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From the Department of Biology,* Boise State University, Boise, Idaho; and the Department of Neurology, Institute for Brain Aging and Dementia, University of California at Irvine, Irvine, California
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Abstract |
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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.19 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).19 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.20
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.21,22
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Methods and Methods |
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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). ß-Amyloid (1-42) (Aß) 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-Aß) antibody was from Senetek PLC (Maryland Heights, MS). Z-Val-Ala-Ala-Asp (OMe)-FMK (Z-VAD) was from Enzyme Systems Products (Livermore, CA).
Generation of Polyclonal Antibodies
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.
Cell Culture
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.
Preparation of Human Brain Lysates
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 x 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.
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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.
Cell-Free Digestion of GFAP
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 2x reaction buffer containing 10 mmol/L dithiothreitol overnight at 37°C. Reactions were terminated by the addition of 5x sample buffer and analyzed by Western blot.
Treatment Protocols for U-87 Cell Experiments
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 Aß 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 5x 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 Aß), and then fixed in ice-cold methanol for 2 minutes. Fluorescence immunocytochemistry was as previously described23 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.
Human Patients
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.
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Free-floating 40-µm-thick serial sections were used for immunohistochemical and immunofluorescence studies as previously described.14 Antibody dilutions were the following: cGFAP-CCP or nGFAP-CCP (1:100), anti-GFAP (1:500), mAb 6E10 (anti-Aß, 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 x20 and x40 objective for image analysis and barrier filters at 510 and 605 nm.
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Results |
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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,24
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.
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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). In Situ Detection of GFAP CCPs in a Model System of Apoptosis
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.25,26
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 Aß (Figure 2B)
. U-87 cells treated with fibrillar Aß exhibited features of apoptosis including cell shrinkage and nuclear condensation, actions of Aß that have been previously reported in neuronal cells.27,28
The immunoreactivity distribution between SST-treated cells and that of Aß-treated was different. Whereas cGFAP-CCP staining was more confined to the cell membrane in SST-treated cells, it appeared more cytoplasmic in Aß-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 Aß may have contributed to the more limited distribution of cGFAP-CCP immunoreactivity.
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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 Aß and tau pathology that closely follows the pathological development of AD in human brain.29,30
In addition, by 18 months of age, 3xTg-AD mice begin to show signs of reactive gliosis in plaque-rich regions.29
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 Figures 1 to 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.
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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.
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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.
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, within beaded processes (Figure 6, B–D
; arrows) and in astrocytes containing what appeared to be autophagic cytoplasmic vacuoles (Figure 6, E–G
; arrows). Although co-localization was evident, there was a clear spatial resolution between the two antibodies in labeled structures, indicating recognition of distinct epitopes within GFAP. Additionally, we were able to determine the co-localization of caspase-cleaved GFAP within Aß plaques after double-label immunofluorescence with the cGFAP-CCP Ab and the monoclonal antibody 6E10, which detects Aß (Figure 6, H–J)
. These data provide further support for the caspase-mediated cleavage of GFAP within damaged astrocytes in the AD brain.
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The in vitro experiments performed in Figures 1 and 2
suggest that caspase-3 may cleave GFAP, generating a 20-kd fragment that is detected by the cGFAP-CCP Ab. To examine a possible relationship between cleaved GFAP and caspase-3 activation in the AD brain, double-labeling experiments were performed using an anti-active caspase-3 antibody and cGFAP-CCP Ab. As shown in Figure 7
, co-localization of these two markers was evident within damaged astrocytes along blood vessels. In general, the staining pattern revealed by these experiments consisted of labeling of the cGFAP-CCP Ab within cell bodies whereas that of active caspase-3 was more homogeneous being found both in the cell body and in astrocytic processes (Figure 7, A and D)
. In addition, many of the astrocytes appeared to have damaged processes that were fragmented or disintegrating (Figure 7, A, B, and D)
. These data showing a relationship of cGFAP-CCP immunoreactivity with a marker for apoptosis support the hypothesis that GFAP is indeed cleaved by caspases in the AD brain and this cleavage event occurs within damaged astrocytes.
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In a final set of experiments, we determined whether the upstream, amino-terminal (N-terminal) fragment of GFAP, nGFAP-CCP, could be detected in the AD brain. To accomplish this, an antibody was designed to the upstream neoepitope region that would be revealed after cleavage of GFAP at D266 (see Materials and Methods). To confirm that this antibody, termed nGFAP-CCP Ab, preferentially recognizes the N-terminal fragment, Western blot analysis was performed after digestion of full-length GFAP with either caspase-3 or -7. In this case, the nGFAP-CCP Ab labeled the predicted 30-kd fragment after GFAP digestion (Figure 8A
, lanes 2 and 3) but did not immunoreact with full-length GFAP (Figure 8A
, lane 1). The nGFAP-CCP Ab labeled several bands 30 kd, most likely representing incomplete cleavage fragments of GFAP. Application of the nGFAP-CCP Ab to control or AD brain sections gave similar results to those of the cGFAP-CCP Ab: a relative paucity of staining in control cases with a total absence of any AD pathology (Figure 8B)
and prominent labeling of damaged astrocytes exhibiting beaded processes in AD cases (Figure 8, C and D)
.
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Discussion |
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The few available studies that have examined a role for apoptosis in astrocytes in AD have been contradictory and, for the most part, inconclusive. Thus, although some studies have supported a role for apoptosis occurring in astrocytes in the AD brain,31,32 others have not.33,34 Evidence for apoptotic cell death in all of these studies consisted of in situ detection of fragmented DNA by terminal dUTP nick-end labeling techniques. Interpretation of terminal dUTP nick-end labeling-positive labeling is a challenge and has come under criticism as to whether or not it is a specific marker for apoptosis,15,35 possibly contributing to the contradictory conclusions regarding whether apoptosis occurs in astrocytes of the AD brain.
An alternative approach for detecting apoptosis is to detect the activation of caspases or their target protein fragments after cleavage. Because caspases are specific in that they cleave only after aspartic residues, cleavage reveals new antigentically distinct sites. These consensus caspase-cleavage sites, therefore, represent desirable targets for cleavage site-directed antibodies. Using such antibodies, numerous studies have demonstrated the activation of caspases10,14,36,37 and the caspase-mediated cleavage of important neuronal proteins in the AD brain, including actin,12 fodrin,11 the amyloid precursor protein,9 and tau.14,16 In the present study, we designed a caspase-cleavage site-directed antibody to GFAP, a protein specifically expressed in astrocytes. Examination of the GFAP sequence revealed a single caspase consensus sequence, DLTD266, based on the general tetrapeptide motif DXXD recognized by caspases.8 Based on this site, we synthesized an antibody to the downstream predicted fragment that would be generated after cleavage of full-length GFAP by caspases at this site. After characterization of this antibody, application to AD brain sections resulted in the labeling of astrocytes that were damaged and displayed features characteristic of apoptosis.
Staining with the cGFAP-CCP Ab occurred within cell bodies, in beaded processes and/or in fragmented processes and co-localized with active caspase-3. It is important to note that the demonstration of caspase-mediated cleavage of GFAP and the activation of caspase-3 do not imply that astrocytes are dying through apoptosis. Indeed, an alternative explanation is that the cleavage of GFAP by caspases reflects the turnover of GFAP in reactive astrocytes, rather than overt apoptosis. This alternative should be considered based on the fact that apoptotic bodies are not observed frequently in sections of the AD brain, and considerable controversy exists on whether cells are in fact dying by this pathway.15 In support of this is the fact that many processes labeled by the GFAP-CCP Abs were not fragmented but were intact and not degenerated. However, the morphological appearance of GFAP-CCP-labeled astrocytes suggested that these cells were severely damaged and exhibited features characteristic of apoptosis. Thus, many of the cGFAP-CCP-positive astrocytes had a punctate appearance with the processes reduced to clusters or lines of dots radiating outward from the cell body. Moreover, many of the labeled astrocytes contained what appeared to be autophagic cytoplasmic vacuoles, another feature indicative of apoptosis. Therefore, taken together, our results suggest that caspase-mediated cleavage of GFAP, activation of apoptotic pathways and degeneration of astrocytes may be linked. This hypothesis is consistent with a previous report that demonstrated a role for astrocytic degeneration, activation of caspase-3, and DNA fragmentation in frontotemporal dementia.38
Evidence for caspase-mediated cleavage of GFAP was also found in control cases, although labeled astrocytes were comparatively less damaged and occurred in those cases that had a final neuropathological diagnosis of senile degenerative changes with mild Braak/Braak changes. The appearance of GFAP-CCP-positive astrocytes in control cases should not be surprising considering that age-related glial changes, which likely represent the normal wear and tear of the aging process, occur in normal elderly individuals.39
An additional finding of the present study was the labeling of GFAP-CCP-positive astrocytes along blood vessels in the AD brain. Many of these labeled astrocytes exhibited processes that were damaged in appearance. One role for astrocytes is in forming the blood-brain barrier. Astrocytes confer a protective role on the blood-brain barrier against hypoxia and aglycemia by extending numerous, long cytoplasmic processes that terminate in end feet and encapsulate brain capillaries.40 Therefore, astrocytes in this role allow the blood-brain barrier to act as a physical and metabolic barrier between the central nervous system and the systemic circulation, regulating and protecting the microenvironment of the brain. Damage of astrocytic processes after activation of apoptotic pathways and cleavage of cytoskeletal proteins, such as GFAP as demonstrated in the present study, may be one of many factors that contribute to the compromised blood-brain barrier observed in AD.21,41
In summary, caspase-mediated cleavage of GFAP, activation of caspase-3, and damage to astrocytes were common features found together in the AD brain. The link between reactive gliosis, caspase activation, and cytoskeletal protein cleavage may arise from extracellular Aß acting to stimulate the activation of apoptotic pathways within astrocytes. The consequences of caspase activation and cleavage of cytoskeletal proteins in astrocytes may lead to the degeneration of astrocytes and loss of blood-brain barrier integrity. Astrocyte degeneration may not only be a common feature found in certain neurodegenerative diseases, including AD and frontotemporal dementia, but may also represent normal age-related processes, although to a lesser degree.
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Footnotes |
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Supported by the National Institutes of Health (National Center for Research Resources grant P20RR016454 to T.T.R.) and the Alzheimer’s Disease Research Center, University of California at Irvine (grant P50 AG16573).
Accepted for publication November 18, 2005.
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