Is DNA repair compromised in Alzheimer’s disease?

Neurobiology of Aging 24 (2003) 953–968

Is DNA repair compromised in Alzheimer’s disease? Vladislav Davydov, Lawrence A. Hansen, Deborah A. Shackelford∗ Department of Neurosciences, University of California at San Diego, La Jolla, CA 92093-0624, USA Received 4 April 2002; received in revised form 27 September 2002; accepted 11 December 2002

Abstract Mammalian cells utilize multiple mechanisms to repair DNA damage that occurs during normal cellular respiration and in response to genotoxic stress. This study sought to determine if chronic oxidative stress proposed to occur during Alzheimer’s disease alters the expression or activity of DNA double-strand break repair or base excision repair proteins. Double-strand break repair requires DNA-dependent protein kinase, composed of a catalytic subunit, DNA-PKcs, and a regulatory component, Ku. Ku DNA binding activity was reduced in extracts of postmortem AD midfrontal cortex, but was not significantly different from the age-matched controls. Decreased Ku DNA binding correlated with reduced protein levels of Ku subunits, DNA-PKcs, and poly(ADP-ribose) polymerase-1. Expression of the base excision repair enzyme Ref-1, however, was significantly increased in AD extracts compared to controls. Ku DNA binding and DNA-PK protein levels in the AD cases correlated significantly with synaptophysin immunoreactivity, which is a measure of synaptic loss, a major correlate of cognitive deficits in AD. Immunohistochemical analysis suggested that DNA-PK protein levels reflected both number of neurons and regulation of cellular expression. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Alzheimer’s disease; DNA damage; DNA repair; DNA-dependent protein kinase; Ku; Redox factor-1; Synaptophysin; Cerebral cortex

1. Introduction Alzheimer’s disease (AD) is a neurodegenerative disease characterized by a progressive decline in cognition and memory. The disease is defined pathologically by extensive neuronal cell loss, extracellular neuritic plaques (NP) composed primarily of ␤-amyloid peptide, and intracellular neurofibrillary tangles (NFT) consisting of paired helical filaments that result from aggregation of hyperphosphorylated tau protein. Cellular damage due to oxidative stress is proposed to contribute to the pathophysiology of neurodegenerative diseases such as Alzheimer’s disease as well as to the process of normal aging. The CNS is thought to be particularly susceptible to oxidative stress due to the high rate of oxygen consumption in the brain and the low level of antioxidant enzymes compared to other somatic tissues. Evidence for increased oxidative stress in AD includes modification of proteins, such as elevated carbonyl and nitrotyrosine content, peroxidation of lipids, and oxidative damage to nucleic acids (for review see [41,42,52]). The cumulative damage ∗ Corresponding author. Tel.: +1-858-534-3459/534-1570; fax: +1-858-822-4106. E-mail address: [email protected] (D.A. Shackelford).

especially to DNA is hypothesized to contribute to progressive neuronal cell loss as unrepaired DNA damage can trigger programmed cell death. Damage to nucleic acids caused by reactive oxygen species includes base modifications, single-strand breaks, and double-strand breaks if single-strand breaks are in close proximity. The predominant marker of oxidative damage is the hydroxylated nucleoside 8-hydroxy-2 -deoxyguanosine (8OHdG). A relatively high basal level of 8OHdG is detected in the brain of control and AD subjects [21,33,41,45]. There are conflicting reports as to whether 8OHdG is elevated in nuclear DNA in any AD brain region [21,33,41,45] but an increase is detected in mitochondrial DNA [45]. In addition, increased oxidative damage to RNA in neurons throughout the brain of AD subjects is observed [49]. A two-fold higher incidence of single-strand breaks and other alkali-labile DNA lesions are detected in the cerebral cortex of AD versus control brains [47]. Evidence for widespread single- and double-strand breaks in AD brains has been provided by in situ labeling methods [1,34,39,59,62,64]. The relative contribution of free radical mediated DNA cleavage, ongoing or incomplete DNA repair processes, or endonuclease cleavage as part of an apoptotic cascade to the generation of DNA strand breaks is

0197-4580/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0197-4580(02)00229-4

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not known. Whether DNA fragmentation accumulates over the course of disease or occurs within in the perimortem period is yet to be established. Mammalian cells utilize multiple mechanisms to repair ongoing DNA damage that occurs during replication and due to reactive oxygen species produced primarily by mitochondria during normal cellular respiration. These include base excision repair, nucleotide excision repair, and double-strand break repair. Evidence for decreased capacity for DNA repair in fibroblasts or lymphocytes derived from some patients with familial AD has been reported [7]. In mammalian cells, DNA double-strand breaks are repaired predominantly by non-homologous end joining (NHEJ). This process requires DNA-dependent protein kinase (DNA-PK), composed of a ∼470,000-Da catalytic subunit, DNA-PKcs, and a regulatory component, Ku antigen [60]. Ku is formed by two proteins of Mr ∼70,000 (Ku70) and 80,000 (Ku80 or Ku86) [17]. The heterodimer binds to free ends of double-stranded DNA and to single-strand nicks and gaps. DNA-PKcs is a serine/threonine kinase with homology to the phosphoinositol-3-phosphate kinase family. The importance of DNA-PK in mammalian cells, not only in DNA repair, but also in V(D)J recombination in lymphogenesis, cell survival in neurogenesis, and chromosome stability has been established by a series of somatic cell mutants and gene-targeted mutation in animals [14,18,22,24,30,60]. DNA-PK is constitutively expressed in most cell types [4,46] and is proposed to play a primary role in detecting DNA damage and signaling downstream repair or apoptotic pathways [15,73]. Acute CNS injury due to reperfusion of ischemic tissue leads to production of reactive oxygen species that cause an immediate increase in DNA damage, including oxidized bases and strand breaks [10]. Ku DNA binding is reduced and protein levels of Ku70, Ku80, DNA-PKcs decrease following severe injury from ischemia and reperfusion that causes extensive neuronal cell death and permanent neurological deficits in a rabbit spinal cord model [57]. In a model of focal cerebral ischemia in mice, a loss of Ku70 and Ku80 immunoreactivity was observed in cells in the ischemic lesion showing apoptotic features and preceded DNA fragmentation [31]. This suggested that after severe acute CNS injury, DNA damage overwhelms repair capabilities and cell death programs are initiated. The present study was undertaken to determine if chronic oxidative stress proposed to occur during AD leads to alterations in the expression or activity of DNA-PK. The DNA binding activity of Ku antigen and protein expression of the Ku subunits and DNA-PKcs were analyzed in postmortem AD and age-matched control brains. Protein levels of the repair enzymes, poly(ADP-ribose) polymerase-1 and Ref-1 (redox factor-1 also known as apurinic/apyrimidinic endonuclease), were also investigated. Correlations of DNA repair enzyme expression with cognitive function or synaptic protein immunoreactivity were analyzed.

2. Methods 2.1. Subjects Frozen human postmortem brain tissue was obtained from the Alzheimer’s Disease Research Consortium (ADRC) at the University of California, San Diego. Tissue from midfrontal cortex of 39 neuropathologically-confirmed AD cases and seven aged-matched, non-demented normal cases were analyzed (Tables 1 and 2). An additional five non-AD cases with short postmortem intervals excluded from the controls were evaluated to ensure that protein and activity levels were not significantly affected by the postmortem delay. These cases included patients with dementia but no plaques or tangles (case no. 11), no dementia but some AD pathology (case nos. 9 and 12), infarct (case no. 8), and Huntington’s disease (case no. 10). During life, most AD subjects in this study underwent one or more of the following cognitive assessments: Mini-Mental State Examination (MMSE; 36 cases), Blessed Information-Memory-Concentration Test (35 cases), and Mattis Dementia Rating Scale (DRS; 27 cases). Seven of the non-AD cases (case nos. 1, 2, 5, 6, 7, 9, and 12) were evaluated. Neocortical plaque and NFT counts from the brains, as well as Braak staging were available as part of the routine ADRC neuropathological evaluation for all AD and non-AD cases. NFT and NP were counted in ×500 and ×100 fields, respectively, to maximize tangle or plaque densities. Quantitation of synaptophysin immmunoreactivity in midfrontal cortex performed by a dot-immunobinding assay [2] was available for 29 of the 39 AD cases. 2.2. Preparation of nuclear extracts Extracts from midfrontal cortex were prepared as described previously [57]. Briefly, tissue (0.1–0.2 g) was homogenized on ice in four volumes of homogenization buffer A (10 mM HEPES, pH 7.9, 0.5 mM dithiothreitol, 10 mM KCl, 1.5 mM MgCl2 , and protease inhibitor cocktail) using a Dounce homogenizer (B-type pestle). The final concentration of protease inhibitors used in the buffers was 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM benzamidine, 0.05 mg/ml leupeptin, 0.025 mg/ml pepstatin A, and 0.05 mg/ml aprotinin. The homogenates were centrifuged at 5000 × g for 15 min at 4 ◦ C. The supernatants were removed and centrifuged again at 16,000 × g for 30 min at 4 ◦ C; the final supernatant was the cytosolic extract. The 5000×g pellet was resuspended in an equal volume of high salt buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl2 , 1 M NaCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, and protease inhibitor cocktail) and was extracted for 30 min at 4 ◦ C with continuous gentle mixing. The extracts were centrifuged at 70,000 × g for 30 min at 4 ◦ C. This supernatant was taken as the nuclear protein fraction. The remaining insoluble particulate fractions were washed with 150 ␮l of buffer A, centrifuged at 16,000 × g for 30 min, and the pellets were

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Table 1 Normal and other cases Case no.

Age (year)/sex

Normal cases 3 69/M 1 79/F 2 83/F 4 77/M 5 81/M 6 83/M 7 76/M Other cases 8 9 10 11 12

65/M 91/M 43/F 82/F 84/F

PMI (h)

Ku DNA (EMSA)

Ku80

DNA-PKcs

Ref-1

Fodrin (250 kDa)

Ku80 (pel)

24 6 20 n/a n/a n/a n/a

0.03 1.09 3.02 4.63 3.12 0.45 0.42

0.50 0.95 1.17 1.20 1.00 1.04 0.83

0.30 0.66 2.01 2.39 2.15 1.10 0.46

0.74 0.83 0.78 0.83 0.46 0.42 0.48

1.16 0.27 0.57 1.17 1.10 0.89 0.47

2.76 0.56 0.64 0.81 0.67 1.70 2.18

3 6 7 7 9

0.02 0.03 0.91 2.63 4.74

0.16 0.42 1.05 1.11 1.22

0.31 0.48 1.54 1.04 2.28

0.73 1.01 1.17 1.28 0.75

2.51 1.14 1.00 1.42 1.68

2.97 2.34 1.44 2.10 0.63

DNA binding of Ku heterodimer (Ku DNA) was quantified in the EMSA. Protein levels of Ku80, DNA-PKcs, Ref-1, and intact ␣-fodrin in the nuclear extracts or Ku80 in the particulate fraction (pel) were determined by immunoblotting. Values for each sample were normalized to standard samples analyzed on each gel; n/a, brain removed within 24 h, specific time not available. Table 2 Alzheimer’s disease cases Case no.

Age/sex (year)

PMI (h)

Ku DNA (EMSA)

Ku80

DNA-PKcs

Ref-1

Fodrin (250 kDa)

Ku80 (pel)

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

75/M 92/F 89/M 77/M 71/M 81/F 88/F 80/F 81/F 66/M 80/F 93/F 100/F 80/F 98/F 85/M 83/F 95/M 77/M 87/M 84/M 89/M 74/M 80/M 65/F 79/F 86/F 76/M 78/F 81/F 69/M 74/M 78/F 81/M 71/M 88/F 83/F 80/F 87/F

2 3 3 3 4 4 4 4 5 5 5 5 5 6 6 6 6 6 7 8 8 8 8 8 8 8 8 8 8 9 9 10 10 11 12 12 12 12 14

3.59 2.28 1.9 0.03 0.03 0.04 0.88 3.6 1.92 1.21 1.13 0.04 1.88 3.4 0.76 2.56 1.02 0.47 0.11 0.02 0.02 0.97 0.15 0.26 0.02 0.09 1.6 0.08 1.95 2.58 0.18 0.05 0.85 0.58 2.29 0.33 3.25 0.06 1.02

2.72 1.9 1.9 0.18 0.11 0.04 0.96 2.66 1.28 0.81 1.05 0.49 3.04 4.1 2.57 3.48 2.59 1.34 0.42 0.31 0.41 0.98 1.06 0.94 0.14 0.87 0.96 0.1 0.71 0.99 0.12 0.39 1.36 0.88 2.57 1.02 3.2 0.32 2.61

1.41 0.81 1.1 0.05 0.02 0 0.28 2.1 0.77 1.07 0.32 0.06 1.84 2.6 0.74 0.29 0.33 0.06 0.05 0.05 0.07 0.2 0.61 0.17 0.01 0.97 0.82 0.05 1.2 1.54 0 0.04 0.92 1.15 1.18 0.72 1.14 0.27 0.95

1.83 1.64 1.5 1.63 2.42 1.19 1.92 2.52 1.32 2.07 1.26 1.56 1.2 1.85 2.13 2.34 1.49 1.68 3.06 1.84 1.97 0.44 0.66 0.67 0.81 1.2 1.18 2.02 1.13 1.3 1.71 1.36 2.28 2.06 2.37 2.28 1.91 0.83 1.25

3.29 0.89 1.8 0.64 0.93 0.03 1.09 1.97 0.45 1.29 0.21 0.37 1.52 2.42 1.13 1.11 0.55 0.23 0.01 0.52 0.33 0.38 0.86 1.09 0.05 1.78 0.97 1.64 1.76 1.56 0.15 0.72 0.66 0.86 1.82 0.92 1.02 0.25 1.92

5.17 1.36 1.5 2.52 2.27 1.43 1.62 2.45 0.64 2.34 0.88 1.59 2.78 2.42 4.43 1.47 0.82 0.89 1.45 2.89 2.08 1.89 2.85 1.9 2.52 3.17 1.56 6.46 2.51 2.34 3.97 1.4 0.52 0.96 1.39 1.27 0.72 0.53 0.52

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resuspended in 500 ␮l of buffer A. The nuclear protein fractions were dialyzed for 6 h at 4 ◦ C against 20 mM HEPES, pH 7.9, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.2 mM PMSF, centrifuged at 16,000 × g for 30 min at 4 ◦ C, aliquoted and frozen at −70 ◦ C. Protein concentrations of the final nuclear, cytosolic and particulate fractions were determined by the BCA protein assay (Pierce, Rockford, IL) using bovine gamma globulin as the standard protein. 2.3. Electrophoretic mobility shift assay (EMSA) Double-stranded oligonucleotide (5 -AGTTGAGGGGACTTTCCCAGGC-3 ) containing the NF-␬B binding motif from the mouse ␬ light chain enhancer (Promega, Madison, WI) was end-labeled using [␥-32 P]-ATP and T4 polynucleotide kinase and purified by centrifugation through a size exclusion column. DNA binding was performed in 20 ␮l reactions containing 25 mM Tris–HCl, pH 7.5, 50 mM NaCl, 3 mM MgCl2 , 1 mM dithiothreitol, 0.01% Nonidet P-40 (NP-40), 2 mM EDTA, 5% glycerol, 50 ␮g/ml bovine serum albumin, 1 ␮g double-stranded poly(dI-dC) (Amersham Pharmacia Biotech, Piscataway, NJ), and 4 × 105 cpm (Cerenkov) labeled probe. Four microgram of nuclear extract was added to the binding mix and incubated at room temperature for 30 min. For some experiments the poly(dI-dC) was added 5 min after the other components as described in Section 3. The reaction mixtures were resolved by electrophoresis through 6% native polyacrylamide gels in buffer containing 50 mM Tris, 0.38 M glycine, and 2 mM EDTA. Gels were dried and subjected to autoradiography with an intensifying screen for 8–24 h. For supershift assays of Ku antigen, 6 ␮g of nuclear extract were incubated on ice for 60 min with 3 ␮g of monoclonal anti-Ku80 antibody (clone Ku15; Sigma, St. Louis, MO) before addition of the complete DNA binding mix and incubation for 30 min at room temperature. For supershift assays of NF-␬B, 6 ␮g of nuclear extract were incubated on ice for 60 min with 2 ␮g of polyclonal antibody to the NF-␬B p50 or RelA(p65) subunit (Santa Cruz Biotechnology, Santa Cruz, CA). 2.4. Immunoblotting Proteins in the homogenate fractions were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on 7% acrylamide/0.19% bisacrylamide or 14% acrylamide/0.09% bisacrylamide gels and transferred to Immobilon-P (Millipore, Bedford, MA) using a semi-dry graphite electroblotter system as described previously [57]. Lysates of HeLa cells treated with or without anisomycin were prepared to use as positive controls for the immunoblots. HeLa cells were incubated with 10 ␮g/ml anisomycin for 4 h at 37 ◦ C and lysed in 1% NP-40, 0.15 M NaCl, 50 mM Tris–HCl, pH 8, 5 mM EDTA,

25 mM ␤-glycerophosphate, 0.1 mM Na3 VO4 , and 0.1 mM PMSF. Blots were blocked for approximately 18 h at 4 ◦ C in 10%. Non-fat dry milk in Tris-buffered saline (TBS; 0.14 M NaCl and 10 mM Tris–HCl, pH 7.4) with 0.1% Tween20 (TBST). Blots were incubated with the primary antibody at room temperature for 1 h, washed with TBST, and incubated for 1 h with goat anti-rabbit IgG or goat anti-mouse IgG coupled to horseradish peroxidase (1:100,000; Zymed, San Francisco, CA), and then washed again with TBST. The bound antibody was detected by enhanced chemiluminescence (SuperSignal; Pierce). Antibodies and dilutions used were: monoclonal anti-DNA-PKcs (clone 42-psc, 1 ␮g/ml; NeoMarkers, Fremont, CA), monoclonal anti-Ku70 (clone N3H10, 0.2 ␮g/ml; NeoMarkers); rabbit anti-Ku80 (0.25 ␮g/ml; Serotec, Indianapolis, IN), rabbit anti-Ref-1 (0.2 ␮g/ml; Santa Cruz Biotechnology), rabbit anti-PARP (1:2000; Boehringer Mannheim), monoclonal anti-PARP (clone C-2-10, 1 ␮g/ml; Oncogene, Boston, MA), monoclonal anti-␣-fodrin (clone 1622, 0.01 ␮g/ml; Chemicon, Temecula, CA). Tau protein was detected with monoclonal anti-tau (T46, 1 ␮g/ml, Zymed) or Tau-1 (1:100 dilution of culture supernatant) followed by rabbit anti-mouse IgG (1:2000; Sigma) and [125 I]-labeled protein A. The Tau-1 epitope maps to unphosphorylated peptide 189–207 in the longest form of human tau [50]. To reveal the Tau-1 epitope, blots were treated with alkaline phosphatase as described previously [56]. Molecular weights were estimated using prestained SDS electrophoresis molecular weight markers from Bio-Rad (Richmond, CA) and Chemicon. 2.5. Immunohistochemistry Formalin-fixed and paraffin-embedded tissue blocks of midfrontal cortex cut at 8 ␮m and mounted on slides were obtained from Dr. Eliezer Masliah (UCSD). Sections from four control and seven AD cases were examined. Sections were deparaffinized and rehydrated in graded alcohols. Tissue sections were microwaved in 0.01 M sodium citrate, pH 6.0 for 5 min, paused for 5 min, and then microwaved for 5 min. The power was reduced to 80% once the solution was brought to a boil. Sections were allowed to cool at room temperature for 1 h. Endogenous peroxidase activity was quenched by incubation with 3% H2 O2 in methanol for 45 min. Sections were blocked in 5% normal horse serum (NHS) for 1 h followed by overnight incubation with primary antibody in 2.5% NHS at 4 ◦ C in a humidified chamber. Monoclonal antibodies used were anti-DNA-PKcs (clone 42-psc, 2 ␮g/ml) and anti-NeuN (2 ␮g/ml; Chemicon). Primary antibody was omitted for some sections as a negative control. Sections were incubated with biotinylated horse anti-mouse IgG for 1 h, followed by 1 h with the avidin–biotin–peroxidase complex (Vectastain ABC Elite kit; Vector, Burlingame, CA). Sections were washed in TBS

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between all incubations. The immunostain was visualized with 0.5 mg/ml 3,3 -diaminobenzidine tetrahydrochloride in 0.03% H2 O2 . Sections were dehydrated in graded alcohol, and coverslipped. 2.6. Data analysis Radioactive EMSA gels were quantified using a Molecular Dynamics PhosphorImager and ImageQuant software (Sunnyvale, CA). Films of 125 I-labeled blots were scanned and quantified using ImageQuant. Chemiluminescence on the immunoblots was quantified using a Syngene GeneGnome gel documentation system and GeneTools image analysis software (K&R Technologies, Frederick, MD). Two non-AD samples were repeatedly analyzed in each gel or assay to provide a normalization standard to allow comparisons between gels. All samples were analyzed at least twice to ensure reproducibility of the results. Statistical comparisons between the control and AD groups were evaluated using the Mann–Whitney U-test. Linear regression analysis and the Spearman’s rank–order correlation coefficient were used to assess correlation between parameters. A value of P < 0.05 was considered significant.

3. Results 3.1. DNA binding activity of Ku in human brain extracts DNA binding activity in nuclear extracts from human midfrontal cortex was analyzed by an EMSA using an oligonucleotide containing the NF-␬B motif (Fig. 1). A minor DNA–protein complex was supershifted by antibodies to NF-␬B subunits p65(RelA) and p50, indicating a low level of NF-␬B activation in the nuclear extracts. The major DNA-binding protein complex observed was supershifted by a monoclonal antibody to Ku80 and a polyclonal antibody to Ku70 (not shown), identifying Ku antigen as part of the complex. The Ku heterodimer binds to free ends of double-stranded DNA independent of sequence [17,32,60]. It has been shown that the order in which components are added in the EMSA binding reaction affects the amount of Ku detected [32,57]. Fig. 2 confirms that adding poly(dI-dC) to the nuclear extract after addition of the labeled oligonucleotide increased the amount of Ku bound to DNA compared to adding a cocktail containing poly(dI-dC) and labeled probe simultaneously. DNA binding activity of Ku antigen was analyzed in midfrontal cortex nuclear extracts from a panel of 39 AD and 7 age-matched normal control brains and 5 additional non-AD cases. The EMSA gels of the controls and 30 of the AD brain extracts are shown in Fig. 3. The additional 9 AD cases were analyzed similarly. Two standard samples were included on each gel and the complex containing Ku was quantified in all samples and normalized to the standard samples. Re-

Fig. 1. DNA binding activity of Ku antigen in human brain. Nuclear extracts from three human midfrontal cortex samples were analyzed by an EMSA using an oligonucleotide containing the NF-␬B binding motif. The major DNA–protein complex (Ku) observed was supershifted (Ku∗ ) by a monoclonal antibody to the Ku80 subunit, identifying Ku antigen as part of the complex. Antibodies to NF-␬B subunits p65 (RelA) and p50 supershifted a minor DNA–protein complex, indicating a low level of activated NF-␬B in the nuclear extracts. The band labeled ns represents variable non-specific binding. Part of the non-complexed oligonucleotide probe (Free Probe) was electrophoresed off the bottom of the gel. Ab, antibody.

sults for the control cases are presented in Table 1 and the AD cases are presented in Table 2. The mean value (±S.D.) of Ku DNA binding activity was reduced in the AD group (1.11 ± 1.14) versus the age-matched controls (1.82 ± 1.76),

Fig. 2. Effect of poly(dI-dC) addition on Ku DNA binding. Midfrontal cortex nuclear extracts from a representative subject with low (1) or high (2) levels of Ku DNA binding were analyzed by the EMSA. Poly(dI-dC) was either (A) added as a cocktail with the binding reaction mix and labeled probe to the aliquot (4 ␮g) of nuclear extract or (B) added 5 min after the binding mix and probe.

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Fig. 3. Ku DNA binding in normal and AD nuclear extracts. Nuclear extracts of midfrontal cortex from a panel of 39 AD and 7 age-matched normal control brains and an additional 5 non-AD cases were analyzed for Ku DNA binding by an EMSA with poly(dI-dC) added 5 min after the binding mix and probe. Gels of the controls (case nos. 1–7), other cases (case nos. 8–12) and 29 of the 39 AD cases (case nos. 13–42) are shown. Replica samples of two cases (case nos. 1 and 10) were included on each gel; the amount Ku DNA complex for each sample was quantified and normalized to these samples.

but was not significantly different. The panel of AD samples was further analyzed to determine if the amount of Ku DNA binding correlated with physical or neuropathological parameters of the cases. There was no significant correlation of Ku DNA binding with postmortem interval (PMI) (r = −0.129; P = 0.433), age (r = 0.161; P = 0.327) or cognitive function as measured by the DRS (r = 0.284; P = 0.152; n = 27) or MMSE (r = 0.258; P = 0.129; n = 36). A negative correlation with the Blessed score did not quite reach significance (r = −0.320; P = 0.061; n = 35). The strongest correlation was between Ku DNA bind-

Fig. 4. Ku DNA binding correlates with synaptophysin immunoreactivity. The panel of AD samples was analyzed to determine correlations of Ku DNA binding measured in the EMSA with physical or mental parameters reported for each case. No significant correlation between Ku DNA binding and postmortem interval (PMI), age, or cognitive function was found. There was, however, a significant correlation between Ku DNA binding and the amount of synaptophysin immunoreactivity measured by dot-immunoblotting of total particulate fractions from 29 of the 39 AD cases. Ku DNA binding also had a negative correlation with the estimated duration of disease and a weaker correlation with the number of neurofibrillary tangles (NFT). Linear regression analyses and Spearman’s rank–order correlation coefficient were used to assess correlations.

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ing and the amount of presynaptic vesicle protein synaptophysin (r = 0.626; P < 0.001; n = 29), determined by a dot-immunobinding assay [2]. Ku DNA binding also had an inverse correlation with the estimated duration of disease (r = −0.464; P = 0.006; n = 34) and the number of tangles in the midfrontal cortex (r = −0.399; P = 0.012; n = 39), but no correlation with the count of neuritic plaques (r = −0.209; P = 0.220; n = 36). Correlations are shown graphically in Fig. 4.

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3.2. Expression of DNA repair proteins in normal and AD brain To determine if DNA-PK protein expression was reduced in samples with lower DNA binding, nuclear extracts were separated by gel electrophoresis and immunoblotted with antibodies to Ku70, Ku80, and the catalytic subunit, DNA-PKcs. In addition, expression of the DNA repair enzymes poly(ADP-ribose) polymerase (PARP) and redox

Fig. 5. Immunoblots of DNA repair proteins in nuclear extracts of normal and AD brain. Aliquots (50 ␮g) of midfrontal cortex nuclear extracts were separated by SDS–PAGE and immunoblotted sequentially with antibodies to DNA-PKcs, Ku80, Ku70, PARP, Ref-1, and ␣-fodrin and detected by enhanced chemiluminescence. Blots of the control and other 5 non-AD cases are shown in (A) and 10 of the 39 AD cases (case nos. 13–22) are shown in (B). Protein expression was quantified on each blot and normalized to standard extracts. The values for Ku80, DNA-PKcs, and Ref-1 are shown in Tables 1 and 2. AD cases with reduced Ku DNA binding also displayed decreased proteins levels of Ku70, Ku80, DNA-PKcs, and PARP, but not Ref-1. Lysates of HeLa cells untreated (H) or treated with anisomycin (H∗ ) to induce apoptosis were included as blotting controls. DNA-PKcs, PARP, and ␣-fodrin are substrates for caspase-3. Cleavage of DNA-PKcs generates fragments of 230–250, 150–165, and 120 kDa; the latter two are recognized by the antibody (42-psc) used as indicated for the anisomycin-treated HeLa sample (H∗ ). Caspase-3 degrades PARP to 89 and 24 kDa peptides. Alpha-fodrin is cleaved in a protease-sensitive region by multiple enzymes including caspase-3 and calpain I, generating a major 150 kDa-breakdown product.

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Fig. 5. (Continued ).

factor-1 (Ref-1), also known as apurinic/apyrimidinic endonuclease (APE), were determined by immunoblotting. PARP is activated by DNA single- and double-strand breaks and promotes their repair (for review see [25]). Ref-1/APE is the rate-limiting enzyme in the base excision repair pathway that removes oxidized and other damaged bases (for review see [16]). Immunoblots of the controls and 10 of the 39 AD cases are shown in Fig. 5A and B. Protein expression was quantified on each blot and normalized to standard extracts. The values for Ku80, DNA-PKcs, and Ref-1 of all samples are shown in Tables 1 and 2. There was a high correlation between Ku DNA binding and the amount of Ku70 (r = 0.827; P < 0.001) or Ku80 (r = 0.814; P < 0.001) in the nuclear extracts of the AD cases (Fig. 6). The mean value of Ku70 (0.89 ± 0.91) or Ku80 (1.32 ± 1.09) protein in the AD samples was actually greater but not significantly different from the controls (Ku70, 0.74 ± 0.37; Ku80,0.95 ± 0.24).

AD cases with decreased amounts of Ku subunits also had reduced expression of DNA-PKcs and PARP. The mean value of DNA-PKcs protein in the AD cases (0.67 ± 0.65) was reduced from the controls (1.30 ± 0.87), but the difference was not quite significant (P = 0.056). DNA-PKcs and PARP are substrates for caspase-3 cleavage during apoptotic cell death. Caspase-3 cleavage of the 470 kDa DNA-PKcs protein generates fragments of 230–250, 150–165, and 120 kDa [9,61]. In Fig. 5 fragments of approximately 150 kDa were observed in most samples and a fragment of approximately 120 kDa was detected in some samples (case nos. 10,14, 19 in Fig. 5 and nine additional AD cases not shown). PARP is degraded during apoptosis to 89- and 24-kDa peptides [25]. No 89-kDa fragment, however, was detected in the AD samples with reduced PARP protein using the polyclonal antibody shown in Fig. 5 or a monoclonal anti-PARP antibody (not shown), both of which

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stained the 89 kDa fragment in apoptotic HeLa cells (H∗ ). Blots also were immunoreacted with antibody to the cytoskeletal protein ␣-fodrin, another high molecular weight protease-sensitive protein. The intact 250-kDa ␣-fodrin was

Fig. 6. Correlation of Ku DNA binding with protein levels of DNA-PK subunits. Ku DNA binding determined by EMSA exhibited a strong correlation with the amount of Ku80 or Ku70 (r = 0.827; P < 0.001; not shown) protein in the nuclear extracts of the AD cases. In addition, reduced Ku DNA binding in the AD cases correlated with reduced expression of DNA-PKcs and full-length ␣-fodrin (250 kDa). There was no correlation between Ku DNA binding and Ref-1 protein expression. No correlation between expression level of any protein and the postmortem interval was observed.

Fig. 7. Correlation of DNA repair protein levels with synaptophysin immunoreactivity. As shown in Fig. 4, Ku DNA binding correlated with synaptophysin immunoreactivity. Protein levels of the Ku subunits and DNA-PKcs determined by immunoblotting the AD nuclear extracts also strongly correlated with the amount of synaptophysin. Levels of intact 250-kDa ␣-fodrin protein showed a weaker, but significant correlation. The expression of Ref-1 protein did not correlate with synaptophysin immunoreactivity.

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detected in most control and AD cases, but was reduced in those samples with decreased levels of DNA-PKcs and Ku subunits. The decrease in DNA-PKcs and intact ␣-fodrin proteins both correlated with decreased Ku DNA binding (Fig. 6). Alpha-fodrin is cleaved in a protease sensitive region by multiple enzymes including calpain I and caspase-3, generating a major 150-kDa breakdown product that was detected in all samples [28,72,77]. Previously, using a polyclonal antibody to fodrin (brain spectrin), it was reported that frontal cortex AD brain extracts had reduced levels of the 250 kDa protein and increased amounts of the 150 kDa breakdown product compared to age-matched controls [43]. High levels of Ref-1 protein were detected in all control and AD cases examined and did not correlate with Ku DNA binding, DNA-PK protein expression, or synaptophysin (Figs. 5–7). In fact, Ref-1 protein was significantly increased in the AD nuclear extracts (1.64 ± 0.58) versus the controls (0.65 ± 0.19; P < 0.001). Protein levels of Ku70/80, DNA-PKcs, and ␣-fodrin in the nuclear extracts of the AD cases each correlated with the amount of synaptophysin immunoreactivity (Fig. 7). Protein levels of Ku70 (r = −0.364; P = 0.035), Ku80 (r = −0.387;

P = 0.024), and DNA-PKcs (r = −0.342; P = 0.048) also showed a negative correlation with the estimated duration of the disease. There was no correlation between decreased expression of any protein and the postmortem interval. No evidence of consistent protein redistribution among cellular fractions was found by immunoblotting the cytosolic, nuclear, and particulate fractions with antibodies to the Ku subunits, DNA-PKcs, PARP, Ref-1, or ␣-fodrin. Ref-1 was predominantly expressed in the nuclear fraction of all samples and ␣-fodrin was distributed in all fractions. Control and AD samples with high levels of Ku subunits, DNA-PKcs and PARP in the nuclear extracts also expressed these proteins in the cytosolic and particulate fractions (data not shown). All four proteins were detected in the particulate fractions even of those samples with reduced expression in the nuclear and cytosolic fractions. Particulate fractions from the entire panel of control and AD cases were immunoblotted for Ku subunits and the blots were quantified. The results for Ku80, displayed in Tables 1 and 2, demonstrated that there was no loss of Ku antigen in the particulate fraction of cases with reduced expression in the

Fig. 8. Subcellular distribution of hyperphosphorylated tau in AD brain samples. (A) Aliquots (50 ␮g) of the nuclear (N), cytosolic (C), and particulate (P) fractions of control and AD brain homogenates were immunoblotted with a monoclonal anti-tau antibody (clone T46) that detects phosphorylated and non-phosphorylated forms of the protein. The hyperphosphorylated isoforms of tau (A68; upper two bands of the triplet are denoted by arrows) characteristic of AD were detected predominantly in the particulate fraction of the AD cases shown. This fraction also contained a continuum of high molecular weight species immunostained by anti-tau consistent with modification by ubiquitin and aggregation. (B) Particulate fractions (50 ␮g aliquots) from control and AD samples were immunoblotted with monoclonal antibody Tau-1 before (−AP) and after treatment with alkaline phosphatase (+AP). Tau-1 does not recognize tau protein phosphorylated on serines in the peptide region 189–207 of the longest human isoform. The A68 isoforms of tau in case nos. 35 and 44 were recognized by Tau-1 only after dephosphorylation, demonstrating that these are hyperphosphorylated forms. Molecular weight standards are indicated on the left.

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nuclear extracts. The particulate fraction contains cellular components insoluble in low or high salt buffers and should include membranes, cytoskeletal structures, and protein aggregates. Reduction in Ku DNA binding exhibited an inverse correlation with the number of neurofibrillary tangles (NFT), one of the hallmarks of AD (Fig. 4). The major protein component of NFT is hyperphosphorylated microtubule-associated

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protein tau (for review see [8]). The cellular fractions were immunoblotted with monoclonal antibody T46 that recognizes all forms of tau to determine its distribution. The higher molecular weight hyperphosphorylated triplet characteristic of AD, referred to as A68 [8], was detected predominantly in the particulate fraction of AD samples, but the amount varied greatly between cases (Fig. 8A). Immunostaining of proteins throughout the higher molecular weight region was probably due to ubiquitination and aggregation of tau [8] in the AD brains and was not detected in the control samples. Phosphorylation of A68 and the high molecular weight tau species was demonstrated by their lack of immunoreaction with the phosphorylation-sensitive antibody Tau-1 until after phosphatase treatment of the blot (Fig. 8B). To determine if the amount of hyperphosphorylated and higher molecular weight tau correlated with loss of DNA-PK subunits, the particulate fractions of control and AD homogenates were immunoblotted with anti-tau antibody T46 (Fig. 9). The amount of A68 tau and aggregated forms correlated with the tangle count (r = 0.545; P < 0.001), but there was no apparent correlation with Ku or DNA-PKcs protein expression. 3.3. Immunohistochemical analysis of DNA-PK expression in normal and AD brain An immunocytochemical survey of DNA-PKcs in human tissue revealed the highest level of expression in testis and in central and autonomic nerve tissue [46]. In Fig. 10, cortical sections from a subset of the control (A–C) and AD cases expressing low (D–F) or high (G–L) amounts of DNA-PKcs, were stained with monoclonal anti-NeuN or anti-DNA-PKcs to determine their cellular localization. As reported [48], NeuN immunostained nuclei and cell bodies of neurons throughout the cortical gray matter. DNA-PKcs was detected predominantly in the nuclei of neurons and other cells in the gray matter (Fig. 10A, B, D, E, G and H). DNA-PKcs was detected also in NeuN-negative cells, presumably glia, in the white matter (J–L) in agreement with a previous study [46]. Immunoreactivity of NeuN and DNA-PKcs varied within the control and AD samples. Three AD cases examined expressing high levels of DNA-PKcs protein exhibited more NeuN-positive cells and cells with more intense DNA-PKcs immunoreactivity than did four AD cases with reduced DNA-PKcs protein.

4. Discussion Fig. 9. Variable expression of hyperphosphorylated isoforms of tau in AD cases. Aliquots (50 ␮g) of particulate fractions from control and AD midfrontal cortex were immunoblotted with monoclonal anti-tau antibody (clone T46). The blots show samples from 20 of the 39 AD cases and 4 of the 12 normal and non-AD cases. The number of tangles in the midfrontal cortex reported in the neuropathological examination is shown below each sample lane (nd, not determined). Amounts of A68 tau isoforms (arrows) and higher molecular weight aggregated/ubiquitinated forms varied between AD samples but were not detected in the non-AD samples. Only one sample (case no. 18) showed substantial proteolytic products of tau.

The present study assessed changes in expression and activity of proteins involved in repair of DNA strand breaks and oxidized bases in Alzheimer’s disease brains versus age-matched non-demented controls. DNA binding activity of the Ku regulatory subunit of DNA-PK was reduced in nuclear extracts of AD midfrontal cortex compared to age-matched normal controls. The small size of the normal control group and the wide variation in DNA binding

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Fig. 10. Immunohistochemical detection of DNA-PKcs and NeuN expression. Midfrontal cortical sections from a control brain (A–C; case 6) and AD brains expressing low (D–F; case 32) or high (G–I; case 26 and J–L; case 49) levels of DNA-PKcs protein were immunostained with monoclonal antibody to DNA-PKcs or NeuN as indicated by the column headings. Panels A–I show staining of neurons and other cells in the cortical gray matter. Panels J and K show expression of DNA-PKcs in NeuN-negative cells (L), presumably glia, in white matter (wm) adjacent to gray matter (gm). Sections from AD brains with high levels of DNA-PKcs (G–I) contain more DNA-PKcs-positive and more NeuN-positive cells than AD brains with low levels of DNA-PKcs (D–F), but also exhibit more intense DNA-PKcs immunoreactivity in expressing cells (H vs. E). The first (A, D, G, J) and last columns (C, F, I, L) represent 100× magnification and the middle column (B, E, H, K) represents 200×.

activity within groups, however, limited the power to detect a significant difference between the two groups. Within the AD group, decreased Ku DNA binding correlated significantly with decreased synaptophysin immunoreactivity determined in midfrontal cortex homogenates prepared independently from 29 of the 39 AD cases. The synaptophysin immunoreactivity assay was shown to correlate with immunocytochemical determinations of synaptic density [2]. Synaptic loss in the frontal cortex, measured by electron microscopy [13] or by immunocytochemical labeling

of synapse-associated proteins [70], is a major correlate of cognitive deficits in AD. A weaker correlation between Ku DNA binding and NFT was observed. A significant but modest correlation between NFT density in the midfrontal cortex and cognitive decline was reported previously [5,70]. A direct significant correlation of Ku DNA binding activity with mental status was not demonstrated in the present study, however, the majority of the AD cases exhibited severe cognitive deficits with MMSE scores less than 3 for 65% of the subjects tested.

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Ku DNA binding activity correlated with protein levels of Ku70, Ku80, and DNA-PKcs. It was reported that DNA-PKcs and Ku80 are expressed in about 50% of cortical gray matter neurons and glial cells [46]. In the current study, immunohistochemical analyses indicated that AD cases with high levels of DNA-PKcs and Ku protein exhibited more NeuN-positive cells and more intense DNA-PKcs staining than those cases with low levels of DNA-PK protein subunits. This suggests that decreased DNA-PK may be due to both neuronal loss and reduced expression. Cell counts, performed as described by Terry et al. [69], were available for 18 of the 39 AD cases spanning the distribution of DNA binding activity. There was no significant correlation between DNA-PKcs and the count of total cells, neurons, glia, or cell density in the midfrontal cortex. This also suggests that neuron or cell loss alone does not account for the reduction in DNA-PKcs and Ku. It is proposed that their protein levels are regulated post-transcriptionally or post-translationally but the mechanisms have not been elucidated [46]. The oligonucleotide used in the EMSA allowed an assessment of NF-␬B activation. A very low level of DNA binding activity attributable to the NF-␬B p65–p50 heterodimer or p50 homodimer was detected in control or AD nuclear extracts. A previous report detected highly variable levels of NF-␬B DNA binding in AD and aged control cases. Elevated DNA binding in either group correlated with upregulated expression of COX-2 mRNA suggesting an association with inflammatory processes [40]. This is consistent with increased immunoreactivity of an antibody to activated NF-␬B in neurons and astroglia in the vicinity of primitive plaques in the cerebral cortex of AD brains [29]. DNA damage responses are crucial for the development and maintenance of the nervous system and genetic defects in repair genes are associated with a range of neurodegenerative phenotypes in man [54]. Targeted inactivation of any of the five genes involved in non-homologous end joining, including Ku70, Ku80, DNA ligase IV, XRCC4, and DNA-PKcs, results in defects in neurogenesis as well as immune deficiencies [6,22,24]. Expression of DNA-PKcs and Ku is estimated to be 50-fold higher in human and other primates than in rodents [4,19]. Levels of DNA-PK are correlated with the species’ life span supporting a role for DNA-PK in maintaining genomic integrity [4,14,18,30,60]. It is proposed that accumulated unrepaired or misrepaired DNA damage contributes to cellular deficits that result in normal aging [3]. Several studies indicate that increased expression of DNA-PK can mitigate the effects of DNA damage caused by agents such as nitric oxide [74] or adriamycin [58], that can produce damage through oxidative stress. Neuronal cultures established from scid (severe combined immunodeficient) mice, harboring a mutation in DNA-PKcs resulting in a truncated protein, also exhibit increased spontaneous cell death among dividing cells [11] and increased susceptibility to oxidative stress and excitotoxicity [12]. It is proposed that neurons in susceptible brain regions in

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AD re-enter the cell cycle but fail to initiate mitosis [76]. Since DNA-PK and other proteins are required to repair DNA lesions that occur spontaneously during the cell cycle [35,67], reduced expression of DNA-PK could contribute to the aborted attempt at cell division in AD. It is proposed that DNA-PKcs plays a primary role in detecting DNA damage and initiating signaling pathways, including programmed cell death in the event of overwhelming DNA damage [60]. Evidence indicates that programmed cell death pathways are operative in Alzheimer’s disease although the number of cells undergoing apoptosis at a given time may be small. Initial studies using in situ detection of DNA fragmentation reported a large number of positive neurons and glia [34,59,64]. Additional studies failed to reveal more than a few neurons with morphological changes and protein expression consistent with classical apoptosis [34,39,62]. More recent reports using antibodies to activated caspase-3 demonstrated increased immunoreactivity in the hippocampus, cortex and other areas of AD brains [55,63,65] but estimates of the frequency of neurons with activated caspase-3 and apoptotic morphology were 1 in 1100–5000 neurons [63]. The infrequent appearance of neurons with apoptotic features may be a consequence of few cells dying acutely at a given moment in a chronic disease with sustained neurotoxicity [51]. The proteins DNA-PKcs, PARP and ␣-fodrin are substrates for the protease caspase-3, considered the central effector enzyme in the apoptotic cascade. Antibodies to caspase-3-generated fragments of ␣-fodrin [53] and actin [75] display elevated immunoreactivity in AD brains supporting the proposal that apoptotic pathways are active during AD. The antibody to caspase-3-cleaved fodrin reacts with 120- and 55-kDa peptides that should share the same N-terminal sequence [53,72]. This antibody immunostains a 55-kDa band in AD extracts and only faintly detects a 120-kDa fragment indicating that the stable endproduct of caspase cleavage in vivo may be the 55-kDa fragment [53]. The monoclonal anti-␣-fodrin antibody used in the current study was shown to react with a 120-kDa but not a 55-kDa caspase-3-generated fragment [28,57,72,77]. In the current study the stable 150-kDa proteolytic breakdown product of ␣-fodrin generated by multiple proteases, including calpain and caspase-3 was observed, but not the 120-kDa peptide specifically produced by caspase-3. If loss of proteins is due to acute activation of caspase-3 and other proteases in a small number of cells but encompassing a larger population of cells over time, sampling at death may reveal only stable proteolytic products. A caspase-3-generated 89-kDa PARP fragment also was not detected. Only 11 of the 39 AD brain extracts exhibited a fragment migrating as expected for the 120-kDa fragment from caspase-3-cleaved DNA-PKcs. Thus, the current study did not provide evidence for caspase-3 activation in a large percentage of cells at the time of death. In contrast to DNA-PK, expression of Ref-1 was significantly increased in nuclear extracts of AD brains versus

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the non-demented controls. Increased expression was not due to translocation from the cytosolic or particulate fraction to the nuclear fraction. The results are consistent with a previous study reporting increased Ref-1 immunoreactivity in the hippocampus of AD brains in areas of neuronal injury and plaque-like structures [68]. Immunoreactivity of the multi-functional proteins ERCC2 and ERCC3, which participate in nucleotide excision repair and transcription, also is reported to be significantly elevated in all brain regions of AD patients [27]. Despite increased levels of Ref-1 in AD brains, two reports indicate that the capacity to remove the hydroxylated base 8OHdG is reduced in AD. An increase in 8OHdG in intact DNA from ventricular cerebrospinal fluid (CSF) and a decrease in free 8OHdG, a byproduct of the repair process, indicates the presence of increased unrepaired DNA in cellular debris filtered through the CSF [37]. A significant decrease in the base-specific glycosylase activity that excises 8OHdG is also observed in all AD brain regions examined except for the cerebellum [38]. Reperfusion of ischemic CNS tissue produces acute injury including DNA damage that, at least in part, is due to oxidative stress. Models of CNS ischemia and reperfusion have shown that severe injury leads to reduction of the DNA repair proteins, DNA-PKcs, Ku, and PARP [31,57]. Expression of Ref-1 [20,23,71] and the base excision repair enzyme, 8-oxoguanine glycosylase [36], also are affected by ischemia and reperfusion injury. Thus, alterations in DNA repair proteins in AD reported in the current study and in previous investigations may be a measure of the sustained oxidative stress generated during the course of the disease. Ku DNA binding did show an inverse correlation with the estimated duration of disease suggesting that a progressive loss of DNA repair capability may occur. It cannot be concluded from this study if loss of certain DNA repair proteins is part of the pathogenesis of AD or a consequence of neurodegeneration. Among the AD cases, 31 were classified as Braak stage V or VI precluding analysis of the relationship between DNA repair protein expression and disease progression. Synaptic alteration in the hippocampus and frontal cortex is proposed to be an early event in AD as indicated by a progressive loss of synaptophysin in subjects rated clinically and pathologically as mild, moderate and severe [26,44,66]. Further studies with a cohort of AD patients representing a spectrum of clinical and pathological stages of the disease should help to clarify the role of the multifunctional DNA repair proteins in the progression of cell loss.

Acknowledgments This study was supported by National Institutes of Health grants NS28121 and AG05131. We thank Takaaki Tobaru, Crystalynn Woodard, and Richard Yeh for technical assistance in these studies, Dr. Gilbert Ho for comments on the manuscript, and Dr. Eliezer Masliah for providing materials.

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