Inflammation Induced by Infection Potentiates Tau Pathological Features in Transgenic Mice
2011; Elsevier BV; Volume: 178; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2011.02.012
ISSN1525-2191
AutoresMichael Sy, Masashi Kitazawa, Rodrigo Medeiros, Lucia Whitman, David Cheng, Thomas E. Lane, Frank M. LaFerla,
Tópico(s)Nuclear Receptors and Signaling
ResumoComorbidities that promote the progression of Alzheimer's disease (AD) remain to be uncovered and evaluated in animal models. Because elderly individuals are vulnerable to viral and bacterial infections, these microbial agents may be considered important comorbidities that could potentiate an already existing and tenuous inflammatory condition in the brain, accelerating cognitive decline, particularly if the cellular and molecular mechanisms can be defined. Researchers have recently demonstrated that triggering inflammation in the brain exacerbates tau pathological characteristics in animal models. Herein, we explore whether inflammation induced via viral infection, compared with inflammation induced via bacterial lipopolysaccharide, modulates AD-like pathological features in the 3xTg-AD mouse model and provide evidence to support the hypothesis that infectious agents may act as a comorbidity for AD. Our study shows that infection-induced acute or chronic inflammation significantly exacerbates tau pathological characteristics, with chronic inflammation leading to impairments in spatial memory. Tau phosphorylation was increased via a glycogen synthase kinase-3β–dependent mechanism, and there was a prominent shift of tau from the detergent-soluble to the detergent-insoluble fraction. During chronic inflammation, we found that inhibiting glycogen synthase kinase-3β activity with lithium reduced tau phosphorylation and the accumulation of insoluble tau and reversed memory impairments. Taken together, infectious agents that trigger central nervous system inflammation may serve as a comorbidity for AD, leading to cognitive impairments by a mechanism that involves exacerbation of tau pathological characteristics. Comorbidities that promote the progression of Alzheimer's disease (AD) remain to be uncovered and evaluated in animal models. Because elderly individuals are vulnerable to viral and bacterial infections, these microbial agents may be considered important comorbidities that could potentiate an already existing and tenuous inflammatory condition in the brain, accelerating cognitive decline, particularly if the cellular and molecular mechanisms can be defined. Researchers have recently demonstrated that triggering inflammation in the brain exacerbates tau pathological characteristics in animal models. Herein, we explore whether inflammation induced via viral infection, compared with inflammation induced via bacterial lipopolysaccharide, modulates AD-like pathological features in the 3xTg-AD mouse model and provide evidence to support the hypothesis that infectious agents may act as a comorbidity for AD. Our study shows that infection-induced acute or chronic inflammation significantly exacerbates tau pathological characteristics, with chronic inflammation leading to impairments in spatial memory. Tau phosphorylation was increased via a glycogen synthase kinase-3β–dependent mechanism, and there was a prominent shift of tau from the detergent-soluble to the detergent-insoluble fraction. During chronic inflammation, we found that inhibiting glycogen synthase kinase-3β activity with lithium reduced tau phosphorylation and the accumulation of insoluble tau and reversed memory impairments. Taken together, infectious agents that trigger central nervous system inflammation may serve as a comorbidity for AD, leading to cognitive impairments by a mechanism that involves exacerbation of tau pathological characteristics. Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the leading cause of dementia, afflicting >35 million individuals worldwide. The AD brain displays several characteristic pathological features, including the buildup of amyloid plaques composed of amyloid-β (Aβ), which can also accumulate intracellularly, and neurofibrillary tangles composed of hyperphosphorylated tau protein.1Selkoe D.J. Toward a remembrance of things past: deciphering Alzheimer disease.Harvey Lect. 2003; 99: 23-45PubMed Google Scholar Neuronal loss, dystrophic neurites, and dendritic spine loss are other critical changes that are well documented in AD. In addition, inflammation, as evidenced by reactive glial cells surrounding amyloid plaques, is consistently observed in the AD brain.2Itagaki S. McGeer P.L. Akiyama H. Zhu S. Selkoe D. Relationship of microglia and astrocytes to amyloid deposits of Alzheimer disease.J Neuroimmunol. 1989; 24: 173-182Abstract Full Text PDF PubMed Scopus (790) Google Scholar, 3McGeer P.L. Rogers J. McGeer E.G. Inflammation, anti-inflammatory agents and Alzheimer disease: the last 12 years.J Alzheimers Dis. 2006; 9: 271-276Crossref PubMed Google Scholar, 4Rogers J. Lue L.F. Microglial chemotaxis, activation, and phagocytosis of amyloid beta-peptide as linked phenomena in Alzheimer's disease.Neurochem Int. 2001; 39: 333-340Crossref PubMed Scopus (206) Google Scholar The factors and molecular mechanisms that affect the pathogenesis of AD still remain largely unknown, although it is widely accepted that this disorder is multifactorial. Certain factors and insults, such as hypoxia, brain ischemia, and stress, that dysregulate brain homeostasis and physiological functions may increase the susceptibility of developing AD (as comorbid factors).5Rothman S.M. Mattson M.P. Adverse stress, hippocampal networks, and Alzheimer's disease.Neuromolecular Med. 2010; 12: 56-70Crossref PubMed Scopus (178) Google Scholar, 6Viswanathan A. Rocca W.A. Tzourio C. Vascular risk factors and dementia: how to move forward?.Neurology. 2009; 72: 368-374Crossref PubMed Scopus (342) Google Scholar Thus, decades of research in epidemiology and postmortem AD brains has suggested that viral or bacterial infections may contribute to the onset of AD.7Holmes C. Cotterell D. Role of infection in the pathogenesis of Alzheimer's disease: implications for treatment.CNS Drugs. 2009; 23: 993-1002Crossref PubMed Scopus (67) Google Scholar, 8Honjo K. van Reekum R. Verhoeff N.P. Alzheimer's disease and infection: do infectious agents contribute to progression of Alzheimer's disease?.Alzheimers Dement. 2009; 5: 348-360Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar With improved quantitative and analytical methods, several viral and bacterial genes, including herpes simplex virus, Chlamydia pneumoniae, and Helicobacter pylori, have been isolated from AD brains; in some reports,9Balin B.J. Gerard H.C. Arking E.J. Appelt D.M. Branigan P.J. Abrams J.T. Whittum-Hudson J.A. Hudson A.P. Identification and localization of Chlamydia pneumoniae in the Alzheimer's brain.Med Microbiol Immunol. 1998; 187: 23-42Crossref PubMed Scopus (431) Google Scholar, 10Kountouras J. Tsolaki M. Gavalas E. Boziki M. Zavos C. Karatzoglou P. Chatzopoulos D. Venizelos I. Relationship between Helicobacter pylori infection and Alzheimer disease.Neurology. 2006; 66: 938-940Crossref PubMed Scopus (205) Google Scholar, 11Itzhaki R.F. Wozniak M.A. Herpes simplex virus type 1 in Alzheimer's disease: the enemy within.J Alzheimers Dis. 2008; 13: 393-405PubMed Google Scholar the presence of these genes in AD brains is statistically significantly higher than in non-AD brains. Evidence from both in vivo and in vitro studies indicates that infections significantly exacerbate AD-like pathological changes, suggesting that infection-mediated alterations (ie, altered immune response) in the brain may increase the susceptibility of developing AD later in life.12Little C.S. Hammond C.J. MacIntyre A. Balin B.J. Appelt D.M. Chlamydia pneumoniae induces Alzheimer-like amyloid plaques in brains of BALB/c mice.Neurobiol Aging. 2004; 25: 419-429Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 13Wozniak M.A. Itzhaki R.F. Shipley S.J. Dobson C.B. Herpes simplex virus infection causes cellular beta-amyloid accumulation and secretase upregulation.Neurosci Lett. 2007; 429: 95-100Crossref PubMed Scopus (254) Google Scholar Brain inflammatory responses may contribute to this pathogenic process.14Brugg B. Dubreuil Y.L. Huber G. Wollman E.E. Delhaye-Bouchaud N. Mariani J. Inflammatory processes induce beta-amyloid precursor protein changes in mouse brain.Proc Natl Acad Sci U S A. 1995; 92: 3032-3035Crossref PubMed Scopus (177) Google Scholar, 15Kitazawa M. Oddo S. Yamasaki T.R. Green K.N. LaFerla F.M. Lipopolysaccharide-induced inflammation exacerbates tau pathology by a cyclin-dependent kinase 5-mediated pathway in a transgenic model of Alzheimer's disease.J Neurosci. 2005; 25: 8843-8853Crossref PubMed Scopus (566) Google Scholar, 16Sheng J.G. Bora S.H. Xu G. Borchelt D.R. Price D.L. Koliatsos V.E. Lipopolysaccharide-induced-neuroinflammation increases intracellular accumulation of amyloid precursor protein and amyloid beta peptide in APPswe transgenic mice.Neurobiol Dis. 2003; 14: 133-145Crossref PubMed Scopus (343) Google Scholar Neuroinflammation in the AD brain likely plays both beneficial and harmful roles.17Wyss-Coray T. Inflammation in Alzheimer disease: driving force, bystander or beneficial response?.Nat Med. 2006; 12: 1005-1015Crossref PubMed Scopus (912) Google Scholar For example, chronic inflammation and cytokine up-regulation induce tau hyperphosphorylation in prepathological 3xTg-AD mice.15Kitazawa M. Oddo S. Yamasaki T.R. Green K.N. LaFerla F.M. Lipopolysaccharide-induced inflammation exacerbates tau pathology by a cyclin-dependent kinase 5-mediated pathway in a transgenic model of Alzheimer's disease.J Neurosci. 2005; 25: 8843-8853Crossref PubMed Scopus (566) Google Scholar In addition, studies18Ard M.D. Cole G.M. Wei J. Mehrle A.P. Fratkin J.D. Scavenging of Alzheimer's amyloid beta-protein by microglia in culture.J Neurosci Res. 1996; 43: 190-202Crossref PubMed Scopus (148) Google Scholar, 19Akiyama H. Schwab C. Kondo H. Mori H. Kametani F. Ikeda K. McGeer P.L. Granules in glial cells of patients with Alzheimer's disease are immunopositive for C-terminal sequences of beta-amyloid protein.Neurosci Lett. 1996; 206: 169-172Crossref PubMed Scopus (70) Google Scholar, 20Herber D.L. Roth L.M. Wilson D. Wilson N. Mason J.E. Morgan D. Gordon M.N. Time-dependent reduction in Abeta levels after intracranial LPS administration in APP transgenic mice.Exp Neurol. 2004; 190: 245-253Crossref PubMed Scopus (134) Google Scholar, 21Herber D.L. Mercer M. Roth L.M. Symmonds K. Maloney J. Wilson N. Freeman M.J. Morgan D. Gordon M.N. Microglial activation is required for Abeta clearance after intracranial injection of lipopolysaccharide in APP transgenic mice.J Neuroimmune Pharmacol. 2007; 2: 222-231Crossref PubMed Scopus (119) Google Scholar indicate that inflammatory processes are involved in clearing or degrading Aβ depositions. The deficiency of CCR2, a chemokine receptor, impairs microglia accumulation and increases Aβ deposition in amyloid precursor protein (APP)-transgenic mice, indicating a role for microglia in regulating Aβ accumulation.22El Khoury J. Toft M. Hickman S.E. Means T.K. Terada K. Geula C. Luster A.D. Ccr2 deficiency impairs microglial accumulation and accelerates progression of Alzheimer-like disease.Nat Med. 2007; 13: 432-438Crossref PubMed Scopus (732) Google Scholar, 23Wyss-Coray T. Yan F. Lin A.H. Lambris J.D. Alexander J.J. Quigg R.J. Masliah E. Prominent neurodegeneration and increased plaque formation in complement-inhibited Alzheimer's mice.Proc Natl Acad Sci U S A. 2002; 99: 10837-10842Crossref PubMed Scopus (389) Google Scholar On the other hand, chronic lipopolysaccharide (LPS)–induced neuroinflammation increases intraneuronal Aβ load in transgenic mice,16Sheng J.G. Bora S.H. Xu G. Borchelt D.R. Price D.L. Koliatsos V.E. Lipopolysaccharide-induced-neuroinflammation increases intracellular accumulation of amyloid precursor protein and amyloid beta peptide in APPswe transgenic mice.Neurobiol Dis. 2003; 14: 133-145Crossref PubMed Scopus (343) Google Scholar, 24Lee J.W. Lee Y.K. Yuk D.Y. Choi D.Y. Ban S.B. Oh K.W. Hong J.T. Neuro-inflammation induced by lipopolysaccharide causes cognitive impairment through enhancement of beta-amyloid generation.J Neuroinflammation. 2008; 5: 37Crossref PubMed Scopus (601) Google Scholar possibly through the release of proinflammatory cytokines and other toxic species25Griffin W.S. Sheng J.G. Royston M.C. Gentleman S.M. McKenzie J.E. Graham D.I. Roberts G.W. Mrak R.E. Glial-neuronal interactions in Alzheimer's disease: the potential role of a “cytokine cycle” in disease progression.Brain Pathol. 1998; 8: 65-72Crossref PubMed Scopus (641) Google Scholar, 26Akiyama H. Barger S. Barnum S. Bradt B. Bauer J. Cole G.M. Cooper N.R. Eikelenboom P. Emmerling M. Fiebich B.L. Finch C.E. Frautschy S. Griffin W.S. Hampel H. Hull M. Landreth G. Lue L. Mrak R. Mackenzie I.R. McGeer P.L. O'Banion M.K. Pachter J. Pasinetti G. Plata-Salaman C. Rogers J. Rydel R. Shen Y. Streit W. Strohmeyer R. Tooyoma I. Van Muiswinkel F.L. Veerhuis R. Walker D. Webster S. Wegrzyniak B. Wenk G. Wyss-Coray T. Inflammation and Alzheimer's disease.Neurobiol Aging. 2000; 21: 383-421Abstract Full Text Full Text PDF PubMed Scopus (3831) Google Scholar and the subsequent exacerbation of AD-related pathological features.27Li Y. Liu L. Barger S.W. Griffin W.S. Interleukin-1 mediates pathological effects of microglia on tau phosphorylation and on synaptophysin synthesis in cortical neurons through a p38-MAPK pathway.J Neurosci. 2003; 23: 1605-1611PubMed Google Scholar, 28Quintanilla R.A. Orellana D.I. Gonzalez-Billault C. Maccioni R.B. Interleukin-6 induces Alzheimer-type phosphorylation of tau protein by deregulating the cdk5/p35 pathway.Exp Cell Res. 2004; 295: 245-257Crossref PubMed Scopus (314) Google Scholar Collectively, infection and neuroinflammation may well be linked to AD and may play key roles in the accelerated onset and development of the disease. In this study, we investigated the role that viral and bacterial infections have on the development of the AD phenotype in the 3xTg-AD mouse model. Viral infection by mouse hepatitis virus (MHV) or LPS to mimic a bacterial infection induced robust, but transient, neuroinflammation; exacerbated tau pathological characteristics; and compromised cognitive function in aged 3xTg-AD mice. LPS injection caused an increase in tau phosphorylation and its partition to the detergent-insoluble fraction, indicating a buildup of aggregated tau in neurons; the aberrant activation of glycogen synthase kinase (GSK)-3β was concomitantly detected in these mice. GSK-3β appears to be one of the main cellular mediators that is activated by infection-induced inflammation, underlying the increased tau pathological characteristics. To determine whether GSK-3β was a necessary mediator of the inflammation-induced changes in tau, we treated mice with lithium, a potent GSK-3β inhibitor, and found that its inhibition reversed both the tau hyperphosphorylation and its shift into the insoluble fraction. Significantly, treatment with lithium also led to an improvement in the cognitive phenotype. Together, our data strongly suggest that viral- or bacterial-mediated infections may act as critical comorbid factors and that tau pathological features are accelerated. 3xTg-AD and nontransgenic (NonTg) mice were maintained on a 12-hour light-dark cycle and had free access to food and water. In this study, 11- to 13-month-old 3xTg-AD or age- and strain-matched NonTg mice were used. LPS (from Escherichia coli 055:B5; Sigma, St Louis, MO) was dissolved in 0.9% NaCl at a concentration of 0.1 mg/mL. LPS was administered i.p. to 12-month-old 3xTg-AD or NonTg mice at a dose of 0.5 mg/kg body weight twice per week for 6 weeks [n = 9 (four females and five males) for 3xtg-AD mice and n = 12 (six females and six males) for NonTg mice)]. A control group of mice received injections in the same manner with 0.9% saline only [n = 10 (six females and four males) for 3xTg-AD mice and n = 9 (four females and five males) for NonTg mice)]. The amount of LPS injected was adjusted according to weight weekly. Mice were euthanized 48 hours after the last injection and perfused with ice-cold PBS, and their brains were isolated. Half of the brain was fixed in 4% paraformaldehyde, and the other half was snap frozen in dry ice and stored at −80°C. Twelve-month-old 3xTg-AD mice were divided into four groups: group 1, received standard rodent chow and saline injections [n = 6 (four females and two males)]; group 2, received standard rodent chow and LPS injections [n = 6 (four females and two males)]; group 3, received lithium chloride (2 g/kg) containing rodent chow (AIN-76A; Research Diets, New Brunswick, NJ) and saline injections [n = 6 (four females and two males)]; and group 4, received lithium chloride containing rodent chow (AIN-76A) and LPS injections [n = 6 (four females and two males)]. LPS (0.5 mg/kg) or saline injections were given twice a week for 6 weeks, as previously described. No obvious weight loss was observed during the 6-week period, and lithium intake was estimated to be 6 to 10 mg/d per mouse based on the assumption that a mouse consumes 3 to 5 g/d of chow. During the last week of injections, mice underwent behavioral testing, as described later. Mice were euthanized 4 days after the last injection and perfused with ice-cold PBS, and their brains were isolated. Half of the brain was fixed in 4% paraformaldehyde, and the other half was snap frozen in dry ice and stored at −80°C. To evaluate the impact of MHV infection on AD pathological characteristics, 11- to 13-month-old 3xTg-AD or NonTg mice were infected with the neuroadapted JHM strain of MHV suspended in HBSS or received an injection of HBSS alone as sham controls.29Stiles L.N. Hardison J.L. Schaumburg C.S. Whitman L.M. Lane T.E. T cell antiviral effector function is not dependent on CXCL10 following murine coronavirus infection.J Immunol. 2006; 177: 8372-8380PubMed Google Scholar, 30Walsh K.B. Lodoen M.B. Edwards R.A. Lanier L.L. Lane T.E. Evidence for differential roles for NKG2D receptor signaling in innate host defense against coronavirus-induced neurological and liver disease.J Virol. 2008; 82: 3021-3030Crossref PubMed Scopus (15) Google Scholar Mice were anesthetized by i.p. injection of ketamine (80 to 100 mg/kg; Phoenix, St. Joseph, MO) and xylazine (5 to 10 mg/kg; MP Biomedicals, LLC, Aurora, OH), diluted in sterile HBSS. Anesthetized mice were injected intracranially with 500 plaque-forming units of the neurotrophic MHV strain J2.2-V.1 (provided by John Fleming, M.D., University of Wisconsin, Madison, WI), diluted in 30 μL of sterile HBSS (n = 34 to 37).31Lane T.E. Asensio V.C. Yu N. Paoletti A.D. Campbell I.L. Buchmeier M.J. Dynamic regulation of alpha- and beta-chemokine expression in the central nervous system during mouse hepatitis virus-induced demyelinating disease.J Immunol. 1998; 160: 970-978PubMed Google Scholar, 32Totoiu M.O. Nistor G.I. Lane T.E. Keirstead H.S. Remyelination, axonal sparing, and locomotor recovery following transplantation of glial-committed progenitor cells into the MHV model of multiple sclerosis.Exp Neurol. 2004; 187: 254-265Crossref PubMed Scopus (88) Google Scholar, 33Wang F.I. Stohlman S.A. Fleming J.O. Demyelination induced by murine hepatitis virus JHM strain (MHV-4) is immunologically mediated.J Neuroimmunol. 1990; 30: 31-41Abstract Full Text PDF PubMed Scopus (147) Google Scholar Sham control animals were injected with 30 μL HBSS alone and did not develop any behavioral deficits. Mice were euthanized 3, 7, and 10 days after MHV infection (n = 6 to 9); and infiltrating leukocytes were immunophenotyped by fluorescence-activated cell sorted staining to define the surface antigens using established methods.29Stiles L.N. Hardison J.L. Schaumburg C.S. Whitman L.M. Lane T.E. T cell antiviral effector function is not dependent on CXCL10 following murine coronavirus infection.J Immunol. 2006; 177: 8372-8380PubMed Google Scholar Brains were removed and stored on ice in 5 mL Dulbecco's modified Eagle's media until processing. The tissue was transferred to a sterile Petri dish and mashed into a single-cell suspension. The cell suspension was transferred to a 15-mL conical tube, and a medium (Percoll; GE Healthcare, Uppsala, Sweden) was added for a final concentration of 30%. The medium (70% Percoll), 1 mL, was underlain; and tubes were centrifuged at 1100 × g for 30 minutes at 4°C. Live cells were collected from the interface, washed twice, and stained for flow cytometric analysis. Isolated cells were Fc blocked with anti-CD16/32, 1:200 (BD Biosciences, San Jose, CA), and immunophenotyed with fluorescent antibodies (BD Biosciences) specific for the following cell surface markers: CD4 (L3T4), CD8a (53-6.7), CD45 (30-F11; eBiosciences, San Diego, CA), I-A/I-E (M5/114.15.2), and F4/80 (CI:A3-1; AbD Serotec, Raleigh, NC). Appropriate isotype antibodies were used for each antibody. Cells were run on a flow cytometer (FACSCalibur; BD Biosciences) and analyzed with software (FlowJo; TreeStar, OR). Frequency data are presented as the percentage of positive cells within the gated population. Total cells were calculated by multiplying these values by the total number of live cells isolated. Intracellular staining for interferon-γ was performed in cells isolated from the brains of MHV-infected control mice and transgenic mice. Cells were stimulated with 5 μmol/L peptide from a control antigen or from the MHV spike glycoprotein (S510, residues 510 to 518). Stimulated cells were incubated for 6 hours at 37°C in media containing Golgi/Stop (Cytofix/Cytoperm kit; BD Biosciences), at which point cells were Fc blocked with anti-CD16/32, 1:200 (BD Biosciences). Cells were then stained with fluorescent antibodies (BD Biosciences) for the following cell surface markers: CD4 (L3T4), CD8a (53-6.7), and CD45 (30-F11; eBiosciences). Spinal cords were obtained from experimental groups at day 14 post infection (p.i.) and fixed by immersion overnight in 10% normal buffered formalin, after which portions of tissue were embedded in paraffin. Spinal cords (7-μm sections) were stained with Luxol fast blue and analyzed by light microscopy. Demyelination was scored as follows: 0, no demyelination; 1, mild inflammation accompanied by loss of myelin integrity; 2, moderate inflammation with increasing myelin damage; 3, numerous inflammatory lesions accompanied by a significant increase in myelin stripping; and 4, intense areas of inflammation accompanied by numerous phagocytic cells engulfing myelin debris. Slides containing stained spinal cord sections were blinded and scored. Frozen brain halves were homogenized in tissue protein extraction reagent (Pierce, Rockford, IL), protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN), and phosphatase inhibitors (5 mmol/L sodium fluoride and 50 μmol/L sodium orthovanadate). Homogenates were centrifuged at 100,000 x g for 1 hour at 4°C. Supernatants were collected as the detergent-soluble fraction. Pellets were resuspended in 70% formic acid and homogenized. After centrifugation at 100,000 x g for 1 hour at 4°C, the resulting supernatants were saved as the formic acid fraction. Protein concentrations were determined by the Bradford method. Equal amounts of protein (20 to 50 μg, depending on the protein of interest) were separated by SDS-PAGE on a 10% Bis-Tris gel (Invitrogen, Carlsbad, CA), transferred to 0.45-μm polyvinylidene difluoride membranes, and blocked for 1 hour in 5% (v/v) nonfat milk in Tris-buffered saline (pH 7.5) supplemented with 0.2% Tween 20. Fractions were immunoblotted with antibodies that recognize APP, total tau (HT7), total endogenous tau (Dako, Carpinteria, CA), phosphorylated tau [AT8, Ser202/Thr205; AT180, Thr231/Ser235; AT100, Thr212/Ser214; and paired helical filament (PHF-1), Ser396/Ser404 (Pierce)], p35/p25 (Santa Cruz Biotechnologies, Santa Cruz, CA), cdk5 (Calbiochem, La Jolla, CA), total GSK-3αβ or phospho-GSK-3β (Ser9) (both from Cell Signaling, Beverly, MA), or total GSK-3β (BD Biosciences). Antibody against β-actin was used as a loading control. Formic acid fractions were neutralized by mixing equal amounts of sample, 10N NaOH, and neutralizing buffer (1 mol/L Tris base and 0.5 mol/L Na2HPO). Equal amounts of protein in the neutralized sample were separated on SDS-PAGE gels in the same manner as the soluble fractions. Quantification of band intensity was measured using software (Scion Image) and was normalized with glyceraldehyde-3-phosphate dehydrogenase, β-actin, or total tau (HT7) levels (for phosphorylated tau analyses). Fixed brain halves were sliced on a vibratome at 50 μmol/L thickness. Before overnight incubation with primary antibody, sections were quenched with 3% hydrogen peroxide plus 10% methanol, permeabilized with 0.1% Triton X-100 Tris-buffered saline, and blocked in solution containing 3% bovine serum albumin. After incubation with primary antibody in Tris-buffered saline containing 3% serum overnight, slices were washed with 0.1% Triton X-100 Tris-buffered saline and incubated with the appropriate secondary antibody. The presence of secondary antibody in tissue was revealed by reaction with diaminobenzidine. Certain antigens required special conditions. Aβ staining required pretreatment with 90% formic acid. CD45 antigen required removal of detergent during antibody incubations. Images of stained hippocampus, entorhinal cortex, and amygdala were acquired by a digital camera (Axiocam) connected to a microscope (Axioskop 50) (Carl Zeiss MicroImaging, Thornwood, NY) and software (AxioVision 4.6). Aβ plaques with a diameter >10 μm were counted in three to four random fields of the CA1 hippocampus in each animal to quantitatively analyze the plaque number. For the analysis of phosphorylated tau-bearing neurons, AT8- or PHF-1–positive neurons in the subiculum and CA1 hippocampus were counted in each animal. To measure Aβ levels, equal amounts of protein (200 μg) from soluble fractions were loaded directly onto enzyme-linked immunosorbent assay (ELISA) plates. Equal amounts of protein (200 μg) from formic acid fractions were diluted 1:20 in neutralization buffer (1 mol/L Tris base and 0.5 mol/L Na2HPO) before loading. Before loading, antibody mAβ20.1 at a concentration of 25 μg/mL in coating buffer (0.1 mol/L NaCO3 buffer, pH 9.6) was coated onto immunoplates (Nunc, Naperville, IL); and plates were blocked with 3% bovine serum albumin. Synthetic Aβ standards of both Aβ40 and Aβ42 were made in antigen capture buffer [20 mmol/L NaH2PO4, 2 mmol/L EDTA, 0.4 mol/L NaCl, 0.5 g (in 1 L total volume) of 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate, and 1% bovine serum albumin (pH 7.0)] and loaded onto ELISA plates, along with soluble and formic acid fractions. Samples and standards were loaded in duplicate, and plates were incubated overnight at 4°C. Plates were washed and probed with either horseradish peroxidase–conjugated anti–Aβ35-40 (MM32-13.1.1 for Aβ1-40) or anti–Aβ35-42 (MM40-21.3.4 for Aβ1-42) overnight at 4°C. The chromogen used was tetramethylbenzidine, and 30% O-phosphoric acid was used to stop the reaction. The concentration of samples was determined from readings at 450 nm. To detect levels of IL-1β and IL-6 in homogenized brains, ELISA kits were purchased (Pierce) and the protocol provided by the manufacturer was followed. Briefly, samples were incubated with biotinylated antibodies against IL-1β and IL-6 on precoated plates. After incubation with a streptavidin–horseradish peroxidase solution, tetramethylbenzidine chromogen was applied and the manufacturer-supplied stop solution was used. The concentration of samples was determined by reading at 450 nm. Brain samples were immunoprecipitated with protein G–agarose and GSK-3β antibody or protein A–agarose and cdk5 antibody. A reaction mixture containing 20 mmol/L 4-morpholinepropanesulfonic acid (pH 7.2), 5 mmol/L MgCl2, 1 mmol/L sodium orthovanadate, 5 mmol/L NaF, 100 μmol/L ATP, 2.5 μCi (γ-32P)ATP, and 0.2 mmol/L cdk5 substrate (Calbiochem) for the cdk5 kinase assay or 0.2 mmol/L GSK-3β substrate (Calbiochem) for the GSK-3β assay. After allowing the reaction to proceed for 1 hour at 30°C, the supernatant was placed on P81 phosphocellulose squares (Upstate, Waltham, MA). After washing in 0.3% phosphoric acid, squares were counted in a scintillation counter to determine the kinase activity. 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 to 24°C. The maze was located in a room containing several simple visual extramaze 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 location was selected randomly for each mouse but was kept constant for each individual mouse throughout training. For each trial, the mouse was placed into the tank at one of four designated starting points in a pseudorandom order. Mice were allowed to find and escape onto the submerged platform. If a mouse failed to find the platform within 60 seconds, the mouse was manually guided to the platform and allowed to remain there for 10 seconds. Afterward, each mouse was placed into a holding cage under a warming lamp for 25 seconds until the start of the next trial. To ensure that memory differences were not because of lack of task learning, mice were given four trials a day for as many days as were required to train the lithium-untreated and lithium-treated 3xTg-AD mice to criterion (<20 seconds mean escape latency before the first probe trial was run). 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. Retention of the spatial training was assessed 1.5 hours and again 24 hours after the last trai
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