Caspase-Mediated Cleavage of Glial Fibrillary Acidic Protein within Degenerating Astrocytes of the Alzheimer's Disease Brain
2006; Elsevier BV; Volume: 168; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2006.050798
ISSN1525-2191
AutoresPeter E. Mouser, Elizabeth Head, Kwang‐Ho Ha, Troy T. Rohn,
Tópico(s)Cell death mechanisms and regulation
ResumoRecent studies demonstrate roles for activation of caspases and cleavage of cellular proteins within neurons of the Alzheimer's disease (AD) brain. To determine whether a similar role for caspases also occurs within glial cells in AD, we designed a site-directed caspase-cleavage antibody specific to glial fibrillary acidic protein (GFAP), a cytoskeleton protein specifically expressed in astrocytes. In vitro characterization of this antibody using both a cell-free system and a cell model system of apoptosis demonstrated that the antibody (termed GFAP caspase-cleavage product antibody or GFAP-CCP Ab) immunolabeled the predicted caspase-cleavage fragment, but not full-length GFAP, by Western blot analysis. To determine whether caspases cleave GFAP in vivo, tissue sections from control and AD brains were examined by immunohistochemistry using the GFAP-CCP Ab. Two prominent features of staining were evident: immunolabeling of degenerating astrocytes in proximity to blood vessels and staining within plaque-rich regions of the AD brain. Furthermore, co-localization of the GFAP-CCP Ab and an antibody specific to active caspase-3 was demonstrated within damaged astrocytes of the AD brain. These data suggest that the activation of caspases and cleavage of cellular proteins such as GFAP may contribute to astrocyte injury and damage in the AD brain. Recent studies demonstrate roles for activation of caspases and cleavage of cellular proteins within neurons of the Alzheimer's disease (AD) brain. To determine whether a similar role for caspases also occurs within glial cells in AD, we designed a site-directed caspase-cleavage antibody specific to glial fibrillary acidic protein (GFAP), a cytoskeleton protein specifically expressed in astrocytes. In vitro characterization of this antibody using both a cell-free system and a cell model system of apoptosis demonstrated that the antibody (termed GFAP caspase-cleavage product antibody or GFAP-CCP Ab) immunolabeled the predicted caspase-cleavage fragment, but not full-length GFAP, by Western blot analysis. To determine whether caspases cleave GFAP in vivo, tissue sections from control and AD brains were examined by immunohistochemistry using the GFAP-CCP Ab. Two prominent features of staining were evident: immunolabeling of degenerating astrocytes in proximity to blood vessels and staining within plaque-rich regions of the AD brain. Furthermore, co-localization of the GFAP-CCP Ab and an antibody specific to active caspase-3 was demonstrated within damaged astrocytes of the AD brain. These data suggest that the activation of caspases and cleavage of cellular proteins such as GFAP may contribute to astrocyte injury and damage in the AD brain. Alzheimer's disease (AD) is characterized by memory loss as well as difficulties with language, visuospatial abilities, and other cognitive domains.1Morris JC Mohs RC Rogers H Fillenbaum G Heyman A Consortium to establish a registry for Alzheimer's disease (CERAD) clinical and neuropsychological assessment of Alzheimer's disease.Psychopharmacol Bull. 1988; 24: 641-644PubMed Google Scholar, 2Carter J Lippa CF Beta-amyloid, neuronal death and Alzheimer's disease.Curr Mol Med. 2001; 1: 733-737Crossref PubMed Scopus (96) Google Scholar The diagnosis of AD is based on the extent of senile plaque and neurofibrillary tangle accumulation.3Mirra SS Heyman A McKeel D Sumi SM Crain BJ Brounlee LM Vogel FS Hughes JP VanBelle G Berg L The consortium to establish a registry for Alzheimer's disease (CERAD) part II. Standardization of the neuropathological assessment of Alzheimer's disease.Neurology. 1991; 41: 479-486Crossref PubMed Google Scholar Senile plaques contain the β-amyloid peptide (Aβ), and evidence suggests that Aβ deposition precedes neurofibrillary tangle formation and may be the earliest event that triggers subsequent downstream molecular events leading to neuronal death and synaptic loss.4Hardy J Selkoe DJ The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.Science. 2002; 297: 353-356Crossref PubMed Scopus (11025) Google Scholar Aβ is known to be toxic to neurons, and one putative mechanism is through the activation of apoptosis.5Pike CJ Cummings BJ Cotman CW Beta-amyloid induces neuritic dystrophy in vitro: similarities with Alzheimer pathology.Neuroreport. 1992; 3: 769-772Crossref PubMed Scopus (125) Google Scholar, 6Cotman CW Anderson AJ A potential role for apoptosis in neurodegeneration and Alzheimer's disease.Mol Neurobiol. 1995; 10: 19-45Crossref PubMed Scopus (358) Google Scholar Apoptosis is characterized by plasma membrane blebbing, nuclear fragmentation, and cell shrinkage7Kerr 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 (12874) Google Scholar and is initiated by the proteolytic enzymes, caspases.8Nicholson DW Caspase structure, proteolytic substrates, and function during apoptotic cell death.Cell Death Differ. 1999; 6: 1028-1042Crossref PubMed Scopus (1305) Google Scholar Numerous studies have documented the activation of caspases in the AD brain as well as the cleavage of critical cellular proteins.9Gervais 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 (716) Google Scholar, 10Rohn 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 (146) Google Scholar, 11Rohn 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 (144) Google Scholar, 12Yang 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, 13Su 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 In addition, recent studies have implicated the activation of caspases and cleavage of tau as early events in the disease process that may link Aβ and neurofibrillary tangle in AD.14Rohn TT Rissman RA Davis MC Kim Y-E Cotman C Head E Caspase-9 activation and caspase cleavage of tau in the Alzheimer's disease brain.Neurobiol Dis. 2002; 11: 341-354Crossref PubMed Scopus (216) Google Scholar, 15Rohn 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, 16Rissman RA Poon WW Blurton-Jones M Oddo S Reidun T Vitek MP LaFerla FM Rohn TT Cotman CW Caspase-cleavage of tau is an early event in Alzheimer's disease pathology.J Clin Invest. 2004; 114: 121-130Crossref PubMed Scopus (463) Google Scholar, 17Guo 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 (236) Google Scholar, 18Gamblin 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 (684) Google Scholar These studies suggest that it is the caspase-mediated cleavage of important cellular proteins, per se, and not the full execution of apoptosis that may be important for driving the pathology associated with AD. Although a role for caspase activation within neurons of the AD brain has been established, whether a similar role can be attributed to caspases in astrocytes has not been explored. Astrocytes outnumber neurons in the brain and play many roles essential for normal brain function including ion buffering, glutamate uptake, and participation in the formation of the blood-brain barrier.19Pekny M Nilsson M Astrocyte activation and reactive gliosis.Glia. 2005; 50: 427-434Crossref PubMed Scopus (1246) Google Scholar During the progression of AD, astrocytes undergo both morphological and functional changes, giving rise to the term "reactive gliosis." Reactive gliosis is characterized by the hypertrophy of astrocytes as well as by proliferation and up-regulation of the intermediate filament protein glial fibrillary acidic protein (GFAP).19Pekny M Nilsson M Astrocyte activation and reactive gliosis.Glia. 2005; 50: 427-434Crossref PubMed Scopus (1246) Google Scholar In AD, reactive astrocytes accumulate in the vicinity of senile plaques and may contribute to maturation from a diffuse to a cored plaque by releasing inflammatory mediators.20Unger JW Glial reaction in aging and Alzheimer's disease.Microsc Res Tech. 1998; 43: 24-28Crossref PubMed Scopus (82) Google Scholar Because previous studies have demonstrated a role for caspases in neurodegeneration and possible neurofibrillary tangle formation in AD, we hypothesized that a similar role for caspases may occur within astrocytes and contribute to astrocytic degeneration. The present study hypothesized that caspase-mediated cleavage of GFAP occurs selectively within reactive astrocytes of the AD brain. A site-directed antibody specific to a putative caspase-cleavage consensus site within GFAP was synthesized and tested for its utility in several in vitro model systems of apoptosis. Application of this antibody to AD brain sections revealed immunolabeling within damaged astrocytes in plaque-rich regions and along blood vessels of the AD brain that co-localized with active caspase-3. These findings not only underscore the involvement of caspases in astrocytic injury but may also provide a possible mechanism for why the blood-brain barrier is compromised in AD.21Huber JD Egleton RD Davis TP Molecular physiology and pathophysiology of tight junctions in the blood-brain barrier.Trends Neurosci. 2001; 24: 719-725Abstract Full Text Full Text PDF PubMed Scopus (640) Google Scholar, 22Farkas E Luiten PG Cerebral microvascular pathology in aging and Alzheimer's disease.Prog Neurobiol. 2001; 64: 575-611Crossref PubMed Scopus (896) Google Scholar Purified GFAP, recombinant human caspase-3 and −7, and staurosporine (SST) were purchased from Calbiochem (La Jolla, CA). The sulfolink coupling kit used to affinity purify antibodies was purchased from Pierce (Rockford, IL). β-Amyloid (1-42) (Aβ) peptide was from Biosource International Inc. (Camarillo, CA). Concanavalin type VI (Con A) was from Sigma (St. Louis, MO). The monoclonal anti-active caspase-3 antibody was from BD Pharmingen (La Jolla, CA). The mouse anti-GFAP antibody (mAb 3402) was purchased from Chemicon International (Temecula, CA). The mouse anti-6E10 (anti-Aβ) antibody was from Senetek PLC (Maryland Heights, MS). Z-Val-Ala-Ala-Asp (OMe)-FMK (Z-VAD) was from Enzyme Systems Products (Livermore, CA). Two sets of polyclonal antibodies were synthesized based on a putative caspase cleavage consensus site (DLTD266) within GFAP: one to the amino-terminal upstream fragment and the other to the downstream carboxy-terminal cleavage fragment that would be generated after cleavage by caspases. For the amino-terminal site, a 16-mer peptide (CGGGGGGRSKFADLTD) corresponding to the upstream neoepitope fragment that would be generated after cleavage was synthesized, coupled to keyhole limpet hemocyanin and injected into rabbits. Resulting sera were used to affinity purify antibodies using a sulfolink column coupled with the peptide CRSKFADLTD. Peptide synthesis and generation of polyclonal antibodies was contracted out to Chemicon International. This antibody is referred to as amino terminal GFAP caspase-cleavage product (nGFAP-CCP) in the current study. For the carboxy-terminal site, an 8-mer peptide (AAARNAEC) was synthesized and injected into rabbits after conjugation to keyhole limpet hemocyanin. This antibody, termed cGFAP-CCP Ab, was purified using a sulfolink column coupled with the peptide AAARNAEC. For this antibody, synthesis of peptides, injections of immunogens, and collection of antisera were contracted out to Bethyl Laboratories (Montgomery, TX). Unless noted otherwise, the cGFAP-CCP Ab was used in all experiments. Human U-87 MG glioblastoma-astrocytoma cells (American Type Culture Collection, Manassas, VA) were grown in Eagle's minimal essential medium (no. 30-2003, American Type Culture Collection) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Case demographics are presented in Table 2. Frozen human temporal cortex tissues from control or AD cases were homogenized in a Tris extraction buffer consisting of 1% sodium dodecyl sulfate and a protease inhibitor cocktail (catalog no. 158837; Biomedicals Inc.). After homogenization, samples were centrifuged for 1 hour at 4°C at 130,000 × g, and the resulting supernatant was collected (representing the soluble fractions). Protein content was measured using the BCA method (Pierce Biotechnology Inc.), and equal protein amounts were then analyzed by Western blot.Table 2Case Demographics for Western Blot AnalysisCaseGroupAge (years)SexPMI (hours)Braak and Braak stageMMSE1Control83M2.3IIN/A2Control88F5II293Control85F4.3III294Early AD81F7V235AD79M4.5VToo impaired6AD96F3.3VToo impaired7AD77M5VIToo impaired8AD76F4.5VIToo impairedPMI, post mortem interval; MMSE, Mini Mental State Examination. Open table in a new tab PMI, post mortem interval; MMSE, Mini Mental State Examination. Purified human GFAP, U-87 cell extracts, or human brain lysates were processed for Western blot analysis. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Membranes were incubated in either cGFAP-CCP (1:500) or anti-GFAP (1:500), and primary antibody was visualized using either goat anti-rabbit or anti-mouse horseradish peroxidase-linked secondary antibody (1:5000; Jackson Laboratory, West Grove, PA), followed by enhanced chemiluminescence detection. To examine whether executioner caspases can cleave GFAP, 15 μg of purified human GFAP was incubated with active human recombinant caspase-3 or −7 (at equivalent specific activities) in 2× reaction buffer containing 10 mmol/L dithiothreitol overnight at 37°C. Reactions were terminated by the addition of 5× sample buffer and analyzed by Western blot. Con A was made as a 25 μmol/L stock in serum-free medium and filter sterilized before use. SST was made as a 5 mmol/L stock in sterile dimethyl sulfoxide and diluted 1:100 in bovine serum albumin/phosphate-buffered saline before addition to cell cultures. Z-VAD was prepared as a 50 mmol/L stock in sterile dimethyl sulfoxide. To permit adequate cellular loading, Z-VAD was added 1 hour before insult. Fibrillar Aβ was prepared by freeze-thawing (three times) followed by incubation at room temperature overnight. After various treatments, U-87 cell extracts were prepared by adding ice-cold lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% Nonidet P-40, 0.25% deoxycholate, 1 mmol/L EGTA, pH 7.4, and protease inhibitor cocktail), followed by centrifugation and addition of 5× sample buffer. For immunocytochemistry, cells were plated on poly-d-lysine/laminin-coated chamber slides (BD Biosciences), treated with various insults (SST, Con A, or Aβ), and then fixed in ice-cold methanol for 2 minutes. Fluorescence immunocytochemistry was as previously described23Rohn TT Cusack SM Kessinger SR Oxford JT Caspase activation independent of cell death is required for proper cell dispersal and correct morphology in PC12 cells.Exp Cell Res. 2004; 295: 215-225Crossref PubMed Scopus (54) Google Scholar using the cGFAP-CCP Ab (1:500). Bound primary antibody was detected using a biotinylated anti-rabbit ABC peroxidase kit (Vector Laboratories, Burlingame, CA) followed by visualization using a tyramide signal amplification kit (Molecular Probes, Eugene, OR) consisting of Alexa Fluor 488-labeled tyramide (excitation/emission = 495/519). To visualize apoptotic cells, the DNA intercalator propidium iodide was used, resulting in red fluorescence at 488 nm. For microscopic observation and photomicrography of fluorescently labeled cells, an Olympus BX60 fluorescence microscope equipped with a PM-10AD system for photomicrography was used. Autopsy brain tissues from the hippocampus and entorhinal cortex of eight neuropathologically confirmed AD cases and six nondemented cases diagnosed as normal were studied. Five of six of the normal cases were identified as having diffuse senile plaques and/or mild Braak/Braak changes within the hippocampus and entorhinal cortex as a final neuropathological diagnosis. Case demographics are presented in Table 1. Age at death was not significantly different between AD (mean, 79.9 ± 5.74 years) and controls (mean, 81.3 ± 10.2 years). Human brain tissues used in this study were provided by the Institute for Brain Aging and Dementia Tissue Repositories at the University of California, Irvine.Table 1Case Demographics for Immunohistochemical AnalysisCaseGroupAge (years)SexPMIMMSEClinical diagnosisNeuropathological diagnosis1AD75M4.521Probable ADAD2AD80F49Probable ADAD3AD73M414Probable ADAD4AD87F2.6718Probable ADAD5AD77M3.5822Probable ADAD6AD85M5.254Probable ADAD7AD87F59Probable ADAD8AD/PD75F2.5N/AProbable AD/PDAD/PD9Ctl84F7.1N/AN/ANormal (MPC)10Ctl74F2.75N/ANormalNormal (MPC)11Ctl68F6N/AN/ANormal (MPC)12Ctl77M6.5N/ANormalNormal (MPC)13Ctl90F6.5N/ANormalNormal (MPC)14Ctl95M3.67N/ANormalNormalPMI, postmortem interval; MMSE, Mini Mental State Examination; MPC, mild pathological changes consistent with Braak and Braak stage I/II; N/A, not available. Open table in a new tab PMI, postmortem interval; MMSE, Mini Mental State Examination; MPC, mild pathological changes consistent with Braak and Braak stage I/II; N/A, not available. Free-floating 40-μm-thick serial sections were used for immunohistochemical and immunofluorescence studies as previously described.14Rohn TT Rissman RA Davis MC Kim Y-E Cotman C Head E Caspase-9 activation and caspase cleavage of tau in the Alzheimer's disease brain.Neurobiol Dis. 2002; 11: 341-354Crossref PubMed Scopus (216) Google Scholar Antibody dilutions were the following: cGFAP-CCP or nGFAP-CCP (1:100), anti-GFAP (1:500), mAb 6E10 (anti-Aβ, 1:10,000). Antigen visualization was determined using ABC complex (ABC Elite immunoperoxidase kit, Vector Laboratories), followed by diaminobenzidine or Blue SG substrate (Vector Laboratories). For immunofluorescence studies, antigen visualization was accomplished using an Alexa Fluor 488-labeled tyramide (green, excitation/emission = 495/519) or streptavidin Cy-3 (red, 1:200). Confocal images were collected on an Olympus IX70 inverted microscope using both a ×20 and ×40 objective for image analysis and barrier filters at 510 and 605 nm. The goal of the present study was to determine whether GFAP, a 50-kd protein expressed specifically in astrocytes, is cleaved by caspases in the AD brain. Because caspases are specific in that they cleave only after aspartic residues,24Nicholson DW Thornberry NA Caspases: killer proteases.Trends Biochem Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2182) Google Scholar cleavage will reveal new, antigenically distinct sites. Examination of the GFAP protein sequence indicated a single putative caspase-cleavage consensus site, DLTD266, that would produce two major predicted fragments on cleavage: an amino-terminal fragment of ∼30 kd and a carboxy-terminal fragment of ∼20 kd. As an initial approach, we synthesized antibodies to the carboxy-terminal (C-terminal) caspase-cleavage site within GFAP and tested its validity as a specific probe for GFAP CCPs using a cell-free system. Purified human GFAP was incubated with or without caspase-3, and samples were immunoblotted with the cGFAP-CCP Ab. Although no immunoreactivity to the antibody was evident in nondigested samples (Figure 1A, lane 1), a prominent band at 20 kd was evident after digestion of GFAP with caspase-3 (Figure 1A, lane 2). To verify that full-length GFAP was indeed present, the same blots were stripped and reprobed with an anti-GFAP antibody that recognizes full-length GFAP. As shown in Figure 1B, strong immunolabeling was evident using this antibody indicating the presence of full-length GFAP in these samples. At longer exposures, we were able to faintly detect the 20-kd fragment of cleaved GFAP using this antibody (data not shown). These initial results suggest that the cGFAP-CCP Ab recognizes the C-terminal fragment of GFAP but does not immunoreact with full-length GFAP. To further characterize the cGFAP-CCP Ab, experiments were performed using human U-87 astrocytoma cells. U-87 cells were incubated overnight in the absence or presence of the classical apoptotic insult SST, and cell extracts were analyzed by Western blot using cGFAP-CCP Ab. A single band corresponding to ∼20 kd was evident after treatment of U-87 cells with SST (Figure 1C, lane 2). The appearance of this SST-induced cleavage fragment was prevented after pretreatment of cells with the caspase inhibitor Z-VAD (Figure 1C, lane 3). As in the cell-free system, the cGFAP-CCP Ab did not appear to strongly label full-length GFAP in U-87 cells even though there were ample levels of full-length GFAP present in these samples (Figure 1D). In addition, the anti-GFAP antibody strongly labeled several caspase cleavage fragments of GFAP, including one at 20 kd (Figure 1D, lane 2), that were completely prevented after preincubation of cells with the caspase inhibitor Z-VAD (Figure 1D, lane 3). Experiments were performed to characterize the cGFAP-CCP Ab by immunocytochemistry. U-87 cells were treated with SST (500 nmol/L) or Con A (1 μmol/L), which have previously been demonstrated to be effective apoptotic stimuli.25Cribbs DH Kreng VM Anderson AJ Cotman CW Crosslinking of membrane glycoproteins by concanavalin A induces apoptosis in cortical neurons.Neuroscience. 1996; 75: 173-185Crossref PubMed Scopus (39) Google Scholar, 26Rohn TT Ivins KJ Bahr BA Cotman CW Cribbs DH A Monoclonal antibody to amyloid precursor protein induces neuronal apoptosis.J Neurochem. 2000; 74: 2331-2342Crossref PubMed Scopus (92) Google Scholar Treatment of U-87 cells with either SST or Con A resulted in the telltale morphological signs of apoptosis including cell shrinkage and nuclear condensation and fragmentation (Figure 2). Application of the cGFAP-CCP Ab resulted in little staining in nontreated cells (Figure 2A). In contrast, strong labeling of fragmented processes and cell bodies was apparent in SST- or Con A-treated cells (Figure 2A). Staining with the DNA intercalator propidium iodide indicated that labeled cells had condensed, fragmented nuclei in contrast to untreated cells (Figure 2A, inset). In a similar set of experiments, in situ detection of the cGFAP-CCP was evident after treatment of U-87 cells with Aβ (Figure 2B). U-87 cells treated with fibrillar Aβ exhibited features of apoptosis including cell shrinkage and nuclear condensation, actions of Aβ that have been previously reported in neuronal cells.27Ivins KJ Thornton PL Rohn TT Cotman CW Neuronal apoptosis induced by beta-amyloid is mediated by caspase-8.Neurobiol Dis. 1999; 6: 440-449Crossref PubMed Scopus (182) Google Scholar, 28Loo DT Copani A Pike CJ Whittemore ER Walencewicz AJ Cotman CW Apoptosis is induced by beta-amyloid in cultured central nervous system neurons.Proc Natl Acad Sci USA. 1993; 90: 7951-7955Crossref PubMed Scopus (1045) Google Scholar The immunoreactivity distribution between SST-treated cells and that of Aβ-treated was different. Whereas cGFAP-CCP staining was more confined to the cell membrane in SST-treated cells, it appeared more cytoplasmic in Aβ-treated cells (Figure 2B). We are unsure of the reason for this difference, but it is possible that the resultant cell shrinkage after treatment of U-87 cells with Aβ may have contributed to the more limited distribution of cGFAP-CCP immunoreactivity. To further verify that cGFAP-CCP Ab can detect CCPs of GFAP, immunohistochemical analysis was performed using tissue sections from triple-transgenic mice (3xTg-AD). The 3xTg-AD mice develop Aβ and tau pathology that closely follows the pathological development of AD in human brain.29Oddo S Caccamo A Shepherd JD Murphy MP Golde TE Kayed R Metherate R Mattson MP Akbari Y LaFerla FM Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Aβ and synaptic dysfunction.Neuron. 2003; 39: 409-421Abstract Full Text Full Text PDF PubMed Scopus (3196) Google Scholar, 30LaFerla FM Oddo S Alzheimer's disease: abeta, tau and synaptic dysfunction.Trends Mol Med. 2005; 11: 170-176Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar In addition, by 18 months of age, 3xTg-AD mice begin to show signs of reactive gliosis in plaque-rich regions.29Oddo S Caccamo A Shepherd JD Murphy MP Golde TE Kayed R Metherate R Mattson MP Akbari Y LaFerla FM Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Aβ and synaptic dysfunction.Neuron. 2003; 39: 409-421Abstract Full Text Full Text PDF PubMed Scopus (3196) Google Scholar No immunoreactivity to the cGFAP-CCP Ab was seen in non-TG control mice (Figure 3A). In contrast, staining of a subset of astrocytes with fragmented processes was observed in the cortex of 25-month-old 3xTg-AD mice after application of the cGFAP-CCP Ab (Figure 3B). Taken together, the results presented in Figure 1, Figure 2, Figure 3 support the conclusion that the cGFAP-CCP Ab is an effective marker for the detection of caspase-cleaved GFAP and therefore, may serve as a useful tool to examine caspase-mediated cleavage of GFAP in the human AD brain. After confirmation of the cGFAP-CCP Ab as a specific probe for the C-terminal caspase-cleavage fragment of GFAP, immunohistochemical experiments were performed using hippocampal sections from AD or age-matched control brains. In control cases examined, we observed staining of astrocytes after application of the cGFAP-CCP Ab (Figure 4A). However, when staining was observed in a normal control case (defined as nondemented at the time of death) the neuropathological diagnosis was inevitably described as having senile degenerative changes with the presence of scattered diffuse senile plaques. In those control patients in which there was a total absence of AD pathology, there was a corresponding lack of immunoreactivity to the cGFAP-CCP Ab (Figure 4A, inset). In contrast, widespread labeling of degenerating astrocytes was observed in all AD brain sections examined after application of the cGFAP-CCP Ab (Figure 4, B–E). In general, staining with the cGFAP-CCP Ab was very heterogeneous in AD cases, for example occurring within beaded astrocytic processes, in cell bodies only, or within small fragmented processes. Evidence for cGFAP-CCP-positive astrocytes was found both in white and gray matter as well as in the hippocampus proper and entorhinal cortex. In AD cases, GFAP-CCP-positive astrocytes displayed many hallmark features of apoptosis including cell shrinkage, swollen varicosities, and fragmented processes (Figure 4, B and C). In addition, in AD sections there were two common features associated with cGFAP-CCP Ab immunoreactivity: staining along blood vessels (Figure 4D) and within plaques (Figure 4E). Figure 4F illustrates that staining with the cGFAP-CCP Ab was completely prevented after preabsorption with free peptide, illustrating the specificity of the cGFAP-CCP Ab. The presence of cGFAP-CCP Ab staining in association with senile plaques supports the hypothesis that extracellular Aβ serves as a stimulus to activate apoptotic pathways in astrocytes as they are recruited to plaque-rich areas. These results demonstrate that GFAP is cleaved by caspases in the AD brain and is confined to those astrocytes that appear damaged. To confirm the immunohistochemical results, Western blot experiments were performed using temporal cortex brain lysates from control or AD brains (see Table 2 for case demographics). Cases were carefully selected based on MMSE (Mini Mental State Examination) scores to clearly define controls from ADs. As depicted in Figure 5, we were able to detect the C-terminal caspase-cleaved fragment of GFAP after application of the cGFAP-CCP Ab in four of four AD cases with only faint labeling of the same band in control cases. It is noteworthy the high degree of specificity exhibited by the cGFAP-CCP Ab by Western blot analysis, further supporting our immunohistochemical findings and suggesting that the C-terminal caspase-cleaved fragment of GFAP is present in the AD brain. Double-label immunofluorescence experiments were also performed to verify that the labeling with the cGFAP-CCP Ab was indeed specific for astrocytes. AD sections were double-labeled with the cGFAP-CCP Ab and the anti-GFAP antibody, which labels full-length GFAP. Co-localization of both antibodies was evident along blood vessels (Figure 6A), wi
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