Accumulation of caspase cleaved amyloid precursor protein represents an early neurodegenerative event in aging and in Alzheimer's disease

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Neurobiology of Disease 14 (2003) 391– 403

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Accumulation of caspase cleaved amyloid precursor protein represents an early neurodegenerative event in aging and in Alzheimer’s disease Ming Zhao,1 Joseph Su,1 Elizabeth Head, and Carl W. Cotman* Institute for Brain Aging and Dementia, University of California Irvine, Irvine, CA 92697-4540, USA Received 11 October 2002; revised 26 June 2003; accepted 31 July 2003

Abstract The activation of caspase-3 and possibly other caspases during apoptosis may lead to the cleavage of the amyloid precursor protein (APP) and subsequent accumulation of APP cleavage products (cAPP). We examined the association between activated caspase-3 and cAPP in human brain by qualitative and quantitative analysis of in situ immunohistochemistry and Western blots. Frontal cortex and hippocampal tissue from age-matched control and Alzheimer’s brains (AD) was used. Both activated caspase-3 and cAPP are increased in AD [Braak and Braak (BB) stage IV–VI] compared to aged control (BB stage 0 –1) and transitional (BB stage II–III) cases in the hippocampal and frontal cortex. Caspase-3 activation and the accumulation of APP cleavage fragments appear to either parallel or precede neurofibrillary tangle formation. These findings raise the possibility that the activation of caspase-3 and cleavage of APP may be involved with neuronal degeneration and that pathways characteristic of apoptosis are activated in AD. © 2003 Elsevier Inc. All rights reserved. Keywords: Alzheimer’s disease (AD); Amyloid precursor protein (APP); Cleavage of APP (cAPP); Caspase-3; Tau protein

Introduction Alzheimer’s disease (AD) is associated with progressive cognitive decline and the accumulation of senile plaque (SP) and neurofibrillary tangle (NFT) neuropathology (Khachaturian, 1985; Mirra et al., 1991). In addition, the brains of individuals with AD develop significant neuron and synapse loss (Terry et al., 1991; West et al., 1994). It is possible that these latter two features account for the clinical signs of AD (Goldman et al., 2001), but the mechanisms underlying cell loss and dysfunction are as yet unclear. There is evidence to suggest that A␤ plays a significant role in the development of AD (LaFerla et al., 1995; Yankner, 1996; Cotman, 1998; Selkoe, 1998). Amyloid ␤-protein (A␤), a 40- to 43-amino acid peptide, is derived from ␤- and ␦-secretase-mediated cleavage of the longer amyloid precursor protein (APP). However, the A␤ fragment may not be the only contributor to neuronal and * Corresponding author. Fax: ⫹1-949-824-2071. E-mail address: [email protected] (C.W. Cotman). 1 These authors contributed equally to the manuscript. 0969-9961/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2003.07.006

synaptic loss in AD. Enzymes that are activated during apoptosis, including caspase-3, 6, 8, and 9, can produce several additional fragments of APP (LeBlanc et al., 1999; Lu et al., 2000). The resulting C-terminal fragments also have proapoptotic activity (Gervais et al., 1999; Lu et al., 2000). The activation of caspases is consistent with evidence of DNA fragmentation, a feature of apoptotic cell death, in AD brain (Smale et al., 1995; Cotman and Su, 1996; Su et al., 1997a). There is also evidence for a role of caspase-3 in neuronal activation and in APP processing and subsequent production of amyloidogenic fragments (Gervais et al., 1999; Kermer et al., 1999; Uetsuki et al., 1999; Lu et al., 2000). Of the human caspases tested, caspase-3 was the most efficient enzyme to cleave APP, although minor cleavage also occurred with caspase-6 and 8 (LeBlanc et al., 1999; Lu et al., 2000). Caspase-3-mediated proteolysis of APP has also been observed in hippocampal neurons in vivo following acute excitotoxic or ischemic brain injury (Gervais et al., 1999; Kermer et al., 1999; Uetsuki et al., 1999). Using an in vitro cleavage assay, caspase-3, 6, and 8 were capable of cleaving APP, and in particular, caspase-3 cleaved at Asp664 to Glu in APP695

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Table 1 Patient demographics (age and PMD in mean ⫾ SE)a Group

N

Age

Sex (no. of cases)

PMD

APOE (no. of cases)

MMSE

Braak

AC TrC AD

5 5 7

74.8 ⫾ 4.1 75.6 ⫾ 1.6 75.3 ⫾ 2.7

F(1)/M(4) F(2)/M(3) F(3)/M(4)

3.9 ⫾ 1.2 3.9 ⫾ 0.8 2.1 ⫾ 0.3

3/3(3); 3/4(1) 3/3(3); 3/4(1) 3/3(5); 3/4(2)

ⱖ30 20–29 ⬍20

0–1 II–III IV–VI

a N, number of cases; PMD, postmortem delay; MMSE, mini-mental state exam; APOE, genotype of apolipoprotein E; Braak, and Braak stage; AC, aged control; AD, Alzheimer’s disease; TrC, transitional case.

(Barnes et al., 1998; Gervais et al., 1999; LeBlanc et al., 1999; Pellegrini et al., 1999; Weidemann et al., 1999; Lu et al., 2000). The presence of both caspase-cleaved APP fragments and activated caspases in the brains of AD patients indicates that this process may also occur in vivo (Gervais et al., 1999; Lu et al., 2000). Since caspase-mediated cleavage of APP into neurotoxic fragments may be an important mechanism underlying neuron dysfunction and death in AD, it is critical to investigate the involvement of specific activated caspases in aging and in AD. The purpose of the current study was to determine the distribution and extent of activated caspase-3 and the accumulation of caspase-mediated APP cleavage fragments in normal aging and in Alzheimer’s disease. To address the issue of whether caspase cleavage of APP occurs prior to or in parallel to neurodegeneration in AD, we compared AD cases with a range of disease severity [based upon Braak and Braak (1991) staging] to nondemented age-matched control cases with little or no neuropathology. Understanding these processes may provide insight into future directions for developing therapeutic strategies based on preventing proteolytic effects of caspases on APP in age-dependent neurodegeneration.

Materials and methods Human tissue Postmortem tissue for immunohistochemistry and Western blot experiments was obtained from the Institute for Brain Aging and Dementia Tissue Repository. AD cases were neuropathologically confirmed based upon CERAD criteria (Khachaturian, 1985; Mirra et al., 1991; The National Institute on Aging, 1997). All individuals were placed into one of three groups: (1) moderate to severe AD neuropathology (AD, n ⫽ 7), (2) individuals with mild NFT and SP neuropathology but at insufficient levels to be diagnosed with AD (TrC, n ⫽ 5), and (3) aged control brains (AC, n ⫽ 5). Disease severity was based on the Braak and Braak NFT staging in the hippocampus, entorhinal, and frontal cortex (Braak et al., 1991) and is listed in Table 1. The groups were matched for age, sex, postmortem delay (PMD), and genotype of apolipoprotein E as closely as

possible. The hippocampus with adjacent entorhinal cortex and midfrontal gyrus were used in this study. The brain samples were fixed in 10% formalin in 0.1 M Sorensen’s buffer, pH 7.3, for 48 h, and subsequently stored in 0.1 M PBS (0.02% sodium azide) at 4°C. Fifty-micrometer-thick free-floating sections were cut on a Vibratome and collected in PBS, pH 7.4. Antibodies Two polyclonal rabbit anti-pro- and active forms of caspase-3 were used in this study. Rabbit anti-caspase-3 (CM1) recognizes a 13-amino acid peptide sequence from the carboxyl terminus of the p18 subunit of activated caspase-3 (IDUN Pharmaceuticals, La Jolla, CA). CM1 does not recognize either the uncleaved p32 procaspase zymogen or the p12 subunit of cleaved caspase-3 (Namura et al., 1998; Srinivasan et al., 1998). Another rabbit antibody was used to recognize both 32-kDa unprocessed (procaspase) and 17-kDa subunit of active caspase-3 (65906E, Pharmingen, San Diego, CA) (Patel et al., 1996; Keane et al., 1997). A neo epitope rabbit antibody was used to identify Cterminal cleavage of APP (cAPP) (Merk Frosst Centre, Quebec, Canada). cAPP was raised against a synthetic peptide corresponding to the nouveau C-terminus of ⌬C-APP ([KLH]-CHGVVEVD), then purified by immunoabsorption to the same peptide corresponding to intact APP (HHGVVEVDAAVTPE). This antibody was confirmed to be highly specific for the caspase-generated neo epitope in APP (Gervais et al., 1999). A mouse anti-human A␤ antibody, 6E10 (Senetek PLC, Maryland Heights, MO), and a mouse anti-APP (22C11, Chemicon International, Inc., Temecula, CA) antibody were used to identify SPs and wildtype APP, respectively. The epitopes recognized by cAPP, 22C11, and A␤ are illustrated in Fig. 1A. A mouse antisynaptophysin (27G12, Novocastra, UK) was used to identify terminal boutons of neurites. To identify the immunoreactive cell types and accurately estimate relative labeling index in the brain, the neuronspecific monoclonal antibody SMI33 was used to label neurofilaments H and M (Sterberger Monoclonals, Lutherville, MD). Double labeling was further used to investigate the possible relationship between active caspase-3, cAPP, or

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Fig. 1. Comparison of cAPP and 22C11 immunostaining. (A) A schematic illustrating the possible locations of caspase-3 cleavage sites in the APP695 sequence. The two potential sites of caspase-3 proteolysis are indicated on the bottom of the figure with the number referring to the Asp residue corresponding to P1. Recognition sites of cAPP and 22C11 are also provided. The pattern of cAPP immunostaining in the subiculum (B) and entorhinal (D) region from AD brain is distinct from full-length APP immunostaining with 22C11 in an adjacent section (C and E, respectively). Note that full-length APP immunoreactivity is primarily observed in plaque-associated dystrophic neurites (C and E, arrowheads) and cAPP was found within cell bodies (B, arrows), proximal neurites (D, arrows), and diffusely associated with plaques (D, arrowhead). (F) Immunoblots with cAPP show two cleaved APP fragment at 80 and 50 kDa from the AC (first 3 lanes), TrC (middle 3 lanes), and AD brains (last 3 lanes). Note the increasing levels of the 50-kDa APP fragments in AD and TrC. (G) Immune blots with 22C11 reveal an 80-kDa cleaved APP band but fail to label the 50-kDa fragment from all the three groups. Thus, the shorter 50-kDa fragment is not recognized by the N-terminal-specific antibody 22C11. Magnification, 100⫻.

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Table 2 Antibodies Antigen SMI33 AT8 6E10 22CII cAPP CPP32 CM Synaptophysin a b

Applicationa

Neurofilaments, H&M PHF-1/tau, ser-202 1–17 amino acid of A␤ N-terminus of pre-A4 C-terminus of APP pro- & active caspase-3 p18 subunit of caspase-3 C-terminal of synaptophysin

IHC IHC IHC IHC&WB IHC&WB WB IHC&WB IHC

Dilutionb and source 1:10,000 Sterberger Monoclonals, Lutherville, MD 1:20,000 Innogenetics, Belgium 1:3,000 Senetek PLC, Maryland Heights, MO 1:50 Chemicon International, Inc., Temecula, CA 1:3,000 Merk Frosst, Quebec, Canada 1:2,000 Pharmingen, San Diego, CA 1:800 IDUNPharmaceuticals, La Jolla, CA 1:100 Novocastra, UK

IHC, immunohistochemistry; WB, Western blot. The dilution listed in the table was optimal for immunohistochemistry; a doubled higher concentration was used in Western blot.

NFTs within neurons. Antibody dilutions optimized for and employed in this study are listed in Table 2. Immunohistochemistry For immunohistochemistry, the tissue sections were treated for 20 min with 1.0% H2O2 to inactivate endogenous peroxidases. For cAPP, A␤, and NFTs immunostaining, the sections were additionally treated with 50% formic acid for 5 min. This treatment has been shown to improve detection of both neurofibrillary and A␤ epitopes (Wang and Lavsson, 1985). Sections were incubated with primary antibodies overnight at room temperature, rinsed, and incubated with biotinylated secondary antibody and avidin-biotin complex for 1 h, respectively (Vector Labs, Burlingame CA). Immunoreactivity was visualized by using diaminobenzidine (DAB) for a brown or SG for a blue-gray reaction product (Vector Labs). In double-labeling experiments, bound antibodies were detected using DAB as a chromagen for the first antigen and SG as chromagen for the second antigen. Sections incubated in parallel without primary antibody failed to develop specific staining. For the purpose of quantitative analysis, double labeling and a series of adjacent sections were used for immunohistochemical staining. All 17 cases were processed in parallel in the same batch of antibody, washing buffers, and detecting reagents, and all staining treatments were simultaneously initiated and processed for all of the selected sections. Finally, immunostained free-floating sections were mounted on vectabonded slides, air-dried overnight, dehydrated through a graded series of alcohols, and coverslipped with DePex (BDH Laboratory Supplies, Poole, England). Imaging, labeling index determination, and statistical analysis Images were captured using an Olympus BX60 microscope with UplanApo 20⫻/0.70 objective and Olympus DP-10 digital camera. The final magnification used was 500⫻ and the capture frame size was 0.3 mm2. The selected anatomical locations included entorhinal cortex, CA3/hilus,

dentate gyrus, CA1/2 field of hippocampus, and frontal cortex. Within each region, five images were captured at evenly spaced intervals to represent the entire anatomic area of interest. In entorhinal cortex, lamina principalis externa of pre-␣, ␤, and ␥, lamina dissecans, and lamina principalis interna of pri-␣, ␤, and ␥ were captured for analysis. In CA3/hilus, the capture frames were evenly distributed in the irregular oval area. In the dentate gyrus, the “C” outline of the narrow band of molecular layer, granule cell, and polymorphic layers was covered. In the sectors of Ammon’s horn 1 (CA1/CA2), the molecular, pyramidal, and polymorphic layers were captured. Final labeling indices were comprised of the 20 images from the four vulnerable anatomic locations including the hippocampal formation (entorhinal cortex, dentate gyrus, CA3/hilus, and CA1/2). To prevent a potential bias toward lower counts of neurons positive for cAPP or activated caspase-3 in severe AD cases due to neuronal loss, all counts were standardized with respect to total number of neurons counted. To do this, sections were first labeled for either CM-1, AT8, or cAPP and then with a second label using SMI-33. Counts were subsequently expressed as the mean number of labeled cells per 1000 SMI-33 immunoreactive neurons in each selected region, termed the “Labeling Index.” The number of CM1or cAPP-positive cells that were also SMI-33 positive in each digitally captured field was determined using ImagePro Plus (version 4.0, Media Cybernetic). Data were analyzed by using parametric statistics of StatView 4.5 (Abacus Concepts), with a two-way analysis of variance (ANOVA), followed by the Bonferroni t post hoc test when significant differences were observed. The experimental design of this study resulted in three groups (AC, Trc, and AD) with four different anatomical regions (entorhinal, CA1/2, CA3/hilus, dentate granule cells for a 3 ⫻ 4 factorial design. Three different markers (cAPP, CM1, and AT8) were examined for each of the groups and regions. Thus, three separate ANOVAs were computed for the group ⫻ region comparisons. Since multiple ANOVAs were computed we reduced the probability of making a type I error by decreasing the acceptable probability to 0.025 for statistical significance.

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Western blots Samples were prepared from fresh-frozen adjacent block brain tissue of the hippocampal formation with attached entorhinal cortex. Tissue blocks of 0.5 g were mechanically homogenized and collected as nuclear and cytoplasmic enriched fractions in Laemmli sample buffer. Protein assays (BCA-Pierce) were performed on the extracts and all samples were adjusted based on protein content to ensure even loading of the gels. Extracts were separated on 12% to 15% sodium dodecyl sulfate–polyacrylamide gels with 4% stacking gel and transferred to polyvinylidene difluride (PVDF) (BioRad) membrane. To block nonspecific binding the membranes were incubated in PBS containing 5% nonfat milk for 60 min at room temperature. The concentration of primary antisera (Table 2) was reduced by one-half for Western blot and was applied to the blots for 60 min at room temperature. Blots were then washed in PBS containing 0.1% Tween, followed by incubation with HRP-conjugated secondary antibody (1:5000, Vector, CA) in blocking solution for 60 min. Blots were then washed five times with PBS containing 0.1% Tween. Specific labeling was detected by enhanced chemiluminescence (ECL, Amersham) according to the manufacturer’s recommended conditions. Immunoreactivity was quantified using densitometric analysis and public domain software (NIH Image 1.62).

Results Specificity of the antibody cAPP for caspase cleavage of amyloid precursor protein (cAPP) cAPP (Merk Frosst Centre, Quebec, Canada) recognizes the C-terminus of ⌬C-APP ([KLH]-CHGVVEVD) and was purified by immunoabsorption to the same peptide to reduce cross reaction to full-length APP. This antibody was confirmed to be highly specific for the caspase-generated neo epitope in APP (Gervais et al., 1999). To compare the appearance of full-length APP with cAPP, cAPP, and 22C11 immunostaining was conducted on the same (double label) or adjacent sections (single label). Different staining patterns were observed for each antibody. cAPP immunostaining was more extensive within neurons (Fig. 1B and D) than 22C11, and the latter exhibited more robust staining for plaque-associated dystrophic neurites (Fig. 1C and E, Table 3). CAPP immunostaining was diffusely associated with senile plaques and was also observed within dystrophic neurites (Fig. 1D) but was not as intense as 22C11 neurite immunostaining (Fig. 1E). Double-labeling studies also indicated that neuronal cAPP did not colocalize with 22C11 in the majority of labeled neurons and immunoreactivity for 22C11 did not colocalize with cAPP in most of the plaqueassociated dystrophic neurites. These results strongly support minimal cross-reaction between cAPP and 22C11 an-

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tibodies in vivo and that the two antibodies can be used in situ to label APP cleavage fragments and full-length APP, respectively. cAPP Western blots revealed two bands at 80 and 50 kDa that correspond to both a single caspase-3 cleavage site at the intracellular region of APP at Asp664 and a double cleavage site at both intra- and extracellular domain of APP Asp664 and 219 amino acids (Fig. 1F). This was in contrast to the pattern observed with 22C11 Western blots that recognize full-length (not shown) and cleaved APP at the N-terminus, and the 80-kDa, but not 50-kDa fragment was observed (Fig. 1G). Expression of caspase cleavage of amyloid precursor protein (cAPP) in aged control (AC), transitional cases (TrC), and Alzheimer’s disease (AD) brains Immunoreactivity patterns for cAPP were examined in 5 aged controls (AC), 5 transitional cases (TrC), and 7 Alzheimer’s disease brains (AD) by qualitative and quantitative analysis of in situ immunohistochemistry and is summarized in Table 3. In AC and TrC brains, cAPP immunoreactivity was present in a subset of neurons in the hippocampal formation and frontal cortex, primarily in pyramidal and granular cells. Neurons positive for cAPP showed weak staining in a fine punctate pattern in the cytoplasm of soma and proximal neurites (Fig. 2A). cAPP-positive neurons were distributed in the entorhinal cortex, subiculum, granular cells of the dentate gyrus, hilus, and CA1 region adjacent to CA2. In the frontal cortex, pyramidal neurons were the dominant reactive cell type for cAPP, particularly in layers II–III. In the seven AD brains, cAPP immunoreactivity was more intense compared to AC and TrC brains (Fig. 2B). cAPP was present within a large subset of neurons in AD brain and distributed throughout all major subregions of the hippocampal formation and pyramidal layers of the frontal cortex. Although the majority of intracellular labeling for cAPP was punctate, primarily in the soma and proximal processes of the neurons, a subset of neurons also exhibited nuclear and tangle-like labeling (left and right inset in Fig. 2B). Furthermore, cAPP immunoreactivity was also detected in the granules resembling granulovacuolar degeneration in CA1 pyramidal neurons. The total labeling index for cAPP was 2.5 to 2.9 times higher in AD cases compared to TrC and AC cases (Fig. 2C). The granule cells and the hilar neurons of the dentate gyrus in AD also showed more intense cAPP immunoreactivity than that observed in AC and TrC cases. cAPP was observed around the neurons in area CA3/ hilus (Fig. 3A) and immunostaining morphologically resembled mossy fiber terminal boutons (Fig. 3B). Double immunostaining for cAPP and synaptophysin, and for cAPP and SMI-33 (neuronal marker), was used to determine whether these deposits were associated with the terminals of neurons. Double labeling confirmed that most of the cAPP

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Table 3 Immunohistochemistry of active caspase-3 and cleavage of APPa Cell type

Staining pattern

Marker

Structure

CM1

pyramidal, astrocyte, granular plaques pyramidal granular synapses neuropil

cAPP

22C11

AT8

plaques & plaqueassociated dystrophic neurites pyramidal, granular, astrocyte

Anatomic locations of the labeled cells and structures ENT

SUB

CAs

HIL

DEG

FTC

punctate & fibrillar in soma & proximal neurites; diffuse in plaques punctate in cytoplasm coarse deposits in layers synaptic structure ‘healthy’ neurites diffuse in plaque fibrillary in neurites

pre-␣ & pri-␣ layer

parvocellular layer

pyramidal layer

hilus/CA4

granular molecular layer

layer 2&3

all the layers

parvocellular layer

pyramidal layer in CA1/2

Hilus/ CA3/4

granular & molecular layer

layer 2&3

superficial & deeper layer

superficial layer & deeper layer

molecular layer

Hilus

granular & molecular layer

all the isocortex layers

fibrillar & punctate in soma & proximal/distal neurites

pri-␣, ␤, ␥ pri-␣, ␤ layer

parvocellular layer

pyramidal & molecular layer

CA3/4

all the three layers

layer 1 to 6

a Anatomic locations: ENT, entorhinal cortex; SUB, subiculum region; CAs, CA1, CA2, CA3; HIL, hilus area; DEG, dentate gyrus; FTC, frontal cortex; marker: CM1, activated caspase-3; cAPP, C-terminus of caspase associated cleavage of APP; AT8, ealy neurofibrillary tangles; 22C11, N-terminus of amyloid precursor protein.

terminals were clustered around neurons positive for SMI-33 (Fig. 3C) and cAPP was colocalized with synaptophysin in bouton structures (Fig. 3C, inset). Neurons that were surrounded by boutons labeled with both cAPP and synaptophysin were positive for SMI-33. Interestingly, SMI-33-positive neurons that were surrounded by cAPPand synaptophysin-immunoreactive products in area CA3/ hilus were themselves generally not reactive for cAPP even in AD brain. Thus, cAPP was within mossy fiber terminals that formed synapses with CA3/hilus neurons. Association between active caspase-3 and cAPP The possible association between active caspase-3 (CM1) and cAPP was studied by two methods: cAPP and activated caspase-3 (CM1) were either used in conjunction in double-label studies on the same tissue section or by using single labels on adjacent sections. A significant number of the cAPP-reactive neurons were positive for CM1 in AD brains (Fig. 4A). However, in AC and TrC brains, a subset of CM1-labeled neurons did not contain the fragment of cAPP suggesting caspase activation may occur prior to the accumulation of caspase-cleaved APP fragments. In contrast, AD brains showed a comparatively larger subset of cAPP-labeled neurons that did not show CM1 reactivity in entorhinal cortex, hippocampal formation, and frontal cortex. This suggests that the accumulation of fragments of APP may follow caspase-3 activation. The proportion of neurons with both cAPP and CM1, or cAPP alone in dou-

ble-label studies, confirmed qualitative observations. The double-labeling index in AD for CM1 and cAPP was about 2.5- to 3-fold higher compared to TrC and AC cases (Fig. 4B). To further confirm the association between the activation of caspase-3 and the accumulation of APP cleavage products, a Western blot analysis was conducted using frozen blocks of hippocampal formation. Activated caspase-3 (20 kDa fragment) was significantly increased in AD and TrC cases compared with AC cases (P ⬍ 0.025, Fig. 4B and Fig. 4C, 20 kDa). In parallel, the levels of the caspase-3 proenzyme decreased (Fig. 4C, 32 kDa fragment). The relative levels of the 80-kDa and particularly the 50-kDa fragment of cAPP also progressively increased from AC to TrC and AD cases (Figs. 4B, C). Association between cAPP and neurofibrillary tangle formation To investigate the association between the accumulation of APP fragments (cAPP) and neurofibrillary tangle degeneration (NFT), a series of double-labeling studies were conducted. Serial hippocampal and frontal cortex sections were immunostained for (1) cAPP and AT8 (Fig. 5A). cAPP immunoreactivity was detected in a large subset of tanglebearing neurons in AD brains. cAPP-positive immunoreactivity was not only seen in pretangle neurons, but was also found in later stage tangle-bearing neurons (Fig. 5A and inset). However, a significant number of neurons contained

Fig. 2. Comparison of cAPP immunoreactivity in control and AD brain. (A) Light to moderate immunoreactivity for cAPP is detected in a subset of neurons in subiculum from aged control brains (arrows), ⫻100. (B) Robust immunoreactivity for cAPP is detected in a subset of neurons of the subiculum from AD brains (arrows), ⫻100. Left inset: high magnification showing that immunoreactivity appears punctate within the cytoplasm (asterisk) and NFT-like neurons (arrows), ⫻300. Right inset: a subset of positive neurons at high magnification shows cAPP immunoreactivity within the nucleus, ⫻300. (C) Total labeling index for cAPP and double-labeling index for cAPP and activated caspase-3 (CM1) in hippocampus and frontal cortex from aged control (AC, white bars), transitional case (TrC, gray bars), and Alzheimer’s disease (AD, black bars). The total-labeling index was derived by counting a total of 1000 SMI-33-positive neurons and expressing the number that were also positive for cAPP or CM-1 as a percentage. The labeling index of cAPP in AD is significantly increased up to 2.4 and 2.9 times higher compared to TrC and AC, respectively (P ⬍ 0.025). There is no significant difference between TrC and AC groups (P ⫽ n.s.). The double-labeling index for cAPP and CM-1 in AD is significantly increased up to 2.5 and 3.0 times higher compared to TrC and AC, respectively (P ⬍ 0.025).

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5B) were also positive for AT8 (Fig. 5C) in TrC brains. The evidence indicates that cAPP is associated with early tangle formation in pre- and early AD brains but can also precede independent of or prior to tangle formation. As tangle formation develops there is a small but insignificant increase in double-labeled cells. The double-labeling index for AT8 and cAPP from hippocampal formation in AD was 1.4 to 1.7 times higher compared to TrC and AC brains, but the

Fig. 3. cAPP immunoreactivity in mossy fiber terminals of AD brain. (A) cAPP immunoreactivity is found in granular cells and in the hilus of the dentate gyrus, ⫻200. Note that the cAPP immunoreactivity found in the hilus (arrowhead) is characterized by coarse deposits that exhibit a morphology consistent with that of terminal boutons as shown at higher magnification in B. High power of (A) shows terminal boutons around the neurons (arrowhead) and boutons contiguous with fine axonal processes (arrows), ⫻400. Right inset: A higher magnification from outlined rectangle. (C) Association of cAPP and synaptic structures in AD brain. cAPP immunoractivity (blue) is observed within mossy fiber terminal boutons in the hippocampus (arrows) as demonstrated by the coarse distribution clustered around neurons (SMI-33-brown), ⫻200. Inset: cAPP-positive coarse deposits (brown) are localized within terminals identified by positive immunostaining for synaptophysin (blue), ⫻300.

only cAPP. AT8-positive neurons without cAPP immunoreactivity were rare. In addition, a subset of cAPP-positive dystrophic neurites that are associated with plaques (Fig.

Fig. 4. (A) Association between active caspase-3 (CM1) and caspasecleaved APP fragments (cAPP) in CA1 region of AD brain. Many pyramidal cells of CA1 that are cAPP positive (blue) are also positive for CM1 (brown) (arrows), ⫻300. (B) Western blot quantification of activated caspase-3 (CM-1), cleaved APP 80 kDa, and cleaved APP 50 kDa in the entorhinal cortex/hippocampus of aged control (AC, white bars), transitional case (TrC, gray bars), and Alzheimer’s disease (AD, black bars). The average relative densities from the Western blot experiments confirm that there is a rise in both cleaved APP (50 and 80 kDa) and active caspase-3 in TrC and AD cases relative to AC. The activated caspase-3 in AD and TrC is significantly higher compared to AC (P ⬍ 0.025). The cleaved APP 80-kDa fragment in AD is higher compared to TrC and AC (P ⬍ 0.025). The cleaved APP 50-kDa fragment is statistically significant among the AC, TrC, and AD cases (P ⬍ 0.025). (C) Immune blot with active caspase-3 (CM1) illustrate that there are parallel rises in both cleaved APP and caspase-3 from AC (first 3 lanes), TrC (middle 3 lanes), and from the AD cases (last 3 lanes) being most affected. The relative density of the activated caspase-3 is statistically significant in AD and TrC cases compared to AC cases (P ⬍ 0.025).

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Fig. 5. (A) Association between hyperphosphorylated tau (AT8, blue) to cAPP (brown) in CA1 region of AD brain illustrates that only a small subset of tangle-bearing neurons is cAPP positive (arrows and asterisk), ⫻200. Right inset: High magnification showing a tangle-bearing neuron (AT8, red fluorescence) colabeled with cAPP (green fluorescence), ⫻300. Left inset: High magnification showing two late-stage tangles (brown, arrow) colabeled with cAPP (blue). (B) cAPP-positive neurites in the AD brain (brown, arrow) are found in association with senile plaques in the hilus (blue), ⫻300. (C) Swollen cAPP-positive dystrophic neurites (brown) in plaques are positive for hyperphosphorylated tau (AT8, blue, arrows), ⫻200. (D) Selective vulnerabilities of cell fields within the hippocampal formation to cAPP. Along with the route of main connections in the hippocampus and the advancement of aging and AD, immunoreactivity is significantly increased in the dentate gyrus of AD cases but not in TrC or AC cases. The labeling index for cAPP in AD is higher than TrC and AC at CA3 of hilus area and CA1/2 field (P ⬍ 0.025).

differences did not reach statistical significance. Thus, it appears as if cAPP is linked to neurofibrillary tangle formation in early AD development. Evidence of sequential involvement of caspase cleavage of amyloid precursor protein (cAPP) in Alzheimer’s disease (AD) cAPP immunoreactivity is increased in the AD brain overall, but the pattern of cAPP immunoreactivity within different regions of the hippocampus and entorhinal cortex,

as a function of disease severity, may provide further insight into the temporal progression of caspase activation. The temporal sequence of caspase cleavage of APP in the AD brain was determined by qualitative and quantitative analysis of immunoreactivity from several vulnerable anatomic locations (Fig. 5D). The entorhinal cortex, one of the earliest sites to undergo tangle formation, projects to the dentate gyrus that in turn connects to area CA3 and these areas project to CA1/2. Thus, the extent of immunoreactivity for CM1 and cAPP in entorhinal cortex, dentate gyrus, hilus/ CA3, and CA1/2 may provide information regarding the

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sequential involvement of caspase activation and the accumulation of APP fragment in the course of aging and AD. The entorhinal cortex (EN) shows similar levels of immunoreactivity for cAPP and CM1 from the AC, TrC, and AD. Along with the route of main connections in the hippocampus and the advancement of aging and AD, immunoreactivity is significantly increased in the dentate gyrus of AD cases but not in TrC or AC cases, suggesting that caspase activation had not proceeded past the EN within the trisynapstic circuit in controls or TrCs. Colabeling index for cAPP and CM1 in AD is higher than TrC and AC at CA3/hilus area and CA1/2 field (P ⬍ 0.025). The fact that the doublelabeling index of CM1 and cAPP was quantitatively similar in the entorhinal cortex among the three groups suggests that all groups were equally affected and may indicate a general age effect. However, immunoreactivity for CM1 and cAPP was significantly increased in granule cells of the dentate gyrus and in the neurons of the CA3/hilus and CA1/CA2 in AD brains (Braak and Braak V and VI) compared to TrC (Braak and Braak III and IV) and AC (Braak and Braak I and II) (Fig. 5D). These results suggest a possible progressive activation of caspase-3 in the hippocampus that was most pronounced in the dentate granule cells and pyramidal neurons of areas CA3/hilus, CA1/2. These results illustrate the progressive vulnerability of the hippocampus proper to the cleavage of APP by caspase-3 or other caspases in the development of AD pathology and suggest that caspase cleavage is an early event in neuronal pathology

Discussion Caspase-3 activation and the cleavage of APP was extensive in AD brain and minimal in cases with no AD pathology. The activation of caspase-3 preceded the accumulation of cleaved APP within the AD brain. Further, caspase-3 activation and the accumulation of cAPP may occur in parallel with the development of early tangles but prior to mature tangle formation. The extent of cAPP accumulation in the hippocampus but not entorhinal cortex differentiated AD from TrC and AC. This suggests that caspase-3 activation and the accumulation of cAPP occurs in the entorhinal cortex at an earlier time than in the hippocampus proper or that entorhinal APP cleavage is age dependent. The hippocampus proper, and in particular area CA1, appears to be vulnerable to AD. Caspase-3 activation and the accumulation of cAPP may be early neurodegenerative events preceding or paralleling the formation of tangle neuropathology. APP is cleaved by caspases in vivo APP belongs to a family of integral membrane proteins that is ubiquitously distributed in many cell types. APP can be cleaved by several types of proteases, namely secretases

(␣-, ␤-, and ␦-secretase), caspases, and calpain. Cleavage of APP by ␤- and ␦-secretases gives rise to the amyloid ␤-protein, the principal constituent of senile plaques and a cytotoxic fragment involved in the pathogenesis of AD. APP may become upregulated in response to injury as elevated levels of APP and the cleavage product, A␤, was observed in motoneurons undergoing cell death resulting from trophic factor deprivation (Barnes et al., 1998). Further, a c-terminal fragment of wild-type APP695 had a proapoptotic effect after staurosporine or tamoxifen induction and the effect was enhanced by caspase expression and activation (Lu et al., 2000). The precise cleavage products of APP depend on the isoform; alternative splicing gives rise to three major APP isoforms, 695, 751, and 770 amino acids. The APP751 and APP770 isoforms are found in both neuronal and nonneuronal cells while the APP695 isoforms are predominantly found in neurons (Koo et al., 1990; Sisodia et al., 1993; Yamazaki et al., 1995). Several studies have shown that APP is a caspase substrate and caspase cleavage of APP at Asp664 generates a cytotoxic C-terminal APP fragment; caspase-3, 6, 8, and 9 were capable of cleaving APP in the brains of AD individuals (Gervais et al., 1999; Lu et al., 2000). Fig. 1A illustrates that two sites of caspase-3 proteolysis in APP were determined to be at amino acids 220 and 700 by recombinant APP analysis (Thornberry et al., 1997; Gervais et al., 1999). Due to the lack of the 56-amino acid protease inhibitor domain in neuronal APP695, these proteolytic sites correspond to Asp664 and 219 in neuronally expressed APP, respectively. For full-length APP in neurons, caspase cleavage would lead to two fragments: an N-terminal fragment of 664 amino acids and a C-terminal fragment of 31 amino acids (Lu et al., 2000). Consistent with recent reports (Barnes et al., 1998; Gervais et al., 1999; LeBlanc et al., 1999; Pellegrini et al., 1999; Weidemann et al., 1999) our data also show the presence of APP fragments consistent with caspase-3 cleavage at Asp664 and Asp219 to produce two predicted fragments at 80 and 50 kDa in the brains of AD and aged control brains. APP can also be proteolytically cleaved by caspases in the C-terminus to generate a second unrelated peptide, called C31 (Lu et al., 2000). C31 is a potent inducer of apoptosis. However, the antibody used in this study would not detect C31 because the epitope was within the N-terminal domain. An antibody specific for this shorter C31 peptide may provide information in the future. Elevation of active caspase-3 and caspase cleavage of APP are early events in aging and AD A close association was observed between the activation of caspase-3 and the accumulation of APP fragments derived from qualitative and quantitative analyses used in this study. Thus, these results in human brain provide further evidence that activated caspase-3 is involved in cleaving

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APP though other caspases may also be involved. Evidence for a role of caspase activation in neurodegeneration characteristic of AD is rapidly accumulating. An increase in APP and subsequent generation of A␤ were suggested to be early events in the development of AD (Selkoe, 1993; Yankner, 1996). The current study further supports the role of APP in the development of AD and provides evidence for a second mechanism in addition to the generation of amyloidogenic fragments, involving the production of other proapoptotic fragments. We detected activated caspase-3 and caspase-induced cleavage of APP in neurons and synaptic structures in aged and AD brains. Furthermore, subsets of neurons positive for cAPP were pre- or early tanglebearing neurons. These findings suggest that activation of caspase-3 and subsequent cleavage of APP into proapoptotic fragments are early events in the progression of neuronal pathology in AD (Rohn et al., 2001). Caspase-3 activation and the accumulation of caspasecleaved APP fragments precede mature neurofibrillary tangle formation in aging and AD A recent study reported that tau protein was also a substrate for caspase-3 cleavage (Canu et al., 1998; Fasulo et al., 2000). In a previous study, we revealed more extensive colocalization of early tau hyperphosphorylation and activated caspase-3 in both control and AD brains and that most of these double-labeled cells were early stage tangles and pretangle neurons (Su et al., 2001). These findings strongly suggest a temporal process in which activated caspase-3 as well as other caspases may be involved in early tau pathology (Cotman and Su, 1996; Canu et al., 1998). Because of the close colocalization of activated caspase-3 with pre-tangle-bearing neurons we have suggested that caspase-3 activation occurs prior to mature tangle formation in aged and AD neurons (Su et al., 2001). However, cAPP may appear in non-tangle-bearing neurons, consistent with an independent or precursor relationship to tau pathology. The presence of a caspase cleavage product of fodrin, building up in parallel with tangles and appearing either in tangle neurons or separately, supports the possibility that caspases are chronically active in AD and may operate along tangle-dependent or independent pathways (Rohn et al., 2000). Additionally, we have previously reported that PHF-1 and TUNEL labeling are related, and that DNA damage may precede tangle formation (Su et al., 1994, 1997a,b). These observations are supported by recent studies showing that caspase-3 is activated in early apoptosis, and that its activation precedes the appearance of DNA damage (Namura et al., 1998; Hartmann et al., 2000). Thus, we suggest that the neurons labeled for active caspase-3 have engaged a neurodegenerative cascade and shift their cellular balance toward a proapoptotic program, including alterations in the expression of TNF pathway death receptor proteins (Zhao and Cotman, 2001). However, the lack of evidence for DNA fragmentation in these cells suggests that

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they are injured but not actively apoptotic or undergoing terminal cell death. It is not surprising that long-lived postmitotic cells have a number of mechanisms in place to delay apoptosis. APP cleavage may contribute to both NFT and SP pathology in AD Amyloid ␤-protein (A␤) is produced from APP by ␤and ␦-secretase (LaFerla et al., 1995; Yankner, 1996; Cotman, 1998; Selkoe, 1998). Secretase-mediated proteolytic processing of APP to generate the A␤ protein is a part of normal processing (Weidemann et al., 1989; Shoji et al., 1992; Busciglio et al., 1993; Selkoe, 1994). This is consistent with reports that soluble A␤ is rapidly cleared from the central nervous system with little detrimental effect (Glabe, 2000). On the other hand, the secretases can cleave APP to yield other forms of A␤ that rapidly fibrillize and are stable, which may promote both neuronal degeneration and gliosis (Weldon et al., 1998). A second mechanism by which APP can be proteolytically cleaved is via caspase cleavage. Not surprisingly, in situ staining was abundant in the perikarya for activated caspase-3 and cAPP; however, proximal as well as distal neuronal processes and synapses were also prominently stained by the antibody, indicating transport of caspase to/or synthesis in distal sites. The presence of cAPP in neurites is consistent with APP cleavage fragments reported to be rich in crude synaptosome preparations (Mattson et al., 1998). This may indicate that caspase activation and subsequent cleavage of APP occurs in neurites and synapses. Therefore, caspase cleavage of APP and other proteins may be a mechanism underlying neuritic and synaptic abnormalities seen in AD brains (Masliah, 1998; Mattson et al., 1998). Because cAPP leaves potentially amyloidogenic fragments, this in turn may contribute to extracellular A␤ deposition, which is consistent with a diffuse association of cAPP in plaques in the current study. Thus, APP cleavage may contribute to both plaque and tangle formation and be an early index of neuronal caspase activation and degeneration.

Acknowledgment The National Institute of Aging (NIA P01 AG00538) supported this work.

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