Activation of the c-Jun N-terminal Kinase Signaling Cascade Mediates the Effect of Amyloid-β on Long Term Potentiation and Cell Death in Hippocampus
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m302530200
ISSN1083-351X
AutoresAedín M. Minogue, Adrien W. Schmid, Marie P. Fogarty, Alison Moore, Veronica A. Campbell, Caroline E. Herron, Marina A. Lynch,
Tópico(s)Cholinesterase and Neurodegenerative Diseases
ResumoAmyloid-β (Aβ) is a major constituent of the neuritic plaque found in the brain of Alzheimer's disease patients, and a great deal of evidence suggests that the neuronal loss that is associated with the disease is a consequence of the actions of Aβ. In the past few years, it has become apparent that activation of c-Jun N-terminal kinase (JNK) mediates some of the effects of Aβ on cultured cells; in particular, the evidence suggests that Aβ-triggered JNK activation leads to cell death. In this study, we investigated the effect of intracerebroventricular injection of Aβ(1–40) on signaling events in the hippocampus and on long term potentiation in Schaffer collateral CA1 pyramidal cell synapses in vivo. We report that Aβ(1–40) induced activation of JNK in CA1 and that this was coupled with expression of the proapoptotic protein, Bax, cytosolic cytochrome c, poly-(ADP-ribose) polymerase cleavage, and Fas ligand expression in the hippocampus. These data indicate that Aβ(1–40) inhibited expression of long term potentiation, and this effect was abrogated by administration of the JNK inhibitor peptide, D-JNKI1. In parallel with these findings, we observed that Aβ-induced changes in caspase-3 activation and TdT-mediated dUTP nick-end labeling staining in neuronal cultured cells were inhibited by D-JNKI1. We present evidence suggesting that interleukin (IL)-1β plays a significant role in mediating the effects of Aβ(1–40) because Aβ(1–40) increased hippocampal IL-1β and because several effects of Aβ(1–40) were inhibited by the caspase-1 inhibitor Ac-YVAD-CMK. On the basis of our findings, we propose that Aβ-induced changes in hippocampal plasticity are likely to be dependent upon IL-1β-triggered activation of JNK. Amyloid-β (Aβ) is a major constituent of the neuritic plaque found in the brain of Alzheimer's disease patients, and a great deal of evidence suggests that the neuronal loss that is associated with the disease is a consequence of the actions of Aβ. In the past few years, it has become apparent that activation of c-Jun N-terminal kinase (JNK) mediates some of the effects of Aβ on cultured cells; in particular, the evidence suggests that Aβ-triggered JNK activation leads to cell death. In this study, we investigated the effect of intracerebroventricular injection of Aβ(1–40) on signaling events in the hippocampus and on long term potentiation in Schaffer collateral CA1 pyramidal cell synapses in vivo. We report that Aβ(1–40) induced activation of JNK in CA1 and that this was coupled with expression of the proapoptotic protein, Bax, cytosolic cytochrome c, poly-(ADP-ribose) polymerase cleavage, and Fas ligand expression in the hippocampus. These data indicate that Aβ(1–40) inhibited expression of long term potentiation, and this effect was abrogated by administration of the JNK inhibitor peptide, D-JNKI1. In parallel with these findings, we observed that Aβ-induced changes in caspase-3 activation and TdT-mediated dUTP nick-end labeling staining in neuronal cultured cells were inhibited by D-JNKI1. We present evidence suggesting that interleukin (IL)-1β plays a significant role in mediating the effects of Aβ(1–40) because Aβ(1–40) increased hippocampal IL-1β and because several effects of Aβ(1–40) were inhibited by the caspase-1 inhibitor Ac-YVAD-CMK. On the basis of our findings, we propose that Aβ-induced changes in hippocampal plasticity are likely to be dependent upon IL-1β-triggered activation of JNK. One of the pathological hallmarks of Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid-β; JNK, c-Jun N-terminal kinase; FasL, Fas ligand; IL, interleukin; LTP, long term potentiation; NBM, neurobasal medium; PARP, poly-(ADP-ribose) polymerase; TBS, Tris-buffered saline; BSA, bovine serum albumin; PBS, phosphate-buffered saline; HFS, high frequency stimuli; EPSP, base-line excitatory postsynaptic potential; TUNEL, TdT-mediated dUTP nick-end labeling; ANOVA, analysis of variance. is an accumulation of plaques consisting predominately of amyloid-β (Aβ) peptide, which is processed from amyloid precursor protein by the action of β- and γ-secretase (1Yankner B.A. Neuron. 1996; 16: 921-932Abstract Full Text Full Text PDF PubMed Scopus (921) Google Scholar). Neuronal cell loss is one feature of AD, and evidence from analysis of changes in cultured cells suggests that Aβ acts as the executioner. Thus, neuronal cultures exposed to Aβ demonstrate signs of apoptosis (2Anderson A.J. Su J.H. Cotman C.W. J. Neurosci. 1996; 16: 1710-1719Crossref PubMed Google Scholar, 3Estus S. Tucker H.M. van Rooyen C. Wright S. Brigham E.F. Wogulis M. Rydel R.E. J. Neurosci. 1997; 17: 7736-7745Crossref PubMed Google Scholar, 4Stadelmann C. Deckwerth T.L. Srinivasan A. Bancher C. Bruck W. Jellinger K. Lassmann H. Am. J. Pathol. 1999; 155: 1459-1466Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar), and previous evidence from this laboratory has revealed that cultured cortical neurons exposed to Aβ(1–40) exhibited increased expression of the tumor suppressor p53; increased activation of caspase-3, a marker of apoptotic cell death; and increased TUNEL reactivity (5Fogarty M.P. Downer E.J. Campbell V.A. Biochem. J. 2003; 371: 789-798Crossref PubMed Scopus (65) Google Scholar). The evidence is consistent with the idea that activation of the stress-activated protein kinase, c-Jun N-terminal kinase (JNK) played a significant role, because depletion of JNK1 following exposure to antisense oligonucleotide prevented the effects of Aβ (5Fogarty M.P. Downer E.J. Campbell V.A. Biochem. J. 2003; 371: 789-798Crossref PubMed Scopus (65) Google Scholar). Similarly, Morishima et al. (6Morishima Y. Gotoh Y. Zieg J. Barrett T. Takano H. Flavell R. Davis R.J. Shirasaki Y. Greenberg M.E. J. Neurosci. 2001; 21: 7551-7560Crossref PubMed Google Scholar) reported that Aβ increased phosphorylation of JNK and c-Jun in cultured cortical neurons and that these changes were associated with expression of the death inducer Fas ligand (FasL). Others have reported findings that support a role for JNK activation in mediating at least certain effects of Aβ. For instance, Aβ-induced parallel increases in JNK activation and TUNEL reactivity in PC12 cells (7Jang J.-H. Surh Y.-J. Ann. N. Y. Acad. Sci. 2002; 973: 228-236Crossref PubMed Scopus (54) Google Scholar), whereas activation of JNK was shown to be localized to amyloid deposits in 7- and 12-month-old mice that overexpress amyloid precursor protein (8Savage M.J. Lin Y.-G. Ciallella J.R. Flood D.G. Scott R.W. J. Neurosci. 2002; 22: 3376-3385Crossref PubMed Google Scholar). It has emerged in several experimental models that increased JNK phosphorylation is associated with deficits in synaptic function; for instance, increased activation of JNK has been reported in the hippocampi of aged rats (9O'Donnell E. Vereker E. Lynch M.A. Eur. J. Neurosci. 2000; 12: 345-352Crossref PubMed Scopus (116) Google Scholar, 10Lynch A.M. Lynch M.A. Eur. J. Neurosci. 2002; 15: 1779-1788Crossref PubMed Scopus (90) Google Scholar), rats exposed to whole body irradiation (11Lonergan P.E. Martin D.S.D. Horrobin D.F. Lynch M.A. J. Biol. Chem. 2002; 277: 20804-20811Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), and rats injected with the proinflammatory cytokine, interleukin (IL)-1β (12Vereker E. O'Donnell E. Lynch M.A. J. Neurosci. 2000; 20: 6811-6819Crossref PubMed Google Scholar) or lipopolysaccharide (13Vereker E. Campbell V. Roche E. McEntee E. Lynch M.A. J. Biol. Chem. 2000; 275: 26252-26258Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), and in all cases glutamate release was decreased. In each of these experimental conditions, long term potentiation (LTP), a model of synaptic plasticity, was markedly impaired, and this impairment was coupled with an increased hippocampal concentration of IL-1β. A number of groups have reported that Aβ administration exerts an inhibitory effect on LTP. For instance, Aβ peptides (14Cullen W.K. Suh Y.H. Anwyl R. Rowan M.J. Neuroreport. 1997; 8: 3213-3217Crossref PubMed Scopus (221) Google Scholar, 15Freir D.B. Holscher C. Herron C.E. J. Neurophysiol. 2001; 85: 708-713Crossref PubMed Scopus (138) Google Scholar, 16Freir D.B. Herron C.E. J. Neurophysiol. 2003; 89: 2917-2922Crossref PubMed Scopus (40) Google Scholar) and naturally secreted Aβ oligomers (17Walsh D.M. Klyubin I. Fadeeva J.V. Cullen W.K. Anwyl R. Wolfe M.S. Rowan M.J. Selkoe D.J. Nature. 2002; 416: 535-539Crossref PubMed Scopus (3721) Google Scholar) inhibited LTP in the CA1 region in vivo, and Aβ peptides also inhibit LTP in dentate gyrus and the CA1 in vitro (18Chen Q.S. Kagan B.L. Hirakura Y. Xie C.W. J. Neurosci. Res. 2000; 60: 65-72Crossref PubMed Scopus (243) Google Scholar, 19Freir D.B. Costello D.A. Herron C.E. J. Neurophysiol. 2003; 89: 3061-3069Crossref PubMed Scopus (60) Google Scholar, 20Wang H.-W. Pasternak J.F. Kuo H. Ristic H. Lambert M.P. Chromy B. Viola K.L. Klein W.L. Stine W.B. Krafft G.A. Trommer B.L. Brain Res. 2002; 924: 133-140Crossref PubMed Scopus (492) Google Scholar, 21Saleshando G. O'Connor J.J. Neurosci. Lett. 2000; 288: 119-122Crossref PubMed Scopus (41) Google Scholar). Similarly, a deficit in LTP was reported in aged mice that overexpress amyloid precursor protein and in which deposition of Aβ was observed (22Chapman P.F. White G.L. Jones M.W. Cooper-Blacketer D. Marshall V.J. Irizarry M. Younkin L. Good M.A. Bliss T.V.P. Hyman B. Younkin S.G. Hsiao K.K. Nat. Neurosci. 1999; 2: 271-276Crossref PubMed Scopus (819) Google Scholar). In this study, we investigated the signaling events induced by Aβ(1–40) that might explain its impact on LTP and report that activation of JNK is a pivotal event in Aβ-induced inhibition of LTP and in Aβ-induced cell death. Preparation of Aβ—Aβ(1–40) (BioSource International) was made up as a 1 mm stock solution in sterile water and allowed to aggregate for 48 h at 30 °C as described previously (5Fogarty M.P. Downer E.J. Campbell V.A. Biochem. J. 2003; 371: 789-798Crossref PubMed Scopus (65) Google Scholar). For treatment of cortical neurons, aggregated Aβ(1–40) was diluted to a final concentration of 2 μm in prewarmed neurobasal medium (NBM; Invitrogen). For analysis of signaling events stimulated by Aβ, aggregated Aβ(1–40) at 37 °C was injected intracerebroventricularly (5 μl; 1nmol in sterile water). This Aβ preparation (and concentration) was adopted because it was shown to produce consistent, reliable, and reproducible results in a number of markers, suggesting that cell death occurred in cultured cells (5Fogarty M.P. Downer E.J. Campbell V.A. Biochem. J. 2003; 371: 789-798Crossref PubMed Scopus (65) Google Scholar). Animals—Groups of young male Wistar rats (200–300 g; Bio Resources Unit, Trinity College, Dublin 2, Ireland), maintained at an ambient temperature of 22–23 °C under a 12 h light-dark schedule, were used in this experiment. The rats were anesthetized by intraperitoneal administration of urethane (1.5 mg/kg) and were injected intracerebroventricularly with either sterile water (5 μl) or Aβ(1–40). 6 h post-injection, the rats were killed by decapitation, the brains were rapidly removed on ice, and area CA1 was dissected free. The tissue was cross-chopped (350 × 350 μm) and frozen in Krebs solution containing 10% Me2SO as previously described (23McGahon B. Lynch M.A. Neuroscience. 1996; 72: 847-855Crossref PubMed Scopus (52) Google Scholar) until required for analysis. Analysis of JNK Phosphorylation, c-Jun Phosphorylation, Cytosolic Cytochrome c Expression, Bax Expression, FasL Expression, and PARP Cleavage—JNK phosphorylation, c-Jun phosphorylation, and expression of Bax, cytosolic cytochrome c, PARP, and FasL were analyzed in samples prepared from CA1 tissue using a method previously described (13Vereker E. Campbell V. Roche E. McEntee E. Lynch M.A. J. Biol. Chem. 2000; 275: 26252-26258Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In the case of JNK, c-Jun, FasL, and PARP, tissue homogenates were diluted to equalize for protein concentration, and aliquots (100 μl, 2 mg/ml) were added to 100 μl of sample buffer (0.5 mm Tris-HCl, pH 6.8, 10% glycerol, 10% SDS, 5% β-mercaptoethanol, 0.05% bromphenol blue (w/v)), boiled for 5 min, and loaded onto 10% SDS-PAGE gels. In the case of cytochrome c, cytosolic fraction was prepared by homogenizing slices of hippocampus in lysis buffer (20 mm HEPES, pH 7.4, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 5 μg/ml pepstatin A, 2 μg/ml leupeptin, 2 μg/ml aprotinin), incubating for 20 min on ice, and centrifuging (15,000 × g for 10 min at 4 °C). The supernatant (i.e. cytosolic fraction) was suspended in sample buffer to a final concentration of 300 μg/ml, boiled for 3 min, and loaded (6 μg/lane) onto 12% SDS-PAGE gels. The pellet (i.e. mitochondrial fraction) was resuspended in sample buffer to a final concentration of 300 μg/ml, boiled for 3 min, and loaded (6 μg/lane) onto 12% SDS-PAGE gels. Bax expression was assessed in the mitochondrial fraction. In all experiments the proteins were separated by application of 32 mA constant current for 25–30 min, transferred onto nitrocellulose strips (225 mA for 90 min), and immunoblotted with the appropriate antibody. For JNK phosphorylation, the proteins were immunoblotted with an antibody that specifically targets phosphorylated JNK (1:300 in Tris-buffered saline (TBS)-Tween (0.05% Tween 20) containing 0.1% BSA; Santa Cruz Biotechnology Inc.) for 2 h at room temperature. The blots were stripped and stained for total JNK. Nitrocellulose strips were probed with a mouse monoclonal IgG1 antibody (1:200; Santa Cruz Biotechnology Inc.) raised against a recombinant protein corresponding to amino acids 1–384 representing full-length JNK1 of human origin. To assess phosphorylation of c-Jun, we immunoblotted with a mouse monoclonal IgG1 antibody (1:400 in PBS-Tween (0.1% Tween 20) containing 2% nonfat dried milk) raised against the peptide corresponding to a short amino acid sequence of phosphorylated c-Jun of human origin (Santa Cruz Biotechnology Inc.). To assess cytoplasmic cytochrome c, a rabbit polyclonal antibody (1:250 in PBS-Tween containing 2% nonfat dried milk; Santa Cruz Biotechnology Inc.) raised against recombinant protein corresponding to amino acids 1–104 of cytochrome c was used. In the case of FasL, we immunoblotted with a rabbit polyclonal antibody (1:500 in TBS-Tween containing 1% BSA; Santa Cruz Biotechnology Inc.) raised against a peptide corresponding to a short amino acid sequence at the N terminus of FasL of human origin. Bax expression was assessed in the mitochondrial fraction using a mouse monoclonal IgG1 antibody (1:200 in TBS-Tween containing 1% BSA; Santa Cruz Biotechnology Inc.). To assess the cleavage of PARP, we immunoblotted with an antibody (1:500 in PBS-Tween (0.1% Tween 20) containing 2% nonfat dried milk) raised against the epitope corresponding to amino acids 764–1014 of PARP of human origin (Santa Cruz Biotechnology Inc.). All of the nitrocellulose strips were reprobed for actin expression to ensure equal loading of protein on all SDS-PAGE gels. Actin expression was assessed using a mouse monoclonal IgG1 antibody (1:300 in PBS-Tween containing 2% nonfat dried milk) corresponding to an amino acid sequence mapping at the C terminus of actin of human origin (Santa Cruz Biotechnology Inc.). Immunoreactive bands were detected as follows: peroxidase-conjugated anti-mouse IgG (Sigma) and Supersignal chemiluminescence (Pierce) for JNK, c-Jun, Bax, and actin and peroxidase-conjugated anti-rabbit IgG (Sigma) and Supersignal (Pierce) for cytochrome c, FasL, and PARP. Induction of LTP in CA1 in Vivo—Male Wistar rats (175–200 g; Biomedical Facility, University College, Dublin, Ireland) were anesthetized with urethane (1.5 mg/kg), placed in a stereotaxic frame, and assessed for LTP as described previously (16Freir D.B. Herron C.E. J. Neurophysiol. 2003; 89: 2917-2922Crossref PubMed Scopus (40) Google Scholar). Small holes were drilled in the skull to allow insertion of a guide cannula to facilitate intracerebroventricular injection and to allow insertion of the reference, stimulating, and recording electrodes. The recording electrode was positioned in the stratum radiatum of area CA1 (3 mm posterior and 2 mm lateral to bregma), and a bipolar stimulating electrode was placed in the Schaffer collateral/commissural pathway distal to the recording electrode (4 mm posterior and 3 mm lateral to bregma). The cannula was positioned above the lateral ventricle in the opposite hemisphere to that of the electrodes (1 mm posterior and 1.2 mm lateral to bregma). Test shocks (0.033 Hz) were delivered to the Schaffer collateral/commissural pathway, and base-line excitatory postsynaptic potentials (EPSPs), recorded at 35–40% of maximal response, were sampled for at least 30 min to allow the response to stabilize. Rats were then injected intracerebroventricularly with either Aβ(1–40) (1nmol in 5 μl), the membrane soluble JNK inhibitor D-JNKI1 (1 nmol in 5 μl), combined Aβ(1–40) and D-JNKI1 (1 nmol of each in 5 μl) or vehicle (5 μl sterile water); and base-line recordings were monitored for a further 60 min before delivery of a series of high frequency stimuli (HFS; 3 × 10 trains of 10 stimuli at 200Hz; intertrain interval, 20 s). Responses to test shock stimulation were recorded for a further 5 h post-HFS, and deep body temperature was maintained at 36.5 ± 0.5 °C using heating pads. Paired pulse facilitation with an interstimulus interval of 50 ms was also examined preinjection, 1 h post-injection of drug/vehicle (prior to HFS), and 5 h following HFS. Deep body temperature was maintained at 36.5 ± 0.5 °C using heating pads. Extracellular field potentials were amplified (×100), filtered at 5 kHz, digitized, and recorded using MacLab software. The EPSP slope was used to measure synaptic efficacy. EPSPs are expressed as percentages of the mean initial slope measured during the first 10 min of the base-line recording period. Analysis of IL-1β Concentration—IL-1β concentration was analyzed in homogenate prepared from CA1 by enzyme-linked immunosorbent assay (R & D Systems) and in supernatants prepared from cultured cells as described below. Antibody-coated (100 μl; final concentration, 1.0 μg/ml; diluted in PBS, pH 7.3; goat anti-rat IL-1β antibody) 96-well plates were incubated overnight at room temperature, washed several times with PBS containing 0.05% Tween 20, blocked for 1 h at room temperature with 300 μl of blocking buffer (PBS, pH 7.3, containing 5% sucrose, 1% BSA, and 0.05% NaN3), and washed. IL-1β standards (100 μl; 0–1000 pg/ml in PBS containing 1% BSA) or samples (homogenized in Krebs solution containing 2 mm CaCl2) were added, and incubation proceeded for 2 h at room temperature. Secondary antibody (100 μl; final concentration, 350 ng/ml in PBS containing 1% BSA and 2% normal goat serum; biotinylated goat anti-rat IL-1β antibody) was added and incubated for 2 h at room temperature. The wells were washed, and detection agent (100 μl; horseradish peroxidase-conjugated streptavidin; 1:200 dilution in PBS containing 1% BSA) was added and incubated continued for 20 min at room temperature. Substrate solution (100 μl; 1:1 mixture of H2O2 and tetramethylbenzidine) was added and incubated at room temperature in the dark for 1 h, after which time the reaction was stopped using 50 μl of 1 m H2SO4. Absorbance was read at 450 nm, and the values were corrected for protein (24Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) and expressed as pg IL-1β/mg protein. Preparation of Cultured Cortical Neurons—Primary cortical neurons were isolated and prepared from 1-day-old Wistar rats (BioResources Unit, Trinity College, Dublin 2, Ireland) and maintained in NBM as previously described (5Fogarty M.P. Downer E.J. Campbell V.A. Biochem. J. 2003; 371: 789-798Crossref PubMed Scopus (65) Google Scholar). The rats were decapitated, the cerebral cortices were dissected, and the meninges were removed. The cortices were incubated in PBS with trypsin (0.25 μg/ml) for 25 min at 37 °C. The cortical tissue was then triturated in PBS containing soy bean trypsin inhibitor (0.2 μg/ml) and DNase (0.2 mg/ml) and gently filtered through a sterile mesh filter (40 μm). The suspension was centrifuged at 2000 × g for 3 min at 20 °C, and the pellet was resuspended in warm NBM, supplemented with heat inactivated horse serum (10%), penicillin (100 units/ml), streptomycin (100 units/ml), and glutamax (2 mm). The suspended cells were plated at a density of 0.25 × 106 cells on circular 10-mm diameter coverslips, coated with poly-l-lysine (60 μg/ml), and incubated in a humidified atmosphere containing 5% CO2:95% air at 37 °C for 2 h prior to being flooded with prewarmed NBM. After 48 h, 5 ng/ml cytosine-arabinofuranoside was added to the culture medium to suppress the proliferation of non-neuronal cells. The media were exchanged for fresh media every 3 days, and the cells were grown in culture for up to 7 days prior to treatment. In one set of experiments the neurons were incubated in the absence/presence of Aβ(1–40) (2 μm in NBM) for 72 h with or without caspase-1 inhibitor (100 nm in NBM; Ac-YVAD-CMK; Calbiochem) or D-JNKI1 (1 μm in NBM; Alexis Biochemicals). In the case of Aβ-treated neurones, the supernatant was removed at 20 h, and IL-1β concentration was assessed. At 72 h, the cells were rinsed in TBS and fixed in 4% paraformaldehyde in TBS for immunohistochemical assessment of JNK phosphorylation, caspase-3 activation, and DNA fragmentation. The cells were incubated in Aβ(1–40) (2 μm) for 18 h for analysis of changes in gene expression. In a second series of experiments, the neurons were incubated in the absence/presence of IL-1β (5 ng/ml in NBM) with or without D-JNKI1 (1 μm in NBM) for 18 h and harvested in lysis buffer (20 mm HEPES, pH 7.4, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 5 μg/ml pepstatin A, 2 μg/ml leupeptin, 2 μg/ml aprotinin) for assessment of c-Jun phosphorylation and FasL expression. Analysis of Bax mRNA and caspase-3 mRNA—Total RNA was extracted from cortical neurones using TRI reagent (Sigma). cDNA synthesis was performed on 1 μg of total RNA using oligo(dT) primer as per the manufacturer's instructions (Superscript reverse transcriptase; Invitrogen). The RNA was treated with RNase-free DNase I (Invitrogen) at 1 unit/μg of RNA for 15 min at 30 °C. Equal amounts of cDNA were used for PCR amplification for a total of 28 cycles. Primers were pretested through an increasing number of amplification cycles to obtain reverse transcriptase-PCR products in the exponential range. In the case of Bax mRNA expression following Aβ treatment primers used were as follows: rat Bax, sense 5′-GCAGAGAGGATGGCTGGGGAGA-3′, and antisense 5′-TCCAGACAAGCAGCCGCTCACG-3′ (25Ray S.K. Fidan M. Nowak M.W. Wilford G.G. Hogan E.L. Banik N.L. Brain Res. 2000; 852: 326-334Crossref PubMed Scopus (182) Google Scholar); rat β-actin, sense 5′-GAAATCGTGCGTGACATTAAAGAGAAGCT and antisense 5′-TCAGGAGGAGCAATGATGATCTTGA-3′. The cycling conditions were 95 °C for 5 min followed by cycles of 95 °C for 75 s, 52 °C for 75 s, and 72 °C for 90 s. A final extension step was carried out at 70 °C for 10 min. These primers generated Bax PCR products of 352 base pairs and β-actin PCR product of 360 base pairs. In the case of caspase-3 mRNA expression following treatment with Aβ(1–40) and Bax mRNA expression following treatment with IL-1β, multiplex PCR was performed using the Quantitative PCR Cytopress detection kit (Rat Apoptosis Set 2; BioSource International) generating caspase-3 PCR products of 320 base pairs, Bax PCR products of 352 base pairs and glyceraldehyde-3-phosphate dehydrogenase PCR product of 532 base pairs. The cycling conditions were 94 °C for 1 min and 58 °C for 2 min. A final extension step was carried out at 70 °C for 10 min. The PCR products were analyzed by electrophoresis on 1.5% agarose gels, photographed, and quantified using densitometry. The target genes were normalized to expression of β-actin or glyceraldehyde-3-phosphate dehydrogenase housekeeping genes. No observable change in β-actin or glyceraldehyde-3-phosphate dehydrogenase mRNA was observed in any of the treatment conditions TUNEL Staining—Apoptotic cell death was assessed using the DeadEnd colorimetric apoptosis detection system (Promega) according to the manufacturer's instructions. Briefly, cultured cortical neurones were prepared from neonatal rats as described above and maintained in NBM for 12 days before incubating in the absence/presence of Aβ(1–40) (1 μm in NBM) for 72 h with or without caspase-1 inhibitor (100 nm in NBM) or D-JNKI1 (1 μm in NBM). Biotinylated nucleotide was incorporated at 3′-OH DNA ends by incubating cells with terminal deoxynucleotidyl transferase for 30 min at 37 °C. The washed cells were incubated in horseradish peroxidase-labeled streptavidin and then incubated in diaminobenzidine chromogen solution, and TUNEL-positive cells were calculated as a proportion of the total cell number. Immunohistochemical Staining for Phosphorylated JNK and Activated Caspase-3—Cultured cortical neurones were prepared from neonatal rats as described previously (5Fogarty M.P. Downer E.J. Campbell V.A. Biochem. J. 2003; 371: 789-798Crossref PubMed Scopus (65) Google Scholar) and maintained in NBM for 12 days before incubating in the absence/presence of Aβ(1–40) (1 μm in NBM) for 72 h with or without caspase-1 inhibitor (100 nm in NBM) or D-JNKI1 (1 μm in NBM). The cells were fixed in 4% paraformaldehyde in TBS, permeabilized in 0.1% Triton containing 0.2% proteinase K, washed in TBS, and refixed in 4% paraformaldehyde. The cells were incubated with 2.5% (v/v) normal goat serum (Vector Laboratories) in TBS. The blocking serum was removed, and the cells were incubated overnight with either antiactive p-JNK (1:200 in TBS containing 2.5% normal goat serum; Santa Cruz Biotechnology Inc.) or antiactive caspase-3 (1:250 in TBS containing 2.5% (v/v) normal goat serum; Promega) in a humidified chamber. The cells were washed in TBS and incubated in the dark for 2 h at room temperature in either fluorescein isothiocyanate-labeled goat anti-mouse IgG or IgM (1:100; Biosource) to immunolabel p-JNK or l-rhodamine-labeled goat anti-rabbit IgG (1: 100; Biosource) to immunolabel active caspase-3. The cells were washed in TBS, mounted with an aqueous mounting medium (Vectastain; Vector Laboratories), and sealed. The slides were examined under a Zeiss fluorescence microscope with the appropriate filter (fluorescein isothiocyanate: excitation, 495 nm, and emission, 525 nm; l-rhodamine: excitation, 540 and 574 nm, and emission, 602 nm). Statistical Analysis—The data are expressed as the means ± S.E. A one-way analysis of variance (ANOVA) was performed to determine whether there were significant differences between conditions. When this analysis indicated significance (at the 0.05 level), post hoc Student Newmann-Keuls test analysis was used to determine which conditions were significantly different from each other. A repeated measures ANOVA was used to compare mean EPSP slopes at different time points in the electrophysiological experiments. When comparisons were being made between two treatments, an unpaired Student's t test for independent means was performed. Fig. 1a shows a sample immunoblot in which a marked increase in p-JNK was observed following intracerebroventricular injection of Aβ(1–40); assessment of the mean data obtained from densitometric analysis revealed a statistically significant increase in JNK phosphorylation induced by Aβ (p < 0.001; Student's t test for independent means; n = 5). In contrast to the change in JNK phosphorylation, total JNK expression was similar in Aβ-treated and control rats as demonstrated in the sample immunoblot and the mean data (Fig. 1b). The Aβ-induced increase in JNK phosphorylation was paralleled by the change in c-Jun phosphorylation; thus, the sample immunoblot shown in Fig. 1c and the mean data obtained from densitometric analysis indicated that Aβ(1–40) induced a marked increase in c-Jun phosphorylation (p < 0.05; Student's t test for independent means; n = 5). Protein loading was checked by reprobing immunoblots for actin, and the data indicate that its expression was similar in samples prepared from control and Aβ-treated rats. We argued that this Aβ-induced increase in JNK activation may contribute to the previously reported Aβ-induced inhibition of LTP (16Freir D.B. Herron C.E. J. Neurophysiol. 2003; 89: 2917-2922Crossref PubMed Scopus (40) Google Scholar), and to assess this, rats were injected intracerebroventricularly with Aβ(1–40) alone or in combination with the peptide inhibitor, D-JNKI1. Fig. 2a shows that, in control rats tetanic stimulation led to an immediate and persistent increase in EPSP slope (p < 0.001; ANOVA); treatment with D-JNKI1 (1nmol) did not significantly affect this change. In contrast, intracerebroventricular injection of Aβ(1–40) inhibited LTP (p < 0.001; ANOVA, Fig. 2b); the effect was observed immediately such that the mean percentage change in the EPSP slope in the 5 min immediately following tetanic stimulation was significantly reduced in Aβ-treated compared with control rats (p < 0.01; ANOVA; Fig. 3a). The Aβ-associated change persisted so that the mean percentage changes in EPSP slopes in the final 5-min period of each hour were also significantly reduced in Aβ-treated rats compared with control animals (***, p < 0.001 in all cases; ANOVA; Fig. 3, b–f). Co-injection of D-JNKI1 and Aβ(1–40) reversed the inhibitory effect of Aβ(1–40) (Fig. 2b), but this effect was not apparent until 2 h after tetanic stimulation (+++, p < 0.001; ANOVA; F
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