JOURNAL OF NEUROCHEMISTRY

| 2015 | 132 | 559–571

doi: 10.1111/jnc.13012

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*Immunology and Infectious Diseases Laboratory, Department of Clinical Biochemistry and Pharmacology, Ben-Gurion University of the Negev and Soroka University Medical Center, BeerSheva, Israel †The Shraga Segal Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, Beer-Sheva, Israel ‡Department of Physiology and cell Biology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Abstract Amyloid-b peptides generated by proteolysis of the b-amyloid precursor protein (APP) play an important role in the pathogenesis of Alzheimer’s disease. The present study aimed to determine whether cytosolic phospholipase A2a (cPLA2a) plays a role in elevated APP protein expression induced by aggregated amyloid-b1-42 (Ab) in cortical neurons and to elucidate its specific role in signal events leading to APP induction. Elevated cPLA2a and its activity determined by phosphorylation on serine 505 as well as elevated APP protein expression, were detected in primary rat cortical neuronal cultures exposed to Ab for 24 h and in cortical neuron of human amyloid-b1-42 brain infused mice. Prevention of cPLA2a up-regulation and its activity by oligonucleotide antisense against cPLA2a (AS) prevented the elevation of APP protein in cortical neuronal cultures and in mouse neuronal cortex. To determine the role of cPLA2a in the signals leading to APP induction, increased cPLA2a expression and activity induced by Ab was prevented by means of

AS in neuronal cortical cultures. Under these conditions, the elevated cyclooxygenase-2 and the production of prostaglandin E2 (PGE2) were prevented. Addition of PGE2 or cyclic AMP analogue (dbcAMP) to neuronal cultures significantly increased the expression of APP protein, while the presence protein kinase A inhibitor (H-89) attenuated the elevation of APP induced by Ab. Inhibition of elevated cPLA2a by AS prevented the activation of cAMP response element binding protein (CREB) as detected by its phosphorylated form, its translocation to the nucleus and its DNA binding induced by Ab which coincided with cPLA2a dependent activation of CREB in the cortex of Ab brain infused mice. Our results show that accumulation of Ab induced elevation of APP protein expression mediated by cPLA2a, PGE2 release, and CREB activation via protein kinase A pathway. Keywords: APP, cortical neurons, cytosolic phospholipase A2a. J. Neurochem. (2015) 132, 559–571.

Neurodegenerative diseases, such as Alzheimer’s disease (AD) are defined by the progressive loss of specific neuronal cell populations coupled by extensive evidence of oxidative stress and inflammatory processes, which might be responsible for the dysfunction or death of neuronal cells (Barnham et al. 2004). Ab is the major constituent of amyloid plaques and is generated through proteolytic processing of the b-amyloid precursor protein (APP) (Haass et al. 1993). APP is widely expressed in all neurons and some glial cells

Received June 24, 2014; revised manuscript received December 02, 2014; accepted December 03, 2014. Address correspondence and reprint requests to Prof Rachel Levy, Infectious Diseases Laboratory, Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel. E-mail: [email protected] 1 These authors contributed equally to this work. Abbreviations used: AD, Alzheimer’s disease; APP, amyloid precursor protein; COX, cyclooxygenase; CREB, cAMP response element binding protein; PBS, phosphate-buffered saline; PKA, protein kinase A.

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in the central and peripheral neuron systems (Card et al. 1988; Kawarabayashi et al. 1991) and is transferred from cell body to nerve ending by fast anterograde axonal transport (Koo et al. 1990). Increased levels of APP gene transcript in specific areas of AD brains (Cohen et al. 1988; Higgins et al. 1988) as well as APP accumulation in dystrophic neurites of the senile plaques have been observed (Shoji et al. 1990). This suggests that over-expression of APP may at least be one of several contributing factors in the formation of amyloid depositions and in the neuropathology associated with AD. Cytosolic phospholipase A2a (cPLA2a) is ubiquitous in brain cells and is essential for their physiological regulation. However, previous findings indicated that cPLA2a is also involved in different forms of neurodegeneration. Increased immunoreactivity of the cPLA2a and of cPLA2a transcript were observed in AD cortex (Clemens et al. 1996; Stephenson et al. 1996, 1999; Colangelo et al. 2002). Furthermore, in AD-affected cortex, cPLA2a was often associated with amyloid deposits (Clemens et al. 1996). cPLA2a specifically hydrolyzes phospholipids containing arachidonic acid (AA) at the sn-2 position (Clark et al. 1990; Kramer and Pepinsky 1991) and is generally thought to be the ratelimiting step in the generation of eicosanoids by cyclooxygenase (COX) and lipoxygenase enzymes. The role of COX in the processing of APP and promoting amyloidosis in the brain was first demonstrated (Xiang et al. 2002) by crossing mice expressing both mutant APP and mutant PS1 with mice expressing human COX-2 selectively in neurons. Similarly, expression of human COXs in human APP-overexpressing Chinese hamster ovary cells or human neuroglioma cells resulted in increased PGE2 concentration in the conditioned medium and coincided with elevation of Ab peptide generation (Qin et al. 2003). The role of EP2 and EP4 in PGE2 mediated production of Ab peptides was also demonstrated (Hoshino et al. 2007). In addition, it was recently reported that 5-lipoxygenase regulates the formation of Ab by activating the cAMP response element binding protein (CREB), which in turn increases transcription of the c-secretase complex (Chu and Pratico 2011). The present study aimed to examine whether cPLA2a regulates the induction of neuronal APP protein expression induced by Ab and to characterize the signaling events leading to its upregulation. To this end, we used Ab in rat primary cortical neuron cultures and in a mouse model of Ab brain infusion (Craft et al. 2004a,b, 2006) as the regulation of APP production cannot be explored in human APP transgenic mice. The mouse model of Ab brain infusion was reported to mimic many features of AD, including robust neuroinflammation, Ab plaques, synaptic, and neuronal damage. Especially important is the significant neuron damage and loss, a feature not generally seen in transgenic mouse models of AD. The quantifiable endpoint pathology is robust, reproducible, and rapid in onset.

Materials and methods Cell cultures Embryos of 18-day-old pregnant Sprague–Dawley rats (Harlan, Israel) were used. Brain cortices were removed and suspended in 1.5 mL of HEPES-buffered salt solution lacking calcium and magnesium (10 mM HEPES, 1 mM pyruvate, 5.6 mM glucose, 0.44 mM KH2PO4, 5.4 mM KCl, 0.18 mM NaH2PO4, 4.2 mM NaHCO3, and 137 mM NaCl) and digested for 7 min at 37°C with trypsin at a final concentration of 0.12%. Then, soybean trypsin inhibitor and 400 units of DNaseI were added, and the tissue was mechanically dissociated with a fire-polished Pasteur pipette. After adding HEPES-buffered salt solution containing Ca2+ and Mg2+ (10 mM HEPES, 1 mM pyruvate, 5.6 mM glucose, 0.44 mM KH2PO4, 5.4 mM KCl, 0.18 mM NaH2PO4, 4.2 mM NaHCO3, 137 mM NaCl, 0.9 mM MgCl2, and 1.1 mM CaCl2) to the suspension and decanting tissue clumps, the cells were plated in poly-L-lysine-coated 6 or 96-well plates at a concentration of 1 9 106 cells/mL. For immunocytochemistry, the cells were seeded on 70% ethanol-washed glass coverslips coated with 0.1 mg/mL poly-L-lysine. The culture medium consisted of Neuro- Basal medium supplemented with B-27, 0.4 mM glutamine, and penicillin/streptomycin. After 7 days in vitro, the cortical population was determined to be at least 96% neuronal by immunostaining. All experiments were performed on 7 days in vitro neurons (Kartvelishvily et al. 2006). The study was approved by Ben-Gurion University Institutional Animal Care and Use Committee (IL-62-11-2009), and was conducted according to the Israeli Animal Welfare Act following the guidelines of the Guide for Care and Use of Laboratory Animal (National Research Council, 1996). Antisense oligonucleotides against cPLA2a Antisense oligonucleotides against cPLA2a were engineered using the computer-based approach RNADraw V1.1 (Mazura Multimedia, Stockholm, Sweden). An oligonucleotide antisense (tcaaaggtctcattccaca) and its corresponding sense with phosphorothioate modifications on the last 3 bases at both 50 and 30 ends were used. The specificity to cPLA2a was analyzed by blast search program, and was demonstrated in our previous study (Liberty et al. 2004). The antisense or sense were dissolved in the medium and added to the cultures. The antisense are efficiently taken up by the cells (Raichel et al. 2008). Animals Male C57Bl/6 mice (Jackson, Maine Bar Harbor, USA) were used. The study was approved by Ben-Gurion University Institutional Animal Care and Use Committee (IL-62-11-2011), and was conducted according to the Israeli Animal Welfare Act following the guidelines of the Guide for Care and Use of Laboratory Animal (National Research Council, 1996). Animals were kept in a 12-h light/12-h dark cycle with food and water ad libitum. Ab brain infusion The infusion of human Ab1-42 into Male C57Bl/6 mice was performed according to (Craft et al. 2004a,b, 2006). A microosmotic pump (Alzet; Durect Corp., Cupertino, CA, USA) was attached to a cannula (Alzet brain infusion kit 3, Durect Corp.) stereotaxically implanted into the right lateral cerebral ventricle

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(1.0 mm mediolateral and 0.5 mm anteroposterior from Bregma). Pumps contained either soluble Ab1-42 (American Peptide, Sunnyvale, CA, USA) in vehicle (4 mM Hepes + 250 lg/mL human high-density lipoprotein) or vehicle alone. High-density lipoprotein, which normally carries Ab in plasma, was used to prevent Ab aggregation and act as an Ab chaperone for better delivery (Frautschy et al. 1996, 2001). The Ab infusion rate was 67 ng/h. Evaluation of behavioral performance in the Y-Maze The Y-maze test of spontaneous alternation was used to evaluate the cognitive performance of the mice as done before for this mouse model (Craft et al. 2004a,b, 2006). Each mouse was placed within the ‘start’ arm and then released to choose one of the other two arms. Once the mouse entered the chosen arm, it was blocked from exiting the arm for 30 s. The mouse was then placed back within the start arm for 30 s and released again to choose one of the two arms. The mouse was returned to its home cage immediately after the second choice was made. The mouse was scored as alternating for the trial, if the second choice was different from the first choice. Mice were tested for 10 days with one trial per day, and a mean of percent alternation was calculated for each mouse. Brain tissue preparation Mice were deeply anesthetized and transcardially perfused with 30 mL of phosphate-buffered saline (PBS) and 30 mL of Paraformaldehyde 4%/PBS. For immunoblot analysis Brains were harvested in Lysis buffer containing 50 mM Tris pH = 8, 150 mM NaCl, 1% NP-40, 0.5% (deoxycholate) DOC, 0.1% sodium dodecyl sulfate (SDS), 10 lg/mL Leupeptin, 1 mM Phenylmethylsulfonyl fluoride, 10 lg/mL Aprotinin, 1 mM Benzamidine, 20 mM P-nitrophenyl phosphate, 5 mM Na3VO4,10 mM NaF, and 50 mM b-glycerol phosphate. Soluble extracts were prepared by centrifugation at 13 000 g for 20 min at 4°C. Lysate protein was separated on gel electrophoresis and transferred to nitrocellulose membranes. Membranes were incubated in Trisbuffered saline (10 mM Tris, 135 mM NaCl, pH = 7.4), with 0.1% Tween 20 (TBS-T) containing 5% non-fat milk for 1.5 h at 25°C. The blots were then incubated with primary antibodies: 1 : 1000 rabbit polyclonal anti-cPLA2a against the carboxytreminal residues (Cell Signaling Technology, Beverly, MA, USA), 1 : 1000 rabbit polyclonal anti-phospho-cPLA2a (Serine 505) against residues surrounding (Cell Signaling Technology), 1 : 1000 rabbit antiAPP against a synthetic peptide corresponding to the N-terminal region amino acid 46-60 (Sigma, Rehovot, Israel), 1 : 1000 rabbit anti-COX-2 (1 : 1000; Abcam, Cambridge, UK), 1 : 500 mouse anti-CD-68 (Abcam), 1 : 1000 mouse anti-MAP-2 (microtubule associated protein 2) (Chemicon, Temecula, CA, USA) or 1 : 500 rabbit anti-Synaptophysin (Abcam) as primary antibodies for overnight at 4°C. After washing with TBS-T, they were incubated with second antibody; peroxidase conjugated goat anti-rabbit or anti-mouse (Amersham Biosciences, Buckinghamshire, UK) for 1 h at 25°C and developed using the enhanced chemiluminescence detection system (PerkinElmer, Waltham, MA, USA). Proteins were quantified using video densitometry analysis (ImageGauge version 4.0 Fuji, Fuji Photo Film Co. Ltd, Tokyo, Japan).

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For immunostaining Brains were post-fixed in the perfusion solution (paraformaldehyde 4%/PBS) overnight at 4°C and then cryoprotected for 24 h in 30% sucrose in PBS. Then brains were embedded in OCT/sucrose (2 : 1), frozen in liquid nitrogen and stored at 80°C. Sections were made by cryostat (Leica Biosystems, Vienna, Austria) at 16 lm thickness and incubated in 3% normal donkey serum and 2% bovine serum albumin at 20°C for 1 h. Then, these sections were incubated with primary antibodies diluted in blocking solution overnight at 4°C 1 : 400 mouse anti-MAP-2 (Chemicon), 1 : 100 rabbit polyclonal anti-cPLA2 against amino acids 1–216 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1 : 150 rabbit anti-COX-2 (Abcam), 1 : 1000 rabbit anti-Iba-1 (Wako Chemicals, Richmond, VA, USA) 1 : 100 mouse monoclonal anti-APP that recognizes amino acid 66-81 of N-terminus on the preA4 molecule (Chemicon), 1 : 100 rabbit anti-APP (described above), 1 : 50 goat anti Iba1 (Novus Biologicals, Littleton, CO, USA), 1 : 100 mouse anti-NeuN (Millipore, Billerica, MA, USA), 1 : 500 rabbit anti-GFAP (Glial Fibrillary acidic protein) (Dako Denmark, Glostrup, Denmark), or 1 : 100 rabbit anti-p-CREB (Cell Signaling Technology). Sections were washed with PBS/tween 0.05%, and incubated with Cy3 or Cy2 conjugated secondary antibodies (1 : 200; Jackson Immunoresearch Laboratories, West Grove, PA, USA) for 1 h at 20°C. Sections were mounted with anti-fading mounting medium (Electron Microscopy Sciences, Hatfield, PA, USA) and photographed with fluorescent microscopy (BX60 Olympus, Hamburg, Germany) or with confocal microscopy (FluoView FV1000 Olympus, Tokyo, Japan). Double Staining of microglia and amyloid beta was performed using Avidin-biotin VECTA-STAIN Kit Elite PK 6102 for anti-mouse and 6101 for anti-rabbit (Vector Laboratories, Burlingame, CA, USA). The cortex slices were incubated with 1 : 1000 rabbit anti-Iba-1 (Wako Chemicals Richmond). After developing the brown microglia staining, the slices were incubated with 1 : 500 mouse anti-human Beta Amyloid Monoclonal antibody 6E10 (Covance, Dedham, MA, USA) and the blue Ab staining was developed using True blue substrate (KPL, Gaithersburg, MD, USA). Cell culture western blot analysis Following specified treatments, primary cortical neuron cultures were washed once with ice-cold PBS and harvested in Lysis buffer containing 50 mM Hepes, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% Glycerol, 1% Triton X-100, 10 lM MgCl2, 10 lg/mL Leupeptin, 1 mM phenylmethylsulfonyl fluoride, 10 lg/mL Aprotinin, 1 mM Benzamidine, 20 mM p-nitrophenyl phosphate, 5 mM Na3VO4, and 10 mM NaF. Soluble extracts were prepared by centrifugation at 13 000 g for 30 min at 4°C. For preparation of nuclear fractions, cells were washed once with ice-cold PBS and homogenized by shearing through a 22-ga needle in HS buffer (250 mM sucrose, 150 mM NaCl, 10 mM Tris-HCl and 5 mM EDTA, pH = 7.4) supplemented with the same protease inhibitors mentioned above. Samples were centrifuged at 1500 9 g for 10 min and the pellet was resuspended in HD buffer (10 mM Tris-HCl, 150 mM NaCl, 1% SDS, and 5 mM EDTA, pH = 7.4) with the protease inhibitors. All steps were carried out on ice. Lysate protein (40 lg) was separated by electrophoresis on 7.5% polyacrylamide SDS gels transferred, immunoblotted and quantified as described above.

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Preparation of aggregated Ab Aggregated Ab (American Peptide) were prepared by incubating freshly solubilized peptides at 250 lM in sterile distilled water at 37°C for 7 days to allow formation of aggregates (Pike et al. 1993; Dahlgren et al. 2002; Szaingurten-Solodkin et al. 2009). The formation of aggregates (data not shown) was determined by western blot analysis on tricine gel using antibody that recognizes 1–16 amino acids of human amyloid 1-42 (1 : 1000; Covance, Princeton, NJ, USA) as shown in our previous study (Sagy-Bross et al. 2013). PGE2 determination PGE2 levels were determined in cell supernatants by using a dextrancoated charcoal radioimmunoassay protocol. The samples were immediately stored at 70°C and analyzed within 1 week from the experiments. Briefly, 100 lL sample of PGE2 standard (Sigma) was incubated in the presence of 500 lL anti-PGE2 antiserum (Sigma) for 30 min. [3H]PGE2 (Amersham Biosciences) was added next for 24 h at 4°C. Twenty-four hours later, 200 lL cold dextran-coated charcoal suspension was added to each tube and incubated for 10 min on ice. The tubes were centrifuged at 15009 g for 15 min at 4°C. A total of 500 lL supernatants-containing [3H] PGE2–anti-PGE2 complexes were counted (Packard Spectrometry 1900CA; Packard Instruments, Company Inc., Meriden, CT, USA), and the amount of PGE2 was calculated (Hadad et al. 2011). Gel mobility shift assay The promoter region was screened for cAMP responsive element (CRE) by the TF program at http://www.cbrc.jp/research/db/ TFSEARCH.html (Heinemeyer et al. 1998). Electrophoretic mobility Fig. 1 The involvement of cPLA2a in elevated amyloid precursor protein (APP) induced by Ab. (a) A representative immunoblot analysis of APP, phosphor-cPLA2a (p-cPLA2a), cPLA2a, and the corresponding calreticulin protein expression in neuronal cell cultures preincubated for 24 h with 1 lM antisense against cPLA2a (AS) or sense (SE) before addition of 1 lM Ab (Ab) for additional 24 h. cPLA2a or APP protein expression was determined by dividing the intensity of each cPLA2a or APP by the intensity of the corresponding calreticulin band after quantitation by densitometry and expressed in the bar graph as arbitrary units. The bar graph is the mean  SE from three independent experiments. *p < 0.001 – significant elevation by Ab treatment; **p < 0.001 significant reduction in the presence of AS compared with its absence in Ab treated cells. #p < 0.01 significant reduction in the presence of AS compared with its absence. (b–e) Brain cPLA2a is upregulated and activated by Ab brain infusion. A representative immunoblot of phosphor-cPLA2a (p-cPLA2a), cPLA2a, and the corresponding calreticulin protein expression of mice cortex lyzates (b) or hippocampus lyzates (d) at 2, 4, and 8 weeks of Ab infusion compared to buffer. cPLA2a was determined as in (a). The bar graph is the mean  SE of five mice. *p < 0.05 between Ab infused mice compared with control mice infused with buffer. Mice cortex sections (c) or hippocampus sections (e) of cPLA2a protein expression at 2, 4, and 8 weeks of Ab infusion compared to buffer. Scale bars = 200 lm. Shown are representative images of five mice in each group. (f) Western blot analysis (left panel) of Ab solution from the infusion pump at the end of the experiment showed that Ab was in its soluble monomeric form (inf Ab). For controls aggregated Ab (aggr. Ab) and monomer (mono Ab) were analyzed. Representative cortex sections

shift assays (EMSAs) were performed using Light Shift Chemiluminescent EMSA kit (Thermo scientific, Rockford, IL USA). The designed double-stranded oligonucleotides containing the consensus sequence for CREB (50 -TGAAGGTGACGTCAAAGTC-30 ) endlabeled with biotin. Nuclear extracts (20 lg of protein) were preincubated with 0.02 A260 units of poly (d(I-C)) for 5 min in nuclear extraction buffer. The extracts were then incubated for an additional 30 min at 20°C with 250 fmol biotin-labeled, doublestranded oligonucleotide. Specificity of CREB binding was estimated by competition with unlabeled wild-type CREB with 50-fold molar excess, added during the preincubation period. The complexes were separated on a 7% non-denaturing polyacrylamide gel in 0.5x TBE buffer with a constant current of 20–25 mA, for 2–3 h at 4°C. The gel was transferred at 380 mA for 30 min and cross-linked using UV cross-linker 230VAC (Hoffer, San Francisco, CA USA). The Biotinlabeled DNA was detected by chemiluminescence. Statistical analysis Data were expressed as mean  SEM. Significant differences from control conditions were determined using either one- or two-way ANOVA followed by a posteriori Bonferroni’s test for multiple comparisons provided by GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA).

Results cPLA2a is involved in induction of APP by Ab In our previous study, we have shown that addition of Ab to rat cortical neuronal cultures induced a dose dependent (right panel) from buffer infused mice and Ab infused mice stained with anti-human Ab to stain amyloid plaques (stained in blue). The microglia in the slices are stained in brown. Scale bars = 5 lm. (g–j) Inhibition of activated and elevated cPLA2a protein expression induced by Ab brain infusion by AS. Mice were infused with either buffer, Ab, Ab+AS, or Ab+sense for 4 weeks and killed at 8 weeks. A representative immunoblot of phosphor-cPLA2a (p-cPLA2a), cPLA2a, and the corresponding calreticulin protein expression in mice cortex lyzates (g) or hippocampus lyzates (i). Densitometry analysis for cPLA2a was preformed as in (a). The bar graph is the mean  SE from five mice. *p < 0.001 significant increase in mice infused with Ab or Ab+sense in comparison to control mice (infused with buffer) or mice infused with Ab+AS. Mice cortex sections (h) or hippocampus sections (j) of cPLA2a protein expression. Scale bars = 200 lm. Shown are representative images of five mice in each group. (k and l) Inhibition of cPLA2a protein expression induced by Ab brain infusion by AS. Mice were infused with either buffer or sense (three mice in each) and six mice were infused with AS for 4 weeks and killed at 8 weeks. A representative immunoblot of phosphor-cPLA2a (p-cPLA2a), cPLA2a, and the corresponding calreticulin protein expression in mice cortex lyzates or hippocampus lyzates (k). The mice treated with buffer of sense showed similar results and Densitometry analysis was performed as in (a). The mice treated with buffer of sense showed similar results. The bar graph is the mean  SE from six mice. #p < 0.01 significant reduction in the presence of AS compared with its absence. Mice cortex sections or hippocampus sections (l) of cPLA2a protein expression. Scale bars = 200 lm. Shown are representative images of six mice in each group.

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apoptotic cell death (Sagy-Bross et al. 2013). Here, we show that prolonged 24 h exposure of rat cortical neuronal cultures to 1 lM Ab induced elevation of APP protein expression that coincided with the elevation of cPLA2a protein expression and its activity as detected by phosphor-cPLA2a on Serine 505 (Fig. 1a). Under these conditions, we have shown a decrease in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction without affecting LDH (Lactate Dehydrogenase) release (Sagy-Bross et al. 2013) similar to the effect of oligomeric Ab (Kriem et al. 2005; He et al. 2011). Pre-incubation with AS against cPLA2a for 24 h prior to addition of Ab, prevented the increased protein expression and activation of cPLA2a as well as the elevation of APP protein expression, suggesting the role of cPLA2a in APP protein induction in cortical neuronal cultures. Pre-incubation of control cells with AS inhibited the expression and activity of cPLA2a. In order to determine whether elevated

cPLA2a protein expression and activity regulate APP overproduction by Ab in vivo as well, the human Ab brain infusion mouse model (Craft et al. 2004a,b, 2006) was studied. To define the temporal onset of cPLA2a in the Ab infusion model, we used the experimental paradigm of implanting human Ab-containing micro-osmotic pumps designed to deliver their contents over 2 or 4 weeks into 3 months old C57Bl/6 mice. The mice were killed at 2, 4, and 8 weeks of Ab infusion. A significant increase in cPLA2a protein expression and its activated form was detected at 2 weeks of Ab infusion and remained elevated during 8 weeks in the cortex (Fig. 1b) and the hippocampus (Fig. 1d) as detected by immunoblot analysis. The elevation of cPLA2a was further confirmed by immune-staining with anti- cPLA2a (Fig. 1c and e). As reported earlier (Craft et al. 2004b) aggregates of human amyloid beta could be detected in the brain of Ab intracerebroventricular infused mice

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(Fig. 1f). Ab was found in its soluble form during the whole infusion period (Fig. 1f), indicating that the soluble Ab formed aggregates in the brain. Infusion of AS (but not sense) together with Ab for 4 weeks prevented the elevated cPLA2a protein expression and activation as analyzed at 8 weeks by both western blot analysis and immunohistochemistry in the cortex (Fig. 1g and h) and in the hippocampus (Fig. 1i and j). Brain infusion of AS alone caused a reduction of cPLA2a expression and activity (Fig. 1k and l). Under these conditions, of 4 weeks infusion of Ab, a significant increase in APP protein expression was detected in the cortex at 8 weeks, by western blot analysis and immunostaining (Fig. 2a and b). The AS treatment that prevented the increased cPLA2a protein expression and activity (Fig. 1g and h) in Ab brain infused cortex as detected at 8 weeks of start of infusion also prevented the increased APP protein expression (Fig. 2a and b), while the sense treatment that did not affect the elevated cPLA2a protein expression and activity, had no effect on the elevated APP protein expression). Brain infusion of AS alone caused a reduction of APP expression in the cortex that coincided with the reduction of cPLA2a expression and activity in these mice (Fig. 1k and l). Although there was a significant elevation of cPLA2a in the hippocampus an elevation of APP protein could not be detected at this stage (Fig. 2c). Double immune-staining analysis (Fig. 2d) showed that APP was expressed mainly in the neurons as detected by anti-NeuN and was hardly expressed in astrocytes or microglia as detected by anti-GFAP and anti-Iba1, respectively. High staining of cPLA2a was detected mainly in the neurons as shown by the double staining of NeuN and cPLA2a (Fig. 2e). Double staining of cPLA2a and APP showed that both proteins are elevated in the Ab infused cortex and this elevation is prevented by AS treatment. Confocal microscopy demonstrated (Fig. 2f) that in the control mice cPLA2a is found in the cortical neuronal cell cytosol, while APP was found in cell membranes. In neuronal cells of mice infused with Ab or with Ab+sense, both cPLA2a and APP were elevated and APP was detected in the membranes as well as in the cytosol overlapping with cPLA2a. In the Ab braininfused mice treated with AS, the protein expression of both cPLA2a and APP in the cortical neurons was lower than in cortical neurons of the Ab brain infused mice. Furthermore, the protein expression and localization of the two proteins was similar to that detected in the neurons of the control mice; cPLA2a in the cytosol and APP in the membranes. These results suggest that the elevated APP protein expression induced by Ab is under cPLA2a regulation in vitro and in vivo. Characterization of the mouse model of Ab brain infusion To validate the mouse model of Ab brain infusion, several features were analyzed. At 8 weeks of Ab brain infusion, a behavioral deficit was detected by reduction in spontaneous

behavioral alterations using Y-maze analysis (Fig. 3a) and neuronal damage was detected by MAP-2 immunostaining that showed a significant reduction in neuronal dendrites length (Fig. 3b) confirmed by immunoblot analysis demonstrating reduced MAP-2 and synaptophysin protein expression (Fig. 3c). Significant microglia activation, determined by CD-68 (Fig. 3d) or Iba-1 (Fig. 3e) was detected at 8 weeks of Ab brain infusion. Prevention of elevated cPLA2a protein expression by brain infusion of AS with Ab (under the conditions described in Fig. 1) prevented the behavioral deficit, neuronal damage as well as microglia activation, while infusion of sense with Ab had no effect (Fig. 3). Brain infusion of AS alone did not affect mice behavior analyzed by Y-maze (Fig. 3a) that is in accordance with the similar behavior in control mice and cPLA2a knockout mice (Sanchez-Mejia et al. 2008). Although the staining of cPLA2a in microglia is much lower than that detected in neurons, it was elevated in Ab infused mice cortex and reduced in the presence of AS but not sense (Fig. 3f). The role of cPLA2a in the signals inducing APP by Ab The mechanism by which cPLA2a over-expression regulates APP protein induction was studied in the cortical neurons in culture. Treatment of cells with 1 lM Ab for 24 h, increased COX-2 expression and PGE2 release, that were prevented by pre-treatment with AS 24 h prior to addition of Ab (Fig. 4a and b). Addition of 5 or 10 lM PGE2 to cortical neurons for 24 h caused a significant increase in APP protein expression (Fig. 4c). Similar to the neuronal cultures, COX-2 was elevated in the mouse cortex at 8 weeks of Ab infusion, while AS (but not sense) infused together with Ab prevented this elevation as shown by immunofluorescence microscopy (Fig. 4d) or immunoblot analysis (Fig. 4e). Brain infusion of AS alone caused a reduction of COX2 expression in the cortex (Fig. 4d) that coincided with the reduction of cPLA2a expression and activity in these mice (Fig. 1k and l). In order to examine whether cPLA2a-dependent APP protein induction is mediated via activation of the cAMP/protein kinase A (PKA) pathway by PGE2, the effect of a COX inhibitor (indometacin), a PKA inhibitor (H89) as well as the cAMP analog (dbcAMP) were studied. The presence of 20 lM H89 or 20 lM indometacin prevented the elevation of APP protein induced by Ab (Fig. 5a), and addition of 100 lM dbcAMP increased APP protein expression similar to the effect of Ab (Fig. 5b). Since our results suggested that PGE2 stimulates the PKA pathway, we speculated that CREB also participates in the induction of APP. Using the TF-search program (Heinemeyer et al. 1998), a CRE sequence similar to the CRE consensus sequence was found in the rat APP gene promoter in chromosome 11, extending between 2720 bp and 2727 bp upstream to the transcription start codon. To determine the role of the transcription factor, CREB, in APP transcription, its activation detected by the

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Fig. 2 cPLA2a regulated elevated amyloid precursor protein (APP) in Ab infused cortical brain. (a and b) Inhibition of elevated APP protein expression induced by Ab infusion by AS against cPLA2a. Mice were treated as in Fig. 1(g)–(l). A representative immunoblot of APP and the corresponding calreticulin protein expression in mice cortex lyzates is presented (a). Densitometry analysis for APP was performed as in Fig. 1(a). The bar graph is the mean  SE from eight mice. *p < 0.001 significant increase in mice infused with Ab or Ab+sense in comparison to control mice (infused with buffer) or mice infused with Ab+AS. # p < 0.01 significant reduction in the presence of AS (in six mice) compared with its absence (as described in Fig. 1k and l). Mice cortex sections (b) of APP protein expression. Scale bar = 200 lm. Shown

are representative images of five mice in each group. (c) APP protein expression in the hippocampus was not affected by Ab infusion in contrast to cPLA2a protein expression. Shown representative results of five mice in each group. Scale bar = 200 lm. (d) Representative Ab infused mice cortex sections (of five mice) double-stained with antiAPP and anti-Iba1, anti-GFAP, or anti-NeuN antibodies. Scale bars = 20 lm. (e) Representative Ab infused mice cortex sections (of five mice) double-stained with anti-cPLA2a and NeuN antibodies. Scale bars = 40 lm. (f) Representative mice cortex sections (treated as in Fig. 1g–j) double-stained by anti-cPLA2a (green) and anti-APP (red) antibodies. Scale bars = 150 lm. Shown are representative images of five mice in each group.

translocation of its phosphorylated form to the nuclear fraction, was analyzed. As shown in Fig. 5(c), treatment of rat cortical neurons with 1 lM Ab for 24 h increased the presence of phospho-CREB on serine 133 in the nucleus. The presence of 20 lM H89 or 20 lM indomethacin prevented this translocation. Similar to the effect of PGE2

in inducing APP elevation (Fig. 4c), PGE2 also induced translocation of phospho-CREB to the nuclear fraction (Fig. 5d). Then, the role of cPLA2a on CREB activation induced by 1 lM Ab was studied. Pre-incubation with AS, but not with sense, inhibited the presence of phosho-CREB in the nucleus at 24 h (Fig. 6a), indicating that cPLA2a is

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Fig. 3 Onset of pathology in the Ab brain infusion mouse model dependent on elevated cPLA2a. Mice were treated as in Fig. 1(g) and (h). (a) Spontaneous behavior alternation was assessed using the Ymaze assay every day from 10 days before mice killed. Each group contained 10 mice **p < 0.05 significant decrease in mice infused with Ab or Ab+sense in comparison to control mice (infused with buffer) or mice infused with Ab+AS. Six mice were brain infused with AS alone and analyzed in parallel to six control mice (as described in Fig. 1k and l). (b and c) Neuronal damage. A representative mouse cortex sections stained with anti-MAP-2 antibody (b). Scale bars = 25 lm. Shown are representative images of eight mice in each group. Representative immunoblot of MAP-2 or Synaptophysin (c) and the corresponding calreticulin protein expression in mouse cortex are presented. MAP-2 or Synaptophysin protein expression was determined by dividing the intensity of each by the intensity of the corresponding calreticulin and after quantitation by densitometry and expressed in the bar graph as arbitrary units The bar graph is the mean  SE from eight mice. **p < 0.05 significant decrease in mice infused with Ab or Ab+sense in comparison to control mice (infused with buffer) or mice infused with Ab+AS. (d–f) Microglia activation – A representative immunoblot of microglial CD-68 and the corresponding calreticulin protein expression in mouse cortex is presented (d). CD-68 protein expression was determined by dividing the intensity of each band by the intensity of the corresponding calreticulin and after quantitation by densitometry and expressed in the bar graph as arbitrary units The bar graph is the mean  SE from eight mice. **p < 0.05 significant increase in mice infused with Ab or Ab+sense in comparison to control mice (infused with buffer) or mice infused with Ab+AS. A representative mouse cortex section stained by anti-Iba-1 antibody (e). Scale bars = 40 lm. Shown are representative images of eight mice in each group. The bar graph presents the mean  SE of the activated cell number/field of three images from each mouse. **p < 0.05 significant increase in mice infused with Ab or Ab+sense in comparison to control mice (infused with buffer) or mice infused with Ab+AS. (f) Representative mice cortex sections (treated as in Fig. 1g–j) double-stained by anti-cPLA2a (green) and anti-Iba1 (red) antibodies. Scale bars = 40 lm. Shown are representative images of five mice in each group.

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involved in the phosphorylation of CREB. DNA binding activity of CREB was analyzed by EMSA, using as a designed double-stranded oligonucleotides probe containing CREB consensus sequence end labeled with Biotin. ProteinDNA binding activation was determined by the intensity of the bands. As shown in Fig. 6(b), significant levels of CREB-DNA complexes could be detected after Ab treatment. Fifty-fold of the unlabeled CRE consensus oligonucleotides competed for the binding of CREB, demonstrating

the specificity of CREB-DNA binding. No elevation in CREB-DNA binding activation was detected in the presence of AS, while the presence of sense did not affect the CREBDNA binding activation by Ab, suggesting the role of cPLA2a in this process. Using the TF-search program (Heinemeyer et al. 1998), a CRE consensus sequence was found in the mouse APP gene promoter in chromosome 11, extending between 631 and 642 bp upstream to start codon. As in cell cultures, Ab brain infusion induced activation of CREB, as detected by its phosphorylated form in the mice cortex and this activation was prevented by co-infusion of Ab with AS (6C).

Discussion The present study shows that Ab induced a neuronal cPLA2a dependent elevation of APP protein expression, in an in vitro system of cortical neuron cultures as well as in cortical

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neurons of an in vivo mouse model of Ab brain infusion. The Ab brain infused mouse model showed a behavioral deficit, neuronal damage and microglia activation as reported earlier

Fig. 4 The involvement of PGE2 in Ab induction of neuronal amyloid precursor protein (APP). (a) A representative immunoblot analysis of cyclooxygenase 2 (COX-2) and the corresponding calreticulin protein expression in neuronal cells treated as in Fig. 1(a). COX-2 protein was determined as in Fig. 1(a). The bar graph is the mean  SE from three independent experiments. (b) The release of PGE2 from cells treated as in Fig. 1(a). The mean  SE from three independent experiments is presented. (c) The effect PGE2 on neuronal APP expression is presented in a representative immunoblot. APP up-regulation was determined as in Fig. 1(a). The mean  SE from three independent experiments is presented. *p < 0.001 – significant up-regulation by Ab treatment; **p < 0.001 significant reduction in the presence of AS compared with its absence. (d) Mice were treated as in Fig. 1(g) and (h). A representative mouse cortex sections stained by anti-COX-2 antibody. Scale bars = 200 lm. Shown are representative images of five mice in each group. (e) Mice were treated as in Fig. 1(g)–(l). A representative immunoblot COX-2 and the corresponding calreticulin protein expression in mouse cortex was determined as in Fig. 1(b). The bar graph is the mean  SE from five mice. #p < 0.01 significant reduction in the presence of AS (in six mice) compared with its absence (as described in Fig. 1k and l).

(Craft et al. 2004a, 2006; Ralay Ranaivo et al. 2006). Although cPLA2a may contribute to the pathogenesis of AD by different mechanisms, including microglia activation (Szaingurten-Solodkin et al. 2009; Sun et al. 2014) and apoptotic neuronal death (Kriem et al. 2005; Sagy-Bross et al. 2013), the focus of the present study was the role of neuronal cPLA2a in induction of APP by Ab. The elevation of APP induced by Ab shown in the present study that is in line with others (Desbene et al. 2012) may cause further amplification of Ab production in AD patients. In addition, recent studies suggested the role of APP protein as a central player in a putative signaling pathway in the pathogenesis of AD. The APP C-terminal fragment interaction with ShcAGrb2 and ERK1/2 activation are strongly enhanced in postmortem AD brains, for review (Schettini et al. 2010). Our previous study showed that addition of 1 lM Ab to rat cortical neuronal cultures induced a rapid activation of cPLA2a as detected by its phosphorylated form on serine 505 (Sagy-Bross et al. 2013) similar to the effect of oligomeric Ab (Shelat et al. 2008). Here, we show that prolonged 24 h exposure of the cultures to 1 lM Ab induced elevation of cPLA2a protein expression and its activity that are responsible for the elevation of APP protein expression (Fig. 1a). cPLA2a up-regulation and activation were also detected in cortical neurons of Ab infused mice and were responsible for elevation of APP in the cortical neurons (Fig. 2). Using confocal microscopy analysis, we show that cortical neurons of control mice expressed basal levels of cPLA2a protein in the cytosol and APP protein in the membranes. Cortical neurons of Ab brain infused mice expressed high levels of cPLA2a protein in the cytosol and around the nucleus, and

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Fig. 5 Involvement of protein kinase A (PKA) in Ab induction of neuronal amyloid precursor protein (APP) up-regulation. (a) A representative immunoblot analysis of APP and the corresponding calreticulin protein expression in lysates of neuronal cells preincubated for 1 h with 20 lM a cyclooxygenase (COX) inhibitor, indometacin (Ind.), or a PKA inhibitor (H89) before addition of 1 lM Ab to neuronal cultures for additional 24 h. APP protein was determined as in Fig. 1(a). The bar graph is the mean  SE from three independent experiments. *p < 0.001 – significant elevation by Ab treatment; **p < 0.001 significant reduction in the presence of Ind. or H89 compared with their absence. (b) Treatment with 100 lM dbcAMP for 24 h increase APP expression similarly to 1 lM Ab. APP elevation was determined as in Fig. 1(a). The bar graph is the mean  SE from three independent experiments. *p < 0.001 – significant elevation by dbcAMP or Ab treatment. (c) A representative immunoblot analysis of nuclear phospho-CREB Ser133 (p-CREB) and the corresponding lamin protein expression in nuclear fraction of neuronal cells treated as in (a). The intensity of each phospho-CREB band was divided by the intensity of each lamin band after quantitation by densitometry and expressed as arbitrary units. The bar graph is the mean  SE from three independent experiments. *p < 0.001 – significant elevation by Ab treatment; **p < 0.001 significant reduction in the presence of Ind. or H89 compared with their absence. (d) The effect 10 lM PGE2 for 24 h on p-CREB nuclear expression is presented in a representative immunoblot. Nuclear p-CREB was determined as in (c). The bar graph is the mean  SE from three independent experiments. *p < 0.001 – significant elevation by Ab treatment.

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robust APP protein in the membranes as well as in the cytosol. The detection of APP in the cytosol may suggest its active synthesis in the endoplasmic reticulum before reaching the membranes via the trans-Golgi-network (Thinakaran and Koo 2008; Zhang et al. 2011). We show here that the elevated COX-2 by Ab in vivo and in vitro is under cPLA2a regulation, in accordance with other reports demonstrating that cPLA2a and COX2 are coregulated (Bosetti and Weerasinghe 2003; Hadad et al. 2011). The results of our present study suggest that the cPLA2a-dependent metabolite that is responsible for the induction of APP protein expression is PGE2. The presence

of AS that prevented the elevation of cPLA2a induced by Ab, prevented both the release of PGE2 and the induction of APP protein expression, while addition of PGE2 induced the production of APP. In line with our studies, the role of PGE2 in APP production in astrocytes (Lee and Trojanowski 1999) and microglia (Pooler et al. 2004) has been reported. Thus, our studies in neuronal cortical cultures suggest that neuronal PGE2 induces the production of APP. However, under in vivo conditions, PGE2 secreted from glia cells (Sun et al. 2014) may also contribute to the production of APP. Previous studies in cell cultures and animal models overexpressing mutant APP reported that COX and PGE2 are involved in cleavage of amyloid beta1-42 peptides from APP (Xiang et al. 2002; Qin et al. 2003; Hoshino et al. 2007). Taken together our findings and these reports, it may be suggested that the accelerated levels of Ab in AD brains is probably because of high production of APP and its increased cleavage both regulated by cPLA2a-dependent PGE2. The cPLA2a-dependent signals involved in induction of APP protein expression are probably through PKA and CREB activation, as has been shown for several proteins in different cell types, including FccRIIA in PLB-985 myeloid cells (Hazan-Eitan et al. 2006), ICAM-1 in endothelial cells (Hadad et al. 2011), and inducible nitric oxide synthetase in microglia (Szaingurten-Solodkin et al. 2009). Addition of a PKA inhibitor to neuronal cortical cultures prevented both;

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Fig. 6 Involvement of cPLA2a in Ab induction cAMP response element binding protein (CREB) activation. (a) Representative Immunoblot analysis of phospho-CREB (Ser-133) in nuclear extracts from cells treated as in Fig. 1(a) Nuclear p-CREB was determined as in Fig. 4(c). *p < 0.001 – significant elevation by Ab treatment; **p < 0.001 significant reduction in the presence of AS compared with its absence. (b) Representative Electrophoretic mobility shift assay (EMSA) for DNA binding activity of CREB. Nuclear extracts of isolated neuron cultures treated as in (a) were incubated with biotinlabeled probe containing CREB consensus sequence from amyloid precursor protein (APP) promoter. For competitive inhibition assay, 50fold molar excess of unlabeled CREB probe were added. DNA-protein complexes were analyzed on a 7% non-denaturing polyacrylamide gel. Three other experiments showed similar results. (c) Mice were infused with either buffer, Ab, Ab+AS, or Ab+sense for 4 weeks and killed at 8 weeks. Mice cortex sections of phospho-CREB (ser133) protein expression. Scale bar = 200 lm. Shown are representative images of five mice in each group.

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Fig. 7 Proposed schematic signaling pathways for the involvement of cPLA2a in amyloid precursor protein (APP) induction induced by Ab1-42 (described in the discussion).

elevated APP expression and CREB activation, while addition of the cAMP analog (dbcAMP) induced an increase in APP protein. A CRE binding domain is found in the rat and mouse promoter on chromosome 11 (between 2720–2727 bp and 610–642 bp, respectively) and in the human APP gene promoter in chromosome 21, between 2798–2806 bp (Salbaum et al. 1988). The presence of AS against cPLA2a that inhibited cPLA2a over-expression and the induction of APP protein in cortical neuronal cultures also prevented the activation of the transcription factors CREB, as determined

by its phosphorylated form in the nuclear fraction and its DNA binding activity. CREB activation, as detected by its phosphorylated form in the cortex of Ab brain infused mice is in contrast to studies demonstrating the reduction of CREB expression and activation in postmortem AD brain and in later stage of the disease in animal models, mainly in the hippocampus and hippocampal neurons (Yamamoto-Sasaki et al. 1999; Vitolo et al. 2002; Ma et al. 2007; Pugazhenthi et al. 2011). The discrepancy is probably because of the different disease stages studied. While others focused on late stages of the disease, in the present study, we used a more relevant model mimicking the early stages of sporadic AD in which behavioral and synaptic impairments are induced upon acute exposure to small amounts of extracellular Ab. Thus, it is possible that CREB activation plays a role in the initial steps of the disease and contributes to APP synthesis mainly

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in the cortex, while in the later stage of the disease its expression as well as its activity are reduced mainly in the hippocampus. It is possible that the reduction of CREB protein expression and phosphorylation in the hippocampus of AD brains and inactivation of PKA/CREB pathway in hippocampal neurons exposed to Ab reported by others (Yamamoto-Sasaki et al. 1999; Vitolo et al. 2002; Pugazhenthi et al. 2011) may offer an explanation for the absence of APP elevation in this brain region. The participation of cPLA2a in behavioral deficit as detected by reduction in spontaneous behavioral alterations using Y-maze analysis shown in our study coincided with earlier studies reporting that a single intracerebroventricular injection of Ab oligomers caused an alteration of cognitive abilities in wild-type mice but not in cPLA2a knockout mice (Desbene et al. 2012) and that elimination or partial reduction of cPLA2 was well tolerated and effectively reduced memory deficits and behavioral alterations in human APP transgenic mice (Sanchez-Mejia et al. 2008). The cPLA2a  dependent activation of microglia in mice receiving Ab brain infusion is in accordance with our previous study demonstrating the role of glial cPLA2a upregulation in activation of primary microglia exposed to Ab (Szaingurten-Solodkin et al. 2009; Sun et al. 2014). In conclusion, we show here that elevation of cPLA2a protein expression and its activity has a profound role in APP over-expression induced by Ab both in vitro and in vivo. cPLA2a provides the substrate for PGE2 production that in turn induces phosphorylation and nuclear translocation of CREB through cAMP/PKA pathway as depicts in Fig. 7.

Acknowledgments and conflict of interest disclosure This research was supported by the Israel Science Foundation (grant No. 1012/09). We thank Dr. Herman Wolosker, Department of Biochemistry, Bruce Rappaport Faculty of Medicine, TechnionIsrael Institute of Technology, Haifa, Israel for his help in the methodology of the primary neuronal cultures. We have no conflict of interest. All experiments were conducted in compliance with the ARRIVE guidelines.

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