Loss of Muscarinic M1 Receptor Exacerbates Alzheimer's Disease–Like Pathology and Cognitive Decline
2011; Elsevier BV; Volume: 179; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2011.04.041
ISSN1525-2191
AutoresRodrigo Medeiros, Masashi Kitazawa, Antonella Caccamo, David Baglietto‐Vargas, Tatiana Estrada-Hernandez, David H. Cribbs, A Fisher, Frank M. LaFerla,
Tópico(s)Neuropeptides and Animal Physiology
ResumoAlzheimer's disease (AD) is pathologically characterized by tau-laden neurofibrillary tangles and β-amyloid deposits. Dysregulation of cholinergic neurotransmission has been implicated in AD pathogenesis, contributing to the associated memory impairments; yet, the exact mechanisms remain to be defined. Activating the muscarinic acetylcholine M1 receptors (M1Rs) reduces AD-like pathological features and enhances cognition in AD transgenic models. To elucidate the molecular mechanisms by which M1Rs affect AD pathophysiological features, we crossed the 3xTgAD and transgenic mice expressing human Swedish, Dutch, and Iowa triple-mutant amyloid precursor protein (Tg-SwDI), two widely used animal models, with the M1R−/− mice. Our data show that M1R deletion in the 3xTgAD and Tg-SwDI mice exacerbates the cognitive impairment through mechanisms dependent on the transcriptional dysregulation of genes required for memory and through acceleration of AD-related synaptotoxicity. Ablating the M1R increased plaque and tangle levels in the brains of 3xTgAD mice and elevated cerebrovascular deposition of fibrillar Aβ in Tg-SwDI mice. Notably, tau hyperphosphorylation and potentiation of amyloidogenic processing in the mice with AD lacking M1R were attributed to changes in the glycogen synthase kinase 3β and protein kinase C activities. Finally, deleting the M1R increased the astrocytic and microglial response associated with Aβ plaques. Our data highlight the significant role that disrupting the M1R plays in exacerbating AD-related cognitive decline and pathological features and provide critical preclinical evidence to justify further development and evaluation of selective M1R agonists for treating AD. Alzheimer's disease (AD) is pathologically characterized by tau-laden neurofibrillary tangles and β-amyloid deposits. Dysregulation of cholinergic neurotransmission has been implicated in AD pathogenesis, contributing to the associated memory impairments; yet, the exact mechanisms remain to be defined. Activating the muscarinic acetylcholine M1 receptors (M1Rs) reduces AD-like pathological features and enhances cognition in AD transgenic models. To elucidate the molecular mechanisms by which M1Rs affect AD pathophysiological features, we crossed the 3xTgAD and transgenic mice expressing human Swedish, Dutch, and Iowa triple-mutant amyloid precursor protein (Tg-SwDI), two widely used animal models, with the M1R−/− mice. Our data show that M1R deletion in the 3xTgAD and Tg-SwDI mice exacerbates the cognitive impairment through mechanisms dependent on the transcriptional dysregulation of genes required for memory and through acceleration of AD-related synaptotoxicity. Ablating the M1R increased plaque and tangle levels in the brains of 3xTgAD mice and elevated cerebrovascular deposition of fibrillar Aβ in Tg-SwDI mice. Notably, tau hyperphosphorylation and potentiation of amyloidogenic processing in the mice with AD lacking M1R were attributed to changes in the glycogen synthase kinase 3β and protein kinase C activities. Finally, deleting the M1R increased the astrocytic and microglial response associated with Aβ plaques. Our data highlight the significant role that disrupting the M1R plays in exacerbating AD-related cognitive decline and pathological features and provide critical preclinical evidence to justify further development and evaluation of selective M1R agonists for treating AD. Alzheimer's disease (AD) is a progressive neurodegenerative disorder that leads to cognitive impairment and dementia. The neuropathological hallmarks of AD are amyloid plaques, composed of β-amyloid (Aβ) peptides, and neurofibrillary tangles, composed of the hyperphosphorylated tau protein. The deposition of fibrillar Aβ in the cerebrovasculature, a condition known as cerebral amyloid angiopathy (CAA), is also a prominent feature of AD. Together with associated processes, such as inflammation and oxidative stress, these pathological cascades contribute to loss of synaptic integrity and progressive neurodegeneration.1Querfurth H.W. LaFerla F.M. Alzheimer's disease.N Engl J Med. 2010; 362: 329-344Crossref PubMed Scopus (3703) Google Scholar Restoring cholinergic dysfunction has been a primary means of improving the cognitive decline in AD because four of the five Food and Drug Administration–approved drugs are acetylcholinesterase inhibitors, with the notable exception of memantine.2Mangialasche F. Solomon A. Winblad B. Mecocci P. Kivipelto M. Alzheimer's disease: clinical trials and drug development.Lancet Neurol. 2010; 9: 702-716Abstract Full Text Full Text PDF PubMed Scopus (967) Google Scholar Acetylcholinesterase inhibitors provide mild symptomatic relief but eventually lose efficacy over time, most likely because they are not disease-modifying agents.1Querfurth H.W. LaFerla F.M. Alzheimer's disease.N Engl J Med. 2010; 362: 329-344Crossref PubMed Scopus (3703) Google Scholar Alternatively, recent evidence3Fisher A. M1 muscarinic agonists target major hallmarks of Alzheimer's disease: the pivotal role of brain M1 receptors.Neurodegener Dis. 2008; 5: 237-240Crossref PubMed Scopus (72) Google Scholar, 4Wess J. Eglen R.M. Gautam D. Muscarinic acetylcholine receptors: mutant mice provide new insights for drug development.Nat Rev Drug Discov. 2007; 6: 721-733Crossref PubMed Scopus (472) Google Scholar indicates that stimulation of muscarinic acetylcholine receptors, in particular the M1 receptor (M1R), restores cognition and attenuates AD-like pathological features in several different animal models, rendering it an attractive therapeutic approach for AD. The M1R is the most abundant muscarinic acetylcholine receptor subtype in the cerebral cortex and hippocampus, the two main brain regions that develop amyloid plaques and neurofibrillary tangles.5Levey A.I. Kitt C.A. Simonds W.F. Price D.L. Brann M.R. 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Corrections for regional brain atrophy and the relative involvement of receptor subtypes are often undefined. Thus, additional studies are necessary to clarify how the activity and levels of M1R are changed in the AD brain. Preclinical data have demonstrated that activation of M1R elevates soluble amyloid precursor protein (APP)α, decreases Aβ and tau pathological features, and blocks Aβ-induced neurotoxicity in vitro.11Nitsch R.M. Slack B.E. Wurtman R.J. Growdon J.H. Release of Alzheimer amyloid precursor derivatives stimulated by activation of muscarinic acetylcholine receptors.Science. 1992; 258: 304-307Crossref PubMed Scopus (849) Google Scholar, 12Sadot E. Gurwitz D. Barg J. Behar L. Ginzburg I. Fisher A. Activation of m1 muscarinic acetylcholine receptor regulates tau phosphorylation in transfected PC12 cells.J Neurochem. 1996; 66: 877-880Crossref PubMed Scopus (139) Google Scholar, 13Haring R. Fisher A. Marciano D. Pittel Z. Kloog Y. Zuckerman A. Eshhar N. Heldman E. 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Reduction of cerebrospinal fluid amyloid beta after systemic administration of M1 muscarinic agonists.Brain Res. 2001; 905: 220-223Crossref PubMed Scopus (66) Google Scholar, 16Beach T.G. Walker D.G. Potter P.E. Sue L.I. Scott S. Layne K.J. Newell A.J. Poston M.E. Webster S.D. Durham R.A. Emmerling M.R. Honer W.G. Fisher A. Roher A.E. Immunotoxin lesion of the cholinergic nucleus basalis causes Abeta deposition: towards a physiologic animal model of Alzheimer's disease.Curr Med Chem Immun Endocr Metab Agents. 2003; 3: 233-243Google Scholar Evidence from our laboratory shows that long-term treatment with this compound reverses cognitive impairments and decreases Aβ and tau pathological features in the 3xTgAD mice.17Caccamo A. Oddo S. Billings L.M. Green K.N. Martinez-Coria H. Fisher A. LaFerla F.M. M1 receptors play a central role in modulating AD-like pathology in transgenic mice.Neuron. 2006; 49: 671-682Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar Similarly, the administration of the muscarinic agonist RS86 to rats decreases APP levels in the cortex and hippocampus and increases the APPα level in the cerebrospinal fluid.18Lin L. Georgievska B. Mattsson A. Isacson O. Cognitive changes and modified processing of amyloid precursor protein in the cortical and hippocampal system after cholinergic synapse loss and muscarinic receptor activation.Proc Natl Acad Sci U S A. 1999; 96: 12108-12113Crossref PubMed Scopus (112) Google Scholar, 19Seo H. Ferree A.W. Isacson O. Cortico-hippocampal APP and NGF levels are dynamically altered by cholinergic muscarinic antagonist or M1 agonist treatment in normal mice.Eur J Neurosci. 2002; 15: 498-506Crossref PubMed Scopus (28) Google Scholar Corroborating these findings, genetic deletion of M1R has recently increased Aβ pathological features in APPSwe/Ind mice.20Davis A.A. Fritz J.J. Wess J. Lah J.J. Levey A.I. Deletion of M1 muscarinic acetylcholine receptors increases amyloid pathology in vitro and in vivo.J Neurosci. 2010; 30: 4190-4196Crossref PubMed Scopus (108) Google Scholar Because of the beneficial effects of M1R agonists in transgenic models, additional studies are necessary to establish the molecular mechanisms through which M1R promotes its neuroprotective effect to justify the translational applicability of using M1R agonists as a therapeutic intervention for AD. To determine the effect of modulating M1R function on the progression of Aβ and tau pathological features and the subsequent effects on cognition, we evaluated the consequences of ablating M1R signaling in the 3xTgAD mice by crossing them with mice with a null mutation of the M1R gene.21Anagnostaras S.G. Murphy G.G. Hamilton S.E. Mitchell S.L. Rahnama N.P. Nathanson N.M. Silva A.J. Selective cognitive dysfunction in acetylcholine M1 muscarinic receptor mutant mice.Nat Neurosci. 2003; 6: 51-58Crossref PubMed Scopus (441) Google Scholar We also assessed the molecular mechanisms by which ablation of M1R affects cerebral microvascular accumulation of Aβ in the transgenic mice expressing human Swedish, Dutch, and Iowa triple-mutant APP (Tg-SwDI), a well-described animal model of CAA.22Davis J. Xu F. Deane R. Romanov G. Previti M.L. Zeigler K. Zlokovic B.V. Van Nostrand W.E. Early-onset and robust cerebral microvascular accumulation of amyloid beta-protein in transgenic mice expressing low levels of a vasculotropic Dutch/Iowa mutant form of amyloid beta-protein precursor.J Biol Chem. 2004; 279: 20296-20306Crossref PubMed Scopus (287) Google Scholar Homozygous M1R−/− mice21Anagnostaras S.G. Murphy G.G. Hamilton S.E. Mitchell S.L. Rahnama N.P. Nathanson N.M. Silva A.J. Selective cognitive dysfunction in acetylcholine M1 muscarinic receptor mutant mice.Nat Neurosci. 2003; 6: 51-58Crossref PubMed Scopus (441) Google Scholar were bred with either homozygous 3xTgAD mice harboring a PS1M146V knock-in and APPSwe and tauP301L transgenes23Oddo S. Caccamo A. Shepherd J.D. Murphy M.P. Golde T.E. Kayed R. Metherate R. Mattson M.P. Akbari Y. LaFerla F.M. Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction.Neuron. 2003; 39: 409-421Abstract Full Text Full Text PDF PubMed Scopus (3158) Google Scholar or heterozygous Tg-SwDI mice that contain the Swedish and the vasculotropic Dutch and Iowa APP mutations.22Davis J. Xu F. Deane R. Romanov G. Previti M.L. Zeigler K. Zlokovic B.V. Van Nostrand W.E. Early-onset and robust cerebral microvascular accumulation of amyloid beta-protein in transgenic mice expressing low levels of a vasculotropic Dutch/Iowa mutant form of amyloid beta-protein precursor.J Biol Chem. 2004; 279: 20296-20306Crossref PubMed Scopus (287) Google Scholar The offspring were intercrossed to yield 3xTgAD+/+/M1R−/− (3xTgAD-M1R−/−) mice and Tg-SwDI+/−/M1R−/− (Tg-SwDI–M1R−/−) mice. Nine-month-old Tg-SwDI and Tg-SwDI–M1R−/− mice and 18-month-old 3xTgAD and 3xTgAD-M1R−/− mice were used in the experiments. Age-matched nontransgenic (nTg) and M1R−/− mice were used as controls. All animals had a C57Bl6/129SvJ background. No obvious histological changes were apparent in the brains of mice lacking the M1R. In addition, no changes in the expression levels and distribution of acetylcholine receptors M2-M5 were found in M1R−/− mice compared with nTg mice. Behavioral, neuropathological, and neurochemical changes in M1R−/− mice have been reviewed elsewhere.4Wess J. Eglen R.M. Gautam D. Muscarinic acetylcholine receptors: mutant mice provide new insights for drug development.Nat Rev Drug Discov. 2007; 6: 721-733Crossref PubMed Scopus (472) Google Scholar All procedures used in the present study followed the Principles of Laboratory Animal Care from NIH publication 85–23 and were approved by the University of California, Irvine, Institutional Animal Care and Use Committee. For open field activity testing, mice were individually placed in the center of a novel open field environment (45 × 45 × 30 cm) and allowed to explore for 5 minutes. The total distance moved in the open field was monitored with the EthoVision XT video-tracking system (Noldus Information Technology, Leesburg, VA). Each mouse was housed individually for 4 days to establish territorial dominance. A juvenile male mouse was used as the intruder. To test for social recognition, the intruder was placed inside a wire cylinder and introduced into the test cage. In the habituation session, mice were allowed to explore the empty wire cylinder for 10 minutes. Subsequently, the intruder mouse was placed into the cylinder and the tested mouse was allowed to freely explore it for 5 minutes. The entire procedure was repeated five times. After the fifth exposure to the same intruder, a novel intruder was added to the wire cylinder. The experiment was videotaped, and the social interaction was measured as the amount of time that animals spent sniffing through the holes of the cylinder (latency). Before testing, each mouse was habituated to an empty Plexiglas arena (45 × 25 × 20 cm) for 3 consecutive days. On the first day of testing, mice were exposed to two identical objects placed at opposite ends of the arena for 5 minutes. Twenty-four hours later, mice were presented for 5 minutes with one of the familiar objects and a novel object of similar dimensions. The arena and the stimulus objects were cleaned thoroughly between trials to ensure the absence of olfactory cues. If an animal did not explore both objects during the training phase, the test was not scored during the test phase. In the scoring procedure, because some mice exhibited freezing or fearful behavior on introduction to the chamber, scoring did not start until the mice physically moved from their initial starting position, which was always in the corner closest to the familiar object. Exploration counted if the mouse's head was within 2.54 cm of the object, with its neck extended and vibrissae moving. Simple proximity, chewing, or standing on the object did not count as exploration. All exploratory segments and tests were videotaped for scoring purposes. The recognition index represents the percentage of the time that mice spend exploring the novel object. The apparatus used for the water maze task was a circular aluminum tank (1.2-m diameter) painted white and filled with water maintained at 22°C ± 2°C (mean ± SEM). The tank was located in a test room containing various prominent visual cues. To reduce stress, mice were placed on the platform for 10 seconds before the first training trial. Mice were trained to swim to a 14-cm-diameter circular clear Plexiglas platform submerged 1.5 cm beneath the surface of the water and invisible to the mice while swimming. The platform was located in a fixed position, equidistant from the center and the wall of the tank. Mice were subjected to four training trials per day (intertrial interval, 5 minutes). During each trial, mice were placed into the tank at one of four designated start points in a pseudorandom order. Mice were allowed to find and escape onto the submerged platform. If they failed to find the platform within 60 seconds, they were manually guided to the platform and allowed to remain there for 10 seconds. Mice were trained for as many days as needed to reach the training criterion of 25 seconds (escape latency). To control for overtraining, probe trials were run for each group, both as soon as they reached group criterion and after all groups had reached criterion. However, the training session was interrupted if mice did not reach the training criterion within 8 days. The probe trial was assessed 24 hours after the last training session and consisted of a 60-second free swim in the pool without the platform. Performance was monitored with the EthoVision XT video-tracking system. During training, mice were placed in the fear conditioning chamber (San Diego Instruments, San Diego, CA) and allowed to explore for 2 minutes before receiving three electric foot shocks (duration, 1 second; intensity, 0.2 mA; intershock interval, 2 minutes). Animals were returned to the home cage 30 seconds after the last foot shock. Twenty-four hours later, behavior in the conditioning chamber was video recorded during 5 minutes and subsequently analyzed for freezing, which was defined as the absence of all movement except for respiration. Mice were deeply anesthetized with sodium pentobarbital and euthanized by perfusion transcardially with 0.1 mol/L PBS solution (pH 7.4). The right brain hemispheres were fixed for 48 hours in 4% paraformaldehyde and cryoprotected in 30% sucrose for immunohistochemical (IHC) analysis. Frozen brains were sectioned coronally into 40 μmol/L sections using a Leica SM2010R freezing microtome (Leica Microsystems, Bannockburn, IL), serially collected in cold 0.02% sodium azide, and stored at 4°C. The left hemispheres were snap frozen on dry ice and subject to protein extraction sequentially using the T-PER tissue protein extraction reagent (Thermo Scientific, Rockford, IL) and 70% formic acid. The supernatant was divided and stored at −80°C. The protein concentration in the supernatant was determined using the Bradford assay. Equal protein amounts were separated on a 4% to 12% SDS-PAGE gradient, transferred to a nitrocellulose membrane, and incubated overnight with primary antibody at 4°C. The following primary antibodies were used in this study: c-Fos (9F6) (1:1000), cAMP response element binding (CREB; 48H2) (1:1000), p-CREB (S133) (87G3) (1:1000), p-glycogen synthase kinase (GSK) 3β (S9) (5B3) (1:3000), postsynaptic density protein 95 (PSD-95, 1:1000) (Cell Signaling Technology, Danvers, MA), human APP-CT20 (751-770) (1:5000), disintegrin and metalloproteinase domain-containing protein (ADAM)10 (735-749) (1:1000), ADAM17 (TACE, 807-823) (1:1000), β-site APP cleaving enzyme (BACE) 1 (485-501) (1:1000), GSK3αβ (1H8) (1:1000), CDK5 (268-283) (1:1000) (Calbiochem, San Diego, CA), human tau (HT7) (159-163) (1:5000), phospho-tau AT8 (p-S202/T205) (1:1000), p-tau AT100 (p-S212/T214) (1:1000), p-tau AT270 (p-T181) (1:1000) (Thermo Scientific), p-tau paired helical filament (PHF)-1 (p-S396/S404) (1:1000) (a gift from Dr. Peter Davies, Albert Einstein College of Medicine, Manhasset, NY), Aβ1-16 (6E10) (1:1000) (Covance Research Products, Denver, PA), PP2A (FL-309) (1:1000), p35 (N-20) (1:200), and glyceraldehyde-3-phosphate dehydrogenase (FL335) (1:5000) (Santa Cruz Biotechnology, Santa Cruz, CA). After washing, the membranes were incubated with adjusted secondary antibodies coupled to horseradish peroxidase. The immunocomplexes were visualized using the SuperSignal West Pico Kit (Thermo Scientific). Band density measurements were obtained using ImageJ 1.36b imaging software (NIH, Bethesda, MD). For the determination of Aβ levels, T-PER soluble fractions were loaded directly onto enzyme-linked immunosorbent assay (ELISA) plates, whereas the formic acid supernatants (insoluble fractions) were diluted 1:20 in a neutralization buffer (1 mol/L Tris base and 0.5 mol/L NaH2PO4) before loading. MaxiSorp immunoplates (Nunc, Rochester, NY) were coated with mAb20.1 antibody (a gift from Dr. William E. Van Nostrand, Stony Brook University, Stony Brook, NY) at a concentration of 25 μg/mL in coating buffer (0.1 mol/L Na2CO3, pH 9.6) and blocked with 3% bovine serum albumin (BSA). Standard solutions for both Aβ40 and Aβ42 were made in the antigen capture buffer (20 mmol/L NaH2PO4, 2 mmol/L EDTA, 0.4 mol/L NaCl, 0.05% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, and 1% BSA, pH 7.0) and loaded onto ELISA plates in duplicate. Samples were then loaded (in duplicate) and incubated overnight at 4°C. Plates were then washed and probed with either horseradish peroxidase–conjugated anti-Aβ40 (C49) or anti-Aβ42 (D32) (a gift from Dr. Vitaly Vasilevko and Dr. David H. Cribbs, University of California, Irvine) overnight at 4°C. The chromogen was 3,3′,5,5′-tetramethylbenzidine, and the reaction was discontinued with 30% phosphoric acid. The plates were read at 450 nm using a plate reader (Molecular Dynamics, Sunnyvale, CA). The readings were then normalized to protein concentrations of the samples. The levels of tumor necrosis factor-α and IL-1β in the T-PER soluble fractions were measured using commercially available ELISA kits (Thermo Scientific), according to the manufacturer's instructions. Antigen retrieval was optimized using a 90% formic acid solution for 7 minutes for Aβ staining. Free-floating sections were pretreated with 3% hydrogen peroxide and 10% methanol in Tris-buffered saline (TBS) for 30 minutes to block endogenous peroxidase activity. After a TBS wash, sections were incubated once in 0.1% Triton X-100 (Fisher Scientific, Pittsburgh, PA) in TBS for 15 minutes and once with 2% BSA in 0.1% Triton-X in TBS for 30 minutes. Sections were then incubated overnight at 4°C with anti-Aβ1-16 (6E10) (1:1000) (Covance Research Products), anti-phospho-tau AT100 (phospho-S212/T214) (1:1000) (Thermo Scientific), anti-glial fibrillary acidic protein (GFAP; Millipore, Billerica, MA), or anti-CD45 (IBL-3/16) (AbD Serotec, Raleigh, NC) with 5% normal serum in TBS. After the appropriate biotinylated secondary antibody (1:200 in TBS, 2% BSA, and 5% normal serum), sections were processed using the Vectastain Elite ABC reagent and 3,3′-diaminobenzidine (Vector Laboratories, Burlingame, CA), according to the manufacturer's instructions. Sections were then mounted on gelatin-coated slides, dehydrated in graded ethanol, cleared in xylene, and coverslipped with DPX mounting medium (BDH Laboratory Supplies, Poole, UK). The immunostaining was assessed at six brain coronal levels. Specifically, six alternate 40-μm sections of the brain, with an individual distance of approximately 160 μm, were obtained between 1.34 and 2.54 mm posterior to the bregma. Images of stained hippocampal, entorhinal cortex, and amygdaloid areas were acquired using an Axiocam digital camera and AxioVision 4.6 software connected to an Axioskop 50 microscope (Carl Zeiss MicroImaging, Thornwood, NY). A threshold optical density that best discriminated staining from the background was obtained using ImageJ 1.36b imaging software. We captured eight images per section: two in the subiculum, two in the CA1, two in the entorhinal cortex, and two in the amygdala (48 images per mouse). For GFAP and CD45 analyses, data are reported as the labeled area captured (positive pixels) divided by the full area captured (total pixels). The data represent the average value obtained by the analysis of images of the hippocampus, entorhinal cortex, and amygdala. All histological assessments were performed by an examiner blinded to sample identities. Sections were first blocked with 3% normal serum, 2% BSA, and 0.1% Triton X-100 in TBS for 1 hour at room temperature. By using the same buffer solution, sections were incubated overnight at 4°C with the following primary antibodies: anti-synaptophysin (SVP-38) (1:250) (Sigma-Aldrich, St Louis, MO), anti-Aβ1-16 (6E10) (1:200) (Covance Research Products), anti-collagen IV (1:250) (Fitzgerald Industries International, Acton, MA), anti-GFAP (1:1000) (Dako, Carpentaria, CA), and/or anti-Iba-1 (1:200) (Wako Chemicals, Richmond, VA). Sections were then rinsed and incubated for 1 hour with secondary Alexa Fluor–conjugated antibodies (Invitrogen, Carlsbad, CA) at room temperature. Finally, sections were mounted onto gelatin-coated slides in Fluoromount-G (Southern Biotech, Birmingham, AL) and examined under a Leica DM2500 confocal laser microscope using Leica Application Suite Advanced Fluorescence software (Leica Microsystems). The immunofluorescence was assessed at the same brain coronal levels previously described. Confocal images were acquired by sequential scanning using a z separation of 1 μm. Three-dimensional reconstruction and three-dimensional–rendered optical sections were generated using Leica Application Suite Advanced Fluorescence software (Leica Microsystems). The vascular Aβ deposition was determined through the analysis of the colocalization of anti-Aβ1-16 (6E10) and anti-collagen IV in the hippocampus, entorhinal cortex, and amygdala. For each section, images were acquired with equal acquisition parameters for both anti-Aβ1-16 (6E10) and anti-collagen IV. Up to 20 vessels were identified using the 6E10 label and then were matched to the corresponding collagen IV staining. Image measurements were obtained using ImageJ 1.36b imaging software. The synaptophysin levels represent the average value obtained by the analysis of images of the hippocampus, entorhinal cortex, and amygdala. The integrated intensity of 6E10 plus collagen IV represents the average optical densities from all of the merged pixels measured. Sections were incubated in 0.5% thioflavin S in 50% ethanol for 10 minutes, differentiated twice in 50% ethanol, and washed in PBS solution. Staining was visualized under a confocal microscope. Image measurements were made using ImageJ 1.36b imaging software. The thioflavin S levels represent the average value obtained by the analysis of images of the hippocampus, entorhinal cortex, and amygdala. The protein kinase (PK) C and A activity levels were measured using the nonradioactive PK assay kit (Calbiochem), according to the manufacturer's instructions. In brief, a fresh half brain sample was homogenized in ice-cold 50 mmol/L Tris-HCl (pH 7.5) containing 10 mmol/L benzamidine, 5 mmol/L EDTA, and 10 mmol/L EGTA. Samples were then sonicated four times (10 seconds each) and centrifuged at 100,000 × g for 60 minutes at 4°C. Fresh supernatant was used for the PK activity assays. The protein concentration in the supernatant was determined using the Bradford assay. The peptide pseudosubstrate RFARKGSLRQKNV that can be phosphorylated by both PKC and PKA was precoated in the plate. A biotinylated monoclonal antibody that recognizes the phosphorylated form of the peptide pseudosubstrate was added to the wells and detected using horseradish peroxidase–conjugated streptavidin. The plates were read at 492 nm using a plate reader (Molecular Dynamics). The readings were then normalized to protein concentrations of the samples. The PKC or PKA activity was directly proportional to the color intensity. The use of specific assay buffer and cofactor provided by the kit enabled us to distinguish PKC an
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