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APP/PS-1 double mutant neuron cultures exhibited increased vulnerab- ility to oxidative stress, mitochondrial dysfunction and apop- tosis induced by Ab(1–42), ...
Journal of Neurochemistry, 2006, 96, 1322–1335

doi:10.1111/j.1471-4159.2005.03647.x

Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid b-peptide (1–42), H2O2 and kainic acid: implications for Alzheimer’s disease Hafiz Mohmmad Abdul,* Rukhsana Sultana,* Jeffrey N. Keller,  Daret K St. Clair,à William R. Markesbery  and D. Allan Butterfield*,  *Department of Chemistry and Center of Membrane Sciences,  Sanders-Brown Center on Aging, àGraduate Center of Toxicology, University of Kentucky, Lexington, Kentucky, USA

Abstract Oxidative stress is observed in Alzheimer’s disease (AD) brain, including protein oxidation and lipid peroxidation. One of the major pathological hallmarks of AD is the brain deposition of amyloid beta-peptide (Ab). This 42-mer peptide is derived from the b-amyloid precursor protein (APP) and is associated with oxidative stress in vitro and in vivo. Mutations in the PS-1 and APP genes, which increase production of the highly amyloidogenic amyloid b-peptide (Ab42), are the major causes of early onset familial AD. Several lines of evidence suggest that enhanced oxidative stress, inflammation, and apoptosis play important roles in the pathogenesis of AD. In the present study, primary neuronal cultures from knock-in mice expressing mutant human PS-1 and APP were compared with those from wild-type mice, in the presence or absence of various oxidizing agents, viz, Ab(1–42), H2O2 and kainic acid (KA). APP/PS-1 double mutant neurons displayed

a significant basal increase in oxidative stress as measured by protein oxidation, lipid peroxidation, and 3-nitrotyrosine when compared with the wild-type neurons (p < 0.0005). Elevated levels of human APP, PS-1 and Ab(1–42) were found in APP/ PS-1 cultures compared with wild-type neurons. APP/PS-1 double mutant neuron cultures exhibited increased vulnerability to oxidative stress, mitochondrial dysfunction and apoptosis induced by Ab(1–42), H2O2 and KA compared with wild-type neuronal cultures. The results are consonant with the hypothesis that Ab(1–42)-associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD. Keywords: Alzheimer’s disease, b-amyloid peptide (1–42), amyloid precursor protein, oxidative stress, presenilin-1. J. Neurochem. (2006) 96, 1322–1335.

Alzheimer’s disease (AD) is one of the most common agerelated neurodegenerative diseases and the most frequent cause of dementia in the elderly (Katzman and Saitoh 1991; Bondi et al. 1995). Mutations in three genes have been shown to cause early onset familial AD (FAD): amyloid precursor protein (APP), presenilin-1 (PS1), and presenilin-2 (PS2) (Drouet et al. 2000). Mutations in these genes lead to increased production of amyloid beta-peptide (Ab), which is

Address correspondence and reprint requests to Professor D. Allan Butterfield, Department of Chemistry and Sanders-Brown Center on Aging, Center of Membrane Sciences, University of Kentucky, Lexington, KY 40506, USA. E-mail: [email protected] Abbreviations used: Ab, amyloid beta peptide; AD, Alzheimer’s disease; APP, amyloid precursor protein; APPsw, Swedish APP; bFGF, basic fibroblast growth factor; BSA, bovine serum albumin; DCF, dichlorofluorescein; DCFH-DA, 2,7-dichlorofluorescin-diacetole; Em, emission wavelength; Ex, excitation wavelength; FAD, familial AD; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HNE, anti-4-hydroxynonenal; KA, kainic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; 3-NT, 3-nitrotyrosine; PBS, phosphatebuffered saline; PI, propidium iodide; PS1, presenilin-1; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; WT, wild type.

Received September 6, 2005; revised manuscript received October 25, 2005; accepted October 31, 2005.

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the main constituent of senile plaques found in AD brain (Selkoe 2001). Ab peptides are 39–43 amino-acid peptides, formed by b- and c-secretase cleavage of APP leads to production of Ab (Selkoe 2001). The role of accumulation of Ab peptides in forming fibrillar deposits, a principal component of senile plaques, has been suggested by two studies (Yankner 1996; Selkoe 1999). However, recent studies suggest that small oligomers of Ab(1–42) are the toxic species of this peptide (Walsh et al. 2002; Drake et al. 2003; Gong et al. 2003). Excess release or decreased reuptake of excitatory neurotransmitters such as glutamate is a cause of neuronal damage (Doble 1999; Mattson 2003; Meldrum 2000). Excessive or persistent activation of glutamate receptorgated ion channels (excitotoxicity) contributes to neuronal degeneration (Choi 1988; Beal 1992). Indeed, there is evidence that agonists specific for the three major glutamate ionotropic receptor sites, namely NMDA, kainic acid (KA), and a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, enhance the rate of reactive oxygen species (ROS) generation (Bondy and Lee 1993; Lafon-Cazal et al. 1993; Bruce and Baudry 1995). KA is a glutamate analog and is 30-fold more potent in neurotoxicity than glutamate (Bleakman and Lodge 1998). In particular, hippocampal pyramidal neurons are highly vulnerable to the excitotoxicity of KA (Sperk et al. 1983). Cell damage is induced by ROS such as hydrogen peroxide (H2O2) (Hensley et al. 1995, 2000). To elucidate potential mechanisms of Ab(1–42)-, H2O2- and KA-induced oxidative stress in primary neuronal cultures from knock-in mice expressing mutant PS-1 and APP, and thereby gain insights into the role of mutations of these genes in oxidative stress associated with FAD, the current study was undertaken. Dying cells display characteristics of apoptosis in AD brains and in cultures of neurons exposed to Ab (Stadelmann et al. 1999). Ab impairs mitochondrial redox activity and increases the generation of ROS (Behl et al. 1994; Hensley et al. 1994; Shearman et al. 1994). Several studies also suggest that Ab-induced oxidative stress leads to apoptotic neuron death that can be inhibited by antioxidants (Behl et al. 1994; Mattson and Goodman 1995; Pillot et al. 1999; Yatin et al. 1999; Boyd-Kimball et al. 2004; Sultana et al. 2004; Sultana et al. 2005). Presenilin is part of the c-secretase complex, which together with b-secretase, cleaves APP to release the Ab peptide. Presenilin and APP mutations sensitize cells to different apoptotic stimuli in vitro (Guo et al. 1999; Popescu and Ankarcrona 2000). The aim of the present study was to test the hypothesis that oxidative stress is increased in neurons from APP/PS-1 double mutant mice. We also tested the hypothesis that there is an increased vulnerability of such neurons to relevant oxidizing agents, i.e. Ab(1–42), H2O2 and KA on oxidative stress. In the current study, protein oxidation, lipid peroxidation and 3-nitrotyrosine formation in primary neuron

cultures from knock-in mice expressing mutant PS-1 and APP were compared with those from wild-type mice, basally and in the presence of Ab(1–42), H2O2 or KA.

Materials and methods Materials All chemicals were of the highest purity and were obtained from Sigma (St Louis, MO, USA) unless otherwise noted. Ab(1–42) peptide was purchased from Anaspec (San Jose, CA, USA). The OxyBlot protein oxidation detection kit and anti-3-nitrotyrosine antibody were purchased from Chemicon (Temecula, CA, USA). Anti-4-hydroxynonenal (HNE) was purchased from Alpha Diagnostic International (San Antonio, TX, USA). Trypsin–EDTA, Dnase-I, trypsin soybean inhibitor, B-27 supplement, basic fibroblast growth factor (bFGF), penicillin–streptomycin–neomycin and glutamax-1 were purchased from Gibco–Invitrogen Corp. (Grand Island, NY, USA). Anti-Ab and anti-APP antibodies were purchased from Stressgen (Victoria, BC, Canada) and anti-PS1 was from Calbiochem (San Diego, CA, USA). APP-PS1 mouse neuronal cultures The University of Kentucky Animal Care and Use Committee approved all the procedures used in this study. The APP/PS-1 mice used in this study are the APPNLh/APPNLh · PS-1P264L./PS-1P264L. double mutant mice generated by using the Cre-loc knock-in technology (Cephalon, Inc., Westchester, PA, USA) to humanize the mouse Ab sequence and to create a PS-1 mutation identified in human AD (Reaume et al. 1996; Siman et al. 2000). These mice were maintained on a CD)1/129 background. Primary neuronal cell cultures were prepared from the brain of APP/PS1 double mutant neonatal pups (born within 24 h) as well as brain from neonatal wild-type mice. In brief, the neonatal mice pups were decapitated and the cerebral cortex was dissected out on ice. Trypsin–EDTA (0.25%) with Dnase-I (10 mg/mL) and trypsin soybean inhibitor (50 mg/mL) was added to the dissected cerebral cortices, and the preparation was gently aspirated with a glass pipette. The cell suspension was incubated in a CO2 incubator for 3 min, followed by gently aspirating it again to obtain a homogenous solution. This solution was then filtered through a 0.22-lm nylon mesh (Falcon, Franklin Lakes, NJ, USA) followed by centrifugation at 272 g for 3 min at 4C. The pellet obtained was washed with neurobasal medium [containing B-27 supplement (20 lL/mL), penicillin– streptomycin–neomycin (20 lL/mL) and glutamax-1 (10 lL/mL)]. The isolated primary cortical neurons were diluted (2 million cells/ mL) in neurobasal medium and seeded onto a 6-well plate precoated (2 h prior to seeding) with poly D-lysine (100 lg/mL). The cells were allowed to incubate in a CO2 incubator for 30 min. The medium was aspirated out completely and a fresh neurobasal medium was added along with bFGF. The neurons were maintained in a humidified incubator with 5% CO2 at 37C and 0.5 mL of fresh neurobasal medium (without bFGF) was added on the 5th day. Experimental treatment of cultures Cortical neuron cultures were used for experimentation on the 10th day in vitro. This time was chosen based on prior studies showing that 10-day-old cultures are mature (Aksenova et al. 1999). Cultures

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were treated independently with three different oxidizing agents, Ab(1–42), H2O2, and KA. Ab(1–42) peptide was dissolved in sterile phosphate-buffered saline (PBS), pH 7.5, and pre-incubated for 24 h at 37C prior to addition to cultures. The final concentrations of the Ab(1–42) peptide in the cell culture were 2, 5, 10, and 25 lM, respectively, and the effects of Ab on the neuronal culture were measured after 24 h of exposure. The effect of oxidizing agents, H2O2 (10, 25, 50, and 75 lM) and KA (50, 100, and 150 lM) were also studied in a dose-dependent manner. The concentrations employed were based on prior literature (Keller et al. 1998). The working solution of H2O2 and KA was prepared in sterile PBS (pH 7.5). Measurement of protein carbonyls Protein carbonyls are an index of protein oxidation (Butterfield and Stadtman 1997). The neuron cultures treated with Ab(1–42), H2O2, and KA, respectively, for 24 h were scraped and used as samples for further analysis. The sample (5 lL) was incubated for 20 min at room temperature (22C) with 5 lL of 12% sodium dodecyl sulfate (SDS) and 10 lL of 2,4-dinitrophenylhydrazine that was diluted 10 times with water from a 200-mM stock. The samples were neutralized with 7.5 lL of neutralization solution (2 M Tris in 30% glycerol). The resulting sample (250 ng) was loaded per well in the slot-blot apparatus. Samples were loaded onto a nitrocellulose membrane under vacuum pressure. The membrane was blocked with 3% bovine serum albumin (BSA) in PBS containing 0.2% (v/v) Tween 20 (wash blot) for 1 h and incubated with a 1 : 100 dilution of anti-DNP polyclonal antibody in wash blot for 1 h. Following completion of the primary antibody incubation, the membranes were washed three times in wash blot for 5 min each. An anti-rabbit IgG alkaline phosphatase secondary antibody was diluted 1 : 8000 in wash blot and added to the membrane for 1 h. The membrane was washed in wash blot three times for 5 min each and developed using Sigmafast tablets (BCIP/NBT substrate). Blots were dried, scanned with Adobe Photoshop (San Jose, CA, USA), and quantitated with Scion Image (PC version of Macintosh-compatible NIH Image; National Institutes of Health, Bethesda, MD, USA) software. Measurement of 4-hydroxy-2-trans-nonenal Levels of HNE, which reflect lipid peroxidation (Butterfield and Stadtman 1997; Lauderback et al. 2001) were quantified by slot-blot analysis as described previously (Lauderback et al. 2001). AntiHNE antibody raised in rabbit was used as the primary antibody (1 : 200 dilution). The membrane was developed using Sigmafast tablets. Blots were dried, scanned with Adobe Photoshop, and quantitated with Scion Image (PC version of Macintosh-compatible NIH Image) software. Measurement of 3-nitrotyrosine (3-NT) The sample (10 lL) was incubated with 10 lL of modified Laemmli buffer containing 0.125 M Tris base, pH 6.8, 4% (v/v) SDS, and 20% (v/v) glycerol. The resulting sample (250 ng) was loaded per well in the slot-blot apparatus. Samples were loaded onto a nitrocellulose membrane under vacuum pressure. The membrane was blocked with 3% (w/v) BSA in wash blot for 1 h and incubated with a 1 : 2000 dilution of 3-NT polyclonal antibody in wash blot for 90 min. The remainder of the procedure was identical to that described for HNE above.

Analysis of DNA fragmentation Murine neuronal cultures were rinsed three times using PBS, fixed with 4% paraformaldehyde for 10 min at 37C, rinsed and stained with Hoechst 332584 (1 mg/mL) and propidium iodide (PI) for 10 min at room temperature. The cells staining were visualized using a fluorescence microscope. The nuclear staining with Hoechst 33258 and PI provided a morphological discrimination between normal and apoptotic cells (Darzynkiewicz et al. 1994). Determination of neuronal mitochondrial function Mitochondrial function was evaluated by the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Briefly, MTT was added to each well with a final concentration of 1.0 mg/mL and incubated for 1 h in a CO2 incubator. The dark blue formazan crystals formed in intact cells were extracted with 250 lL of dimethylsulfoxide, and the absorbance was read at 595 nm with a microtiter plate reader (Bio-Tek Instruments, Winooski, VT, USA). Results were expressed as the per cent MTT reduction (Liu et al. 1997) of control cells (without any treatment). Western blot analysis Neuron cultures were incubated with Ab(1–42), H2O2, or KA for 24 h. The protein content of the supernatants (culture medium) and the cells was determined using the Bio-Rad protein assay reagent (bicinchoninic acid). Equal amounts of protein were separated on 4–20% SDS–polyacrylamide gels, transferred to nitrocellulose membranes, and then exposed to the appropriate antibodies. Ab, cytochrome c and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were detected with anti-Ab, anti-cytochrome c and anti-GAPDH specific antibodies, respectively. After incubation with the primary antibodies, the nitrocellulose membranes were incubated with a secondary alkaline phosphatase-conjugated antibody. Proteins were visualized by developing with Sigmafast tablets (BCIP/NBT substrate). Blots were dried, scanned with Adobe Photoshop, and quantitated with Scion Image (PC version of Macintosh-compatible NIH Image) software. Measurement of intracellular ROS Because it is a non-polar diester, 2,7-dichlorofluorescin-diacetole (DCFH-DA) crosses the neuronal membrane, where cytosolic esterases cleave the ester functionality, forming an anion that is trapped within the neuron. Reaction of ROS, especially H2O2 or peroxyl free radicals, with DCFH yields fluorescent dichlorofluorescein (DCF). The intracellular accumulation of ROS in neuronal cell culture, following 24 h of Ab(1–42) or KA or H2O2 treatment, was measured in cells that were rinsed with Krebs’ ringer solution (100 mM NaCl, 2.6 mM KCl, 25 mM NaHCO3, 1.2 mM MgSO4, 1.2 mM KH2PO4 and 11 mM glucose), and 10 mM DCFH-DA was loaded. After 1 h incubation at 37C, cells were washed twice with the same buffer and examined under a confocal fluorescence microscope equipped with an argon laser [excitation wavelength (Ex)] 485 nm, [emission wavelength (Em) 530 nm]. Statistical analysis Data are presented as the means ± SEM. One-way ANOVA was used to determine the effect of different stress-inducing agents (dose dependent) on the murine neuronal culture and release of cyto-

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chrome c. Two-way ANOVA (Prism, GraphPad Software, San Diego, CA, USA) was used to determine the effect of genotype (i.e. wildtype and APP/PS1) on different treatments with stress-inducing agents (at different concentrations) to test whether there was a significant interaction between these variables. p-values of < 0.05 were considered significant.

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APP/PS1 mutations increase the basal level of oxidative stress in neurons Wild-type and APP/PS1 cortical neuronal cultures were used for studying the oxidative stress parameters by measuring the levels of protein carbonyls, HNE and 3-nitrotyrosine formations. The results suggest that there is a significant basal increase (approximately 40%) in protein carbonyls (Fig. 1a), HNE (Fig. 1b), and 3-nitrotyrosine (Fig. 1c) in APP/PS1 neurons when compared with wild-type neurons. Protein carbonyls are elevated in vulnerable regions of AD brain (Hensley et al. 1995; Butterfield and Lauderback 2002; Castegna et al. 2002a,b), in brain of APP/PS1 double mutant mice (Mohmmad Abdul et al. 2004), and in brain treated in vivo with Ab(1–42) (Boyd-Kimball et al. 2005). 3-NT is formed by the reaction of reactive nitrogen species with proteins and is reported to be elevated in AD brain (Smith et al. 1997; Castegna et al. 2003). A dose-dependent study of oxidants was carried out in the wild-type and APP/PS1 neurons. Treatment of wild-type (Fig. 2a) neurons or APP/ PS1 double mutant neurons (Fig. 2a) revealed increased vulnerability of APP/PS1 neurons to protein oxidation induced by Ab()42). KA or H2O2 treatment of wild-type or APP/PS1 double mutant (Fig. 2a) mice neuronal cultures also demonstrated increased vulnerability of the latter cultures to protein oxidation. Similar conclusions were revealed by examination of the levels of Ab(1–42) or KA or H2O2-induced lipid peroxidation indexed by HNE (Fig. 2b) and 3-NT (Fig. 2c).

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Ab(1–42), H2O2, or KA-induced intracellular ROS formation Intracellular ROS was measured using cell permeable DCFDA. This dye is hydrolyzed to DCFH by intracellular esterase activity once it gets into the cell. DCFH interacts with peroxides to form fluorescent DCF. Ab(1–42)-, H2O2-, or KA-treated APP/PS1 neuronal cells displayed increased fluorescence staining with the DCF dye compared with that of the wild type (p < 0.001) (Fig. 3). Moreover, studies suggest that, in the presence of various oxidative damage inducing agents, Ab(1–42), H2O2 or KA (at lower concentrations), the primary neurons from knock-in mice expressing mutant APP and PS-1 displayed increased fluorescence staining with the DCF dye compared with wild-type cells (Fig. 3).

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Fig. 1 Mutations in APP and PS1 induces oxidative stress. (a) Protein oxidation (protein carbonyls). *p < 0.0001, wild type versus APP and PS1. (b) 4-Hydroxy-2-trans-nonenal (HNE). Results shown are mean ± SEM obtained for five independent preparations. Significance was assessed by one-way ANOVA. *p < 0.0005, wild type (WT) versus APP and PS1. (c) 3-Nitrotyrosine. *p < 0.0005, wild type versus APP and PS1.

Expression of APP and PS1 in the neuronal culture Untreated wild-type and APP/PS1 cortical neuronal cultures (10-day-old cultures) were used for studying the expression profile of APP and PS1, and the results suggest that there is an increase in the APP (Fig. 4a) and PS1 (Fig. 4b) levels in the APP/PS1 neuronal cultures when compared with the

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wild-type cultures. To test the specificity of anti-APP and anti-PS1 primary antibodies, we probed BSA and cytochrome c (both from Sigma) on western blots. Neither antibody detected either protein, which suggests a specificity of the antibodies employed in these studies to APP and PS-1, respectively (data not shown).

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Fig. 2 Dose-dependent increase in (a) protein carbonyls, (b) HNE and (c) 3-NT after treating the wild-type and APP/PS1 neurons with Ab(1– 42), H2O2 and kainic acid. Significance was assessed by one-way ANOVA. AB, Ab(1–42); KA, kainic acid. Plain bars refer to the wild-type and grid bars to APP/PS1 neurons. All the treatments were compared independently with their controls (no treatment). Results shown are mean ± SEM obtained for five independent preparations. p-value for protein carbonyls is **p < 0.0001 and *p < 0.0005 versus control, and, for HNE and 3-NT, **p < 0.0001 and *p < 0.001 versus control.

Increased vulnerability of APP/PS-1 double mutant neurons to Ab(1–42)-, H2O2- or KA-induced neuronal apoptosis A dose-response study with varying concentrations of Ab(1– 42) (2, 5,10, and 25 lM), H2O2 (10, 25, 50, and 75 lM) and KA (50, 100, and 150 lM) was carried out to determine the concentration of the above-mentioned oxidative stress-inducing agents that led to approximately equivalent cytotoxicity. Morphological studies were carried out by phase-contrast microscopy. The wild-type (Fig. 5) or APP/PS1 double mutant (Fig. 5) control neuronal cells exhibited healthy morphology and a strong neuronal network. The wild-type neurons did not show any signs of cytotoxicity, whereas the wild-type neuronal cells treated for 24 h with 10 lM Ab(1– 42), 150 lM KA, or 75 lM H2O2 (Fig. 5), exhibited membrane blebbings and cell shrinkage, with the loss of neuronal processes that are normally associated with apoptotic cell death. APP/PS1 neurons treated for 24 h with 2 lM Ab(1– 42), 50 lM KA, or 10 lM H2O2 (Fig. 5) showed, at a much lower concentration of oxidant, similar morphological changes to those observed in wild-type neurons at a higher concentration of the same oxidants. However, the wild-type neuronal cells treated for 24 h with the lower level of oxidants [2 lM Ab(1–42), 50 lM KA, or 10 lM H2O2 (Fig. 5)] exhibited healthy morphology and a strong neuronal network. Hoechst and PI staining was further used for detection of apoptotic bodies inside the neuronal cells. Twenty-four-hour treatment of the wild-type neuronal cells with 10 lM Ab(1–42), 150 lM KA, or 75 lM H2O2 (Fig. 6), or APP/PS1 neurons with 2 lM Ab(1–42), 50 lM KA, or 10 lM H2O2 (Fig. 6) significantly increased the number of apoptotic bodies (indicated by arrowheads). We observed that there was a different threshold for the effect of Ab(1–42), H2O2, and KA on cell viability. The MTT assay suggests that a 24-h exposure of wild-type or APP/PS1 cortical neurons to Ab(1–42) at 10 or 2 lM (Fig. 7a), respectively, resulted in significant loss of cell viability by 30%. Similarly, a 24-h exposure of wild-type or APP/PS1 cortical neurons to KA led to 50% survivability at concentrations of 150 or 50 lM, respectively (Fig. 7b), again suggesting an increased vulnerability to loss of cell viability in APP/PS1 neurons. Addition of H2O2 to wildtype or APP/PS1 cortical neurons at concentrations of 75 or 25 lM, respectively (Fig. 7c), led to 40% loss of cell viability, demonstrating with a third oxidative stress inducer, the increased cytotoxic vulnerability of APP/PS1 neurons.

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Fig. 3 Ab(1–42), H2O2 and KA-induced intracellular ROS accumulation. ROS levels were determined as described in Materials and methods. The treatment of cell culture is the same as in Fig. 2. Plane bars refer to the wild-type and grid bars to APP/PS1 neurons. The data are the mean ± SEM expressed as a percentage of control values (untreated wild type). Statistical comparison was made using two-way ANOVA. n ¼ 5; p < 0.0001.

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Fig. 4 Expression profile of APP and PS1 on the 10th day in vitro. Total protein (100 lg) from untreated wild-type and APP/PS1 cortical neuronal cultures, respectively, was analyzed by western blot analysis. (a) APP blot: lane 1, wild type; lane 2, APP/PS1. (b) PS1 blot: lane 1, wild type; lane 2, APP/PS1. Please note the increase in expression profile of APP levels in the APP/PS1 neuronal cultures when compared with the wild type.

To further investigate and confirm the results obtained by the MTT assay, the level of cytochrome c release was determined in the supernatants of the neuronal cultures treated with different concentrations of Ab(1–42), H2O2, or KA (Fig. 8). The results suggest that the amount of cytochrome c released in the supernatants of wild-type neuronal cultures treated with 10 lM of Ab(1–42) is 4–5fold elevated (Fig. 8c) (p < 0.005) compared with the control (wild-type neurons without any treatment). In contrast, this amount of cytochrome c released in the supernatants of APP/PS1 neuronal cells was achieved with only 2 lM Ab(1–42). With KA, wild-type neurons treated with 150 lM KA released 5-fold elevated cytochrome c (Fig. 8c) (p < 0.005), whereas 75 lM H2O2 led to a 6-fold elevation of cytochrome c release (Fig. 8c) (p < 0.005)

compared with wild-type untreated control. The amount of cytochrome c released in the supernatants of the APP/PS1 neuronal cultures treated with 50 lM KA is about three times its control (which had a higher basal level than wild type) (Fig. 8d) (p < 0.05), and H2O2 (10 lM) led to about 3-fold elevation of cytochrome c release (Fig. 8d) (p < 0.05) compared with the control (APP/PS1 neuronal cells without any treatment). All the above cytotoxicity studies suggest that, in the presence of various oxidative damage-inducing agents, Ab(1–42), H2O2 or KA (all at lower concentrations), the primary neurons from knock-in mice expressing mutant PS-1 and APP were more vulnerable to apoptosis compared with wild-type cells. Intracellular and extracellular Ab(1–42) levels in the neuronal cultures In order to determine if the increased basal oxidative damage in APP/PS-1 double mutant neurons was associated with elevated human Ab(1–42), we measured neuronal levels of this peptide in the untreated cultures (Fig. 9). The results showed significantly elevated human Ab(1–42) in 10-dayold APP/PS-1 cultures compared with 10-day-old wild-type cultures. Similarly, secreted Ab(1–42) was measured in APP/ PS-1 cultures compared with wild-type cultures (Fig. 9). Anti-Ab primary antibody was specific to Ab as it did not react with bicinchoninic acid and cytochrome c (both from Sigma) on western blots, suggesting this antibody is specific for Ab (data not shown). Discussion

Oxidative stress is extensive in AD, a neurodegenerative disease associated with cognitive decline and aging (Subbarao et al. 1990; Hensley et al. 1995; Markesbery 1997; Butterfield et al. 2001, 2002). The high oxygen consumption

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Fig. 5 Effect of Ab(1–42), H2O2 and KA on cortical neuron viability. Different concentrations of Ab(1–42), H2O2 and KA were added to the cell culture and incubated for 24 h, followed by which cell morphology

was visualized by phase-contrast microscopy (magnification · 100). The images displayed exhibit morphological changes at the respective concentrations as indicated in Results.

rate, abundant unsaturated lipid content, and relatively low availability of antioxidant enzymes compared with other tissues make brain susceptible to oxidative stress (Coyle and Puttfarcken 1993; Markesbery 1997, 1999). In the present

study, we investigated the effects of Ab(1–42), H2O2, and KA on primary neuronal cell cultures from knock-in mice expressing mutant PS-1 and APP and neurons from wildtype mice. In particular, we tested the hypothesis that basal

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Fig. 6 Ab(1–42), H2O2 and KA-induced DNA fragmentation as determined by Hoechst PI staining. The arrowheads indicate the apoptotic bodies.

levels of oxidative stress are increased in APP/PS1 double mutant neurons. We also tested the hypothesis that there is an increased vulnerability of oxidative stress and neurotoxicity in APP/PS1 neuronal cultures induced by Ab(1–42), H2O2, and KA. The results of this study strongly support both hypotheses. Mitochondria are particularly vulnerable to oxidative damage. Mitochondrial dysfunction has been observed in AD brain (Hirai et al. 2001). It has been reported that Swedish APP (APPsw)-bearing cells show decreased mitochondrial membrane potential after exposure to H2O2 (Eckert et al. 2003). In addition, the activity of the executor caspase 3 after treatment with H2O2 was elevated in APPsw double mutation cells, which seems to be the result of an enhanced activation of both intrinsic and extrinsic apoptotic pathways (Eckert et al. 2001). Apoptosis is attributed, not only to normal development, but also to neurodegenerative diseases and neurological disorders, such as AD and Parkinson’s disease (Mattson 2003). There is evidence suggesting that oxidative stress also plays a key role in the Ab-mediated neurotoxicity because

increased levels of H2O2 were detected (Behl et al. 1994; Schubert et al. 1995; Huang et al. 1999). Ab peptide induces apoptosis in mouse neuronal cultures (Loo et al. 1993). Su et al. (1994) reported evidence for DNA fragmentation in neurons from patients with AD. Studies in AD have established that high levels of Ab(1–42) result in neuron death via apoptosis (Selkoe 1997). Several lines of evidence suggest that b-amyloid is involved in the neurodegenerative cascade of AD (Lue et al. 1999; Butterfield and Lauderback 2002). In the present study, Ab(1–42), H2O2, and KA were used to study morphological changes by examining the dynamic neuronal-network processing in developing cortical neurons, and the results are in agreement with the above findings, suggesting that the neuronal cells bearing APP and PS1 (Figs 5 and 6) mutations are more vulnerable to apoptosis compared with wild-type cells. One of the mitochondrial specific events that has been well defined in apoptosis is the cytosolic translocation of apoptogenic factors such as cytochrome c and apoptosis-inducing factor (BossyWetzel et al. 1998; Fulda et al. 1998). Our results (Fig. 7) are consistent with this notion because we found a significant

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Fig. 7 The effect of different neurotoxic agents on neuronal cell culture in a dose–response manner as assessed by MTT reduction assay. (a) Ab(1–42), (b) KA, (c) H2O2. HO/HO refers to APP/PS1 neurons. Results shown are mean ± SEM obtained for five independent preparations. Significance was assessed by two-way ANOVA; p < 0.0001. Note the lower concentration of added neurotoxic agents necessary for inducing cell death in APP/PS1 double mutant neurons.

increase (dose-dependent) in released cytochrome c levels, both in the wild-type and APP/PS1 neuron cultures treated with Ab(1–42), H2O2, or KA. However, for APP/PS1 cells, cytochrome c release occurred at lower levels of each of the three oxidative stress-inducing agents, consonant with the hypothesis of increased vulnerability of APP/PS1 neurons to

apoptotic insults. Presumably, the elevated levels of Ab(1– 42) in APP/PS1 cells (Fig. 9) led to this increased vulnerability. Increased markers of lipid peroxidation, including 4-hydroxynonenal and acrolein, occur in AD brain (Markesbery and Lovell 1998; Calingasan et al. 1999; Lauderback et al. 2001; Lovell et al. 2001). There is increased protein oxidation in the brain in AD (Smith et al. 1991; Hensley et al. 1995). Additionally, markers of oxidative stress in AD include nitrotyrosine (Smith et al. 1997; Castegna et al. 2003). Earlier studies suggest that primary hippocampal neurons from PS1 mutant knock-in mice exhibit increased vulnerability to Ab-peptide toxicity (Guo et al. 1999). The current study showed a significant basal increase in protein oxidation, lipid peroxidation, and 3-nitrotyrosine formations in primary neuron cultures from knock-in mice expressing mutant APP/PS1 over that of wild-type mice (Fig. 1). This result suggests that the mutations in APP and PS1 in the absence of external stressors significantly increase oxidative stress. Similar results were found in the brain of APP/PS1 mice (Mohmmad Abdul et al. 2004). Increased RNA oxidation was also observed in the frontal cortex of FAD associated with a mutation in the PS-1 or APP gene (Nunomura et al. 2004). There is an increased oxidative stress in synaptosomal proteins from mutant PS-1 knock-in mice (LaFontaine et al. 2002). Moreover, in this present study, in the presence of three oxidative stress-inducing agents, Ab(1–42), H2O2, and KA, neuronal cells bearing mutations in both APP and PS1 are more vulnerable to oxidative stress (at lower concentrations of these respective agents) compared with wild-type cells. The polyunsaturated fatty acids of membrane phospholipids are highly susceptible to peroxidation by ROS and a self-propagating chain of free radical reactions can produce various aldehydes, alkenals and hydroxyalkenals, including malondialdehyde and HNE (Schneider et al. 2001; Butterfield et al. 2002). These aldehydes are cytotoxic and generally more stable than ROS and can cause extensive damage to proteins and other cellular constituents. Addition of H2O2 to cells in culture can lead to transition metal iondependent hydroxyl radical-mediated oxidative DNA damage (Spencer et al. 1996). H2O2 can also react with superoxide radicals to form more reactive hydroxyl radicals in the presence of trace amounts of Fe2+ or Cu2+ (Thompson et al. 1987). The hydroxyl radicals initiate self-propagating reactions leading to peroxidation of membrane lipids and destruction of proteins (Asada and Takahashi 1987; Halliwell 1987; Bowler et al. 1992). Hence, ROS and the resulting oxidative stress play a key role in apoptosis. Our results (Fig. 3) are consistent with this notion because we found a significant increase (dose dependent) in DCF fluorescence levels in the APP/PS1 neuron cultures treated with Ab(1–42), H2O2, or KA compared with wild type. However, for APP/PS1 cells, the increase in the DCF fluorescence

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Fig. 8 Wild-type and APP/PS1 neuronal cultures were treated with different concentrations of Ab(1–42), H2O2 and KA, incubated for 24 h, following which cytochrome c release in the culture supernatants was analyzed by western blot analysis. AB, Ab(1–42); KA, kainic acid. (a) Representative western blot of wild-type neuron culture supernatants. Lane 1, control (without any treatment); lane 2, 2 lM Ab (1–42); lane 3, 5 lM Ab(1–42); lane 4, 10 lM Ab(1–42); lane 5, 25 lM Ab(1– 42); lane 6, 50 lM kainic acid; lane 7, 100 lM kainic acid; lane 8, 150 lM kainic acid; lane 9, 10 lM H2O2; lane 10, 25 lM H2O2; lane 11, 50 lM H2O2; lane 12, 75 lM H2O2. The blots were probed with anticytochrome c monoclonal antibody (Sigma). (b) Representative western blotting of APP/PS1 neuron culture supernatants. The treatment of samples in each of these lanes is in the same order as Fig. 8(a). The blots were probed with anti-cytochrome c monoclonal antibody (Sigma). (c) Bar graph representation of cytochrome c release from the data obtained from western blot densitometry analysis of wild-type neurons (Fig. 8a). Results shown are mean ± SEM

obtained for five independent preparations. Significance was assessed by one-way ANOVA; *p < 0.05 and **p < 0.005 versus control. (d) Bar graph representation of cytochrome c release from the data obtained from western blot densitometry analysis of APP/PS1 double mutant neurons (Fig. 8b). Results shown are mean ± SEM obtained for five independent preparations. Significant differences were assessed by one-way ANOVA; *p < 0.05 and **p < 0.005 versus control. (e) Representative western blot to verify equal loading of protein (from wild-type culture supernatant) by analyzing the levels of GAPDH: After respective treatments, as in Fig. 8(a), 100 lg of the proteins in the culture supernatants were separated by 4–15% SDS–polyarylamide gel electrophoresis, blotted onto a nitrocellulose membrane and probed with anti-GAPDH monoclonal antibody (Stressgen Bioreagents). (f) Representative western blot to verify equal loading of protein (from APP/PS1 culture supernatant) by analyzing the levels of GAPDH: After respective treatments as in Fig. 8(b), the remaining methodology is the same as followed in Fig. 8(e).

occurred at lower concentrations of each of the three oxidative stress-inducing agents, suggesting increased vulnerability of APP/PS1 neurons. Kainic acid, which binds to kainate receptors and is an agonist for kainate and a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors, is an excitotoxin in the hippocampus (Campochiaro and Coyle 1978; Nadler et al. 1978; Cook and Crutcher 1986). KA is a potent excitatory analog of glutamic acid, which targets a subset of glutamatergic receptors, resulting in hyperstimulation and eventual death of neurons, predominantly the pyramidal neurons in the CA3 region of the hippocampus (Schwob et al. 1980; Ben-Ari 1985). The mode of cell death

appears to be apoptotic (Pollard et al. 1994). KA hyperstimulates hippocampal neurons via KA receptors, inducing depolarization (Sari and Kerr 2001). This strong and persistent depolarization also results in NMDA receptor stimulation, leading to an influx of calcium ions. The increase in cytoplasmic calcium ions (Lafon-Cazal et al. 1993) triggers a variety of intracellular cascades through stimulation of enzymes, including proteases, phospholipase A2, and nitric oxide synthase, which also lead to increased levels of free radical species (Farooqui et al. 2001). In rats injected with KA, about 30% of enthorinal cortical cells were stained for specific DNA fragmentation, 5% exhibited apoptotic morphology, 5–40% over-expressed Fas-L, and

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2% of dying cells expressed cleaved, i.e. activated, caspase 3 (Puig and Ferrer 2002). This type of overexcitation-induced neuronal damage is accompanied by calcium influx and the generation of ROS, which in turn results in damage of intracellular membranes and subsequently delayed cell death (Sun and Chen 1998). Our present study with KA is consistent with these studies, as suggested by the increase in protein carbonyls, HNE, 3-NT and DCF levels in the neuronal cultures. Rare point mutations in APP cause early-onset FAD by deviating APP into an amyloidogenic direction with increased amyloid peptides, especially the longer, lesssoluble Ab(1–42) form (Younkin 1995; Selkoe 1996). Mutations in presenilins cause most early onset FAD cases and similarly increase amyloid peptides (Borchelt et al. 1996; Scheuner et al. 1996). Earlier studies suggest that there is increased oxidative stress in the temporal inferior cortex from patients with Swedish FAD bearing the APP670/671 mutation (Bogdanovic et al. 2001). One potential mechanism by which APP mutations enhance this process could be the increased production of Ab, which is able to induce apoptotic cell death (Siman et al. 1996). Our studies regarding determining the intracellular

levels Ab are consistent with the notion that increased production of Ab at physiological levels primes APP/PS1 neurons to undergo death and leads to increased cell death only after additional oxidative stress in the presence of Ab(1–42), H2O2, and KA, a scenario which may occur in the brain in AD. Our results suggest that already the basal Ab levels in APP/PS1 cells are probably sufficient to induce significant oxidative stress and prime cells to undergo apoptosis. Hence, exposure of increased concentrations of Ab(1–42), H2O2, and KA to wild-type and APP/PS1 cells further strengthen the effects of oxidative stress. Moreover, Ab is produced intracellularly and can accumulate within cells (Hartmann 1999; Wirths et al. 2001). Intracellular accumulation of Ab, soluble or insoluble, might impair cellular functions and may represent the primary event within the neurotoxic Ab cascade. Our results of cytochrome c release provide evidence that the massive neurodegeneration in APP/PS1 neurons could be a consequence of an increased vulnerability of neurons to mitochondrial abnormalities, resulting in activation of apoptotic pathways as a consequence to elevated oxidative stress levels. It is possible that the vulnerability of neurons in FAD may lead to an earlier onset and more aggressive form of the disease. Although Ab(1–42)-, H2O2-, and KA-mediated cell death is observed both in vitro and in vivo and may be apoptotic in nature, the underlying mechanisms triggering neuronal death as a result of oxidative stress are still not completely understood. Overall, our results indicate that neuronal cells bearing APP and PS1 mutations have a higher basal oxidative stress and are more vulnerable to oxidative stress compared with wild-type cells. These results are in agreement with the notion that APP and PS1 mutations cause the aggressive form of FAD, leading to a more rapid disease process. Identification of molecular targets involved in the increased basal levels of oxidative stress and increased APP/PS1 neurons to oxidative insults may provide insight into the mechanisms underlying these effects and may be useful for development of new therapeutic strategies in FAD. Such studies are underway in our laboratory. Acknowledgements This research was supported in part by NIH grants AG-10836 to DAB and AG-05119 to DAB, JNK, DSC., and WRM. We thank Cephalon Inc., Frazer, PA, USA for the donation of the animals used in this study.

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