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• W2000854765 abstract "There is now clear evidence that the Complement anaphylatoxin C3a and C5a receptors (C3aR and C5aR) are expressed in glial cells, notably in astrocytes and microglia. In contrast, very few data are available concerning the possible expression of these receptors in neurons. Here, we show that transient expression of C3aR and C5aR occurs in cerebellar granule neurons in vivo with a maximal density in 12-day-old rat, suggesting a role of these receptors during development of the cerebellum. Expression of C3aR and C5aR mRNAs and proteins was also observed in vitro in cultured cerebellar granule cells. Quantification of the mRNAs by real-time reverse transcription-PCR showed a peak of expression at day 2 in vitro (DIV 2); the C3aR and C5aR proteins were detected by Western blot analysis at DIV 4 and by flow cytometry and immunocytochemistry in differentiating neurons with a maximum density at DIV 4–9. Apoptosis of granule cells plays a crucial role for the harmonious development of the cerebellar cortex. We found that, in cultured granule neurons in which apoptosis was induced by serum deprivation and low potassium concentration, a C5aR agonist promoted cell survival and inhibited caspase-3 activation and DNA fragmentation. The neuroprotective effect of the C5aR agonist was associated with a marked inhibition of caspase-9 activity and partial restoration of mitochondrial integrity. Our results provide the first evidence that C3aR and C5aR are both expressed in cerebellar granule cells during development and that C5a, but not C3a, is a potent inhibitor of apoptotic cell death in cultured granule neurons. There is now clear evidence that the Complement anaphylatoxin C3a and C5a receptors (C3aR and C5aR) are expressed in glial cells, notably in astrocytes and microglia. In contrast, very few data are available concerning the possible expression of these receptors in neurons. Here, we show that transient expression of C3aR and C5aR occurs in cerebellar granule neurons in vivo with a maximal density in 12-day-old rat, suggesting a role of these receptors during development of the cerebellum. Expression of C3aR and C5aR mRNAs and proteins was also observed in vitro in cultured cerebellar granule cells. Quantification of the mRNAs by real-time reverse transcription-PCR showed a peak of expression at day 2 in vitro (DIV 2); the C3aR and C5aR proteins were detected by Western blot analysis at DIV 4 and by flow cytometry and immunocytochemistry in differentiating neurons with a maximum density at DIV 4–9. Apoptosis of granule cells plays a crucial role for the harmonious development of the cerebellar cortex. We found that, in cultured granule neurons in which apoptosis was induced by serum deprivation and low potassium concentration, a C5aR agonist promoted cell survival and inhibited caspase-3 activation and DNA fragmentation. The neuroprotective effect of the C5aR agonist was associated with a marked inhibition of caspase-9 activity and partial restoration of mitochondrial integrity. Our results provide the first evidence that C3aR and C5aR are both expressed in cerebellar granule cells during development and that C5a, but not C3a, is a potent inhibitor of apoptotic cell death in cultured granule neurons. The Complement is an important component of the immune system that has the capacity to recognize and eliminate a large range of pathogens. Activation of the Complement cascade results in the generation of several fragments, especially the anaphylatoxins C3a and C5a, which share several biological activities including mast cell degranulation, vasodilation, smooth muscle contraction, and recruitment of immune cells to the site of inflammation (1Frank M.M. Fries L.F. Immunol. Today. 1991; 12: 322-326Abstract Full Text PDF PubMed Scopus (413) Google Scholar). C3a and C5a exert their biological effects through specific binding to membrane receptors, named, respectively, C3aR 1The abbreviations used are: C3aR, C3a receptor; C5aR, C5a receptor; EGL, external granular layer; MAP-C3a/C5a, multiple-associated peptide C3a/C5a; DIV, day(s) in vitro; S+K25, medium with fetal calf serum containing 25 mm KCl; S-K5, serum-free medium containing 5 mm KCl; RT, reverse transcription; PBS, phosphate-buffered saline; BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. and C5aR (CD88). These two receptors belong to the seven-transmembrane receptor superfamily and are coupled to a Gi protein (2Rollins T.E. Siciliano S. Kobayashi S. Cianciarulo D.N. Bonilla-Argudo V. Collier K. Springer M.S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 971-975Crossref PubMed Scopus (72) Google Scholar, 3Norgauer J. Dobos G. Kownatzki E. Dahinden C. Burger R. Kupper R. Gierschik P. Eur. J. Biochem. 1993; 217: 289-294Crossref PubMed Scopus (93) Google Scholar). C3aR and C5aR are expressed in myeloid cells (4Gerard N.P. Hodges M.K. Drazen J.M. Weller P.F. Gerard C. J. Biol. Chem. 1989; 264: 1760-1766Abstract Full Text PDF PubMed Google Scholar, 5Oppermann M. Raedt U. Hebell T. Schimidt B. Zimmermann B. Götze O. J. Immunol. 1993; 151: 3785-3794PubMed Google Scholar, 6Gerard C. Gerard N.P. Annu. Rev. Immunol. 1994; 12: 775-808Crossref PubMed Scopus (388) Google Scholar, 7Ames R.S. Li Y. Sarau H.M. Nuthulaganti P. Foley J.J. Ellis C. Kumar C. J. Biol. Chem. 1996; 271: 20231-20234Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 8Crass T. Raffetseder U. Martin U. Grove M. Klos A. Köhl J. Bautsch W. Eur. J. Immunol. 1996; 26: 1944-1950Crossref PubMed Scopus (157) Google Scholar) and, more surprisingly, in non-myeloid cells and tissues (7Ames R.S. Li Y. Sarau H.M. Nuthulaganti P. Foley J.J. Ellis C. Kumar C. J. Biol. Chem. 1996; 271: 20231-20234Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 8Crass T. Raffetseder U. Martin U. Grove M. Klos A. Köhl J. Bautsch W. Eur. J. Immunol. 1996; 26: 1944-1950Crossref PubMed Scopus (157) Google Scholar, 9Haviland D.L. Mc Coy R.L. Whitehead W.T. Akama H. Molenti E.P. Brown A. Haviland J.C. Parks W.C. Perlmutter D.H. Wetsel R.A. J. Immunol. 1995; 154: 1861-1869PubMed Google Scholar, 10Foreman K.E. Vaporciyan A.A. Bonish B.K. Jones M.L. Johnson K.J. Glovsky M.M. Eddy S.M. Ward P.A. J. Clin. Investig. 1994; 94: 1147-1155Crossref PubMed Scopus (415) Google Scholar). In the central nervous system it has been shown that C3aR and C5aR are constitutively expressed by glial cells in vitro (11Ischenko A. Sayah S. Patte C. Andreev S. Gasque P. Schouft M.T. Vaudry H. Fontaine M. J. Neurochem. 1998; 71: 2487-2496Crossref PubMed Scopus (44) Google Scholar, 12Gasque P. Chan P. Fontaine M. Ischenko A. Lamacz M. Götze O. Morgan B.P. J. Immunol. 1995; 155: 4882-4889PubMed Google Scholar, 13Sayah S. Patte C. Gasque P. Chan P. Ischenko A. Vaudry H. Fontaine M. Mol. Brain Res. 1997; 48: 215-222Crossref PubMed Scopus (28) Google Scholar, 14Lacy M. Jones J. Whitemore S.R. Haviland D.L. Wetsel R.A. Barnum S.R. J. Immunol. 1995; 61: 71-78Scopus (164) Google Scholar) and in vivo (15Müller-Ladder U. Jones J. Gay S. Wetsel R.A. Raine C. Barnum S.R. J. Neurol. Sci. 1996; 144: 135-144Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and that the expression of C3aR and C5aR is increased in inflammatory conditions (15Müller-Ladder U. Jones J. Gay S. Wetsel R.A. Raine C. Barnum S.R. J. Neurol. Sci. 1996; 144: 135-144Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 16Gasque P. Singhrao S.K. Neal J.W. Gotze O. Morgan B.P. Am. J. Pathol. 1997; 150: 31-41PubMed Google Scholar, 17Van Beek J. Bernaudin M. Petit E. Gasque P. Nouvelot A. MacKenzie E.T. Fontaine M. Exp. Neurobiol. 2000; 161: 373-382Crossref PubMed Scopus (132) Google Scholar). Low constitutive expression of C3aR and/or C5aR in neurons has been recently demonstrated by in situ hybridization and immunohistochemistry analysis in the cerebral cortex (18Stahel P.F. Frei K. Eugster H.P. Fontana A. Hummel K.M. Wetsel R.A. Ames R.S. Barnum S.R. J. Immunol. 1997; 159: 861-869PubMed Google Scholar, 19Davoust N. Jones J. Stahel P.F. Ames R.S. Barnum S.R. Glia. 1999; 26: 201-211Crossref PubMed Scopus (120) Google Scholar), spinal cord (20Nataf S. Davoust N. Barnum S.R. J. Neuroimmunol. 1998; 91: 147-155Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), hippocampus (19Davoust N. Jones J. Stahel P.F. Ames R.S. Barnum S.R. Glia. 1999; 26: 201-211Crossref PubMed Scopus (120) Google Scholar), and Purkinje cells in the cerebellum (19Davoust N. Jones J. Stahel P.F. Ames R.S. Barnum S.R. Glia. 1999; 26: 201-211Crossref PubMed Scopus (120) Google Scholar). A strong up-regulation of C5aR expression in neurons has been observed under various pathological conditions such as experimental excitotoxic neurodegeneration (21Osaka H. Mukherjee P. Aisen P.S. Pasinetti G.M. J. Cell. Biochem. 1999; 73: 303-311Crossref PubMed Scopus (120) Google Scholar), bacterial meningitis (18Stahel P.F. Frei K. Eugster H.P. Fontana A. Hummel K.M. Wetsel R.A. Ames R.S. Barnum S.R. J. Immunol. 1997; 159: 861-869PubMed Google Scholar), multiple sclerosis (15Müller-Ladder U. Jones J. Gay S. Wetsel R.A. Raine C. Barnum S.R. J. Neurol. Sci. 1996; 144: 135-144Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 16Gasque P. Singhrao S.K. Neal J.W. Gotze O. Morgan B.P. Am. J. Pathol. 1997; 150: 31-41PubMed Google Scholar), experimental autoimmune encephalomyelitis (20Nataf S. Davoust N. Barnum S.R. J. Neuroimmunol. 1998; 91: 147-155Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and traumatic axonal injury (22Stahel P.F. Kossmann T. Morganti-Kossmann M.C. Hans V.H.J. Barnum S.R. Mol. Brain Res. 1997; 50: 205-212Crossref PubMed Scopus (63) Google Scholar). Functional roles for C3a and C5a have been described in neuronal cell death. For instance, C3a exerts a neuroprotective effect against excitotoxicity-induced death of neurons that are cultured with astrocytes (23Van Beek J. Nicole O. Ali C. Ischenko A. MacKenzie E.T. Buisson A. Fontaine M. Neuroreport. 2001; 12: 789-793Crossref Scopus (92) Google Scholar). Recently, it has been reported that C5a mediates apoptosis in neuroblastoma cells (24Farkas I. Baranyi L. Liposits Z.S. Yamamoto T. Okada H. Neuroscience. 1998; 86: 903-911Crossref PubMed Scopus (81) Google Scholar, 25Farkas I. Takahashi M. Fukuda A. Yamamoto N. Akatsu H. Baranyi L. Tateyama H. Yamamoto T. Okada N. Okada H. J. Immunol. 2003; 170: 5764-5771Crossref PubMed Scopus (63) Google Scholar). In contrast, C5a protects differentiated human neuroblastoma cells from the neurotoxic effect of the amyloid Aβ peptide (26O'Barr S.A. Caguioa J. Gruol D. Perkins G. Ember J.A. Hugli T. Cooper N.R. J. Immunol. 2001; 166: 4154-4162Crossref PubMed Scopus (126) Google Scholar), and a possible neuroprotective role for C5a has been suggested in Alzheimer's disease (27Mukherjee P. Pasinetti G.M. J. Neuroimmunol. 2000; 105: 124-130Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Moreover, C5-deficient mice are more sensitive to kainic acid excitotoxicity (28Pasinetti G.M. Neurobiol. Aging. 1996; 17: 707-716Crossref PubMed Scopus (109) Google Scholar), and C5a has been found to protect neurons from apoptotic cell death in vitro and in vivo in a model of intracerebroventricular kainic injection in mice (21Osaka H. Mukherjee P. Aisen P.S. Pasinetti G.M. J. Cell. Biochem. 1999; 73: 303-311Crossref PubMed Scopus (120) Google Scholar). These observations suggest that the neurotoxic or neuroprotective effects of anaphylatoxins depend on many factors such as the neuronal population considered, the state of neuronal differentiation, and the pathological context. Development of the cerebellum involves a delicate balance between proliferation, differentiation, and programmed cell death (29Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2409) Google Scholar, 30Pettmann B. Henderson C.E. Neuron. 1998; 20: 633-647Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar). In the developing brain programmed cell death contributes to the removal of neuronal precursors that fail to establish appropriate synaptic connections (31Oppenheim R.W. Annu. Rev. Neurosci. 1991; 14: 453-501Crossref PubMed Scopus (2759) Google Scholar) and, thus, plays a crucial role in morphogenesis of the cerebellum. During the first weeks of postnatal life, immature neurons generated in the external granular layer (EGL) migrate along radial processes of glial cells through the molecular layer to reach their final destination within the internal granular layer (32Altman J. J. Comp. Neurol. 1972; 145: 353-514Crossref PubMed Scopus (819) Google Scholar, 33Wood K.A. Dipasquale B. Youle R.J. Neuron. 1993; 11: 621-632Abstract Full Text PDF PubMed Scopus (318) Google Scholar). Only half of granule cells give rise to mature neurons, because massive cell loss occurs in the EGL and, later on, in the internal granular layer (33Wood K.A. Dipasquale B. Youle R.J. Neuron. 1993; 11: 621-632Abstract Full Text PDF PubMed Scopus (318) Google Scholar, 34Raff M.C. Barres B.A. Burne J.F. Coles H.S. Ishizaki Y. Jacobson M.D. Science. 1993; 262: 695-700Crossref PubMed Scopus (1353) Google Scholar). Immature cerebellar granule neurons from early postnatal rats can be maintained alive in serum-containing medium by elevating extracellular potassium concentration (25 mm) (35Gallo V. Kingsbury A. Balàzs R. Jorgensen O.S. J. Neurosci. 1987; 7: 2203-2213Crossref PubMed Google Scholar), i.e. in conditions that mimic in vitro the innervation that neurons receive in vivo from mossy fibers (36Balàzs R. Hack N. Jorgensen O.S. Neurosci. Lett. 1988; 87: 80-86Crossref PubMed Scopus (200) Google Scholar). This state of depolarization improves survival of granular neurons and allows neuronal differentiation and maturation in vitro. Apoptosis of cultured cerebellar granule cells can be reliably induced by removing serum and lowering the extracellular potassium concentration (37D'Mello S.R. Galli C. Ciotti T. Calissano P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10989-10993Crossref PubMed Scopus (851) Google Scholar, 38Galli C. Meucci O. Scorziello A. Werge T.M. Calissano P. Schettini G. J. Neurosci. 1995; 25: 1172-1179Crossref Google Scholar, 39Miller T.M. Johnson Jr., E.M. J. Neurosci. 1996; 16: 7487-7495Crossref PubMed Google Scholar). Cerebellar granule neurons is, thus, a very suitable model to study the functions of anaphylatoxin receptors (40Vaudry D. Falluel-Morel A. Leuillet S. Vaudry H. Gonzalez B.J. Science. 2003; 300: 1532-1534Crossref PubMed Scopus (54) Google Scholar). The aim of the present study was to investigate the expression of C3aR and C5aR during maturation of cerebellar granule neurons and the possible effect of their ligands C3a or C5a on apoptosis induced by serum deprivation and low potassium concentration. Animals—Wistar rats (Charles River Laboratories, Arbresle, France) were kept in a temperature-controlled room (21 ± 1 °C) under an established photoperiod (lights on 07.00–19.00 h) with free access to food and tap water. Animal manipulations were performed according to the recommendations of the French Ethics Committee and under the supervision of authorized investigators. Reagents—Multiple-associated peptide (MAP)-C3a and MAP-C5a are eight peptidic monomers corresponding to the last 13 amino acids in the C-terminal region of the anaphylatoxins attached to a polylysine comb (a gift of Dr. Ischenko). These peptides were previously used and were shown to exhibit the same activity as recombinant or natural anaphylatoxins (41Sayah S. Ischenko A. Zhaklov A. Bonnard A.S. Fontaine M. J. Neurochem. 1999; 72: 2426-2436Crossref PubMed Scopus (73) Google Scholar, 42Monsinjon T. Gasque P. Ischenko A. Fontaine M. FEBS Lett. 2001; 487: 339-346Crossref PubMed Scopus (41) Google Scholar, 43Jauneau A.C. Ischenko A. Chan P. Fontaine M. FEBS Lett. 2003; 537: 17-22Crossref PubMed Scopus (45) Google Scholar). C5aR antagonist (44Finch A.M. Wong A.K. Paczkowski N.J. Wadi S.K. Craik D.J. Fairlie D.P. Taylor S.M. J. Med. Chem. 1999; 42: 1965-1974Crossref PubMed Scopus (216) Google Scholar) was synthesized by Dr J. Leprince (INSERM U413). Anti-mouse C3aR and anti-rat C5aR were obtained by immunization of rabbits with recombinant fusion proteins consisting of glutathione S-transferase fused to the region 161–333 of the large extracellular loop of mouse C3aR and to the 29-amino acid peptide corresponding to the 8–36 sequence of the N-terminal region of rat C5aR (according to EMBL library index accession numbers NM_009779 and Y09613, respectively). Cell Culture—Granule cell suspensions were prepared from cerebelli of 8-day-old Wistar rats, as described previously (45Gonzalez B.J. Leroux P. Lamacz M. Bodenant C. Balazs R. Vaudry H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9627-9631Crossref PubMed Scopus (96) Google Scholar). Dispersed cells were seeded in multiwell plates or dishes (Falcon, BD Biosciences) coated with 10 μm poly-l-lysine (Sigma) at a density of 2.5 × 105 cells per cm2. Granule neurons were plated in a medium consisting of 75% Dulbecco's modified Eagle's medium (Invitrogen) and 25% Ham's F-12 medium (Sigma) containing 2 mm glutamine, 1 mm sodium pyruvate, 1% antibiotic-antimycotic solution (BioWhittaker, Verviers, Belgium) supplemented with 25 mm KCl and 10% fetal calf serum (S+K25). Proliferation of non-neuronal cells was blocked by the addition of 10 μm cytosine β-d-arabinofuranoside (Sigma) on day 1 in vitro (DIV 1). Cultures prepared by this method were enriched in granule neurons by more than 95%; the cell population was negative for glial fibrillary acidic protein (Sigma), OX 42 (ATCC, Manassas, VA), and calbindin D28K (Sigma) staining (data not shown). Cells were grown at 37 °C in a humidified incubator with an atmosphere of 5% CO2. RNA Isolation and Real-time Quantitative Reverse Transcription (RT)-PCR—Total RNAs were extracted from cultured granule cells by the guanidinium isothiocyanate method followed by ultracentrifugation onto a CsCl cushion as previously described (46Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 7.19-7.21Google Scholar). Total RNAs (2 μg) were incubated at 70 °C for 10 min, and RT was carried out in a thermocycler (Hybaid, Cera-Labo, Ecquevilly, France) at 37 °C for 1 h with 5 mm dithiothreitol, 1 mm dNTPs, 500 pmol of random hexamer primers pdN6 (Amersham Biosciences), 120 units of RNasin (Promega, Charbonnières, France), and 400 units of Moloney murine leukemia virus (M-MLV) RT (Promega) in the reaction buffer (50 mm Tris-HCl, 75 mm KCl, 3 mm MgCl2, and 5 m m dithiothreitol). The absence of genomic contaminant was checked routinely by RT-PCR in negative control samples in which either the RNA samples were replaced with sterile water or the Moloney murine leukemia virus RT was omitted. PCR was carried out by using the LightCycler FastStart DNA Master SYBR Green I kit (Roche Applied Science), which is specially adapted for the LightCycler instrument (Roche Applied Science), and the PCR reaction was performed in a final volume of 20 μl in the LightCycler glass capillaries according to the manufacturer's recommendations. The primers used in our study were: RC3a forward (GAC CTA CAC TCA GGG C), RC3a reverse (ATG ACG GAC GGG ATA AG), RC5a forward (ATG CCT GCA GAT GGC GTT TA), and RC5a reverse (CAG AAA CCA AAT GGC GTT GAC). The housekeeping gene primers were glyceraldehyde-3-phosphate dehydrogenase forward (TGC CAT CAA CGA CCC CTT CA) and glyceraldehyde-3-phosphate dehydrogenase reverse (TGA CCT TGC CCA CAG CCT TG). Primers were chosen according to their cDNA sequences reported in the EMBL data library index accession numbers M33197 for glyceraldehyde-3-phosphate dehydrogenase, NM_032060 for C3aR, and Y09613 for C5aR. Southern Blot Analysis—RT-PCR products (5 μl) were loaded onto a 1% agarose gel and separated by electrophoresis. The gel was treated for 10 min in 0.15 m HCl and neutralized for 30 min in 0.4 m NaOH. Southern blotting was performed by capillary transfer for 16 h in 0.4 m NaOH onto Nylon Plus membranes (Amersham Biosciences). The blot was neutralized for 10 min in 2× SSPE (0.3 m NaCl, 17 mm Na2HPO4, and 50 mm EDTA). Rat C5aR and mouse C3aR cDNA probes were labeled by using Rediprime Kit (Amersham Biosciences) with [32P]dCTP (Amersham Biosciences) at a specific activity of 2 × 109 cpm/μg. Membranes for C5aR were pre-hybridized in homologous conditions at 42 °C for 4 h in a solution containing 50% formamide, 5× SSPE, 1% SDS, 5× Denhardt's, 5% dextran sulfate, and 100 μg/ml herring sperm DNA, whereas membranes for C3aR were pre-hybridized in heterologous conditions in a solution containing 20% formamide, 4× SSPE, 0.05 m Tris-HCl, pH 7.5, 1 m NaCl, 0.1% SDS, 10× Denhardt's, and 100 μg/ml herring sperm DNA. Hybridization was performed at 42 °C for 16 h in the same solutions supplemented with 108 cpm of labeled probe. The membranes were washed briefly 3 times at room temperature in 2 × SSPE, 0.1% SDS, for 1 h at 68 °C for C5aR and 60 °C for C3aR in 2× SSPE, 0.1% SDS, and for 1 h at 68 °C for C5aR and 60 °C for C3aR in 1× SSPE, 1% SDS and exposed for 4 h at room temperature onto X-Omat film (Eastman Kodak Co.). Flow Cytometry Analysis—Neurons were harvested from cultures by incubation in phosphate-buffered saline (PBS) containing 10 mm EDTA. Cells were washed and resuspended in PBS containing 1% bovine serum albumin (BSA) and incubated with 10 μg/ml non-immune mouse IgG for 15 min. After washing, cells were incubated with 2 μg/ml anti-C3aR or anti-C5aR IgG or anti-γ-aminobutyric acid, type A receptor α6 subunit (Chemicon, Euromedex, Souffelweyersheim, France) diluted 1:100 in PBS containing 1% BSA at 4 °C for 30 min. After washing, cells were incubated at 4 °C for 30 min with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Jackson Immuno Research, Interchim, Montluçon, France) diluted at 1:200 and measured in a FL-1 channel (530 ± 15 nm bandpass filter). For two-color flow cytometric analysis, cells were incubated in PBS containing 1% BSA and 0.1% saponin for 30 min with biotinylated anti-C3aR or biotinylated anti-C5aR (2 μg/ml) and monoclonal anti-neurofilament 68 clone NR4 (Sigma) diluted 1:100. After washing, cells were incubated for 30 min with streptavidin cyanine 5 (measured in FL-4 channel, 661 ± 8 nm bandpass filter) diluted 1:500 and with fluorescein isothiocyanateconjugated goat anti-mouse IgG (FL-1) diluted 1:200. Cells were washed before analysis on a FACScalibur flow cytometer (BD Biosciences) operating with the Cell Quest™ software. Dead cells and debris were excluded from the analysis by gating living neurons from size/structure density plots. Data were displayed on a logarithmic scale with increasing fluorescence intensity. Each histogram plot was recorded for at least 10,000 gated events. Immunocytochemistry—Granule cells were fixed with 1% formaldehyde in PBS at room temperature for 20 min. Fixed cells were incubated 1 h at room temperature with 2 μg/ml anti-C3aR or anti-C5aR or with anti-neurofilament 68 diluted 1:100 in PBS containing 1% BSA. After several rinses in PBS, cells were incubated at room temperature for 2 h with peroxidase-conjugated Affinipure goat anti-rabbit or anti-mouse IgG (Jackson Immuno Research) diluted 1:1000 in PBS containing 1% bovine serum albumin. Cells were washed twice with PBS. The immunoreactivity was revealed by adding 0.5 mg/ml diaminobenzidine (Sigma) and 0.015% H2O2 in PBS. The reaction was stopped by removing the diaminobenzidine solution and by rinsing in a large volume of water. The immunolabeling was analyzed using a computer-assisted microscope AxioVert (Zeiss, Le Pecq, France). Immunohistochemistry—Rats were deeply anesthetized with chloral hydrate (400 mg/kg intraperitoneal) and transcardially perfused and fixed with 4% paraformaldehyde in PBS. The cerebellum was removed, post-fixed for 12 h in 4% paraformaldehyde, and placed in 15 and 30% sucrose until they sank. Tissue sections (10-μm thick) were cut with a cryostat (Leica, Rueil-Malmaison, France). For immunohistochemical detection, endogenous peroxidase was blocked by incubating sections in 3% H2O2 in methanol at room temperature for 10 min. Then fixed tissues were incubated at 4 °C overnight with 2 μg/ml anti-C3aR or anti-C5aR or anti-neurofilament 68 in PBS containing 0.3% Triton X-100 and 1% BSA. After several rinses in PBS, the tissues were incubated at room temperature for 2 h with peroxidase-conjugated Affinipure goat anti-rabbit or anti-mouse IgG diluted 1:1000. After several rinses in PBS, the sections were incubated at room temperature for 2 h with the peroxidase-anti peroxidase complex (Sigma) diluted 1:200 in PBS. The sections were washed twice with PBS and rinsed with 0.1 m Tris-HCl buffer. The immunoreactivity was revealed by using a 3,3′-diaminobenzidine substrate kit containing 0.02% H2O2 (Sigma) and nickel ammonium sulfate. The reaction was stopped by removing the diaminobenzidine solution and by rinsing with a large volume of water. The specificity of immunolabeling was checked by omitting the primary antibody and by replacing the antiserum with pre-immune serum. Microphotographs were acquired on a computer-assisted image analyzer (Biocom 2000, Les Ulis, France). Immunoprecipitation and Western Blot—Total cellular proteins were extracted from DIV 4 neurons in lysis buffer containing 0.15 m NaCl, 0.05 m EDTA, 4% CHAPS, and 0.2% of the protease inhibitor mixture (0.5 m EDTA, 2.5 m benzamidine, 2.18 mm pepstatin A, 11.7 m leupeptin) for C3aR detection and 1% Triton X-100, 25 mm Tris-HCl, 5 mm EDTA, 250 mm NaCl, 10% glycerol and 0.2% of the protease inhibitor mixture for C5aR, Bax, and Bcl-2 detections. Spleen or cerebellum of 12-day-old rat were lyophilized, and the dry powders were resuspended in the corresponding lysis buffer (10 ml per g of lyophilized tissue) and incubated overnight at 4 °C. These extracts were centrifuged (15,000 × g, 4 °C, 15 min), and the supernatant was immunoprecipitated before Western blotting to enrich extracts in C3aR or C5aR proteins. Freshly made cell lysates were incubated with anti-C3aR or anti-C5aR at 4 °C for 30 min. The lysates were then incubated with protein A-Sepharose at 4 °C for 1 h under gentle rotation. After centrifugation at 10,000 g for 5 min, the supernatant was discarded. Immunoprecipitates were washed 5 times with PBS. The resulting pellet was solubilized in Laemmli sample buffer, reduced with the addition of mercaptoethanol (47Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207227) Google Scholar), and boiled for 5 min. The immunoprecipitate proteins were separated by 10% SDS-PAGE on 12% gels and electrotransferred to polyvinylidene difluoride membranes (Immobilon-P Millipore, Saint-Quentin en Yvelines, France). Nonspecific sites were blocked with 5% nonfat milk at room temperature for 30 min, and the membrane was incubated overnight at 4 °C with biotinylated anti-C3aR, biotinylated anti-C5aR (2 μg/ml), anti-Bax (1:200, sc-493, Santa Cruz Biotechnology, CA), or anti-Bcl-2 (1:200, sc-492, Santa Cruz Biotechnology) with rocking. After washing, bands were visualized by incubation with streptavidin-horseradish peroxidase or conjugated Affinipure goat anti-rabbit IgG (1:1000) for 2 h followed by chemiluminescence detection (ECL detection kit, Amersham Biosciences). Measurement of Calcium Flux—Cells were loaded with 4 μm fluo-4AM (Molecular Probes, Interchim) in HBK buffer (120 mm NaCl, 5 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 6 mm glucose, 10 mm HEPES, pH 7.4) for 30 min at 37 °C. After washing twice with HBK buffer, fluorescence intensity was measured with a FL600 microplate reader (Bio-Tek Instruments, Winooski, VT) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm every 15 s for 6 min. Apoptosis Induction—Cerebellar granule neurons were cultured for 4 days in S+K25. The cells were washed twice and cultured in serum-free medium containing low (5 mm) potassium concentration (S-K5). Under these conditions cerebellar granule neurons degenerate and die by apoptotic cell death within 8–24 h (37D'Mello S.R. Galli C. Ciotti T. Calissano P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10989-10993Crossref PubMed Scopus (851) Google Scholar, 38Galli C. Meucci O. Scorziello A. Werge T.M. Calissano P. Schettini G. J. Neurosci. 1995; 25: 1172-1179Crossref Google Scholar, 39Miller T.M. Johnson Jr., E.M. J. Neurosci. 1996; 16: 7487-7495Crossref PubMed Google Scholar). Cell Viability—Cells were incubated with 1.3 μm calcein AM (Molecular Probes) in PBS for 15 min. After washing twice with PBS, fluorescence intensity was measured with a FL600 microplate reader (Bio-Tek Instruments) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Pilot experiments have shown that the fluorescence intensity is proportional to the number of viable cells (in the range 5 × 104 to 1 × 106 cells/ml). DNA Fragmentation—Internucleosomal DNA cleavage was assessed by conventional gel electrophoresis after extraction of nuclear DNA by using the Wizard Plus Minipreps DNA purification system (Promega). Granule neurons were incubated for 10 min at room temperature in a lysis buffer consisting of 50 mm Tris-HCl, 10 mm EDTA, 1% Triton X-100, and 50 μg/ml RNase A. After centrifugation at 14,000 × g for 15 min, the cleared lysates were mixed with the Wizard resin and transferred into a vacuum manifold column. Three washes with the columnwash solution were performed, and DNA fragments were eluted with 50 μl of water at 60 °C. DNA ladders were visualized on a 1.5% agarose gel. Measurement of Caspase Activity—For measurement of caspase-3 activity, cultured cells were washed twice with PBS at 37 °C, resuspended in Dulbecco's modified Eagle's medium (1" @default.
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• W2000854765 date "2004-10-01" @default.
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• W2000854765 title "Characterization of C3a and C5a Receptors in Rat Cerebellar Granule Neurons during Maturation" @default.
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• W2000854765 doi "https://doi.org/10.1074/jbc.m404124200" @default.
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