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From the Institute for Brain Aging and Dementia,*
University of California at Irvine, Irvine, California; and the
Department of Pharmaceutical Sciences and the Neurosciences
Program,
University of Connecticut,
Storrs, Connecticut
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Abstract |
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Introduction |
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To address the question of whether there is a relationship between NFTs and neuronal apoptosis in AD, we designed a caspase-cleavage site-directed antibody to fodrin, a major constituent of the membrane cytoskeleton in neurons.19 We chose fodrin as our marker for apoptosis because it is expressed predominantly and abundantly in neurons19 and it is an excellent substrate for caspase-3 cleavage.20-23 Using this novel probe, we demonstrate widespread caspase activation and accumulation of stable caspase cleavage products in AD brain, and provide support for the hypothesis that activation of apoptotic mechanisms may contribute to disease progression.
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Materials and Methods |
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All chemicals used were of the highest grade available. Poly-D-lysine and concanavalin A (Con A) were obtained from Sigma Chemical Co. (St. Louis, MO). Staurosporine was from Calbiochem-Novabiochem (La Jolla, CA). Z-Val-Ala-Ala-Asp (OMe)-FMK (Z-VAD) was from Enzyme Systems Products (Livermore, CA). A multiple antigen peptide (MAP) containing the SVEALI sequence that was used for generation of polyclonal antibodies was synthesized by Research Genetics, Inc. (Huntsville, AL). Apopain/YAMA/CPP32/caspase-3-agarose was from Upstate Biotechnology (Lake Placid, NY). The sulfolink kit used to affinity purify antibody was purchased from Pierce (Rockford, IL).
Cell Culture
Human neuroblastoma SH-SY5Y cells (SY5Y) were grown to confluence
on 12-well plates (
2 x 106
cells/well)
in Dulbecco’s modified Eagle’s medium supplemented with 10%
fetal bovine serum and 100 U/ml penicillin and 100 µg/ml
streptomycin. Sprague-Dawley rat embryos (day 18 of gestation) were
used to produce short-term cultures of cortical neurons as previously
described.24
For all morphological studies, cells were
plated at a density of 1.5 x 105
cells/ml
in 24-well poly-D-lysine-coated plates. For Western blot
analysis, neurons were plated at 3.75 x 105
cells/ml in 12-well poly-D-lysine-coated plates. All
experiments were performed on day 4 of the neuronal cultures.
Treatment Protocols
Con A was made up as a 25 µmol/L stock in Dulbecco’s modified Eagle’s medium and filter sterilized before use. 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. Cultures were used for experimentation on day 4 in vitro.
Immunocytochemistry in Cell Cultures
Immunocytochemistry studies in cortical neurons were performed as previously described.25 The primary antibody, fodrin caspase cleavage product (CCP), was incubated overnight at 4°C at a final concentration of 0.4 to 0.5 µg/ml. Bound antibody was detected using a biotinylated anti-rabbit ABC peroxidase kit (Vector Laboratories, Burlingame, CA) followed by color development using diaminobenzidine (DAB kit; Vector Laboratories) as the chromogenic substrate. For fluorescence labeling of cells, bound antibody was detected using a biotin-SP-conjugated goat anti-rabbit IgG (1:200 for 1 hour at room temperature; Jackson ImmunoResearch, West Grove, PA), followed by Cy-3 conjugated streptavidin for 1 hour at room temperature (1:800).
Western Blot Analysis
Western blot analysis was performed as previously described.23 Briefly, for analysis of fodrin CCPs, cortical neurons, treated under various conditions, were extracted with 2x sample buffer and subjected to sodium dodecyl sulfate electrophoresis on 7.5% gels, transferred to nitrocellulose, and blocked for 1 hour at room temperature with 5% nonfat dried milk. Blots were incubated with fodrin CCP antibody (1 to 2 µg/ml) overnight at 4°C, followed by horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:5,000) for 1 hour at room temperature. Blots were developed using an enhanced chemiluminescence system (Amersham, Arlington Heights, IL). Transferred gel slabs were silver-stained for total protein to ensure equal protein loading.
Human and Animal Subjects
Autopsy brain tissue from the hippocampus and entorhinal cortex of
eight neuropathologically confirmed AD cases and two aged nondemented
cases with no evidence of senile plaques or NFTs were studied. In
addition, seven aged nondemented cases with early senile degenerative
changes (senile plaques and NFTs), termed mild pathology controls, that
did not reach numbers to be consistent with a diagnosis of AD were
compared. Case demographics for these cases are presented in Table 1
. Braak and Braak staging was based on
PHF-1 immunostaining (see below).26
Age at death was not
significantly different between AD (mean, 76.8 ± 2.21) and
controls (mean, 74.1 ± 6.96). To determine whether fodrin CCP
staining was associated only with NFT formation, immunostaining in the
prefrontal cortex of an aged dog (12.2 years) was compared to that of a
young dog (1.6 years). Human and canine brain tissues used in this
project were provided by The Institute for Brain Aging and Dementia
Tissue Repositories at the University of California, Irvine.
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Free-floating 50-µm-thick serial sections from the hippocampus with entorhinal cortex were used for immunocytochemical studies as previously described.27 For single-labeling experiments, sections were incubated overnight in either anti-fodrin CCP (1:100), anti-PHF-1 (1:1,000; kindly provided by Dr. P. Davies, Albert Einstein College of Medicine, Bronx, NY) or anti-phosphorylated tau at serine 202 (AT8, 1:30,000; Innogenetics, Ghent, Belgium) at room temperature. Controls were performed to rule out nonspecific immunostaining by eliminating the primary or secondary antibody. To determine whether cross-reactivity to reagents was a factor in double-labeling experiments, experiments were replicated with the antibodies reversed. All controls were negative.
Immunofluorescence and Confocal Microscopy
For fodrin CCP and PHF-1 co-localization studies, sections were incubated with anti-fodrin CCP (0.4 to 0.5 µg/ml) overnight at room temperature. Subsequently, sections were incubated for 1 hour at room temperature in biotin-SP-conjugated goat anti-rabbit IgG (1:200; Jackson ImmunoResearch) followed by Cy-3 conjugated streptavidin for 1 hour at room temperature (1:250). For the second label, sections were incubated with anti-PHF-1 (1:1,000) followed by 1-hour incubations in succession with biotin-SP-conjugated goat anti-mouse IgG and Cy-2-conjugated streptavidin (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. Channel 1 was used to acquire Cy2 fluorescence and channel 2 was used to collect Cy-3 immunofluorescence. A z-series at 1-µm intervals was captured to determine the spatial co-localization characteristics of fodrin CCP and NFTs staining within individual neurons.
Quantification
The number of PHF-1, and fodrin CCP-positive-only neurons were
counted using a x10 objective while excluding double-labeled
structures in five regions of double-immunostained sections: area CA1,
hilus, and subiculum of the hippocampus and the superficial and deep
layers of entorhinal cortex. Raw counts for each region and for each
immunostain were summed and analyzed using SPSS for Windows
(SPSS, Chicago, IL) with an 
of 0.05 followed by calculation of
either a Pearson or Spearman rank correlation coefficient. Similarly, a
multiple linear regression analysis was used to determine the
relationship between fodrin CCP, dementia severity (Mini Mental State
Examination scores), postmortem interval, and age at death for total
counts.
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Results |
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The goal of the present study was to develop a neuron selective
marker for apoptosis. Because caspases are only activated during
apoptosis, we took the approach of designing a cleavage site-directed
antibody against caspase-mediated cleavage products of fodrin. Our
analysis of the amino acid sequence for the -subunit of fodrin
yielded a number of potential caspase cleavage sites. As an initial
approach, we chose the six-amino acid sequence, SVEALI, to immunize
rabbits using the general guidelines established for calpain cleavage
sites in proteins.28
The rationale for picking this
sequence was based on the fact that this site represents the newly
generated N-terminal neoepitope generated after active caspase cleavage
and not the C-terminal fragment containing the caspase consensus
recognition site. Thus, this approach reduces the potential of an
antibody to cross-react with other caspase cleavage fragments. In
addition, this site, when cleaved by caspases, would give predicted
CCPs of 120 and 55 kd, which are the major CCPs of fodrin cleaved by
caspase-3 that have been characterized in the
literature.20-22
The initial screening of the antibodies was performed by enzyme-linked
immunosorbent assay using the MAP peptides adsorbed to the
enzyme-linked immunosorbent assay plate. Once a sufficient titer of
antibody was obtained, the antibody was purified using a sulfolink
column (Pierce) coupled with the peptide SVEALIC. Purified antibody
showed strong immunoreactivity at dilutions up to 1:4,000 in
enzyme-linked immunosorbent assays (data not shown). To validate this
antibody as a specific probe for apoptosis, a series of in
vitro experiments was undertaken, beginning with testing the
ability of this antibody to recognize CCPs of fodrin after digestion
with caspase-3 in a cell-free system. Purified fodrin, prepared as
previously described,29
or rat cortical neuron extracts
were incubated with or without caspase-3 for 24 hours at room
temperature. As shown in Figure 1A
, the
CCP antibody recognized two primary CCPs of fodrin corresponding to
molecular weights of 120 and 55 kd, but did not recognize full-length
fodrin, which runs at 240 kd, thus illustrating the specificity of the
fodrin CCP antibody for caspase-3 cleaved fragments. As shown in Figure 1B
, purified fodrin when probed with a full-length anti-fodrin antibody
gave two bands corresponding to 240 and 150 kd, respectively. The
150-kd band most likely represents a breakdown product of fodrin as a
result of calpain activation during the purification process. The
fodrin CCP antibody did not label this band either in the control or
after digestion with caspase-3 (Figure 2A)
. Figure 1B
also shows the numerous
fragments generated, including the 120- and 55-kd fragments, after
digestion of purified fodrin with caspase-3. This result is in
accordance with the multiple predicted caspase cleavage sites in
fodrin.
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When rat cortical neuronal extracts were digested with caspase-3,
major bands corresponding to 120 and 55 kd were labeled by fodrin CCP,
whereas no band corresponding to full-length fodrin (240 kd) was
observed (Figure 1A)
. Although the antibody recognized the major 55-
and 120-kd CCPs, other caspase-3 cleavage products of fodrin were also
labeled. These results suggest that fodrin CCP identifies the major
120- and 55-kd CCPs of fodrin after caspase-3 cleavage that have been
characterized in the literature, and therefore, this antibody may serve
as a useful apoptotic marker for certain neurodegenerative diseases.
To further validate fodrin CCP as an apoptotic marker, a model system
of apoptosis consisting of human neuroblastoma SH-SY5Y cells was used.
Treatment of SH-SY5Y cells with the apoptotic insult, staurosporine,
resulted in the generation of a 120-kd band after immunoblotting with
fodrin CCP. This band was completely abolished if SH-SY5Y cells were
pretreated with the caspase inhibitor, Z-VAD (Figure 1C)
.
Fodrin is not only a target for certain caspases, but is also a
sensitive target for calpain.30
In contrast to the 120-
and 55-kd cleavage products produced by caspase-mediated cleavage,
calpain-mediated cleavage of fodrin produces a predominant 150-kd
fragment.31
To rule out the possibility that fodrin CCP
may recognize calpain cleavage products of fodrin, primary cultured
neurons were treated with the calcium ionophore, A23187 to induce
necrosis and activate calpain. After treatment, Western blot analysis
was performed and neuronal extracts were probed with fodrin CCP. As
shown in Figure 1E
(i), fodrin CCP did not recognize a 150-kd fragment,
or any other fragments, after treatment of neurons with A23187 (lane
marked A23187). To ensure that the 150-kd calpain cleavage product was
generated under these experimental conditions, we probed the same
neuronal extracts with a site-directed antibody that specifically
recognizes the 150-kd calpain cleavage product of fodrin but not
full-length fodrin.28
Accordingly, Figure 1E
(ii) shows a
significant increase in the 150-kd band recognized by this antibody
after treatment of neurons with A23187. Thus, taken together, these
results demonstrate that fodrin CCP recognizes cleavage products
generated after the activation of apoptotic, but not calpain-mediated
or necrotic, pathways.
Experiments were also undertaken to characterize the fodrin CCP
antibody by immunocytochemistry using rat cortical neurons as our model
apoptotic system. In this case, we used the lectin toxin, Con A, which
has previously been shown to be an effective apoptotic insult in this
model system.25
As previously demonstrated, treatment of
neurons with Con A caused a majority of the neurons to show
morphological characteristics of apoptosis including neurite
degeneration, nuclear condensation, and internucleosomal DNA
cleavage25
(data not shown). To examine fodrin CCP
antibody staining in situ, cortical neurons were treated
with Con A, fixed in 4% paraformaldehyde, and immunolabeled with
fodrin CCP. Although very little immunoreactivity was observed in
control cells, extensive punctate process and perikaryal labeling was
observed in Con A-treated neurons (Figure 2, A and B)
. The pronounced
neurite staining seen after treatment with Con A is consistent with
reports demonstrating that fodrin is localized in both the neuronal
processes as well as in neuronal cell bodies.32
This
neurite staining was examined further by low-field magnification.
Extensive neurite staining was observed after treatment of cortical
neurons with Con A (Figure 2C)
. No staining was observed in untreated
neurons or neurons treated with Z-VAD only (data not shown).
Importantly, treatment with the caspase inhibitor Z-VAD under identical
experimental conditions primarily abolished fodrin CCP neurite labeling
(Figure 2D)
, indicating the involvement of caspases in this
neurodegenerative process. Although these data support a role for
caspase activation in fodrin cleavage, it is possible that one or more
downstream proteases may also result in fodrin cleavage, because Z-VAD
has also been shown to block a variety of cysteine proteases including
members of the cathepsin family at concentrations used to inhibit
caspase activity in cell cultures.33
Taken together, these
results support the conclusion that fodrin CCP recognizes CCPs of
fodrin after cleavage by caspases, and therefore, may be a useful
marker for following the activation of apoptotic pathways in certain
neurodegenerative diseases.
Fodrin CCP as a Marker for Apoptosis in AD
We next examined whether this novel antibody probe could provide
insights into a possible relationship between the activation of
neuronal apoptotic pathways and NFTs in the AD brain. Tissue sections
from the hippocampus of a severe AD case immunostained with fodrin CCP
showed extensive labeling of neurons throughout the hippocampus (Figure 3, B and D)
. No staining was observed
when preimmune serum was used in place of the primary antibody,
illustrating the specificity of the fodrin CCP antibody (Figure 3C)
. In
addition, staining with fodrin CCP was prevented after preabsorption
with free peptide (data not shown). Moreover, no staining was observed
in aged-matched nondemented controls without neuropathology after
incubation of hippocampal sections with anti-fodrin CCP (Figure 3A)
.
These results were confirmed after Western blot analysis using brain
extracts from representative control and AD cases. As shown in Figure 1D
, fodrin CCP recognized a predominant band at 55 kd in two
representative AD cases that was absent in controls. Also note that
full-length fodrin (240 kd) was not recognized by the anti-fodrin CCP
providing additional evidence of the specificity for caspase-cleaved
fragments. In some instances, we also were able to detect a faint band
at 120 kd in extracts from severe AD (data not shown). Evidently, the
55-kd band is perhaps the most stable cleavage product of fodrin
in vivo. Therefore, results from both immunohistochemistry
and Western blot analysis support the conclusion that activation of
apoptotic pathways occurs in a subpopulation of neurons in the AD brain
but not in normal aged-matched controls.
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, shows the low- and high-magnification characteristics,
respectively, of fodrin CCP staining in the hippocampus of a
representative AD case. Immunolabeling of neurons was not restricted to
the hippocampus: significant neuronal labeling was also observed in the
subiculum, entorhinal cortex, and frontal cortex of AD. To assess
whether the number of fodrin CCP-positive neurons was greater in the AD
cases (mean = 134.2, SEM = ±25.1, n = 5)
than in nondemented controls (both mild and no pathology, mean =
2.0, SEM = ±1.29, n = 7), an independent
t-test was used to indicate that there were significantly
more fodrin CCP-positive neurons in AD cases (t(10) = 6.34,
P < 0.0004). No significant correlation with
postmortem interval was found. In addition to neuronal cell soma being
labeled, numerous neuropil threads and processes of various lengths
were fodrin CCP-positive in the hippocampus (data not shown), including
areas with extensive plaque formation (Figure 3E
, arrowheads). It is
noteworthy that the antibody against fodrin CCPs was sensitive to
tissue fixation; more extensive neuronal immunolabeling was observed in
4% paraformaldehyde-fixed than 10% formalin-fixed tissues. Relationship between Caspase Activation and NFT Formation in AD
Figure 3F
illustrates a high magnification image of typical fodrin
CCP staining in the hippocampus from a severe AD case. Of interest was
that the appearance of neurons labeled with fodrin CCP was similar to
that of NFTs (Figure 3F
, arrowhead). To examine a possible relationship
between NFT-bearing neurons and caspase activation, double-labeling
experiments were undertaken using anti-fodrin CCP and AT8/PHF-1 as
markers for NFT formation. As shown in Figure 4, A and B
, a subset of neurons was
fodrin CCP-positive and could be clearly differentiated from
NFT-containing neurons. In addition, co-localization of PHF-1 and
fodrin CCP was evident within individual neurons (Figure 4B
, arrow).
Figure 4, C and D
, provides further evidence that not all fodrin
CCP-positive neurons contain NFT pathology (arrowheads). Taken
together, these results suggest that caspase activation may occur in
NFT-bearing neurons.
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shows a single pyramidal
neuron from a severe AD case with co-localization of both fodrin CCP
(red) and PHF-1 (yellow). A series of scans were taken at 1-µm
intervals and clearly demonstrates that fodrin CCP labeling is
primarily localized to or near the plasma membrane whereas that of
PHF-1 is cytoplasmic. This profile is predicted based on the
localization of each structure in neurons: fodrin is a
membrane-associated protein, whereas NFTs are predominantly localized
intracellularly in association with microtubules.
To examine a possible association between caspase activation and NFT
pathology, a statistical analysis was performed by counting neurons
with positive immunostaining for PHF-1 and fodrin CCP after
double-labeling experiments (Table 1)
. The number of neurons with NFT
(r = 0.801, n = 12,
P < 0.002), the number of neurons with fodrin CCP
(r = 0.935, n = 12,
P < 0.0001), and the number of neurons where these two
markers were co-localized (r = 0.879,
n = 12, P < 0.001) were significantly
associated with Braak and Braak stage. Moreover, as the extent of NFT
formation (number of neurons with NFT) increased, there was a
significant corresponding increase in the number of neurons positive
for fodrin CCP (r = 0.840, n =
12, P < 0.001). The number of neurons exhibiting both
fodrin CCP and PHF-1 immunolabeling in AD cases (mean = 81.4,
SEM = ±23.4, n = 5) was significantly higher than
in the control cases (mean = 1.7, SEM =±1.0, n =
7) [t(10) = 4.097, P < 0.002]. In addition, a
multiple linear regression analysis using a stepwise procedure that
included extent of NFT formation, postmortem interval, and disease
severity (Mini Mental State Examination) indicates that the best
predictor of the extent of fodrin CCP neuronal labeling was the extent
of NFT formation [F(1,7) = 9.941, P <
0.022]. Therefore, there does seem to be a significant association
between NFT formation and caspase activation in AD.
In the next set of experiments, we assessed the temporal relationship
between NFT formation and caspase activation in hippocampal sections
from seven cases of AD with increasing dementia severity as determined
by Mini Mental State Examination scores (ranging from 0 to 30). NFT
formation, identified by using anti-PHF-1 immunostaining (blue), was
evident in mild to severe cases of AD in a greater frequency than that
of fodrin CCP (brown) staining (Figure 4, H and I)
. In contrast,
although the number of fodrin CCP-positive neurons was considerable in
more severe cases of AD (Figure 4J)
, staining was absent in nondemented
control cases and infrequent in nondemented mild pathology cases
(Figure 4H)
, and extensive in moderate AD cases (Figure 4I
and Table 1
). These results suggest that NFT formation may precede the activation
of neuronal apoptotic pathways in a subpopulation of neurons in AD.
To confirm and extend these findings, we also explored whether the
generation of fodrin CCP was dependent on NFT formation by studying the
canine model of brain aging. In the canine model, animals show
extensive Aß deposition as a function of age, but do not exhibit any
NFT pathology.34,35
Figure 4, F and G
, illustrates the
results of experiments in an aged canine. Numerous fodrin CCP-positive
neurons were present in the prefrontal cortex of an aged canine that
was not present in young dogs. Deep-layer pyramidal neurons were fodrin
CCP-positive in the prefrontal cortex sections (Figure 4F)
, which was
completely devoid of NFTs (Figure 4G)
. These results suggest that PHF
formation may be one, but not the only factor, involved in the
activation of an apoptotic program in AD brain. In addition, postmortem
intervals were no longer than 30 minutes and allowed us to further rule
out the possibility of confounding postmortem artifacts.
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Discussion |
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-subunit of fodrin has been found to be a
substrate for the caspase family of proteases.20,22
Caspase cleavage of critical cellular proteins is believed to be
responsible for the morphological and functional changes that occur
when cells undergo apoptosis.36
Fodrin is one of the first
substrates to be cleaved by caspases during the initiation phase of
apoptosis.37,38
In addition, accumulation of abnormal
deposits of fodrin in neurons in AD31,39
and an
increase in fodrin proteolysis in fibroblasts from aged and AD donors
has been reported,40
indicating that there is a widespread
alteration in the proteolytic processing of this protein that occurs
with aging. Because fodrin caspase cleavage products (CCPs) accumulate
as stable intracellular deposits, they represent death products with
novel antigenic epitopes that provide a signature of the contribution
of caspases to neurodegeneration. In this study, we designed a
site-directed antibody based on the unique antigenic epitopes that are
generated by caspase cleavage of fodrin. This strategy has been
successfully used for the cytoskeleton protein, actin, to demonstrate
apoptosis-like mechanisms in plaque-associated neurons and microglia in
AD.7 Our antibody, which we term fodrin CCP, specifically recognized the major 120- and 55-kd caspase cleavage products of fodrin characterized in the literature, but did not recognize full-length fodrin. Detailed characterization of this antibody in vitro suggested that this antibody may provide a diagnostic tool to better assess the contribution of apoptotic mechanisms (caspase activation and accumulation of cleavage products) in neurodegenerative diseases. Fodrin CCP was present within numerous neurons throughout the hippocampus and entorhinal cortex in AD brain. Neuronal labeling by fodrin CCP supports the conclusion that widespread activation of neuronal apoptotic-like mechanisms occurs in AD, and that this is likely to be a significant cell death pathway active during disease progression.
Fodrin is a major constituent of the cytoskeleton of neurons19,41 and has been implicated in transport of synaptic vesicles and organelles within the axon,42 and coupling membrane-spanning cell surface proteins to cytoplasmic elements.43 Therefore, the cleavage of fodrin by caspase would be expected to have dire consequences for the neuron, disrupting these important functions of fodrin as well as altering cell morphology. Altering cell morphology, in turn, may either activate a death signal or disrupt the survival signal necessary to suppress the intrinsic cell death signal.44
Although unequivocal evidence has not been presented in the current
study demonstrating that caspase-3 is the caspase responsible for the
cleavage of fodrin-producing cleavage products that are recognized by
the fodrin CCP antibody, we hypothesize that this is indeed the
effector caspase involved. First, digestion of fodrin by caspase-3
generated cleavage products that were strongly immunoreactive with the
fodrin CCP antibody. Second, a recent study has demonstrated that
caspase-3 is required for the cleavage of fodrin but dispensable for
the cleavage of other substrates.22
Finally, in a study by
Wang et al20
the cleavage of fodrin by caspase-3 was
examined in detail. By using a series of recombinant GST-fodrin fusion
peptides, they were able to map the consensus caspase cleavage site
responsible for producing an 50- and 120-kd products they observed
after digestion with caspase-3. They determined this sequence to be
SVEALI, the sequence we chose to immunize rabbits to generate the
fodrin CCP antibody. Thus, the results of the current study suggest
that caspase-3 is the effector caspase responsible for the generation
of cleavage products immunolabeled with fodrin CCP antibody in AD.
Consistent with this is other evidence for caspase activation in AD
detected by an antibody against amyloid precursor protein fragments
cleaved predominantly by caspase-3.5
Therefore,
pharmacological blockade of caspase-3 may be an effective therapeutic
strategy for the treatment of AD.
Interestingly, the majority of fodrin CCP-positive neurons were closely associated with NFT formation. Furthermore, as the extent of NFT formation increased, there was a corresponding increase in fodrin CCP immunolabeling (r = 0.840). Thus, there does seem to be a significant correlation between NFT formation and the activation of neuronal apoptotic mechanisms associated with AD. In addition, a multiple regression analysis indicates that NFT formation is the best predictor of the presence of fodrin CCP. The results of the current study contrasts with other studies by Stadelmann et al15 and Selznick et al.14 These authors did not find an association between activated caspase-3 with NFT but rather with granulovacuolar degeneration.14,15 One possible difference in these studies and the current study is the use of thicker, free-floating sections as compared with paraffin-embedded sections. Further, we hypothesize that fodrin-CCP serves as a marker that is able to accumulate over a period of time after cleavage by caspase-3.
It should be emphasized that even though we have demonstrated that NFT formation and fodrin CCP are frequently present within the same neurons and are correlated, this does not imply that they are causally interrelated events. Indeed, we were also able to detect neurons that labeled only with fodrin CCP or with NFT. These may, therefore, represent two independent processes. However, our results do not rule out the possibility of a causal relationship.
To determine a possible temporal relationship between NFTs and
accumulation of caspase cleavage products, we analyzed AD cases with a
range of dementia severity based on Mini Mental State Examination
scores (Table 1)
. Double-labeling experiments using fodrin CCP and
PHF-1 revealed an overall pattern for NFT pathology preceding the
accumulation of caspase-mediated break down products of fodrin. These
results suggest an accumulation of caspase breakdown products
throughout time. However, whether activation of caspases itself
precedes or follows NFT formation has yet to be determined. This is a
difficult question to address using the current sample of cases because
the assumption cannot be made that mild pathology controls will
transition to AD. Other model systems, in which NFT pathology
accumulates in an age-dependent manner, such as the development of AD
pathology in individuals with Down syndrome will help to clarify this
issue.
To further test the role of NFT formation in promoting pathways associated with apoptosis and also to rule out confounding postmortem artifacts, we used tissues obtained from aged canines. Aged canines develop extensive Aß with increasing age that is associated with cognitive decline.45,46 On the other hand, aged canines do not develop NFTs.34,35 Thus, if NFT formation was exclusively responsible for apoptotic cell death we predicted that fodrin CCP would not be present in the brains of aged canines. However, we found that subpopulations of neurons in the prefrontal cortex of aged canines were immunopositive for fodrin CCP suggesting that NFT may serve as one stimulus, but is not the only stimulus, promoting the activation of apoptotic pathways.
An interesting aspect to the current study is the extensive accumulation of cleaved fodrin in a large number of neurons. This suggests that caspase activation in AD neurons may not immediately lead to cell death. In contrast, our evidence seems to indicate a slow, apoptotic-like degenerative process that is profoundly different from the rather rapid, classical apoptotic pathway. One interpretation of the data are that neurons implement a number of survival mechanisms, such as the up-regulation of bcl-2, to prevent engaging the full apoptotic program.47,48 However, consideration should be given to the fact that postmortem analyses reflect a single time point and thus, provide limited information regarding the rate of accumulation of specific proteins.
In conclusion, we have developed a caspase cleavage site-directed antibody to fodrin and have demonstrated widespread neuronal activation of apoptotic-like pathways in AD. This antibody should be useful for investigating in more detail the temporal and spatial relationship between caspase activation and other events associated with AD including Aß deposition. The accumulation of cleavage products mediated by caspases and the commitment to an apoptotic program may be a point of convergence for a number of stimuli, such as NFT or Aß, leading to neurodegeneration. A recent report in transgenic mice expressing mutant human SOD1, which serve as a model of amyotrophic lateral sclerosis, shows that inhibiting caspase activation by ZVAD-fmk delays disease onset and mortality.49 This study suggests that preventing caspase activation, a downstream event in an apoptotic program, regardless of the stimuli promoting apoptosis, may be one aspect of neurodegeneration amenable to therapeutic intervention. These are promising leads into the evaluation of new therapeutics for the treatment of AD.
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Acknowledgements |
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Footnotes |
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Supported in part by National Institute on Aging grants 5T32AG00096, 5R01AG13007, and AG12694.
T. T. R. and E. H. contributed equally to this work.
Present address of T. T. R.: Department of Biology, Science/Nursing Building, Room 228, Boise State University, Boise, Idaho 83725.
Accepted for publication September 14, 2000.
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