Quinolinic acid selectively induces apoptosis of human astrocytes: potential role in AIDS dementia complex
© Guillemin et al; licensee BioMed Central Ltd. 2005
Received: 02 March 2005
Accepted: 26 July 2005
Published: 26 July 2005
There is evidence that the kynurenine pathway (KP) and particularly one of its end products, quinolinic acid (QUIN) play a role in the pathogenesis of several major neuroinflammatory diseases, and more particularly AIDS dementia complex (ADC). We hypothesized that QUIN may be involved in astrocyte apoptosis because: 1) apoptotic astrocytes have been observed in the brains of ADC patients, 2) ADC patients have elevated cerebrospinal fluid QUIN concentrations, and 3) QUIN can induce astrocyte death. Primary cultures of human fetal astrocytes were treated with three pathophysiological concentrations of QUIN. Numeration of apoptotic cells was assessed using double immunocytochemistry for expression of active caspase 3 and for nucleus condensation. We found that treatment of human astrocytes with QUIN induced morphological (cell body shrinking) and biochemical changes (nucleus condensation and over-expression of active caspase 3) of apoptosis. After 24 hours of treatment with QUIN 500 nM and 1200 nM respectively 10 and 14% of astrocytes were undergoing apoptosis. This would be expected to lead to a relative lack of trophic support factors with consequent neuronal dysfunction and possibly death. Astroglial apoptosis induced by QUIN provides another potential mechanism for the neurotoxicity of QUIN during ADC.
KeywordsHuman astrocyte apoptosis quinolinic acid caspase 3 AIDS dementia complex
The kynurenine pathway (KP) is a major route of L-tryptophan catabolism, resulting in the production of nicotinamide adenine dinucleotide and other neuroactive intermediates . Of the metabolites, the N-methyl-D-aspartate (NMDA) receptor agonist and neurotoxin quinolinic acid (QUIN) is likely to be one of the most important. There is evidence that QUIN is involved in the neurocytotoxicity associated with several major inflammatory brain diseases [2, 3] such as AIDS dementia complex (ADC) [4, 5] and other viral brain infections . In the central nervous system (CNS), astrocytes play essential roles including metabolic homeostasis especially providing neurotrophic support, detoxification, maintenance of the blood brain barrier and immune response. During the brain inflammation associated with ADC, many mediators are released and astrocytes are activated leading to their cellular hypertrophy and/or proliferation . For some astrocytes, this prolonged activation may induce apoptosis and the death of these reactive astrocytes can directly and/or indirectly affect functions and survival of the neighbouring neurons and astrocytes . The consequences of astrocyte apoptosis could be either neuroprotective  or neurodamaging [7, 9]. Apoptosis of astrocytes has been described in the brains of patients with ADC [10–12]. Furthermore, in ADC both brain parenchyma and cerebrospinal fluid (CSF) concentrations of QUIN are strongly elevated [4, 5, 13] respectively 300 and 100 fold compared to controls. The HIV-1 proteins Nef and Tat induce macrophages to produce QUIN . The association between brain cell apoptosis and increased levels of QUIN have been found in various other neurodegenerative diseases. We therefore hypothesized that QUIN could be linked with astrocyte apoptosis.
This study demonstrated that treatments with QUIN in pathophysiological concentrations induced morphological and biochemical changes of apoptosis in a sub-group of human astrocytes in a dose dependent manner. Some issues can be raised in term of potential experimental limitations of our in vitro results. Firstly, there are two known subtypes of astrocytes  but only type1 astrocytes can be grown in the primary cultures obtained by our method . Differences between these subtypes are not well known and it is possible that they may have a different sensitivity to QUIN. Secondly, it can be argued that fetal and adult astrocytes may have a different sensitivity to QUIN. This is unlikely because we previously showed the high degree of similarity between immature and mature astrocytes [18, 19]. Moreover, in ADC brains high levels of QUIN  are associated with a high rate of apoptosis of adult astrocytes . Finally, the limited number of apoptotic astrocytes (<15%) implies that only a subset of astrocytes is susceptible to QUIN toxicity. The receptors and signalling pathways that initiate astroglial apoptosis has not been identify yet. However, two main mechanisms may potentially be involved. The first is the activation of NMDA receptors by QUIN , which is already known to mediate neuronal apoptosis with caspase-3 activation [20–23]. However, the presence of NMDA receptors on astrocytes is very controversial [24, 25]. A similar pathway may be involved in neurons and astrocytes, but the presence of functional NMDA receptors on astrocytes still has to be proved. The second possibility, which represent another important aspect of QUIN toxicity, is lipid peroxidation . QUIN in concentration as low as 120 nM induces lipid peroxidation and formation of free radicals leading to neuronal death . This second mechanism is more likely to be involved astrocyte apoptosis induced by QUIN.
Interestingly, our in vitro results on QUIN toxicity in human astrocytes correlate well with previous in vivo studies using animal models. After QUIN injection into the rat brain, a reduction in the density of GFAP-immunoreactivity of astrocytes occurs early (within 6 hours of injection) suggesting astrocytic death; which could be associated with necrosis rather than apoptosis [28, 29]. Another in vivo study using fetuses derived from ewes and damaged by hypoxia and hypoglycemia showed a significant increase in QUIN concentration associated with reduction in GFAP in fetal brain . However, whether QUIN is able to induce astrogliosis and/or astrocytosis is still controversial [28, 29]. QUIN can increase local inflammation by inducing production of cytokines and chemokines by human astrocytes . QUIN increases neuronal release of glutamate and inhibits its uptake by astrocytes, leading to an excessive glutamate concentration in the neuronal microenvironment and secondly to neurotoxicity .
It is still debated whether astroglial activation is beneficial or detrimental to neurons . For some astrocytes, this activation will lead to an early cellular death, as we showed here with QUIN. This may be part of a selective neuroprotective mechanism because on one hand reactive astrocytes may contribute to a decline of neurological function through various mechanisms . On the other hand, the loss of astrocytes compromises their beneficial effects on neuronal function and survival . The results of this study are biologically relevant because brain cell apoptosis (including neurons and astrocytes) and increased levels of QUIN have been found associated in various neurodegenerative diseases and brain disorders such as ADC , Alzheimer's disease [32, 33], cerebral malaria , and traumatic brain injury .
A large majority of the in vivo or ex vivo studies concerning astrocyte apoptosis are related to HIV brain infection and ADC . Thompson et al.  found that there is a correlation between an increased number of HIV DNA-positive astrocytes and an increased number of apoptotic astrocyte and rapid progression of patients to dementia. Furthermore, QUIN production is directly related to the viral load in patients with ADC . There is strong evidence that ADC is associated with NMDA receptor activation [37, 38]. We previously demonstrated that HIV-1 proteins Nef and Tat lead to production of high levels of QUIN by human macrophages ; that inhibition of QUIN production by HIV-1 infected macrophages strongly reduces neurotoxicity ; and finally that QUIN can amplify neuroinflammation and increase astroglial expression of HIV-1 co-receptors . Interestingly, viral induction of IDO in human macrophages differs according to particular HIV-1 isolates  and similarly diverse HIV-1 primary isolates have different effects on astrocyte apoptosis . The present study provides a direct link between the astrocyte apoptosis and high levels of QUIN in ADC brains. These data provide a new mechanism by which HIV-1 may be involved in astrocyte apoptosis directly and indirectly via QUIN production. The characterisation of molecular pathways leading to QUIN-induced astrocyte apoptosis has the potential for developing agents that reduce glial and neuronal death related to QUIN-toxicity. Development of pharmacological agents targeting specific KP enzymes was reviewed recently .
All cell culture media and additives were from Invitrogen (Melbourne, VIC, Australia) unless otherwise stated. QUIN, DAPI, cycloheximide was obtained from Sigma-Aldrich Chemical Co. (Sydney, NSW, Australia). anti-active caspase-3 antibody (polyclonal) was from BD Pharmingen. Mouse mAb anti-GFAP (clone GA-5) was obtained from Novacostra (Newcastle, UK). Secondary goat anti-mouse IgG and anti-rabbit Alexa 488 (green) or Alexa 594 (red) conjugated antibodies were purchased from Molecular Probes (Eugene, OR, USA). All commercial antibodies were used at the concentrations recommended by the manufacturer. Human fetal brains were obtained from 16 to 19 week old fetuses collected following informed consent. Astrocytes were prepared using a protocol adapted from previously described methods . The experiments were carried out in triplicate throughout. Initially, cells were incubated in serum-free AIM-V. The negative control cells were incubated in AIM-V only. The QUIN group was incubated in 350, 500 and 1200 nM QUIN in AIM-V. The positive control cells were incubated in cycloheximide (20 μg/ml)  in AIM-V. Dose-response and time course (3, 6, 12, 24, 48, 72 hours) have been done for QUIN and cycloheximide (data not shown). The optimal time of incubation was 24 hours for the active caspase 3 detection. QUIN concentrations between 350 nM to 1200 nM are known to be found in patients with ADC . The characterization of human brain cell using immunocytochemistry was previously described . The following three controls were performed for each labelling experiment: 1) isotypic antibody controls, 2) incubation with only the secondary labelled antibodies, and 3) estimation of auto-fluorescence of unlabelled cells. The cell counting was performed in a blinded manner. The whole controls and treated chamber slides were counted. Enumeration for each slide was rechecked by the experimenter and cells classified according to the following scheme: DAPI staining for total cell number, GFAP immunoreactivity for astrocytes, and active caspase 3 immunoreactivity together with DAPI for apoptotic cells. Experiments were done in triplicates using brain cells from three different brain tissues. Mean values and standard errors were calculated for each treatment and the results were plotted on a histogram (Fig. 2). Unpaired t tests were performed on the results obtained at 24 hours. Student's t-test was used to analyse the significance of differences between pairs of the three treatments. A p value of <0.05 was regarded as statistically significant.
List of abbreviations
AIDS dementia complex
Glial fibrillary acid protein
human immunodeficiency virus
The St Vincent's Clinic Foundation, The Australian Alzheimer Foundation, the National Health and Medical Research Council, the NSW Health Dept and the University of NSW have supported this work. We thank Ms Belinda Creighton (UNSW) for the advice and aliquot of anti caspase 3 pAb.
- Stone TW: Neuropharmacology of quinolinic and kynurenic acids. Pharmacol Rev. 1993, 45: 309-379.PubMedGoogle Scholar
- Heyes MP, Saito K, Crowley JS, Davis LE, Demitrack MA, Der M, Dilling LA, Elia J, Kruesi MJ, Lackner A: Quinolinic acid and kynurenine pathway metabolism in inflammatory and non-inflammatory neurological disease. Brain. 1992, 115 ( Pt 5): 1249-1273.View ArticleGoogle Scholar
- Stone TW: Endogenous neurotoxins from tryptophan. Toxicon. 2001, 39: 61-73. 10.1016/S0041-0101(00)00156-2.View ArticlePubMedGoogle Scholar
- Guillemin GJ, Kerr SJ, Brew BJ: Involvement of quinolinic acid in AIDS dementia complex. Neurotox Res. 2004, 7: 103-124.View ArticleGoogle Scholar
- Heyes MP, Brew BJ, Martin A, Price RW, Salazar AM, Sidtis JJ, Yergey JA, Mouradian MM, Sadler AE, Keilp J: Quinolinic acid in cerebrospinal fluid and serum in HIV-1 infection: relationship to clinical and neurological status. Ann Neurol. 1991, 29: 202-209. 10.1002/ana.410290215.View ArticlePubMedGoogle Scholar
- Eddelston M, Mucke L: Molecular profile of reactive astrocytes-implications for their role in neurologic disease. Neuroscience. 1993, 54: 15-36. 10.1016/0306-4522(93)90380-X.View ArticleGoogle Scholar
- Tacconi MT: Neuronal death: is there a role for astrocytes?. Neurochem Res. 1998, 23: 759-765. 10.1023/A:1022463527474.View ArticlePubMedGoogle Scholar
- Takuma K, Baba A, Matsuda T: Astrocyte apoptosis: implications for neuroprotection. Prog Neurobiol. 2004, 72: 111-127. 10.1016/j.pneurobio.2004.02.001.View ArticlePubMedGoogle Scholar
- Tavares RG, Tasca CI, Santos CE, Alves LB, Porciuncula LO, Emanuelli T, Souza DO: Quinolinic acid stimulates synaptosomal glutamate release and inhibits glutamate uptake into astrocytes. Neurochem Int. 2002, 40: 621-627. 10.1016/S0197-0186(01)00133-4.View ArticlePubMedGoogle Scholar
- Thompson KA, McArthur JC, Wesselingh SL: Correlation between neurological progression and astrocyte apoptosis in HIV-associated dementia. Ann Neurol. 2001, 49: 745-752. 10.1002/ana.1011.View ArticlePubMedGoogle Scholar
- Ohagen A, Ghosh S, He J, Huang K, Chen Y, Yuan M, Osathanondh R, Gartner S, Shi B, Shaw G, Gabuzda D: Apoptosis induced by infection of primary brain cultures with diverse human immunodeficiency virus type 1 isolates: evidence for a role of the envelope. J Virol. 1999, 73: 897-906.PubMed CentralPubMedGoogle Scholar
- Sabri F, Titanji K, De Milito A, Chiodi F: Astrocyte activation and apoptosis: their roles in the neuropathology of HIV infection. Brain Pathol. 2003, 13: 84-94.View ArticlePubMedGoogle Scholar
- Heyes MP, Ellis RJ, Ryan L, Childers ME, Grant I, Wolfson T, Archibald S, Jernigan TL: Elevated cerebrospinal fluid quinolinic acid levels are associated with region-specific cerebral volume loss in HIV infection. Brain. 2001, 124: 1033-1042. 10.1093/brain/124.5.1033.View ArticlePubMedGoogle Scholar
- Smith DG, Guillemin GJ, Pemberton L, Kerr S, Nath A, Smythe GA, Brew BJ: Quinolinic acid is produced by macrophages stimulated by platelet activating factor, Nef and Tat. J Neurovirol. 2001, 7: 56-60. 10.1080/135502801300069692.View ArticlePubMedGoogle Scholar
- Heyes MP, Saito K, Lackner A, Wiley CA, Achim CL, Markey SP: Sources of the neurotoxin quinolinic acid in the brain of HIV-1- infected patients and retrovirus-infected macaques. Faseb J. 1998, 12: 881-896.PubMedGoogle Scholar
- Raff MC, Abney ER, Cohen J, Lindsay R, Noble M: Two types of astrocytes in cultures of developing rat white matter: differences in morphology, surface gangliosides and growth characteristics. J Neurosc. 1983, 3: 1289-1300.Google Scholar
- Guillemin G, Boussin FD, Croitoru J, Franck-Duchenne M, Le Grand R, Lazarini F, Dormont D: Obtention and characterization of primary astrocyte and microglial cultures from adult monkey brains. J Neurosci Res. 1997, 49: 576-591. 10.1002/(SICI)1097-4547(19970901)49:5<576::AID-JNR8>3.0.CO;2-8.View ArticlePubMedGoogle Scholar
- Guillemin GJ, Croitoru-Lamoury J, Dormont D, Armati PJ, Brew BJ: Quinolinic acid upregulates chemokine production and chemokine receptor expression in astrocytes. Glia. 2003, 41: 371-381. 10.1002/glia.10175.View ArticlePubMedGoogle Scholar
- Croitoru-Lamoury J, Guillemin GJ, Boussin FD, Mognetti B, Gigout LI, Cheret A, Vaslin B, Le Grand R, Brew BJ, Dormont D: Expression of chemokines and their receptors in human and simian astrocytes: Evidence for a central role of TNFalpha and IFNgamma in CXCR4 and CCR5 modulation. Glia. 2003, 41: 354-370. 10.1002/glia.10181.View ArticlePubMedGoogle Scholar
- Yu SP, Yeh CH, Gottron F, Wang X, Grabb MC, Choi DW: Role of the outward delayed rectifier K+ current in ceramide-induced caspase activation and apoptosis in cultured cortical neurons. J Neurochem. 1999, 73: 933-941. 10.1046/j.1471-4159.1999.0730933.x.View ArticlePubMedGoogle Scholar
- Tenneti L, Lipton SA: Involvement of activated caspase-3-like proteases in N-methyl-D-aspartate-induced apoptosis in cerebrocortical neurons. J Neurochem. 2000, 74: 134-142. 10.1046/j.1471-4159.2000.0740134.x.View ArticlePubMedGoogle Scholar
- Kerr SJ, Armati PJ, Brew BJ: Neurocytotoxity of quinolinic acid in human brain cultures. J Neurovirol. 1995, 1: 375-380.View ArticlePubMedGoogle Scholar
- Stone TW: Kynurenines in the CNS: from endogenous obscurity to therapeutic importance. Prog Neurobiol. 2001, 64: 185-218. 10.1016/S0301-0082(00)00032-0.View ArticlePubMedGoogle Scholar
- Schipke CG, Ohlemeyer C, Matyash M, Nolte C, Kettenmann H, Kirchhoff F: Astrocytes of the mouse neocortex express functional N-methyl-D-aspartate receptors. FASEB J. 2001, 00-0439fje.Google Scholar
- Conti F, Minelli A, DeBiasi S, Melone M: Neuronal and glial localization of NMDA receptors in the cerebral cortex. Mol Neurobiol. 1997, 14: 1-18.View ArticlePubMedGoogle Scholar
- Rios C, Santamaria A: Quinolinic acid is a potent lipid peroxidant in rat brain homogenates. Neurochem Res. 1991, 16: 1139-1143. 10.1007/BF00966592.View ArticlePubMedGoogle Scholar
- Behan WM, McDonald M, Darlington LG, Stone TW: Oxidative stress as a mechanism for quinolinic acid-induced hippocampal damage: protection by melatonin and deprenyl. Br J Pharmacol. 1999, 128: 1754-1760. 10.1038/sj.bjp.0702940.PubMed CentralView ArticlePubMedGoogle Scholar
- Bjorklund H, Olson L, Dahl D, Schwarcz R: Short- and long-term consequences of intracranial injections of the excitotoxin, quinolinic acid, as evidenced by GFA immunohistochemistry of astrocytes. Brain Res. 1986, 371: 267-277. 10.1016/0006-8993(86)90362-8.View ArticlePubMedGoogle Scholar
- Dihne M, Block F, Korr H, Topper R: Time course of glial proliferation and glial apoptosis following excitotoxic CNS injury. Brain Res. 2001, 902: 178-189. 10.1016/S0006-8993(01)02378-2.View ArticlePubMedGoogle Scholar
- Nicholls T, Lacey B, Nitsos I, Smythe G, Walker DW: Regional changes in kynurenic acid, quinolinic acid, and glial fibrillary acidic protein concentrations in the fetal sheep brain after experimentally induced placental insufficiency. Am J Obstet Gynecol. 2001, 184: 203-208. 10.1067/mob.2001.108862.View ArticlePubMedGoogle Scholar
- Srebro Z, Dziobek K: Neuroprotection: the role of neuroglia. Folia Med Cracov. 2001, 42: 113-121.PubMedGoogle Scholar
- Barinaga M: Is apoptosis key in Alzheimer's disease?. Science. 1998, 281: 1303-1304. 10.1126/science.281.5381.1303.View ArticlePubMedGoogle Scholar
- Guillemin GJ, Brew BJ, Noonan C, Cullen K: Indoleamine 2,3 dioxygenase and quinolinic acid in plaque and neurons in post-mortem brain tissue from Alzheimer’s disease and control cases. Neuropathol Appl Neurobiol. 2005, in press:Google Scholar
- Sanni LA, Thomas SR, Tattam BN, Moore DE, Chaudhri G, Stocker R, Hunt NH: Dramatic changes in oxidative tryptophan metabolism along the kynurenine pathway in experimental cerebral and noncerebral malaria. Am J Pathol. 1998, 152: 611-619.PubMed CentralPubMedGoogle Scholar
- Sinz EH, Kochanek PM, Heyes MP, Wisniewski SR, Bell MJ, Clark RS, DeKosky ST, Blight AR, Marion DW: Quinolinic acid is increased in CSF and associated with mortality after traumatic brain injury in humans. J Cereb Blood Flow Metab. 1998, 18: 610-615. 10.1097/00004647-199806000-00002.View ArticlePubMedGoogle Scholar
- Brew BJ, Rosenblum M, Cronin K, Price RW: AIDS dementia complex and HIV-1 brain infection: clinical-virological correlations. Ann Neurol. 1995, 38: 563-570. 10.1002/ana.410380404.View ArticlePubMedGoogle Scholar
- Lipton SA: Neuronal injury associated with HIV-1: approaches to treatment. Annu Rev Pharmacol Toxicol. 1998, 38: 159-177. 10.1146/annurev.pharmtox.38.1.159.View ArticlePubMedGoogle Scholar
- Chao CC, Hu S, Peterson PK: Glia: the not so innocent bystanders. J Neurovirol. 1996, 2: 234-239.View ArticlePubMedGoogle Scholar
- Kerr SJ, Armati PJ, Pemberton LA, Smythe G, Tattam B, Brew BJ: Kynurenine pathway inhibition reduces neurotoxicity of HIV-1-infected macrophages. Neurology. 1997, 49: 1671-1681.View ArticlePubMedGoogle Scholar
- Grant RS, Naif H, Thuruthyil SJ, Nasr N, Littlejohn T, Takikawa O, Kapoor V: Induction of indolamine 2,3-dioxygenase in primary human macrophages by human immunodeficiency virus type 1 is strain dependent. J Virol. 2000, 74: 4110-4115. 10.1128/JVI.74.9.4110-4115.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Stone TW, Darlington LG: Endogenous kynurenines as targets for drug discovery and development. Nat Rev Drug Discov. 2002, 1: 609-620. 10.1038/nrd870.View ArticlePubMedGoogle Scholar
- Tsuchida T, Kato T, Yamada A, Kawamoto K: Cycloheximide induces apoptosis of astrocytes. Pathol Int. 2002, 52: 181-185. 10.1046/j.1440-1827.2002.01336.x.View ArticlePubMedGoogle Scholar
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