Actions of β-Amyloid Protein on Human Neurons Are Expressed through the Amylin Receptor
2011; Elsevier BV; Volume: 178; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2010.11.022
ISSN1525-2191
AutoresJack H. Jhamandas, Zong‐Ming Li, David Westaway, Jing Yang, Simran Jassar, David MacTavish,
Tópico(s)Receptor Mechanisms and Signaling
ResumoDisruption of neurotoxic effects of amyloid β protein (Aβ) is one of the major, but as yet elusive, goals in the treatment of Alzheimer's disease (AD). The amylin receptor, activated by a pancreatic polypeptide isolated from diabetic patients, is a putative target for the actions of Aβ in the brain. Here we show that in primary cultures of human fetal neurons (HFNs), AC253, an amylin receptor antagonist, blocks electrophysiological effects of Aβ. Pharmacological blockade of the amylin receptor or its down-regulation using siRNA in HFNs confers neuroprotection against oligomeric Aβ-induced caspase-dependent and caspase-independent apoptotic cell death. In transgenic mice (TgCRND8) that overexpress amyloid precursor protein, amylin receptor is up-regulated in specific brain regions that also demonstrate an elevated amyloid burden. The expression of Aβ actions through the amylin receptor in human neurons and temporospatial interrelationship of Aβ and the amylin receptor in an in vivo model of AD together provide a persuasive rationale for this receptor as a novel therapeutic target in the treatment of AD. Disruption of neurotoxic effects of amyloid β protein (Aβ) is one of the major, but as yet elusive, goals in the treatment of Alzheimer's disease (AD). The amylin receptor, activated by a pancreatic polypeptide isolated from diabetic patients, is a putative target for the actions of Aβ in the brain. Here we show that in primary cultures of human fetal neurons (HFNs), AC253, an amylin receptor antagonist, blocks electrophysiological effects of Aβ. Pharmacological blockade of the amylin receptor or its down-regulation using siRNA in HFNs confers neuroprotection against oligomeric Aβ-induced caspase-dependent and caspase-independent apoptotic cell death. In transgenic mice (TgCRND8) that overexpress amyloid precursor protein, amylin receptor is up-regulated in specific brain regions that also demonstrate an elevated amyloid burden. The expression of Aβ actions through the amylin receptor in human neurons and temporospatial interrelationship of Aβ and the amylin receptor in an in vivo model of AD together provide a persuasive rationale for this receptor as a novel therapeutic target in the treatment of AD. Several lines of evidence support a role for amyloid β-protein (Aβ) in the pathogenesis of Alzheimer's disease (AD). The genetic data include the occurrence of AD with inherited amyloid precursor protein (APP) mutations adjacent to the β- and γ-secretase cleavage sites, trisomy 21 with the APP gene, and early-onset PS1 and PS2 mutations in the γ-secretase catalytic subunit.1Brouwers N. Sleegers K. Van Broeckhoven C. Molecular genetics of Alzheimer's disease: an update.Ann Med. 2008; 40: 562-583Crossref PubMed Scopus (178) Google Scholar Other data include neurotoxicity of soluble oligomeric Aβ when applied to neurons2Yankner B.A. Lu T. Amyloid beta-protein toxicity and the pathogenesis of Alzheimer disease.J Biol Chem. 2009; 284: 4755-4759Crossref PubMed Scopus (169) Google Scholar and the generation of APP-overexpressing mice that recapitulate certain neuropathological and behavioral features of AD.3Price D.L. Wong P.C. Markowska A.L. Lee M.K. Thinakaren G. Cleveland D.W. Sisodia S.S. Borchelt D.R. The value of transgenic models for the study of neurodegenerative diseases.Ann N Y Acad Sci. 2000; 920: 179-191Crossref PubMed Scopus (47) Google Scholar Although Aβ exerts a wide range of biological effects and is potently neurotoxic, there is as yet no unequivocally identified receptor for Aβ. Several putative receptor candidates for Aβ have been reported (eg, RAGE receptors, the p75NTR receptor, scavenger receptors, neuronal nicotinic receptors, and the tachykinin and serpin-enzyme complex receptors), but the functional significance of Aβ interactions with such receptors in the brain has yet to be identified or remains controversial.4Chen X. Walker D.G. Schmidt A.M. Arancio O. Lue L.F. Yan S.D. RAGE: a potential target for Abeta-mediated cellular perturbation in Alzheimer's disease.Curr Mol Med. 2007; 7: 735-742Crossref PubMed Scopus (115) Google Scholar, 5Salminen A. Ojala J. Kauppinen A. Kaarniranta K. Suuronen T. Inflammation in Alzheimer's disease: amyloid-beta oligomers trigger innate immunity defence via pattern recognition receptors.Prog Neurobiol. 2009; 87: 181-194Crossref PubMed Scopus (284) Google Scholar, 6Sotthibundhu A. Sykes A.M. Fox B. Underwood C.K. Thangnipon W. Coulson E.J. 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Cellular mechanisms for amyloid-beta protein activation of rat cholinergic basal forebrain neurons.J Neurophysiol. 2001; 86: 1312-1320Crossref PubMed Scopus (65) Google Scholar, 17Jhamandas J.H. Harris K.H. Cho C. Fu W. MacTavish D. Human amylin actions on rat cholinergic basal forebrain neurons: antagonism of beta-amyloid effects.J Neurophysiol. 2003; 90: 3130-3136Crossref PubMed Scopus (52) Google Scholar Furthermore, Aβ and human amylin not only induce apoptotic cell death in cultured neurons and pancreatic β-islet cells, but demonstrate a neurotoxicity profile that is identical, including time- and concentration-dependent induction of apoptotic genes.14May P.C. Boggs L.N. Fuson K.S. Neurotoxicity of human amylin in rat primary hippocampal cultures: similarity to Alzheimer's disease amyloid-beta neurotoxicity.J Neurochem. 1993; 61: 2330-2333Crossref PubMed Scopus (132) Google Scholar, 15Tucker H.M. Rydel R.E. Wright S. Estus S. Human amylin induces “apoptotic” pattern of gene expression concomitant with cortical neuronal atrophy.J Neurochem. 1998; 71: 506-516Crossref PubMed Scopus (59) Google Scholar, 18Lim Y.A. Ittner L.M. Lim Y.L. Götz J. Human but not rat amylin shares neurotoxic properties with Abeta42 in long-term hippocampal and cortical cultures.FEBS Lett. 2008; 582: 2188-2194Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Recent data using quantitative iTRAC proteomics analysis (iTRAC stands for “isobaric tag for relative and absolute quantitation”) reveal that human amylin and Aβ deregulate identical mitochondrial proteins, further supporting the notion that both amyloidoses have common targets.19Lim Y.A. Rhein V. Baysang G. Meier F. Poljak A. Raftery M.J. Guilhaus M. Ittner L.M. Eckert A. Götz J. Abeta and human amylin share a common toxicity pathway via mitochondrial dysfunction.Proteomics. 2010; 10: 1621-1633Crossref PubMed Scopus (113) Google Scholar Collectively, these observations suggest that the human amylin receptor, which serves as the endogenous receptor for the pancreatic amylin peptide, could also serves as a putative receptor for the expression of the biological effects of Aβ. Dimerization of the calcitonin receptor (CTR) with RAMP3 yields a receptor that binds amylin with a significantly higher affinity than CGRP and adrenomedullin, two other peptides belonging to this family.20Poyner D.R. Sexton P.M. Marshall I. Smith D.M. Quirion R. Born W. Muff R. Fischer J.A. Foord S.M. International Union of Pharmacology. XXXII. The mammalian calcitonin gene-related peptides, adrenomedullin, amylin, and calcitonin receptors.Pharmacol Rev. 2002; 54: 233-246Crossref PubMed Scopus (686) Google Scholar, 21Young A. Receptor pharmacology.Adv Pharmacol. 2005; 52: 47-65Crossref PubMed Scopus (32) Google Scholar Several peptides, typically analogs of salmon calcitonin, have been developed as amylin receptor antagonists, chiefly with a view to treating diabetes mellitus.22Hay D.L. Christopoulos G. Christopoulos A. Sexton P.M. Amylin receptors: molecular composition and pharmacology.Biochem Soc Trans. 2004; 32: 865-867Crossref PubMed Scopus (74) Google Scholar, 23Hay D.L. Christopoulos G. Christopoulos A. Poyner D.R. Sexton P.M. Pharmacological discrimination of calcitonin receptor: receptor activity-modifying protein complexes.Mol Pharmacol. 2005; 67: 1655-1665Crossref PubMed Scopus (163) Google Scholar Of these, AC187 and AC253 are highly selective and potent antagonists at the amylin receptor.21Young A. Receptor pharmacology.Adv Pharmacol. 2005; 52: 47-65Crossref PubMed Scopus (32) Google Scholar, 23Hay D.L. Christopoulos G. Christopoulos A. Poyner D.R. Sexton P.M. 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Harris K.H. Cho C. Fu W. MacTavish D. Human amylin actions on rat cholinergic basal forebrain neurons: antagonism of beta-amyloid effects.J Neurophysiol. 2003; 90: 3130-3136Crossref PubMed Scopus (52) Google Scholar We have shown that both the acute electrophysiological and neurotoxic effects of amylin and Aβ in the rat cholinergic basal forebrain neurons can be blocked using amylin receptor antagonists.17Jhamandas J.H. Harris K.H. Cho C. Fu W. MacTavish D. Human amylin actions on rat cholinergic basal forebrain neurons: antagonism of beta-amyloid effects.J Neurophysiol. 2003; 90: 3130-3136Crossref PubMed Scopus (52) Google Scholar, 26Jhamandas J.H. MacTavish D. Antagonist of the amylin receptor blocks beta-amyloid toxicity in rat cholinergic basal forebrain neurons.J Neurosci. 2004; 24: 5579-5584Crossref PubMed Scopus (61) Google Scholar An important question raised by our observations is whether blockade of the amylin receptor confers neuroprotection against Aβ toxicity in cultures of human neurons. This is a critical issue, because rodents (rats, mice, hamsters), the species in which the effects of Aβ have been most widely studied, do not develop an age-related human equivalent of AD. In the present study, using whole-cell patch clamp recordings from primary cultures of human fetal neurons (HFNs), we found that acute applications of nanomolar concentrations of Aβ result in an activation of a suite of potassium conductances, which can be blocked by exposure to the amylin receptor antagonist AC253. Furthermore, the amyloid-induced toxicity mediated via caspase-dependent and -independent pathways in HFNs can be attenuated with pretreatment of cultures with AC253 or through down-regulation of the amylin receptor gene expression with small interfering RNA (siRNA). Finally, we demonstrate that in transgenic mice that overexpress APP (TgCRND8), amylin receptor expression in the brain is up-regulated in an age-dependent manner, but only within specific brain regions that demonstrate an increased amyloid burden. All experiments were conducted in compliance with the relevant laws and the guidelines set by the Canadian Council for Animal Care and with the approval of the Human Research Ethics Board and Animal Care Use Committee (Health Sciences) at the University of Alberta. HFNs, grown on coverslips, were bathed with oxygenated artificial cerebrospinal fluid that contained (in mmol/L) 140 NaCl, 2.5 KCl, 1.5 CaCl2, 1.2 MgCl2, 10 HEPES, and 33 d-glucose (pH 7.4). Whole cell patch-clamp recordings were performed at room temperature (20°C–22°C) using an Axopatch-200B amplifier in combination with a 1200A interface (Axon Instruments, Foster City, CA). Patch electrodes (thin wall with filament, 1.5-mm diameter; World Precision Instruments, Sarasota, FL) were pulled (PP-83 electrode puller; Narishige Scientific Instrument Lab, Tokyo, Japan) to yield resistances of 3–6 MΩ. The internal patch pipette solution contained (in mM) 140 K-methylsulfate, 10 EGTA, 5 MgCl2, 1 CaCl2, 10 HEPES, 2.2 Na2-ATP, and 0.3 Na-GTP (pH 7.2). All whole-cell recordings were made in current-clamp and voltage-clamp mode, and bridge balance and capacitance compensation were used. After whole-cell configuration was established with voltage-clamp mode (holding potential = −80 mV), we waited at least 5 minutes for the cell to stabilize, then started either voltage-clamp studies or switched to current-clamp recording mode. The current and membrane voltages were recorded using a low-pass filter at 5 kHz and were digitized at 10 kHz. All data were acquired and analyzed using pClamp8 software v8.0 (Axon Instruments). Whole-cell currents were activated by a voltage-ramp protocol, where the cells were held at −80 mV and subjected to voltage ramps from −110 to +30 mV at the rate of 20 mV/s. A 1-second-long hyperpolarizing command to −110 mV was applied to remove inactivation of K+ channels so that maximum current could be activated during the subsequent slow voltage ramp to +30 mV. Ca2+-dependent K+ conductances were isolated using the BK channel-specific blocker iberiotoxin. Drugs were either bath-applied or delivered via a focal applicator. Neuronal cultures were prepared from 12- to 15-gestational week fetuses with approval of the Human Ethics Research Board at the University of Alberta. The meninges and blood vessels were removed, the brain tissue was washed in minimum essential medium and mechanically dissociated by repeated trituration through a 20-gauge needle. Cells were centrifuged at 1500 g for 10 minutes and resuspended in minimum essential medium with 10% heat-inactivated fetal bovine serum, 0.2% N2 supplement, and 1% antibiotic solution (104 U of penicillin G per milliliter, 10 mg streptomycin per milliliter, and 25 mg amphotericin B per milliliter in 0.9% NaCl).27Power C. McArthur J.C. Nath A. Wehrly K. Mayne M. Nishio J. Langelier T. Johnson R.T. Chesebro B. Neuronal death induced by brain-derived human immunodeficiency virus type 1 envelope genes differs between demented and nondemented AIDS patients.J Virol. 1998; 72: 9045-9053Crossref PubMed Google Scholar Subsequently, cultures were treated with arabinofuranosylcytosine (25 μmol/L) for 2 weeks and were plated (at a density of 5 × 10−5 per well) on 96-well plates. HFN cultures were grown in a 5% CO2 humidified incubator at 37°C. Sample wells were immunostained for the neuronal marker microtubule-associated protein 2, and only cultures in which >70% of the cells stained positive for the marker were used for experiments. All reagents were obtained from Invitrogen (Burlington, ON, Canada) and antibodies from Sigma-Aldrich (Oakville, ON, Canada). Soluble oligomeric Aβ1-42 or the inverse sequence peptide Aβ42-1, human amylin, and AC253 were prepared according to published protocols.28Stine Jr, W.B. Dahlgren K.N. Krafft G.A. LaDu M.J. In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis.J Biol Chem. 2003; 278: 11612-11622Crossref PubMed Scopus (826) Google Scholar, 29White J.A. Manelli A.M. Holmberg K.H. Van Eldik L.J. Ladu M.J. Differential effects of oligomeric and fibrillar amyloid-beta 1-42 on astrocyte-mediated inflammation.Neurobiol Dis. 2005; 18: 459-465Crossref PubMed Scopus (193) Google Scholar All peptides were purchased from American Peptide (Sunnyvale, CA). To determine the dose-dependent toxicity of Aβ peptides and human amylin. HFNs were treated with different concentrations of the peptides (0.5–50 μmol/L) (see Supplemental Figure S1 at http://ajp.amjpathol.org). In each experiment and in subsequent ones described below, two rows of eight wells each (of a 96-well plate) received the same treatment, and each experiment was repeated a minimum of four times. To evaluate the neuroprotective effects of the amylin receptor antagonist AC253 against Aβ toxicity, cultured HFNs were preexposed to AC253 (10 μmol/L) for 24 hours and then to soluble oligomeric 20 μmol/L Aβ1-42 or human amylin (2 μmol/L). Cells in adjacent rows of wells received applications of either 20 μmol/L Aβ1-42, 2 μmol/L human amylin, or 10 μmol/L or AC253. Controls included applications of the inverse sequence Aβ peptide Aβ42-1 or no drug. After 48 hours, the control and treated cultures were examined for neuronal survival using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich).26Jhamandas J.H. MacTavish D. Antagonist of the amylin receptor blocks beta-amyloid toxicity in rat cholinergic basal forebrain neurons.J Neurosci. 2004; 24: 5579-5584Crossref PubMed Scopus (61) Google Scholar, 30Ding X. MacTavish D. Kar S. Jhamandas J.H. Galanin attenuates beta- amyloid (Abeta) toxicity in rat cholinergic basal forebrain neurons.Neurobiol Dis. 2006; 21: 413-420Crossref PubMed Scopus (64) Google Scholar For experiments examining the effects of caspase inhibition on Aβ toxicity, the cell cultures were pretreated for 2 hours with individual caspase inhibitors (20 μmol/L; kit from RD Systems, Minneapolis, MN), followed by exposure to 20 μmol/L Aβ1-42 for 48 hours. At the end of this period, the cultures were processed for MTT assay. Each experiment was conducted four times. Western blotting was performed as described previously.26Jhamandas J.H. MacTavish D. Antagonist of the amylin receptor blocks beta-amyloid toxicity in rat cholinergic basal forebrain neurons.J Neurosci. 2004; 24: 5579-5584Crossref PubMed Scopus (61) Google Scholar, 30Ding X. MacTavish D. Kar S. Jhamandas J.H. Galanin attenuates beta- amyloid (Abeta) toxicity in rat cholinergic basal forebrain neurons.Neurobiol Dis. 2006; 21: 413-420Crossref PubMed Scopus (64) Google Scholar Briefly, samples of control and treated groups of cultured cells (with AC253, Aβ1-42, or AC253 pretreatment followed by Aβ1-42) with equal amounts of protein were separated by 12% polyacrylamide gel electrophoresis and the resolved proteins were transferred onto nitrocellulose membranes and probed with anti-caspase 3, 6, and 9 antibodies and also with antibodies to pro- and antiapoptotic intermediaries (cytochrome c, SMAC, PUMA, Bax, and Bcl-2; New England Biolabs, Ipswich, MA). Blots were also probed with anti-β actin (Abcam, Cambridge, MA) as loading control. To assess whether AC253 also blocked Aβ-induced caspase-independent cell death, we performed Western blot and immunohistochemistry to detect cleavage and translocation of apoptosis inducing factor (AIF) from the activated mitochondria using AIF antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and MitoTracker Red dye (Molecular Devices, Sunnyvale, CA). Each experiment was repeated four times. For all quantitative analysis of Western blots, Image J software v1.44j (National Institutes of Health, Bethesda, MD) was used. Relative intensities were calculated by comparing the intensity of protein bands of interest to control β-actin loading bands. siRNA corresponding to the human CTR (NM_001742) and RAMP3 (NM_005856) genes, purchased from Open BioSystems (Huntsville, AL), were inserted into a vector (pGIPz) that produced green fluorescent protein. HFNs were plated at a density of 2 million cells/ml in a 6-well plate and cultured for 24 hours before transfection. On the day of transfection, culture media was replaced with OptiMEM (Invitrogen). Four micrograms of plasmid siRNA mixed with Lipofectamine 2000 (Invitrogen) was incubated for 30 minutes and then added to the cultures. Cultures were gently mixed and placed in an incubator at 37°C with 5% CO2 for 10 hours, after which normal culture media was replaced. Subsequently, transfected and non-transfected HFN cultures in 96-well plates were exposed to soluble oligomeric Aβ1-42 (20 μmol/L) or the inverse sequence peptide, Aβ42-1 (20 μmol/L), and cell survival was measured using the MTT assay. Brains were quickly removed from 1-, 4-, and 6-month-old TgCRND8 APP mice (human APP695 transgene array incorporating Swedish K670M/N671L and Indiana V717F mutations superimposed on a C57BL6/C3H genetic background) and from control nontransgenic C57BL6/C3H littermate mice. The cortex, hippocampus, diagonal band of Broca, brainstem, and cerebellum were dissected and weighed before the preparation of normalized lysates. Proteins from each dissected region were isolated according to the protocol provided with the Aβ1-42 enzyme-linked immunosorbent assay kit (BioSource, Burlington, ON, Canada). In brief, in this protocol a monoclonal antibody specific to the NH-2 terminus of the Aβ1-42 is coated to the wells of microtiter strips. Proteins are incubated in the well. A secondary antibody specific to the COOH-terminus of the Aβ1-42 sequence is then incubated in the wells. Bound rabbit antibody is detected with a horseradish peroxidase-labeled anti-rabbit antibody. Excess anti-rabbit antibody is removed by washing, and then a substrate solution is added and is converted, producing a color. The color is measured at 450 nm and the intensity of color is directly proportional to the amount of Aβ1-42 in the tissue. Under halothane anesthesia, TgCRND8 APP mice (1, 4, and 6 months of age, n = 5 for each age group) and age-matched control mice (n = 5 for each age group) were decapitated and brains were quickly removed. Brains were hemisected and one half of the brain was used for dissecting the cortex, hippocampus, diagonal band of Broca, cerebellum, and brainstem. Tissue was weighed and the placed in cold RIPA (radio-immunoprecipitation assay) buffer with protease inhibitors and proteins were isolated and measured using a Bio-Rad protein assay kit (Mississauga, ON, Canada). Proteins were diluted with SDS sample buffer (New England Biolabs) and were loaded at 30 μg per lane on a 12% agarose gel. Proteins were transferred to nitrocellulose blocked with nonfat milk. Nitrocellulose membranes were incubated with antibodies overnight at 4°C on a shaker. Blots were incubated in the following antibodies, one at a time, and were stripped between each application: CTR (1:1000 rabbit; kindly supplied by Professor P. Sexton, Monash University, VIC, Australia), RAMP3 (1:1000 goat; Santa Cruz Biotechnology), 6E10 for Aβ (1:5000 mouse; 6E10; Signet Laboratories, Dedham, MA), and β-actin (1:1000 mouse; Sigma-Aldrich). Blots were subsequently incubated with corresponding secondary antibodies labeled with horseradish peroxidase and were washed with Immobilon Western chemiluminescent substrate (Millipore, Billerica, MA). Blots were exposed to X-ray film (Kodak BioMax MR) for 10 seconds to 5 minutes. X-ray blots were then scanned and analyzed with Image J software. The other half of the brains from TgCRND8 and control mice was fixed for immunohistochemical analysis of brain sections from cortex and hippocampus to determine Aβ plaque deposition, and the same sections were also double-stained using the CTR antibody. Data are presented as means ± SEM. Unless otherwise indicated, group data were compared using one-way analysis of variance followed by Newman-Keuls post hoc test with the P-value set at 0.05. In HFNs, applications of oligomeric Aβ1-42 caused a time-independent and concentration-dependent reduction in whole-cell outward currents (WCCs) in the voltage range −30 to +30 mV (see Supplemental Figure S2 at http://ajp.amjpathol.org). WCCs were not affected by applications of the inverse sequence Aβ42-1 peptide (data not shown). AC253 is a selective peptidergic amylin receptor antagonist that, applied in the concentration range 10−5 to 10−6 mol/L, has been shown to block the actions of human amylin.17Jhamandas J.H. Harris K.H. Cho C. Fu W. MacTavish D. Human amylin actions on rat cholinergic basal forebrain neurons: antagonism of beta-amyloid effects.J Neurophysiol. 2003; 90: 3130-3136Crossref PubMed Scopus (52) Google Scholar, 31Riediger T. Rauch M. Schmid H.A. Actions of amylin on subfornical organ neurons and on drinking behaviour in rats.Am J Physiol. 1999; 276: R514-R521PubMed Google Scholar We investigated whether the effects of Aβ could also be blocked using this amylin receptor antagonist. Application of 10 nmol/L AC253 blocked Aβ1-42-induced reduction in WCCs (20 nmol/L, n = 8, P < 0.01) (Figure 1, A and B). Peak WCC (at +30 mV) in the presence of Aβ1-42 was significantly reduced, compared with the control level of current (27.7%, n = 8, P < 0.01), but in the presence of AC253, the Aβ1-42-evoked response was abolished (Figure 1C). When the high conductance Ca2+-activated K+ channels Ic (BK channels) were inhibited with iberiotoxin (25 nmol/L), Aβ1-42-induced reduction in WCCs was significantly blocked (Figure 1D), indicating that Aβ effects are expressed via this ionic conductance. Under current-clamp conditions, the number of spikes generated by injection of a current pulse in HFNs was significantly increased in the presence of Aβ1-42 (10 nmol/L), compared with control conditions (Figure 2A). Application of AC253 (10 nmol/L) blocked the increase in excitability induced by Aβ1-42. The average number of spikes elicited by current injection was 1.2 ± 0.3 Hz under control conditions, which significantly increased to 16.6 ± 3.5 Hz in the presence of Aβ1-42 (Figure 2B) (n = 5, P < 0.01). In the presence of AC253, application of Aβ1-42 did not result in a significant increase in firing (n = 5, P > 0.05), compared with control. Under resting conditions, application of Aβ1-42 (10 nmol/L) to HFNs resulted in membrane depolarization and an increase in action potential firing, which were blocked in the presence of AC253 (Figure 2C). To determine whether Aβ toxicity is expressed via amylin receptors, we used primary cultures of HFNs to initially establish that application of oligomeric Aβ1-42 (see Supplemental Figure S3 at http://ajp.amjpathol.org and also the Materials and Methods section) or human amylin evokes concentration-dependent cell death in such neurons (see Supplemental Figure S1 at http://ajp.amjpathol.org). Pretreatment of HFNs for 24 hours with the amylin receptor antagonist AC253 (10 μmol/L) resulted in a significant improvement in neuronal survival of HFNs exposed to either Aβ1-42 (20 μmol/L) or human amylin (2 μmol/L) (Figure 3, A and B). Concentrations of extracellularly applied oligomeric Aβ1-42 used were based on our data (see Supplemental Figure S1 at http://ajp.amjpathol.org) and data reported in the literature for studies on human neurons.32Mattson M.P. Cheng B. Davis D. Bryant K. Lieberburg I. 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