- Short report
- Open Access
Complement anaphylatoxin C5a neuroprotects through regulation of glutamate receptor subunit 2 in vitro and in vivo
Journal of Neuroinflammation volume 5, Article number: 5 (2008)
The complement system is thought to be involved in the pathogenesis of numerous neurological diseases. We previously reported that pre-treatment of murine cortico-hippocampal neuronal cultures with the complement derived anaphylatoxin C5a, protects against glutamate mediated apoptosis. Our present study with C5a receptor knock out (C5aRKO) mice corroborates that the deficiency of C5a renders C5aRKO mouse more susceptible to apoptotic injury in vivo. In this study we explored potential upstream mechanisms involved in C5a mediated neuroprotection in vivo and in vitro.
Based on evidence suggesting that reduced expression of glutamate receptor subunit 2 (GluR2) may influence apoptosis in neurons, we studied the effect of human recombinant C5a on GluR2 expression in response to glutamate neurotoxicity. Glutamate analogs were injected into C5aRKO mice or used to treat in vitro neuronal culture and GluR2 expression were assessed in respect with cell death.
In C5aRKO mice we found that the neurons are more susceptible to excitotoxicity resulting in apoptotic injury in the absence of the C5a receptor compared to WT control mice. Our results suggest that C5a protects against apoptotic pathways in neurons in vitro and in vivo through regulation of GluR2 receptor expression.
Complement C5a neuroprotects through regulation of GluR2 receptor subunit.
The complement system is an essential effector of the humoral and cellular immunity involved in cytolysis and immune inflammatory responses. There is now compelling evidence that complement activation in the brain is a double-edged sword in that it can exert beneficial or detrimental effects depending on the pathophysiological context . Complement has been implicated in diverse human neurodegenerative disorders such as Alzheimer's, Huntington's and Pick's disease [2, 3]
The complement system composed of more than 30 proteins is not only activated by antigen-antibody complexes but also by other molecules found in the brain, e.g. myelin and neurofilaments [4, 5]. Activation of the complement cascade results in the release of several anaphylatoxins, notable being C3a and C5a, leading to inflammation. C3a and C5a exert their functions by binding to specific receptors, C3aR and C5aR respectively .
Functional roles for C3a and C5a have been described during development of cerebellum , tissue regeneration  and neuronal death . C3a exerts a neuroprotective effect against excitotoxicity-induced death of neurons that are cultured with astrocytes . C5a mediates apoptosis in neuroblastoma cells , whereas, it is a potent inhibitor of apoptotic cell death in cultured granule neurons .
Neuronal excitation involving the excitatory glutamate receptors is recognized as an important underlying mechanism in neurodegenerative disorders. Excitation resulting from stimulation of the ionotropic glutamate receptors is known to cause neuronal apoptosis. Kainic acid (KA) is an agonist for a subtype of ionotropic glutamate receptor, and administration of KA has been shown to increase production of reactive oxygen species, mitochondrial dysfunction, and apoptosis in neurons in brain . We had earlier reported that the complement component C5 neuroprotects against excitotoxicity; further we showed that mice genetically deficient of complement component C5 revealed a higher susceptibility to KA neurodegeneration [11, 12] suggesting that in addition to their pro-inflammatory mechanisms, specific complement components may also mediate neuroprotection. This hypothesis was further supported by evidence showing that C5a may neuroprotect against glutamate mediated apoptosis through the regulation of mitogen activated protein kinase (MAPK) signal transduction pathways [13, 14] or by inhibition of caspase-3 activity .
Based on the evidence that neuronal death in response to excitotoxic insult involves the regulation of GluR2 receptor expression  and that GluR2 receptor expression is reduced coincidental to increase in expression of apoptotic markers like caspase 3 in Alzheimer's brain  we decided to explore the role of GluR2 receptors in C5a mediated protection in vivo and in vitro. In the present study using C5a receptor knockout (C5aRKO) mice we found that neurons were more susceptible to excitotoxicity resulting in apoptotic injury in the absence of the C5a receptor. Our study suggests that C5a may protect against neurodegenerative excitotoxicity and apoptosis in neuronal cells through the regulation of GluR2 receptor expression in vitro and in vivo.
Primary neuron cultures
Primary cortico-hippocampal cultures of mouse embryonic neurons (gestational day 14–16) were prepared as previously described . Briefly, neurons were seeded at 2 × 105 cells/well in poly-D-lysine (Sigma) coated 96-well plates or at 106 cells/well in poly-D-lysine coated 6-well plates and cultured in serum-free chemically defined medium Neurobasal/B27 (2%) supplement and 1% Penicillin-Streptomycin (Gibco-BRL). The absence of astrocytes (<1–2%) was confirmed by the lack of glial fibrillary acidic protein (GFAP) immunostaining verified in parallel studies (data not shown). Northern blot hybridization of total RNA confirmed C5a receptor (C5aR) expression in these primary cortico-hippocampal neurons as previously described .
Human recombinant (hr) C5a and glutamate toxicity
The hrC5a (Sigma) was solubilized in phosphate buffered saline (PBS) and stored at -20°C in disposable 50 μM aliquots; purity was verified by PAGE-Coomassie blue (BRL) as previously described . Chemokinetic potency of hrC5a (EC50: 1.2 × 10-10 M in human neutrophil) was assessed for biological activity (H. Osaka and G.M. Pasinetti, unpublished observations). In our studies, l-glutamate (Sigma) was dissolved in PBS (pH 7.4) and stored at 4°C in 500 mM aliquots, the C5aR antagonist C177 (gift of Dr. Martin Springer, Merck, NJ; 18), was stored in disposable 1 mM aliquots in PBS at -20°C and the MAPK pathway inhibitor PD98059 (Calbiochem) was solubilized in DMSO and stored at -20°C in 10 mM aliquots. Disposable aliquots of DMSO were also stored at -20°C to mimic freeze thaw conditions in vehicle treated cultures. Cultures were treated with glutamate, hrC5a, C177, PD98059 or vehicle (0.001% PBS or 0.01% DMSO, final concentration), as indicated. Glutamate exposure was performed in 7 days old cultures by adding 50 μM glutamate from concentrated stocks into the existing culture media for 24 hr until neuron cultures were collected for viability assays. All cultures and reagents were demonstrated to be free of endotoxin (<10 pg/ml) by Limulus lysate assay (Sigma).
Kainic Acid (KA) mediated lesions
All procedures involving animals met the guidelines described in the NIH Guide for the Care and Use of Laboratory Animals and had been approved by the Animal Facility of the Mount Sinai School of Medicine. Adult male C57B6 mice (25–36 g) (n = 4) were injected intraventricularly with KA (Sigma) to induce hippocampal lesions as previously described . Mice were anesthetized using Avertin (tribromoethanol, 125 mg/kg body weight). KA (80 ng/0.5 μl volume) or vehicle (0.5 μl PBS) was injected unilaterally into wild type (WT) (control mice) (n = 4) and C5aRKO mice (n = 4) in the lateral ventricle using a 5 μl Hamilton syringe attached to stereotaxic apparatus. Mice were sacrificed 72 hr after injection and brains were quickly removed, rinsed in cold PBS and immersed in methylbutane at -25°C for 3 min. 10 micron slices from the frozen brains were mounted on polylysine-coated slides and stored at -70°C.
Lactic acid dehydrogenase (LDH) Assay
Glutamate neurotoxicity was assessed by measuring the lactate dehydrogenase (LDH) released in culture media 24 hr after glutamate treatment using the Cytotox 96 non-radioactive cytotoxicity assay kit (Promega) (n = 6, experiment repeated three times). Results were quantitated by measuring the wavelength absorbance at 490 nm and were normalized to total LDH in the cells. LDH release was also measured in vehicle treated cultures to control for the effects of DMSO or PBS on cell viability. Data are expressed as percentage of control.
Hematoxylin and eosin (H&E) staining
Assessment of morphological features of apoptotic damage was done by counting neurons with evident pyknotic condensed nuclei surrounded by cytoplasmic eosinophilia using H&E staining as previously described (12). Primary neuronal cultures plated on chambered slides were fixed in 100% methanol for 10 min., and air-dried. Damaged neurons were quantified in vitro from 8–10 randomly selected fields. 10 μm slices from KA and vehicle injected brains of C5aRKO and WT mice were mounted on polylysine-coated slides and damaged neurons were quantified in vivo from the different anatomical regions of the hippocampal formation in each brain slice.
In situ labeling of fragmented DNA (TUNEL assay)
Apoptotic neurons were identified using the terminal deoxynucleotidyl transferase nick end labeling (TUNEL) method of detecting fragmented DNA using the ApopTag kit (Intergen) (n = 4). Briefly, primary cortico-hippocampal cultures on chamber slides were fixed in 100% methanol (15 min) and air-dried following appropriate glutamate and/or hrC5a treatment. The fixed slide cultures were then re-hydrated in PBS and incubated in 0.3% H2O2 for 5 min. at room temperature. Next, the slides were incubated with TdT enzyme in a humidified chamber at 37°C for 1 hr, followed by 30 min. incubation with anti-Digoxigenin conjugate. TUNEL positive cells were visualized using the chromogen 3-3-Diaminobenzidine (DAB) using the ABC substrate kit (Vector) and mounted for microscopy. Results were quantitated from 8–10 randomly selected fields per well (n = 4 wells).
Immunocytochemical (ICC) detection of GluR2
For ICC, slides were re-hydrated in phosphate buffered saline (PBS) and incubated in 0.3% H2O2 for 30 min to block endogenous peroxidase activity. The slides were subsequently incubated with a monoclonal antibody against GluR2 (1:100) (gift of Dr. JH Morrison, Mount Sinai School of Medicine, NY)  overnight at 4°C, followed by incubation with secondary antibody (goat anti-mouse HRP; Pharmingen, CA) (1:200) for 60 min. at room temperature. Neurons positive for GluR2 were visualized with DAB using the ABC kit (Vector) (n = 6 slides per group). The slides were then dehydrated in ascending ethanol series, cleared in xylene and mounted with Permount (Sigma) for microscopy. The immunostaining densities were digitized with a high-resolution charge-coupled-device camera (Sony, Tokyo, Japan) and semi-quantified using Bioquant computer-assisted densitometry (Biometrics, Nashville, TN). Camera aperture and focus were adjusted to provide an optimal image. The overall illumination was also adjusted so that the distribution of relative gray values, ie., number of pixels in the image as a function of gray value (0–255), fell within the limits of the system, typically within 30 to 220 gray value units, avoiding a floor or ceiling effect. Once established, the setting remained constant for all the images acquired for all the ICC experiments. Therefore, when all the parameters were fixed, only tissue staining intensities influenced the measured gray value. GluR2 immunoreactive neurons were quantitated from 8–10 randomly selected fields per well (n = 6 wells) and results were expressed as a percent of control.
Difference between groups was assessed by t-test. One way ANOVA was used to compare three or more treatments. Bonferroni's multiple comparison tests was used to detect differences between treatments. For all statistical analyses, the null hypothesis was rejected at p < 0.05.
C5aRKO mice are more susceptible to KA excitotoxicity and apoptosis than WT littermates
The excitatory neurotransmitter glutamate is known to play an important role in the induction of excitotoxic neurodegeneration through activation of its receptors. Kainic acid (KA) is a potent glutamate receptor agonist with selectivity towards non-NMDA type glutamate receptors. Both apoptotic and necrotic death of neurons are associated with KA-induced excitotoxicity, suggesting the existence of multiple death pathways induced by glutamate receptor neurotoxicity. Histology performed with hematoxylin/eosin on brain slices from C5aRKO and WT C57B6 mice suggested that C5aRKO mice are more susceptible to KA induced neurodegeneration than age and gender matched WT mice. Furthermore, KA lesions in C5aRKO mice resulted in greater number of TUNEL positive neurons than in KA injected WT littermates (Fig. 1) (ANOVA: Hematoxylin/eosin WT and C5aRKO – KA induced: p < 0.01; TUNEL WT and C5aRKO – KA induced: p < 0.006). This result suggests that C5aR exerts a neuroprotective role against KA-induced neurotoxicity.
C5aRKO mice show decreased GluR2 expression in vivoin response to KA compared to WT littermates
Non-NMDA receptor ion channels that can be gated by KA are formed by the glutamate receptor subunits GluR1-GluR4. GluR2 is downregulated in neurons following a wide range of neurological insults  and our subsequent study focused on the role of KA on GluR2 in C5aRKO and its WT control. When KA was administered intraventricularly in C5aRKO and wild type mice, we found that the immunostaining of GluR2 receptor protein expression was decreased in C5aRKO mice in response to KA lesions as assessed by immunocytochemical analysis (Fig. 2). Quantitation of GluR2 immunoreactivity after treatment with KA showed a significant decrease in expression of GluR2 in the C5aRKO mice compared to the vehicle treated littermates (t-test: C5aRKO vehicle vs KA induced: p < 0.05). The results suggest that C5aR plays a neuroprotective role by upregulating GluR2 against KA-induced neurotoxicity possibly by making the cells impermeable to Ca2+ influx.
Glutamate induces a receptor specific decrease in neuronal GluR2 expression in vitro
Previous experiments performed to characterize glutamate excitotoxicity in primary cortico-hippocampal neuronal cultures showed that concentrations of glutamate ranging from 25–100 μM cause neuronal toxicity after 12–24 hr in approximately 30–75% cells in in vitro cell culture experiments . Subsequent experiments were performed using a dose of 50 μM glutamate for 24 hr, which achieved a significant amount of neuronal death within the linear range of increasing glutamate toxicity assessed by LDH assay in our culture. Moreover, treatment of neurons with the non-competitive NMDA receptor antagonist MK801 (10 μM) blocked glutamate toxicity (not shown). WT primary cultures treated with glutamate and hrC5a (100 nM) which is able to neuroprotect significantly against glutamate neurotoxicity in WT cultures did not show significant decrease in GluR2, whereas, primary cultures from C5aR KO mice co-treated with glutamate and hrC5a showed significant decrease in the expression of GluR2 (Fig. 3) (ANOVA: WT Control vs Glutamate p < 0.01; WT Control vs Glutamate+hrC5a p < 0.0001; C5aRKO Control vs Glutamate p < 0.006; C5aRKO Control vs Glutamate+hrC5a p < 0.006). The result suggests that addition of hrC5a to C5aRKO mice neurons do not decrease glutamate toxicity due to the absence of specific C5a receptors.
C5a protection against glutamate mediated GluR2 depletion in vitrois specific for both C5a and glutamate receptors
Previous studies had determined that an optimal dose of 100 nM hrC5a was required to achieve a maximal neuroprotection when pretreated 24 hr before glutamate exposure in primary murine cortico-hippocampal neurons [14, 15]. In our final experiment (repeated thrice) designed to determine the specificity of both C5aR and glutamate receptor we treated WT neurons with the antagonists of C5a. C5a alone enhanced the expression of GluR2 and the C5a antagonist C177 per se had no effect on GluR2 expression. Glutamate alone had a negative effect on GluR2, whereas hrC5a in the presence of glutamate had a positive effect, to the extent that there was an increase in expression of GluR2. However, the hrC5a mediated protection of GluR2 (Fig. 4) in the presence of glutamate in primary neuronal cultures from WT mice was absent when co-treated with C177 (ANOVA: WT Control vs Glutamate p < 0.0001; WT Control vs Glutamate+C5a p < 0.01; WT Glutamate vs Glutamate+C5a p < 0.05; Control vs Glutamate+C177+C5a p < 0.03).
Complement has long been hypothesized to play a role in neuroinflammation and C5a has been postulated to have several different roles in central nervous system disease . Though it has been suggested that C5a-C5aR interaction may lead to increased neuronal cell death in Alzheimer's disease  our group has demonstrated a novel protective role for C5a in inflammation models [14, 15]. Reiman et al.  developed a transgenic mouse that produces C5a exclusively in the brain using the astrocyte-specific, murine glial fibrillary acidic protein (GFAP) promoter. C5a/GFAP mice develop normally and do not demonstrate any signs of spontaneous inflammation or neurodegeneration with age. C5a also plays an important protective role in allergic lung disease by suppressing inflammatory responses and Th2 effector functions as observed in an experimental model of asthma . The C5aRKO mice also develop normally and do not demonstrate any signs of inflammation or neurodegeneration. In this study we observed that C5aRKO mice are more susceptible to apoptosis induced by KA (an analog of glutamate) stimulation compared to the WT littermates.
Neuronal injury mediated by overstimulation of receptors for the major excitatory transmitter, glutamate (Glu), has been implicated in a variety of neurodegenerative conditions . Exposure to neurotoxic concentrations of Glu leads to necrosis via the NMDA receptors , while overstimulation of the non-NMDA receptors, KA and AMPA, commonly produces a pattern of cell death characteristic of apoptosis [25, 26]. Activation of the NMDA receptor stimulates JNK and p38 MAP kinases in cultured cerebellar granule cells (CGCs) , and in hippocampal neurons AMPA and KA receptors stimulate ERKs, JNK and p38 kinases . KA and/or AMPA receptor stimulation results in the marked activation of the ERK kinases in oligodendrocytes  and striatal slices . There is also evidence that C5a inhibits the spontaneous apoptosis of neutrophils, extending their lifespan after recruitment to sites of inflammation . Other inflammatory mediators including perforin may facilitate this protective function of C5a . We had earlier shown that C5a may protect against glutamate-induced apoptosis in neurons through MAPK-mediated regulation of caspase cascades . In this study we found that C5a may protect against apoptotic pathways in neurons in vitro and in vivo in part through regulation of GluR2 receptor expression in the brain.
The relative presence of the GluR2 subunit determines the functional properties of AMPA receptors, the mediators of fast excitatory neurotransmission, play a crucial role in synaptogenesis and formation of neuronal circuitry, as well as in synaptic plasticity. Studies show the levels of GluR2 mRNA and protein in CA3 and/or CA1 are downregulated after KA-induced seizures [16, 32], indicating that KA-induced reduction of GluR2 is related to GluR2 gene transcription. The mechanism of GluR2 downregulation during KA exposure is still not established. Jia et al.  reported that suppression of GluR2 gene promoter activity is associated with KA induced downregulation of GluR2 subunit levels in primary cultured cortical neurons. In our studies we observed that when KA was administered in C5aRKO, there was a reduction of GluR2 receptor protein expression. We hypothesize that during KA induced sensitization in the absence of C5aR, GluR2 is downregulated leading to apoptosis. Animal models of global ischemia and induction of status epilepticus have demonstrated such a downregulation of GluR2 mRNA and protein expression in the CA1 and CA3 regions of rat hippocampus [34, 35].
It is widely known that C5a functions by binding to its specific receptor-C5aR. Our results show that exogenous addition of hrC5a alone increases GluR2 protein expression in WT neurons, whereas there was no increase in GluR2 protein levels in C5aRKO mouse neuron. Glutamate alone was toxic for both WT and C5aRKO mouse neuron, but the level of toxicity was lower in WT mouse neuron in the presence of hrC5a since it could access specific C5a receptors. To prove that C5a protection against glutamate mediated GluR2 depletion was specific for both C5a and glutamate receptors we used the C5a antagonist-C177. Our results show that the complement C5a neuroprotects through regulation of GluR2, in addition to mitogen activated protein kinase (MAPK) signal transduction pathways [13, 14], and inhibition of caspase-3 activity .
In conclusion we found that the complement C5a protects against apoptotic pathways in neurons in vitro and in vivo through regulation of GluR2 receptor subunit.
Glutamate Receptor 2
C5a Receptor Knock Out.
van Beek J, Elward K, Gasque P: Activation of complement in the central nervous system: roles in neurodegeneration and neuroprotection. Ann NY Acad Sci. 2003, 992: 56-71.
Morgan BP, Gasque P, Singharo S, Piddlesden SJ: The role of complement in disorders of the nervous system. Immunopharmacology. 1997, 38: 43-50. 10.1016/S0162-3109(97)00059-3.
Mukherjee P, Pasinetti GM: The role of complement anaphylatoxin C5a in neurodegeneration: implications in Alzheimer's disease. J Neuroimmunol. 2000, 105: 124-130. 10.1016/S0165-5728(99)00261-1.
Pasinetti GM: Inflammatory mechanisms in neurodegeneration and Alzheimer's disease: the role of the complement system. Neurobiol Aging. 1996, 17: 707-716. 10.1016/0197-4580(96)00113-3.
Gasque P, Neal JW, Singhrao SK, McGreal EP, Dean YD, Van BJ, Morgan BP: Roles of the complement system in human neurodegenerative disorders: pro-inflammatory and tissue remodeling activities. Mol Neurobiol. 2002, 25: 1-17. 10.1385/MN:25:1:001.
Benard M, Gonzalez BJ, Schouft MT, Falluel-Morel A, Vaudry D, Chan P, Vaudry H, Fontaine M: Characterization of C3a and C5a receptors in rat cerebellar granule neurons during maturation. Neuroprotective effect of C5a against apoptotic cell death. J Biol Chem. 2004, 279: 43487-43496. 10.1074/jbc.M404124200.
Kimura Y, Madhavan M, Call MK, Santiago W, Tsonis PA, Lambris JD, Del Rio-Tsonis K: Expression of complement 3 and complement 5 in newt limb and lens regeneration. J Immunol. 2003, 170: 2331-2339.
Farkas I, Takahashi M, Fukuda A, Yamamoto N, Akatsu H, Baranyi L, Tateyama H, Yamamoto T, Okada N, Okada H: Complement C5a receptor-mediated signaling may be involved in neurodegeneration in Alzheimer's disease. J Immunol. 2003, 170: 5764-5771.
van Beek J, Nicole O, Ali C, Ischenko A, MacKenzie ET, Buisson A, Fontaine M: Complement anaphylatoxin C3a is selectively protective against NMDA-induced neuronal cell death. Neuroreport. 2001, 12: 289-293. 10.1097/00001756-200102120-00022.
Wang Q, Yu S, Simonyi A, Sun GY, Sun AY: Kainic acid-mediated excitotoxicity as a model for neurodegeneration. Mol Neurobiol. 2005, 31: 3-16. 10.1385/MN:31:1-3:003.
Pasinetti GM, Tocco G, Sakhi S, Musleh WD, DeSimoni MG, Mascarucci P, Schreiber S, Baudry M, Finch CE: Hereditary deficiencies in complement C5 are associated with intensified neurodegenerative responses that implicate new roles for the C-system in neuronal and astrocytic functions. Neurobiol Dis. 1996, 3: 197-204. 10.1006/nbdi.1996.0020.
Tocco G, Musleh W, Sakhi S, Schreiber SS, Baudry M, Pasinetti GM: Complement and glutamate neurotoxicity. Genotypic influences of C5 in a mouse model of hippocampal neurodegeneration. Mol Chem Neuropathol. 1997, 31: 289-300. 10.1007/BF02815131.
Osaka H, McGinty A, Hoepken UE, Lu B, Gerard C, Pasinetti GM: Expression of C5a receptor in mouse brain: role in signal transduction and neurodegeneration. Neuroscience. 1999, 88: 1073-1082. 10.1016/S0306-4522(98)00372-8.
Mukherjee P, Pasinetti GM: Complement anaphylatoxin C5a neuroprotects through mitogen-activated protein kinase-dependent inhibition of caspase 3. J Neurochem. 2001, 77: 43-49. 10.1046/j.1471-4159.2001.00167.x.
Osaka H, Mukherjee P, Aisen PS, Pasinetti GM: Complement-derived anaphylatoxin C5a protects against glutamate-mediated neurotoxicity. J Cell Biochem. 1999, 73: 303-311. 10.1002/(SICI)1097-4644(19990601)73:3<303::AID-JCB2>3.0.CO;2-2.
Friedman LK: Selective reduction of GluR2 protein in adult hippocampal CA3 neurons following status epilepticus but prior to cell loss. Hippocampus. 1998, 8: 511-525. 10.1002/(SICI)1098-1063(1998)8:5<511::AID-HIPO9>3.0.CO;2-W.
Chan SL, Griffin WS, Mattson MP: Evidence for caspase-mediated cleavage of AMPA receptor subunits in neuronal apoptosis and Alzheimer's disease. J Neurosci Res. 1999, 57: 315-323. 10.1002/(SICI)1097-4547(19990801)57:3<315::AID-JNR3>3.0.CO;2-#.
Konteatis ZD, Siciliano SJ, Van Riper G, Molineaux CJ, Pandya S, Fischer P, Rosen H, Mumford RA, Springer MS: Development of C5a receptor antagonists. Differential loss of functional responses. J Immunol. 1994, 153: 4200-4205.
Vissavajjhala P, Janssen WG, Hu Y, Gazzaley AH, Moran T, Hof PR, Morrison JH: Synaptic distribution of the AMPA-GluR2 subunit and its colocalization with calcium-binding proteins in rat cerebral cortex: an immunohistochemical study using a GluR2-specific monoclonal antibody. Exp Neurol. 1996, 142: 296-312. 10.1006/exnr.1996.0199.
Barnum SR: Complement in central nervous system inflammation. Immunol Res. 2002, 26: 7-13. 10.1385/IR:26:1-3:007.
Reiman R, Torres AC, Martin BK, Ting JP, Campbell IL, Barnum SR: Expression of C5a in the brain does not exacerbate experimental autoimmune encephalomyelitis. Neurosci Lett. 2005, 390: 134-138. 10.1016/j.neulet.2005.08.022.
Drouin SM, Sinha M, Sfyroera G, Lambris JD, Wetsel RA: A protective role for the fifth complement component (c5) in allergic airway disease. Am J. 2006, 173: 852-857.
Leist M, Nicotera P: Apoptosis, excitotoxicity, and neuropathology. Exp Cell Res. 1998, 239: 183-201. 10.1006/excr.1997.4026.
Choi DW, Koh JY, Peters S: Pharmacology of glutamate neurotoxicity in cortical cell culture: attenuation by NMDA antagonists. J Neurosci. 1988, 8: 185-196.
Larm JA, Cheung NS, Beart PM: Apoptosis induced via AMPA-selective glutamate receptors in cultured murine cortical neurons. J Neurochem. 1997, 69: 617-622.
Cheung NS, Carroll FY, Larm JA, Beart PM, Giardina SF: Kainate-induced apoptosis correlates with c-Jun activation in cultured cerebellar granule cells. J Neurosci. 1998, 52: 69-82. 10.1002/(SICI)1097-4547(19980401)52:1<69::AID-JNR7>3.0.CO;2-I.
Kawasaki H, Morooka T, Shimohama S, Kimura J, Hirano T, Gotoh Y, Nishida E: Activation and involvement of p38 mitogen-activated protein kinase in glutamate-induced apoptosis in rat cerebellar granule cells. J Biol Chem. 1997, 272: 18518-18521. 10.1074/jbc.272.30.18518.
Liu HN, Larocca JN, Almazan G: Molecular pathways mediating activation by kainate of mitogen-activated protein kinase in oligodendrocyte progenitors. Brain Res Mol Brain Res. 1999, 66: 50-61. 10.1016/S0169-328X(99)00009-1.
Fuller G, Veitch K, Ho LK, Cruise L, Morris BJ: Activation of p44/p42 MAP kinase in striatal neurons via kainate receptors and PI3 kinase. Brain Res Mol Brain Res. 2001, 89: 126-132. 10.1016/S0169-328X(01)00071-7.
Lee A, Whyte MK, Haslett C: Inhibition of apoptosis and prolongation of neutrophil functional longevity by inflammatory mediators. J Leukocyte Biol. 1993, 54: 283-288.
Hopkins JI, Jones J, Morgan BP: Non-lethal effects of perforin on polymorphonuclear leukocytes. Biochem Soc Trans. 1998, 26: S50.
Friedman LK, Veliskova J, Kaur J, Magrys BW, Liu H: GluR2(B) knockdown accelerates CA3 injury after kainate seizures. J Neuropathol Exp Neurol. 2003, 62: 733-750.
Jia YH, Zhu X, Li SY, Ni JH, Jia HT: Kainate exposure suppresses activation of GluR2 subunit promoter in primary cultured cerebral cortical neurons through induction of RE1-silencing transcription factor. Neurosci Lett. 2006, 403: 103-108. 10.1016/j.neulet.2006.04.027.
Gorter JA, Petrozzino JJ, Aronica EM, Rosenbaum DM, Opitz T, Bennett MV, Connor JA, Zukin RS: Global ischemia induces downregulation of Glur2 mRNA and increases AMPA receptor-mediated Ca2+ influx in hippocampal CA1 neurons of gerbil. J Neurosci. 1997, 17: 6179-6188.
Grooms SY, Opitz T, Bennett MV, Zukin RS: Status epilepticus decreases glutamate receptor 2 mRNA and protein expression in hippocampal pyramidal cells before neuronal death. Proc Natl Acad Sci USA. 2000, 97: 3631-3636. 10.1073/pnas.050586497.
Supported by NIA grants AG13799, AG14239 and AG14766 to GMP.
The author(s) declare that they have no competing interests.