Neuronal Apoptosis and Autophagy Cross Talk in Aging PS/APP Mice, a Model of Alzheimer's Disease
2008; Elsevier BV; Volume: 173; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2008.071176
ISSN1525-2191
AutoresDun‐Sheng Yang, Asok Kumar, Philip Stavrides, Jesse Peterson, Corrine Peterhoff, Monika Pawlik, Efrat Levy, Anne M. Cataldo, Ralph A. Nixon,
Tópico(s)Cell death mechanisms and regulation
ResumoMechanisms of neuronal loss in Alzheimer's disease (AD) are poorly understood. Here we show that apoptosis is a major form of neuronal cell death in PS/APP mice modeling AD-like neurodegeneration. Pyknotic neurons in adult PS/APP mice exhibited apoptotic changes, including DNA fragmentation, caspase-3 activation, and caspase-cleaved α-spectrin generation, identical to developmental neuronal apoptosis in wild-type mice. Ultrastructural examination using immunogold cytochemistry confirmed that activated caspase-3-positive neurons also exhibited chromatin margination and condensation, chromatin balls, and nuclear membrane fragmentation. Numbers of apoptotic profiles in both cortex and hippocampus of PS/APP mice compared with age-matched controls were twofold to threefold higher at 6 months of age and eightfold higher at 21 to 26 months of age. Additional neurons undergoing dark cell degeneration exhibited none of these apoptotic features. Activated caspase-3 and caspase-3-cleaved spectrin were abundant in autophagic vacuoles, accumulating in dystrophic neurites of PS/APP mice similar to AD brains. Administration of the cysteine protease inhibitor, leupeptin, promoted accumulation of autophagic vacuoles containing activated caspase-3 in axons of PS/APP mice and, to a lesser extent, in those of wild-type mice, implying that this pro-apoptotic factor is degraded by autophagy. Leupeptin-induced autophagic impairment increased the number of apoptotic neurons in PS/APP mice. Our findings establish apoptosis as a mode of neuronal cell death in aging PS/APP mice and identify the cross talk between autophagy and apoptosis, which influences neuronal survival in AD-related neurodegeneration. Mechanisms of neuronal loss in Alzheimer's disease (AD) are poorly understood. Here we show that apoptosis is a major form of neuronal cell death in PS/APP mice modeling AD-like neurodegeneration. Pyknotic neurons in adult PS/APP mice exhibited apoptotic changes, including DNA fragmentation, caspase-3 activation, and caspase-cleaved α-spectrin generation, identical to developmental neuronal apoptosis in wild-type mice. Ultrastructural examination using immunogold cytochemistry confirmed that activated caspase-3-positive neurons also exhibited chromatin margination and condensation, chromatin balls, and nuclear membrane fragmentation. Numbers of apoptotic profiles in both cortex and hippocampus of PS/APP mice compared with age-matched controls were twofold to threefold higher at 6 months of age and eightfold higher at 21 to 26 months of age. Additional neurons undergoing dark cell degeneration exhibited none of these apoptotic features. Activated caspase-3 and caspase-3-cleaved spectrin were abundant in autophagic vacuoles, accumulating in dystrophic neurites of PS/APP mice similar to AD brains. Administration of the cysteine protease inhibitor, leupeptin, promoted accumulation of autophagic vacuoles containing activated caspase-3 in axons of PS/APP mice and, to a lesser extent, in those of wild-type mice, implying that this pro-apoptotic factor is degraded by autophagy. Leupeptin-induced autophagic impairment increased the number of apoptotic neurons in PS/APP mice. Our findings establish apoptosis as a mode of neuronal cell death in aging PS/APP mice and identify the cross talk between autophagy and apoptosis, which influences neuronal survival in AD-related neurodegeneration. Alzheimer's disease (AD) is associated with the widespread loss of neurons, which correlates with the severity of clinical symptoms.1Price JL Ko AI Wade MJ Tsou SK McKeel DW Morris JC Neuron number in the entorhinal cortex and CA1 in preclinical Alzheimer disease.Arch Neurol. 2001; 58: 1395-1402Crossref PubMed Scopus (444) Google Scholar, 2Giannakopoulos P Herrmann FR Bussiere T Bouras C Kovari E Perl DP Morrison JH Gold G Hof PR Tangle and neuron numbers, but not amyloid load, predict cognitive status in Alzheimer's disease.Neurology. 2003; 60: 1495-1500Crossref PubMed Scopus (760) Google Scholar, 3Gómez-Isla T Price JL McKeel Jr, DW Morris JC Growdon JH Hyman BT Profound loss of layer II entorhinal cortex neurons occurs in very mild Alzheimer's disease.J Neurosci. 1996; 16: 4491-4500PubMed Google Scholar, 4West MJ Coleman PD Flood DG Troncoso JC Differences in the pattern of hippocampal neuronal loss in normal ageing and Alzheimer's disease.Lancet. 1994; 344: 769-772Abstract PubMed Scopus (981) Google Scholar Little is known, however, about the molecular mechanisms that mediate neuronal cell death in AD. Complicating this analysis are observations that multiple proteolytic systems are activated as neurons slowly degenerate in AD brain, including calpains,5Saito K Elce JS Hamos JE Nixon RA Widespread activation of calcium-activated neutral proteinase (calpain) in the brain in Alzheimer disease: a potential molecular basis for neuronal degeneration.Proc Natl Acad Sci USA. 1993; 90: 2628-2632Crossref PubMed Scopus (526) Google Scholar, 6Grynspan F Griffin WR Cataldo A Katayama S Nixon RA Active site-directed antibodies identify calpain II as an early-appearing and pervasive component of neurofibrillary pathology in Alzheimer's disease.Brain Res. 1997; 763: 145-158Crossref PubMed Scopus (129) Google Scholar the lysosomal system (cathepsins),7Nixon RA Cataldo AM Lysosomal system pathways: genes to neurodegeneration in Alzheimer's disease.J Alzheimers Dis. 2006; 9: 277-289Crossref PubMed Google Scholar and caspases.8Rohn TT Rissman RA Head E Cotman CW Caspase activation in the Alzheimer's disease brain: tortuous and torturous.Drug News Perspect. 2002; 15: 549-557Crossref PubMed Scopus (38) Google Scholar, 9Cribbs DH Poon WW Rissman RA Blurton-Jones M Caspase-mediated degeneration in Alzheimer's disease.Am J Pathol. 2004; 165: 353-355Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Apoptosis, a highly regulated process of cell death, is often proposed as a possible mode of neuronal death in AD10Cotman CW Su JH Mechanisms of neuronal death in Alzheimer's disease.Brain Pathol. 1996; 6: 493-506Crossref PubMed Scopus (234) Google Scholar based on the presence in affected neurons of fragmented DNA detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL),11Su JH Anderson AJ Cummings BJ Cotman CW Immunohistochemical evidence for apoptosis in Alzheimer's disease.Neuroreport. 1994; 5: 2529-2533Crossref PubMed Scopus (555) Google Scholar, 12Lassmann H Bancher C Breitschopf H Wegiel J Bobinski M Jellinger K Wisniewski HM Cell death in Alzheimer's disease evaluated by DNA fragmentation in situ.Acta Neuropathol (Berl). 1995; 89: 35-41Crossref PubMed Scopus (420) Google Scholar, 13Anderson AJ Su JH Cotman CW DNA damage and apoptosis in Alzheimer's disease: colocalization with c-Jun immunoreactivity, relationship to brain area, and effect of postmortem delay.J Neurosci. 1996; 16: 1710-1719Crossref PubMed Google Scholar, 14Li WP Chan WY Lai HW Yew DT Terminal dUTP nick end labeling (TUNEL) positive cells in the different regions of the brain in normal aging and Alzheimer patients.J Mol Neurosci. 1997; 8: 75-82Crossref PubMed Scopus (118) Google Scholar, 15Lucassen PJ Chung WC Kamphorst W Swaab DF DNA damage distribution in the human brain as shown by in situ end labeling; area-specific differences in aging and Alzheimer disease in the absence of apoptotic morphology.J Neuropathol Exp Neurol. 1997; 56: 887-900Crossref PubMed Scopus (125) Google Scholar activated caspases,16Stadelmann C Deckwerth TL Srinivasan A Bancher C Bruck W Jellinger K Lassmann H Activation of caspase-3 in single neurons and autophagic granules of granulovacuolar degeneration in Alzheimer's disease. Evidence for apoptotic cell death.Am J Pathol. 1999; 155: 1459-1466Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 17Rohn TT Head E Nesse WH Cotman CW Cribbs DH Activation of caspase-8 in the Alzheimer's disease brain.Neurobiol Dis. 2001; 8: 1006-1016Crossref PubMed Scopus (138) Google Scholar, 18Rohn TT Rissman RA Davis MC Kim YE Cotman CW Head E Caspase-9 activation and caspase cleavage of tau in the Alzheimer's disease brain.Neurobiol Dis. 2002; 11: 341-354Crossref PubMed Scopus (207) Google Scholar, 19Su JH Kesslak JP Head E Cotman CW Caspase-cleaved amyloid precursor protein and activated caspase-3 are co-localized in the granules of granulovacuolar degeneration in Alzheimer's disease and Down's syndrome brain.Acta Neuropathol (Berl). 2002; 104: 1-6Crossref PubMed Scopus (71) Google Scholar, 20Guo H Albrecht S Bourdeau M Petzke T Bergeron C LeBlanc AC Active caspase-6 and caspase-6-cleaved tau in neuropil threads, neuritic plaques, and neurofibrillary tangles of Alzheimer's disease.Am J Pathol. 2004; 165: 523-531Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar and products of caspase-specific protein cleavage.21Gamblin TC Chen F Zambrano A Abraha A Lagalwar S Guillozet AL Lu M Fu Y Garcia-Sierra F LaPointe N Miller R Berry RW Binder LI Cryns VL Caspase cleavage of tau: linking amyloid and neurofibrillary tangles in Alzheimer's disease.Proc Natl Acad Sci USA. 2003; 100: 10032-10037Crossref PubMed Scopus (664) Google Scholar, 22Cotman CW Poon WW Rissman RA Blurton-Jones M The role of caspase cleavage of tau in Alzheimer disease neuropathology.J Neuropathol Exp Neurol. 2005; 64: 104-112Crossref PubMed Scopus (156) Google Scholar, 23Rohn TT Head E Su JH Anderson AJ Bahr BA Cotman CW Cribbs DH Correlation between caspase activation and neurofibrillary tangle formation in Alzheimer's disease.Am J Pathol. 2001; 158: 189-198Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 24Yang F Sun X Beech W Teter B Wu S Sigel J Vinters HV Frautschy SA Cole GM Antibody to caspase-cleaved actin detects apoptosis in differentiated neuroblastoma and plaque-associated neurons and microglia in Alzheimer's disease.Am J Pathol. 1998; 152: 379-389PubMed Google Scholar, 25Gervais FG Xu D Robertson GS Vaillancourt JP Zhu Y Huang J LeBlanc A Smith D Rigby M Shearman MS Clarke EE Zheng H Van Der Ploeg LH Ruffolo SC Thornberry NA Xanthoudakis S Zamboni RJ Roy S Nicholson DW Involvement of caspases in proteolytic cleavage of Alzheimer's amyloid-beta precursor protein and amyloidogenic A beta peptide formation.Cell. 1999; 97: 395-406Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar, 26Ayala-Grosso C Ng G Roy S Robertson GS Caspase-cleaved amyloid precursor protein in Alzheimer's disease.Brain Pathol. 2002; 12: 430-441Crossref PubMed Scopus (37) Google Scholar Apoptosis is characterized morphologically by cytoplasmic shrinkage, chromatin condensation, nuclear and cellular fragmentation, and the formation of apoptotic bodies. Typical nuclear changes include highly compact chromatin masses with geometrically regular shapes. Cell membrane integrity and mitochondria are primarily unchanged morphologically in the early stage. The period from initiation to execution of apoptosis has been estimated to be hours or days, and apoptotic bodies are rapidly phagocytosed by adjacent cells or macrophages. Therefore, at a single moment in time in a chronic neurodegenerative disease, only a few cells that are undergoing apoptosis can be detected.10Cotman CW Su JH Mechanisms of neuronal death in Alzheimer's disease.Brain Pathol. 1996; 6: 493-506Crossref PubMed Scopus (234) Google Scholar, 27Kerr JF Wyllie AH Currie AR Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.Br J Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12616) Google Scholar, 28Kroemer G Dallaporta B Resche-Rigon M The mitochondrial death/life regulator in apoptosis and necrosis.Annu Rev Physiol. 1998; 60: 619-642Crossref PubMed Scopus (1744) Google Scholar, 29Saraste A Pulkki K Morphologic and biochemical hallmarks of apoptosis.Cardiovasc Res. 2000; 45: 528-537Crossref PubMed Scopus (974) Google Scholar, 30Leist M Jaattela M Four deaths and a funeral: from caspases to alternative mechanisms.Nat Rev Mol Cell Biol. 2001; 2: 589-598Crossref PubMed Scopus (1365) Google Scholar, 31Ziegler U Groscurth P Morphological features of cell death.News Physiol Sci. 2004; 19: 124-128Crossref PubMed Scopus (395) Google Scholar, 32Jellinger KA Challenges in neuronal apoptosis.Curr Alzheimer Res. 2006; 3: 377-391Crossref PubMed Scopus (65) Google Scholar, 33Roth KA Caspases, apoptosis, and Alzheimer disease: causation, correlation, and confusion.J Neuropathol Exp Neurol. 2001; 60: 829-838Crossref PubMed Scopus (159) Google Scholar Ultrastructural features of in vivo neuronal apoptosis in mammalian brains have been recently described in detail by Dikranian and colleagues34Dikranian K Ishimaru MJ Tenkova T Labruyere J Qin YQ Ikonomidou C Olney JW Apoptosis in the in vivo mammalian forebrain.Neurobiol Dis. 2001; 8: 359-379Crossref PubMed Scopus (157) Google Scholar including formation of condensed chromatin balls (CBs) and apoptotic bodies, fragmentation of nuclear membranes, disassociation of the nucleolus, and either intact cytoplasmic organelles or transformation of cytoplasmic contents into nondescript particulate or membranous debris, depending on the stages in the apoptotic process. Activation of the caspase cascade is the most central and pathognomonic biochemical event in apoptosis. Various stimuli trigger the death receptor, the mitochondrial or the endoplasmic reticulum stress pathway to activate initiator caspases such as caspase-8, -9, or -12, which, in turn, activate effector caspases including caspases-3, -6, and -7. Activated effector caspases cleave various vital cellular substrates including structural proteins such as lamin, actin, and spectrin resulting in characteristic features of apoptotic morphology.35Budihardjo I Oliver H Lutter M Luo X Wang X Biochemical pathways of caspase activation during apoptosis.Annu Rev Cell Dev Biol. 1999; 15: 269-290Crossref PubMed Scopus (2240) Google Scholar, 36Hengartner MO The biochemistry of apoptosis.Nature. 2000; 407: 770-776Crossref PubMed Scopus (6173) Google Scholar, 37Guimarães CA Linden R Programmed cell death: apoptosis and alternative deathstyles.Eur J Biochem. 2004; 271: 1638-1650Crossref PubMed Scopus (249) Google Scholar, 38Stefanis L Caspase-dependent and -independent neuronal death: two distinct pathways to neuronal injury.Neuroscientist. 2005; 11: 50-62Crossref PubMed Scopus (128) Google Scholar, 39Chang HY Yang X Proteases for cell suicide: functions and regulation of caspases.Microbiol Mol Biol Rev. 2000; 64: 821-846Crossref PubMed Scopus (565) Google Scholar DNA fragmentation—cleavage of chromosomal DNA into mono- and oligonucleosome-sized fragments—is another biochemical hallmark in apoptosis40Zhang J Xu M Apoptotic DNA fragmentation and tissue homeostasis.Trends Cell Biol. 2002; 12: 84-89Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar that can be detected biochemically by agarose gel electrophoresis41Wyllie AH Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation.Nature. 1980; 284: 555-556Crossref PubMed Scopus (4129) Google Scholar or morphologically by the incorporation of labeled dUTP by the TUNEL method.42Gavrieli Y Sherman Y Ben-Sasson SA Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.J Cell Biol. 1992; 119: 493-501Crossref PubMed Scopus (9100) Google Scholar A possible opposing influence on apoptosis is autophagy,43Baehrecke EH Autophagy: dual roles in life and death?.Nat Rev Mol Cell Biol. 2005; 6: 505-510Crossref PubMed Scopus (827) Google Scholar, 44Levine B Yuan J Autophagy in cell death: an innocent convict?.J Clin Invest. 2005; 115: 2679-2688Crossref PubMed Scopus (1447) Google Scholar, 45Nixon RA Autophagy in neurodegenerative disease: friend, foe or turncoat?.Trends Neurosci. 2006; 29: 528-535Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar a major lysosomal pathway for the turnover of organelles and cytoplasmic constituents, including pathological protein aggregates.46Blommaart EF Luiken JJ Meijer AJ Autophagic proteolysis: control and specificity.Histochem J. 1997; 29: 365-385Crossref PubMed Scopus (216) Google Scholar, 47Klionsky DJ Ohsumi Y Vacuolar import of proteins and organelles from the cytoplasm.Annu Rev Cell Dev Biol. 1999; 15: 1-32Crossref PubMed Scopus (385) Google Scholar, 48Klionsky DJ Emr SD Autophagy as a regulated pathway of cellular degradation.Science. 2000; 290: 1717-1721Crossref PubMed Scopus (2901) Google Scholar, 49Larsen KE Sulzer D Autophagy in neurons: a review.Histol Histopathol. 2002; 17: 897-908PubMed Google Scholar, 50Cuervo AM Autophagy: in sickness and in health.Trends Cell Biol. 2004; 14: 70-77Abstract Full Text Full Text PDF PubMed Scopus (699) Google Scholar During states of nutritional deprivation or trophic factor withdrawal, autophagy delays or prevents apoptosis by turning over nonessential cell constituents to provide substrates for energy.51Lum JJ Bauer DE Kong M Harris MH Li C Lindsten T Thompson CB Growth factor regulation of autophagy and cell survival in the absence of apoptosis.Cell. 2005; 120: 237-248Abstract Full Text Full Text PDF PubMed Scopus (1242) Google Scholar Autophagy induction in injury states eliminates damaged organelles that could trigger cell death. Autophagy is, therefore, commonly considered a cytoprotective response, which can become permissive or facilitative for apoptosis or necrosis if autophagy fails in pathological states.44Levine B Yuan J Autophagy in cell death: an innocent convict?.J Clin Invest. 2005; 115: 2679-2688Crossref PubMed Scopus (1447) Google Scholar, 45Nixon RA Autophagy in neurodegenerative disease: friend, foe or turncoat?.Trends Neurosci. 2006; 29: 528-535Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar In certain conditions, overactivated autophagy can also initiate an autophagic pattern of programmed cell death distinct from apoptosis.52Clarke PG Developmental cell death: morphological diversity and multiple mechanisms.Anat Embryol. 1990; 181: 195-213Crossref PubMed Scopus (1521) Google Scholar, 53Edinger AL Thompson CB Death by design: apoptosis, necrosis and autophagy.Curr Opin Cell Biol. 2004; 16: 663-669Crossref PubMed Scopus (1095) Google Scholar, 54Matyja E Taraszewska A Naganska E Rafalowska J Autophagic degeneration of motor neurons in a model of slow glutamate excitotoxicity in vitro.Ultrastruct Pathol. 2005; 29: 331-339Crossref PubMed Scopus (25) Google Scholar Autophagy is induced but impaired in affected neurons in AD brain55Nixon RA Wegiel J Kumar A Yu WH Peterhoff C Cataldo A Cuervo AM Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study.J Neuropathol Exp Neurol. 2005; 64: 113-122Crossref PubMed Scopus (1097) Google Scholar, 56Yu WH Cuervo AM Kumar A Peterhoff CM Schmidt SD Lee JH Mohan PS Mercken M Farmery MR Tjernberg LO Jiang Y Duff K Uchiyama Y Naslund J Mathews PM Cataldo AM Nixon RA Macroautophagy—a novel beta-amyloid peptide-generating pathway activated in Alzheimer's disease.J Cell Biol. 2005; 171: 87-98Crossref PubMed Scopus (797) Google Scholar causing autophagic vacuoles (AVs) to accumulate profusely in dystrophic neurites. The relationship of autophagy to neuronal survival or death, however, remains unclear. Neuron loss is limited in most mouse models of β-amyloidosis, although it is significant in some animal models, particularly those expressing mutations in two AD-related genes. In PS/APP mice57Holcomb L Gordon MN McGowan E Yu X Benkovic S Jantzen P Wright K Saad I Mueller R Morgan D Sanders S Zehr C O'Campo K Hardy J Prada CM Eckman C Younkin S Hsiao K Duff K Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes.Nat Med. 1998; 4: 97-100Crossref PubMed Scopus (1145) Google Scholar that express mutant human presenilin 1 (PS) and mutant amyloid precursor protein (APP), β-amyloid is deposited progressively in the brain beginning at 8 weeks of age,58Kurt MA Davies DC Kidd M Duff K Rolph SC Jennings KH Howlett DR Neurodegenerative changes associated with beta-amyloid deposition in the brains of mice carrying mutant amyloid precursor protein and mutant presenilin-1 transgenes.Exp Neurol. 2001; 171: 59-71Crossref PubMed Scopus (87) Google Scholar and is associated with the extensive dystrophy of neurites containing marked accumulations of AVs.56Yu WH Cuervo AM Kumar A Peterhoff CM Schmidt SD Lee JH Mohan PS Mercken M Farmery MR Tjernberg LO Jiang Y Duff K Uchiyama Y Naslund J Mathews PM Cataldo AM Nixon RA Macroautophagy—a novel beta-amyloid peptide-generating pathway activated in Alzheimer's disease.J Cell Biol. 2005; 171: 87-98Crossref PubMed Scopus (797) Google Scholar, 58Kurt MA Davies DC Kidd M Duff K Rolph SC Jennings KH Howlett DR Neurodegenerative changes associated with beta-amyloid deposition in the brains of mice carrying mutant amyloid precursor protein and mutant presenilin-1 transgenes.Exp Neurol. 2001; 171: 59-71Crossref PubMed Scopus (87) Google Scholar Stereological analyses have demonstrated significant hippocampal neuronal cell loss in PS/APP mice at 22 months of age,59Sadowski M Pankiewicz J Scholtzova H Ji Y Quartermain D Jensen CH Duff K Nixon RA Gruen RJ Wisniewski T Amyloid-beta deposition is associated with decreased hippocampal glucose metabolism and spatial memory impairment in APP/PS1 mice.J Neuropathol Exp Neurol. 2004; 63: 418-428PubMed Google Scholar which have not been seen in young adult PS/APP mice60Takeuchi A Irizarry MC Duff K Saido TC Hsiao Ashe K Hasegawa M Mann DM Hyman BT Iwatsubo T Age-related amyloid beta deposition in transgenic mice overexpressing both Alzheimer mutant presenilin 1 and amyloid beta precursor protein Swedish mutant is not associated with global neuronal loss.Am J Pathol. 2000; 157: 331-339Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar; however, molecular events involved in the compromise and loss of these cells are unexplained. Here we investigated apoptosis as a possible basis for neuronal cell death in the PS/APP mouse using, for the first time, concurrent morphological and biochemical criteria for apoptosis validated in the well-established model of programmed neuronal cell death that occurs in early postnatal mouse brain development. This combined approach demonstrated unequivocal apoptosis of neurons in the PS/APP cortex and hippocampus and provided evidence for cross talk between apoptosis and autophagy in the neurodegenerative process. All procedures were performed following the National Institutes of Health Guidelines for the Humane Treatment of Animals, with approval from the Institutional Animal Care and Use Committee at the Nathan Kline Institute. Animals of both sexes were used in this study. The transgenic PS/APP mice, which expressed both the Swedish double mutations of APP (K670N/M671L) and mutant PS1 (PS1M146L), were generated as previously described.57Holcomb L Gordon MN McGowan E Yu X Benkovic S Jantzen P Wright K Saad I Mueller R Morgan D Sanders S Zehr C O'Campo K Hardy J Prada CM Eckman C Younkin S Hsiao K Duff K Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes.Nat Med. 1998; 4: 97-100Crossref PubMed Scopus (1145) Google Scholar An equal number of age-matched wild-type (WT) mice were used as controls. Additional neonatal mouse brains were obtained from normal C57BL/6J mice on postnatal day 5. All of the animals were anesthetized with a mixture (0.01 ml/g body weight, i.p.) of ketamine (10 mg/ml) and xylazine (1 mg/ml) and fixed by perfusion with aldehydes. PS/APP and WT mice (n = 4 for each of 6 months old or 16 months old of each genotype; n = 8 for 21 to 26 months old of each genotype) or neonatal C57BL/6J mice (n = 5) were fixed by cardiac perfusion using 4% paraformaldehyde in 0.1 mol/L sodium cacodylate buffer. After perfusion fixation, the brains were immersion-fixed in the same fixative overnight at 4°C. Forty-μm-thick vibratome sections were cut and processed for immunocytochemistry using the following primary antibodies: polyclonal anti-activated caspase-3 (catalog no. 9661; Cell Signaling, Beverly, MA), rabbit antibody Ab246 (recognizing caspase-cleaved α-spectrin fragments, a kind gift from Dr. Robert Siman, University of Pennsylvania, Philadelphia, PA),61Oo TF Siman R Burke RE Distinct nuclear and cytoplasmic localization of caspase cleavage products in two models of induced apoptotic death in dopamine neurons of the substantia nigra.Exp Neurol. 2002; 175: 1-9Crossref PubMed Scopus (32) Google Scholar, 62Zhang C Siman R Xu YA Mills AM Frederick JR Neumar RW Comparison of calpain and caspase activities in the adult rat brain after transient forebrain ischemia.Neurobiol Dis. 2002; 10: 289-305Crossref PubMed Scopus (56) Google Scholar and monoclonal anti-NeuN (catalog no. MAB377; Chemicon International, Temecula, CA). After overnight incubation with primary antibodies at 4°C, sections were washed and processed using an avidin-biotin complex method. Signal was detected with 3,3′-diaminobenzidine tetrahydrochloride. Sections were counterstained with cresyl violet. Immunocytochemical controls consisted of either incubating tissue in nonimmune sera or omitting incubation in primary antisera (Supplemental Figure 1, see http://ajp.amjpathol.org). TUNEL was performed as described elsewhere.63Tatton NA lean-Fraser A Tatton WG Perl DP Olanow CW A fluorescent double-labeling method to detect and confirm apoptotic nuclei in Parkinson's disease.Ann Neurol. 1998; 44: S142-S148Crossref PubMed Scopus (188) Google Scholar Briefly, the sections were permeabilized with proteinase K (20 μg/ml), treated with RNAase A (100 μg/ml), and incubated with an equilibration buffer. The enzymatic reaction was performed for 1 hour at 37°C with the terminal deoxynucleotidyl transferase (TdT; catalog no. 3333566, Roche, Indianapolis, IN) and BODIPY TR-14-coupled dUTP (catalog no. C7618; Molecular Probe, Eugene, OR). The cyanine dye YOYO-1 (catalog no. Y3601, Molecular Probe) was used as a counterstain. For conventional electron microscopy (EM), PS/APP mice brains (n = 4, 16 months of age) were fixed by cardiac perfusion using 2% glutaraldehyde-4% paraformaldehyde in 0.1 mol/L sodium cacodylate buffer and postfixed in 1% osmium tetroxide. After alcohol dehydration, sections were embedded and ultrathin sections prepared and stained with uranyl acetate and lead citrate. Material was viewed with a Philips (Eindoven, Netherlands) CM 10 EM equipped with a Hamamatsu (Shizuoka, Japan) C4742-95 digital camera aided by AMT (Danvers, MA) Image Capture Engine software (version 5.42.443a). One-μm-thick sections were stained with toluidine blue for light microscopic examination. A modified pre-embedding staining technique64Totterdell S Ingham CA Bolam JP Immunocytochemistry I: pre-embedding staining.in: Bolam JP Experimental Neuroanatomy: A Practical Approach. Oxford University Press, Oxford1992: 103-128Google Scholar, 65Teclemariam-Mesbah R Wortel J Romijn HJ Buijs RM A simple silver-gold intensification procedure for double DAB labeling studies in electron microscopy.J Histochem Cytochem. 1997; 45: 619-621Crossref PubMed Scopus (25) Google Scholar, 66Talbot K Cho DS Ong WY Benson MA Han LY Kazi HA Kamins J Hahn CG Blake DJ Arnold SE Dysbindin-1 is a synaptic and microtubular protein that binds brain snapin.Hum Mol Genet. 2006; 15: 3041-3054Crossref PubMed Scopus (134) Google Scholar was used for immunoelectron microscopy (IEM). PS/APP, WT mice of 24 months of age (n = 4 for each genotype), or neonatal C57BL/6J mice (n = 3) were perfused-fixed with 0.1% glutaraldehyde-4% paraformaldehyde in 0.1 mol/L sodium cacodylate buffer. The brains were removed and immersion-fixed for 4 hours at 4°C and subsequently transferred to 4% paraformaldehyde overnight at 4°C. Seventy-μm-thick vibratome sections were cut into phosphate-buffered saline (PBS), and treated with the following solutions alternating with PBS washes: 50% ethanol in PBS for 20 minutes, 0.05% Triton X-100 in PBS for 20 minutes, freshly made 1% sodium borohydride in PBS for 10 minutes, and 3% H2O2 for 10 minutes. After blocking the sections in 10% normal goat serum for 1 hour at room temperature, they were briefly rinsed and incubated in the anti-activated caspase-3 antibody [catalog no. 9661, Cell Signaling; diluted 1:200 in 1% bovine serum albumin (BSA)/PBS] overnight at 4°C. After washing in PBS, the sections were incubated in a biotinylated goat anti-rabbit secondary antibody (diluted 1:250 in 1% BSA/PBS; Vector Laboratories, Burlingame, CA) for 1 hour, then incubated in a Vector standard ABC solution (Vector Laboratories) for 2 hours, and reacted with 0.025% 3,3′-diaminobenzidine tetrahydrochloride in PBS in the presence of 0.006% H2O2 for 10 minutes. The 3,3′-diaminobenzidine tetrahydrochloride reaction product was then intensified with silver-gold treatment following the protocol from Teclemariam-Mesbah and colleagues.65Teclemariam-Mesbah R Wortel J Romijn HJ Buijs RM A simple silver-gold intensification procedure for double DAB labeling studies in electron microscopy.J Histochem Cytochem. 1997; 45: 619-621Crossref PubMed Scopus (25) Google Scholar The sections were then postfixed in 1% osmium tetroxide in 0.1 mol/L cacodylate buffer for 30 minutes, dehydrated in ethanol, and flat-embedded in resin. Areas of interest containing caspase-3-positive neuron(s) were first identified by light microscopy and ultrathin sections were placed on grids and not poststained. Note that optimization of immunolabeling by this technique requires fixation with very a low percentage of glutaraldehyde and treatment with Triton X-100, which results in suboptimal structural preservation. Postembedding IEM with gold-conjugated secondary antibody was used to detec
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