The spleen tyrosine kinase (Syk) regulates Alzheimer amyloid-β production and Tau hyperphosphorylation.

Molecular Bases of Disease: The Spleen Tyrosine Kinase (Syk) Regulates Alzheimer Amyloid- β Production and Tau Hyperphosphorylation

J. Biol. Chem. 2014, 289:33927-33944. doi: 10.1074/jbc.M114.608091 originally published online October 20, 2014

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Daniel Paris, Ghania Ait-Ghezala, Corbin Bachmeier, Gary Laco, David Beaulieu-Abdelahad, Yong Lin, Chao Jin, Fiona Crawford and Michael Mullan

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 49, pp. 33927–33944, December 5, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

The Spleen Tyrosine Kinase (Syk) Regulates Alzheimer Amyloid-␤ Production and Tau Hyperphosphorylation* Received for publication, August 29, 2014, and in revised form, October 1, 2014 Published, JBC Papers in Press, October 20, 2014, DOI 10.1074/jbc.M114.608091

Daniel Paris1, Ghania Ait-Ghezala, Corbin Bachmeier, Gary Laco, David Beaulieu-Abdelahad, Yong Lin, Chao Jin, Fiona Crawford, and Michael Mullan From the Roskamp Institute, Sarasota, Florida 34243

We have previously shown that the L-type calcium channel (LCC) antagonist nilvadipine reduces brain amyloid-␤ (A␤) accumulation by affecting both A␤ production and A␤ clearance across the blood-brain barrier (BBB). Nilvadipine consists of a mixture of two enantiomers, (ⴙ)-nilvadipine and (ⴚ)-nilvadipine, in equal proportion. (ⴙ)-Nilvadipine is the active enantiomer responsible for the inhibition of LCC, whereas (ⴚ)-nilvadipine is considered inactive. Both nilvadipine enantiomers inhibit A␤ production and improve the clearance of A␤ across the BBB showing that these effects are not related to LCC inhibition. In addition, treatment of P301S mutant human Tau transgenic mice (transgenic Tau P301S) with (ⴚ)-nilvadipine reduces Tau hyperphosphorylation at several Alzheimer disease (AD) pertinent epitopes. A search for the mechanism of action of (ⴚ)-nilvadipine revealed that this compound inhibits the spleen tyrosine kinase (Syk). We further validated Syk as a target-regulating A␤ by showing that pharmacological inhibition of Syk or down-regulation of Syk expression reduces A␤ production and increases the clearance of A␤ across the BBB mimicking (ⴚ)-nilvadipine effects. Moreover, treatment of transgenic mice overexpressing A␤ and transgenic Tau P301S mice with a selective Syk inhibitor respectively decreased brain A␤ accumulation and Tau hyperphosphorylation at multiple AD relevant epitopes. We show that Syk inhibition induces an increased phosphorylation of the inhibitory Ser-9 residue of glycogen synthase kinase-3␤, a primary Tau kinase involved in Tau phosphorylation, by activating protein kinase A, providing a mechanism explaining the reduction of Tau phosphorylation at GSK3␤-dependent epitopes following Syk inhibition. Altogether our data highlight Syk as a promising target for preventing both A␤ accumulation and Tau hyperphosphorylation in AD.

* This work was supported by the Roskamp Foundation. 1

To whom correspondence should be addressed: The Roskamp Institute, 2040 Whitfield Ave., Sarasota, FL 34243. Tel.: 941-752-2949; Fax: 941-7522948; E-mail: [email protected].

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Alzheimer disease (AD)2 is the most prevalent form of dementia in the elderly. AD pathology is characterized by the presence of extracellular deposits of amyloid ␤ (A␤) peptides and by the accumulation of intraneuronal tangles made of hyperphosphorylated Tau. Although the amount of fibrillar A␤ peptides deposited as senile plaques at autopsy does not correlate with AD cognitive impairment (1), the levels of the soluble forms of the peptide appear to correlate better with the severity of the dementia (2). Recent longitudinal positron emission tomography imaging studies aimed at detecting A␤ in the brain of subjects with mild cognitive impairment, AD, and normal controls revealed that elevated brain A␤ levels at baseline are associated with greater cognitive worsening in both AD, mild cognitive impairment, and normal individuals over a period of 18 months (3). In addition, all familial forms of AD identified so far are caused by mutations in the amyloid precursor protein (APP) or in presenilin proteins resulting in increased brain A␤ accumulation and alteration of A␤ production (4, 5), suggesting that A␤ plays an important role in the development of AD. Interestingly, a coding mutation found in the APP gene resulting in a reduction of A␤ production is protective against AD and cognitive decline in the elderly (6, 7) further supporting the view that A␤ is a key culprit in the pathobiology of AD. A␤ is generated by the sequential proteolytic cleavage of APP by ␤and ␥-secretase. The ␥-secretase cut is imprecise and leads to the formation of A␤ peptides of different size, mainly A␤38, A␤40, and A␤42. Soluble A␤ oligomers are now widely recognized as pathogenic because they have been shown to inhibit synaptic function, to cause synaptic degeneration, to induce Tau hyperphosphorylation, and to impair memory (8 –11). 2

The abbreviations used are: AD, Alzheimer disease; Syk, spleen tyrosine kinase; APP, amyloid precursor protein; BACE-1, ␤-site amyloid precursor protein cleaving enzyme 1; GSK3␤, glycogen synthase 3␤; sAPP, secreted amyloid precursor protein; PMA, phorbol 12-myristate 13-acetate; AKT, protein kinase B; RAF kinase, rapidly accelerated fibrosarcoma kinase; CREB, cyclic adenosine monophosphate response element-binding protein; FU, fluorescence unit; CCB, calcium channel blocker; BBB, blood-brain barrier; ANOVA, analysis of variance; Tg, transgenic; FlA␤, fluorescein-labeled A␤; ROCK, Rho-associated coiled-coil containing protein kinase; BTK, Bruton’s tyrosine kinase.

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Background: Spleen tyrosine kinase (Syk) mediates microglial activation and neurotoxicity elicited by Alzheimer A␤ peptides. Results: Syk regulates A␤ production via NF␬B-dependent mechanisms and Tau phosphorylation by controlling the activation of glycogen synthase 3␤. Conclusion: Inhibition of Syk can interrupt the neuroinflammation, pathological A␤ accumulation, and Tau hyperphosphorylation in AD. Significance: Syk represents a therapeutic target for the key pathological lesions that define AD.

Syk Controls Alzheimer Disease Pathological Lesions

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EXPERIMENTAL PROCEDURES Chemicals and Reagents—Racemic nilvadipine, (⫺)-nilvadipine, and (⫹)-nilvadipine were synthesized as described previously (26) and were obtained from Archer Pharmaceuticals. BAY61-3606, phorbol 12-myristate 13-acetate (PMA), human recombinant TNF-␣, dimethyl sulfoxide (DMSO), 2-mercaptoethanol, imidazole, sodium chloride, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma. KT5270 was obtained from R&D Systems. All antibiotics, fungizone, PBS, culture media, and fetal bovine serum were purchased from Invitrogen. Fluorescein-A␤(1– 42) was purchased from rPeptide (Bogart, GA). The MPER reagent and the protease/phosphatase inhibitors mixture were purchased from Thermo Fisher Scientific. Guanidine hydrochloride was obtained from EMD Millipore. In Vitro Assays for A␤ Production and sAPP␤ Secretion—7W CHO (29) cells stably transfected with human APP751 were maintained in DMEM containing 10% fetal bovine serum, 1⫻ mixture of penicillin/streptomycin/fungizone mixture, and 0.3% geneticin as a selecting agent. Cells were cultured in 96-well culture plates and treated for 24 h with a dose range of BAY61-3606 (0.5, 1, 5, and 10 ␮M), a dose range of (⫺)-nilvadipine (1, 5, 10, and 20 ␮M), (⫹)-nilvadipine, and a racemic mixture of nilvadipine consisting of an equal amount of (⫹)- and (⫺)-nilvadipine. Potential cytotoxicity of the different treatments was routinely evaluated using the cytotoxicity detection kit (Roche Diagnostics), and no significant toxicity was observed for the different treatments (data not shown). Following the treatments with nilvadipine enantiomers, A␤40 and A␤42 were analyzed in the culture medium by using commercially available sandwich ELISAs (Invitrogen) according to recommendations of the manufacturer. Following the treatments with a dose range of BAY61-3606, A␤38, A␤40, and A␤42 were quantified by electrochemiluminescence using multiplex A␤ assays according to the manufacturer’s recommendations (Meso Scale Discovery, MD). All experiments were performed at least in quadruplicate for each treatment dose. Additionally, sAPP␣ was detected by Western blot in the culture medium surrounding 7W CHO cells using the antibody 6E10 (Signet Laboratories Inc.), which recognizes amino acids 1–17 of A␤, and sAPP␤ was detected in the culture medium using an anti-human sAPP␤ antibody (Immuno-Biological Laboratories Co. Ltd., Gunma, Japan) as we described previously (30). In Vitro Blood-Brain Barrier Model—The in vitro model of the BBB consisting of a polarized human brain microvascular endothelial cell monolayer grown on cell culture inserts that separate into apical (“blood”) and basolateral (“brain”) compartments was established as described previously by our group (18, 19, 38 – 41). A␤ exchange dynamics across the BBB model were examined using a fluorometric A␤42 assay as we described previously (18, 19, 31–34). Briefly, the apical (receiver) side of the membrane was exposed to various concentrations of racemic nilvadipine, (⫺)-nilvadipine, (⫹)-nilvadipine, or BAY613606. The donor compartment was sampled at time 0 to establish the initial concentration of fluorescein-labeled A␤(1– 42) (FlA␤(1– 42)) in each group. Following exposure of the insert to the well containing FlA␤(1– 42), samples were collected from VOLUME 289 • NUMBER 49 • DECEMBER 5, 2014


The Tau pathology in AD is also the subject of intensive research, and many aberrantly hyperphosphorylated sites on Tau have been identified in AD. Tau is known to bind to microtubules and to promote their polymerization and stabilization. Tau hyperphosphorylation results in its dissociation from microtubules, which affects microtubule-dependent axonal transport and promotes Tau oligomerization and aggregation, eventually leading to neurodegeneration. Several kinases responsible for Tau hyperphosphorylation have been identified; among them cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 (GSK3) are considered important candidates responsible for pathological Tau phosphorylation in AD (12–14). Cdk5 has been shown to indirectly affect Tau hyperphosphorylation by regulating GSK3␤ activity establishing GSK3␤ as a key mediator of Tau phosphorylation at diseaseassociated sites (12). GSK3␤ appears to be a pivotal enzyme in AD as it regulates A␤ production and mediates pathological Tau hyperphosphorylation induced by A␤ (15–17). We have shown previously that a subset of L-type calcium channel blockers (CCB) belonging to the class of antihypertensive dihydropyridines display some A␤-lowering properties by affecting both the processing of APP and the clearance of A␤ across the BBB (18, 19), properties that are unrelated to the inhibition of calcium channels or to their anti-hypertensive activity (18). Among the dihydropyridine CCB tested, we identified nilvadipine for its ability to readily cross the BBB, to lower brain A␤ levels, to improve cognition, and to prevent cerebral blood flow deficits in a transgenic mouse model of AD overexpressing A␤ (18, 20). Nilvadipine is a clinically used antihypertensive that we have shown to be well tolerated and to stabilize cognition in AD patients compared with untreated patients (21, 22). Interestingly, long term use of nilvadipine in subjects with mild cognitive impairment has also been shown to prevent cognitive decline and to reduce the incidence of AD conversion (23) suggesting that nilvadipine may have disease-modifying benefits. Nilvadipine consists of a mixture of two enantiomers, (⫹)nilvadipine and (⫺)-nilvadipine, in equal proportion. (⫹)-Nilvadipine is known to be the active isomer responsible for the CCB/ anti-hypertensive properties of nilvadipine, whereas (⫺)nilvadipine is a much weaker CCB (24 –28). As nilvadipine is an anti-hypertensive compound, the dosage of nilvadipine in AD patients may be limited as nilvadipine may induce an unwanted drop in blood pressure in these patients possibly resulting in adverse events. For these reasons, we explored the potential A␤-lowering properties of (⫺)-nilvadipine and (⫹)-nilvadipine in vitro using a cell line overexpressing A␤. We tested the impact of (⫹)- and (⫺)-nilvadipine on the clearance of A␤ peptides across the blood-brain barrier (BBB) in vitro and in vivo because we have shown previously that racemic nilvadipine stimulates the BBB clearance of A␤ (18). Additionally, we evaluated the A␤-lowering properties of (⫹) and (⫺)-nilvadipine in vivo following an acute treatment in Tg PS1/APPsw mice. We also tested the impact of (⫺)-nilvadipine on Tau phosphorylation using a transgenic mouse model of tauopathy. Finally, we searched for a possible mechanism of action of nilvadipine that can explain the impact of nilvadipine on A␤ and Tau phosphorylation.



days. Mice were humanely euthanized 30 min after the last injection, and their brains (without cerebella) were snap-frozen in liquid nitrogen until analysis for A␤38, A␤40, and A␤42 levels by electrochemiluminescence using multiplex A␤ assays according to the manufacturer’s recommendations (Meso Scale Discovery). Brain homogenates for the quantification of A␤ peptides were prepared as described above. Silencing of the SYK Gene by shRNA in NF␬B Luciferase Reporter Cells and in 7W CHO APP-overexpressing Cells— Short hairpin RNAs (shRNAs) were used to stably knockdown SYK gene expression in HEK-NF␬B luciferase reporter cells (Panomics, CA). SYK shRNAs cloned into the GIPZ lentiviral vector were purchased from Origene (Rockville, MD). Five different SYK shRNAs and a scrambled control shRNA were used separately for transfection. Approximately 1 million HEKNF␬B luciferase reporter cells detached with TripLE (Invitrogen) were mixed together with 10 ␮g of shRNA in genepulser cuvettes (0.4 cm) (Bio-Rad) and electroporated using a square wave protocol, 110 V, one pulse length of 25 ms using a Bio-Rad gene pulser, and were seeded in 6-well plates. 48 h later, the cell culture media were replaced by selective media containing 6 mg/ml puromycin for stable selection of transfected cells. Single colonies resistant to puromycin were isolated. Western blots were run with cell lysates from several of these clones, including the control shRNA (control) to confirm silencing of the SYK gene using the anti-Syk antibody from Santa Cruz Biotechnology (1:500 dilution). The clones (83D04, 38G10, and 69F04) with the most profound silencing were chosen to evaluate the effect of Syk deficiency on NF␬B luciferase activity. Briefly, NF␬B activation was triggered with 10 ng/ml PMA for 24 h, and NF␬B luciferase activity was quantified by chemiluminescence using the Luc-Screen Extended Glow from Applied Biosystems with a Synergy HT chemiluminescence reader (Biotek) as we described previously (36, 37). Similarly, a clone of 7W CHO cells overexpressing APP stably transfected with SYK shRNAs to knock down Syk expression was selected. Functional inactivation of Syk activity in 7W CHO cells was characterized by quantifying RAF phosphorylation (Ser-338) in response to PMA in regular 7W CHO cells and in 7W CHO cells transfected with SYK shRNAs by Western blot using a phospho-RAF (56A6) rabbit antibody (Cell Signaling Technology). Expression and Purification of Human Biotinylated Syk—The plasmid pDW445 (38) containing the BirA biotin-protein ligase gene was obtained from Addgene and digested with BamHI and HindIII restriction enzymes to remove the BirA gene. The BirA gene was then ligated into pBac-1 (EMD Millipore), which had been digested with the same restriction enzymes to give pBacBirA. The human SYK gene was synthesized and had added to the 5⬘ end a BamHI restriction site followed by the ATG start codon. A Gly-4 –Ser linker followed by an AviTag (Avidity) GLNDIFEAQKIEWHE (which is biotinylated on the Lys by the BirA enzyme (39)) and a His6 tag followed by a stop codon and an EcoRI restriction site (GenScript) were added to the 3⬘ end of the human SYK gene. The synthesized gene (SykA6H) was blunt-end cloned into pUC57 using an EcoRV site to give pSykAvi6H; the gene sequence was verified by DNA sequencing (GenScript). The pSykAvi6H plasmid was digested with BamHI and EcoRI, and the SykAvi6H gene was JOURNAL OF BIOLOGICAL CHEMISTRY

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the apical compartment at various time points up to 90 min to assess the movement of FlA␤(1– 42) across the human brain microvascular endothelial cell monolayer (basolateral-to-apical). The samples were analyzed (␭ex ⫽ 485 nm and ␭em ⫽ 516 nm) for FlA␤(1– 42) using a BioTek Synergy HT multidetection microplate reader (Winooski, VT). The apparent permeability (Papp) of FlA␤(1– 42) was determined using the following equation: Papp ⫽ 1/AC0䡠(dQ/dt), where A represents the surface area of the membrane; C0 is the initial concentration of FlA␤(1– 42) in the basolateral compartment, and dQ/dt is the amount of FlA␤(1– 42) appearing in the apical compartment in the given time period. The apparent permeability of FlA␤(1– 42) in the presence of drug was compared with control (i.e. no drug exposure) and expressed as a percentage. We corrected for permeability resistance associated with the blank membrane as reported previously (31). Treatments of Tg PS1/APPsw Mice with Nilvadipine Stereoisomers and Quantification of Brain A␤ Levels—Mice were maintained under specific pathogen-free conditions in ventilated racks at the Association for Assessment and Accreditation of Laboratory Animal Care International accredited vivarium of the Roskamp Institute. All the experimentations involving mice were reviewed and approved by the Institutional Animal Care and Use Committee of the Roskamp Institute before implementation and were conducted in compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. (⫺)-Nilvadipine and (⫹)-nilvadipine were administered intraperitoneally to 26-week-old Tg PS1/APPsw mice (35) for 4 consecutive days at a dosage of 2 and 4 mg/kg of body weight. (⫺)-Nilvadipine and (⫹)-nilvadipine were dissolved in 50% DMSO/PBS immediately before the injection. Vehicle-treated Tg PS1/APPsw mice received an intraperitoneal injection of 50% DMSO in PBS for 4 days. Within 1 h after the last injection, animals were humanely euthanized, and their brains (without cerebella) were snap-frozen in liquid nitrogen until analyzed for A␤40 and A␤42 levels using commercially available ELISA kits (Invitrogen). Briefly, brains were homogenized at 4 °C in MPER reagent (Thermo Fisher Scientific Inc.) containing 1 mM PMSF and 1⫻ protease/phosphatase inhibitors mixture (Thermo Fisher Scientific Inc.) and centrifuged at 15,000 ⫻ g for 30 min at 4 °C. The supernatant-containing detergent-solubilized A␤ was collected and denatured in 5 M guanidine for 1 h before being diluted with the sample diluent provided in the A␤ ELISA kits and assessed for A␤40 and A␤42 according to the manufacturer’s protocol. The pellet containing detergentinsoluble material was resuspended in 5 M guanidine and diluted with the sample diluent provided in the A␤ ELISA kits before assaying for A␤. Protein concentrations were measured in the guanidine extracts using the BCA method (Invitrogen), and results of brain A␤40 and A␤42 were calculated in picograms/mg of protein. Treatment of Tg PS1/APPsw Mice with BAY61-3606 and Brain A␤ Quantification—BAY61-3606 was administered intraperitoneally to 28-week-old Tg PS1/APPsw mice for 4 consecutive days at a dosage of 2 mg/kg of body weight. BAY613606 was dissolved in sterile PBS. Vehicle-treated Tg PS1/ APPsw mice received an intraperitoneal injection of PBS for 4



formed for 120 s, the dissociation step for 180 s followed by an additional wash step of 30 s in kinetic buffer. SYK-loaded biosensors and reference sensors were gradually subjected to the same dose range of (⫺)-nilvadipine (2.5, 5, 10, 20, 40, and 80 ␮M) or BAY61-3606 (0.625, 1.25, 2.5, 5, and 10 ␮M). Data analysis and curve fitting were done using the Octet software version 6.4 (fortéBio Corp.). Steady-state kinetic analyses were performed for every data set to calculate the KD value using the estimated response at the equilibrium for each concentration of (⫺)-nilvadipine and BAY61-3606. An average KD value (⫾ S.E.) representative of six experiments with (⫺)-nilvadipine and BAY61-3606 was calculated. Treatment of Tg Tau P301S Mice with (⫺)-Nilvadipine and BAY61-3606 —Tg Tau P301S mice (line PS19) (40) were purchased from the Jackson Laboratory. (⫺)-Nilvadipine was administered intraperitoneally at a dosage of 2 mg/kg of body weight to 25-week-old Tg Tau P301S mice for 10 consecutive days. Vehicle-treated Tg Tau P301S received an intraperitoneal injection of 50% DMSO in PBS (vehicle used to dissolve (⫺)nilvadipine) for 10 consecutive days. Tau phosphorylation was quantified by Western blots using the following Tau antibodies: AT8 (Thermo Fisher Scientific Inc., IL) and PHF-1 (41) as we described previously (42). BAY61-3606 (dissolved in PBS) was administered intraperitoneally at a dosage of 2 mg/kg of body weight to 25-week-old Tg Tau P301S mice for 5 consecutive days. Tau phosphorylation at multiple epitopes was quantified in brain homogenates by dot blots as we described previously (42) using PHF-1, RZ3, CP13 (48), and 9G3 (MédiMabs, Canada) antibodies. SH-SY5Y Cell Culture Experiments—SH-SY5Y cells were purchased from American Type Culture Collection (Manassas, VA). SH-SY5Y cells were grown in DMEM/F-12 medium supplemented with 10% serum and 1% penicillin/streptomycin/ fungizone. BACE-1 mRNA quantifications were performed by real time quantitative RT-PCR using the housekeeping gene GAPDH as a reference mRNA, and BACE-1 protein expression was analyzed by Western blots as we described previously (36, 37). SH-SY5Y cells were treated with a dose range of BAY613606 for 30 min and 24 h to assess the impact of Syk inhibition on AKT phosphorylation and GSK3␤ Ser-9 phosphorylation by Western blots using phospho-selective Ser-9 GSK3␤ and phospho-Ser-473 AKT antibodies (Cell Signaling Technology) at 1:1,000 dilutions. The effects of BAY61-3606 on Tau phosphorylation at multiple AD-relevant epitopes (9G3 (phospho-Tyr18), PHF-1, RZ3, and CP13) were studied by Western blot as we described previously (42). The involvement of PKA in mediating the stimulation of Ser-9 phosphorylation of GSK3␤ was tested by using the selective PKA inhibitor KT5270 (Tocris, CA). SH-SY5Y cells were pretreated with KT5270 for 5 min prior to being challenged with 10 ␮M BAY61-3606 for a period of 30 min. The impact of these treatments on Ser-9 GSK3␤ phosphorylation and on CREB phosphorylation (Ser-133) was followed by Western blots using phospho-selective antibodies from Cell Signaling Technology at 1:1,000 dilutions. Statistical Analyses—Results are expressed as mean ⫾ S.E. Statistical analyses were performed using SPSS Version 12.0.1 for Windows. Data were examined for assumption of normality using the Shapiro-Wilk statistic and for homogeneity of variVOLUME 289 • NUMBER 49 • DECEMBER 5, 2014


ligated into pBac-1 that had been digested with the same restriction enzymes to give pBacSykAvi6H. Both pBacBirA and pBacSykAvi6H were transfected into Sf9 insect cells as described previously (39) with the following modification: the flashBAC plasmid (Oxford Expression Technologies, Inc.) was used to generate the recombinant baculoviruses fBAC-BirA and fBAC-SykAvi6H. The recombinant baculoviruses were amplified and used to infect Sf9 insect cells grown in shaker flasks at 27 °C with GlutaMAX media (Invitrogen) supplemented with 100 ␮M biotin. The fBAC-BirA and fBAC-SykAvi6H co-infected insect cells were harvested after 60 –70 h and centrifuged at 2,000 ⫻ g. The pellet was then resuspended in ice-cold lysis buffer (50 mM sodium phosphate, pH 7.4, 100 mM NaCl, 0.5% Nonidet P-40, 5 mM 2-mercaptoethanol, and complete EDTA-free protease inhibitor mixture (Roche Applied Science and Invitrogen)) and then incubated on ice for 15 min with gentle mixing every 3 min to keep cells suspended. The sample was then centrifuged at 6,000 ⫻ g for 10 min at 5 °C, and the supernatant was removed, placed in a new tube, and centrifuged at 15,000 ⫻ g for 10 min at 5 °C. The supernatant was collected and placed in a new tube on ice. The crude lysate was batch-bound to Talon Superflow resin (GE Healthcare) with inversion for 1 h at 5 °C, batched washed with Buffer A (50 mM sodium phosphate, pH 7.4, 300 mM sodium chloride), supplemented with 8 mM imidazole, and then poured into an FPLC column. The column was washed with Buffer A and 8 mM imidazole, and then the imidazole concentration was increased to 150 mM to elute SykAvi6H. The column fractions were immediately made with 5 mM EDTA and then 50% glycerol and stored at ⫺20 °C. SykAvi6H was incubated in 50 mM sodium phosphate, pH 7.4, 50 mM sodium chloride at 30 °C for 30 min and found to remain at full length (data not shown). The SykAvi6H was ⬎95% biotinylated as determined using streptavidin-agarose resin (Thermo Fisher Scientific Inc., IL) in a pulldown assay (data not shown). Syk Enzymatic Assay—The Syk enzymatic reactions were performed using human full-length recombinant Syk prepared as described above and the Omnia Y peptide 7 kit following the recommendations of the manufacturer (Invitrogen). Enzymatic reactions were carried out at 30 °C using a Biotek Synergy HT plate reader. Fluorescence intensity readings (␭ex ⫽ 360 nm/␭em ⫽ 485) were collected every 39 s for 40 min. Biolayer Interferometry—A fortéBio Octet Red platform (fortéBio Corp., Menlo Park, CA) was used to study the binding kinetics of (⫺)-nilvadipine and BAY61-3606 to immobilized human full-length SYK that was biotinylated in situ as described above. All the binding assays were performed at 30 °C with an agitation set at 1,000 rpm using solid black 96-well plates (Grieger Bio-One) in fortéBio kinetic buffer to minimize nonspecific interactions. The final volume for all the solutions was 200 ␮l/well. Human biotinylated SYK was loaded on the surface of super-streptavidin biosensors (fortéBio Corp.). SYK biosensors were washed several times in kinetic buffer prior to performing the binding experiments. Reference super-streptavidin biosensors loaded with biotinylated PEG4 (Thermo Fisher Scientific Inc.) were used to correct for nonspecific binding of (⫺)-nilvadipine and BAY61-3606 to the biosensors. For (⫺)nilvadipine and BAY61-3606, the association step was per-


ance using the Levene’s test. Statistical significance was determined by Student’s t test, univariate, or repeated measures analysis of variance (ANOVA) where appropriate followed by post hoc comparisons with the Bonferroni method. For data not satisfying assumptions of normality and homogeneity of variance, a nonparametric Mann-Whitney test was used. p values ⬍ 0.05 were considered significant. DECEMBER 5, 2014 • VOLUME 289 • NUMBER 49

RESULTS In Vitro Effects of Nilvadipine Enantiomers on A␤ Production and A␤ Transport across the Blood-Brain Barrier—The impact of a dose range of (⫺)-nilvadipine and (⫹)-nilvadipine and a racemic mixture of nilvadipine containing an equal amount of each enantiomer on A␤ production was tested in vitro using 7W CHO cells overproducing A␤. Following 24 h of treatment JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 1. Effects of nilvadipine enantiomers on A␤ production, sAPP␤ secretion, and BACE-1 expression. A, dose-dependent inhibition of A␤40 production by 7W CHO cells overexpressing A␤. ANOVA reveals a significant main effect of (⫺)-nilvadipine (p ⬍ 0.001), (⫹)-nilvadipine (p ⬍ 0.001), and racemic nilvadipine (p ⬍ 0.001) on A␤40 production. Post hoc comparisons show significant differences in A␤40 levels between control and (⫺)-nilvadipine (p ⬍ 0.001), control and (⫹)-nilvadipine (p ⬍ 0.001), and control and racemic nilvadipine (p ⬍ 0.001) for doses of nilvadipine greater than 1 ␮M. No significant difference in A␤40 values was observed among (⫺)-nilvadipine, (⫹)-nilvadipine, and racemic nilvadipine treatments for all doses tested (p ⬎ 0.05) showing that (⫺)nilvadipine, (⫹)-nilvadipine, and racemic nilvadipine dose-dependently inhibit A␤40 to the same extent. B, representative Western blots showing that (⫺)nilvadipine reduces the secretion of sAPP␤ in 7W CHO cells overexpressing APP showed an inhibition of the ␤-cleavage of APP, whereas sAPP␣ secretion is not significantly impacted. C, effects of (⫺)-nilvadipine and racemic nilvadipine on TNF␣-induced BACE-1 transcription in human neuron-like cells SH-SY5Y. SH-SY5Y cells were treated with (⫺)-nilvadipine, racemic nilvadipine, and TNF␣ alone and in combination for the period of time indicated in the bar graph. BACE-1 transcription was quantified by quantitative real time RT-PCR using the housekeeping GAPDH mRNA as a reference. ANOVA reveals a significant main effect of treatment duration (p ⬍ 0.001), (⫺)-nilvadipine (p ⬍ 0.001) and racemic nilvadipine (p ⬍ 0.001) on BACE-1 mRNA levels as well as an interactive term between treatment duration and (⫺)-nilvadipine (p ⬍ 0.006) and between treatment duration and racemic nilvadipine (p ⬍ 0.003). Post hoc comparisons show significant differences between the control conditions and TNF␣ treatment for all the time points studied (p ⬍ 0.001) showing that TNF␣ is stimulating BACE-1 transcription. BACE-1 mRNA levels are also significantly reduced in SH-SY5Y cells co-treated with TNF␣ and (⫺)-nilvadipine (p ⬍ 0.01) and in cells co-treated with TNF␣ and racemic nilvadipine (p ⬍ 0.001) compared with the TNF␣ treatment conditions for all the time points tested showing that (⫺)-nilvadipine and racemic nilvadipine antagonize BACE-1 transcription induced by TNF␣. * denotes statistically significant differences compared with the control conditions, and $ indicates statistically significant differences compared with the TNF␣ treatment conditions. D, effects of racemic nilvadipine and (⫺)-nilvadipine on basal BACE-1 protein expression. HEK293 cells were treated for 24 h with 20 ␮M racemic nilvadipine or (⫺)-nilvadipine. BACE-1 protein expression was analyzed by Western blots in cellular lysates, and actin was used as a reference protein. Quantification of BACE-1/actin chemiluminescent signals reveal a significant decrease in BACE-1 protein expression following (⫺)-nilvadipine (p ⬍ 0.01) and racemic nilvadipine (p ⬍ 0.01) treatments.


with the pure enantiomers or the racemic mixture of nilvadipine, a dose-dependent inhibition of A␤ production was observed (Fig. 1A). We found overall that the (⫹)- and (⫺)-


enantiomers or the racemic mixture of nilvadipine have similar effects on A␤ production in vitro. We have previously shown that racemic nilvadipine affects the ␤-cleavage of APP and VOLUME 289 • NUMBER 49 • DECEMBER 5, 2014


FIGURE 2. Effects of (⫺)-nilvadipine on the clearance of A␤ across the BBB and on brain A␤ levels in Tg PS1/APPsw mice. A, (⫺)-nilvadipine and (⫹)-nilvadipine stimulate the transport of A␤(1– 42) in an in vitro BBB model employing human brain microvascular endothelial cells. The histogram represents the apparent permeability of A␤(1– 42) calculated for a period of 90 min in response to a dose range of (⫹)-nilvadipine and (⫺)-nilvadipine depicting the transport of A␤(1– 42) from the basolateral (brain) to the apical (blood) compartment of the BBB model. Results are expressed as the percentage of A␤(1– 42) apparent permeability observed in the control conditions. ANOVA show a significant main effect of (⫹)-nilvadipine (p ⬍ 0.05) and (⫺)-nilvadipine doses (p ⬍ 0.05) on A␤(1– 42) levels transported to the apical side of the BBB model. Post hoc comparisons reveal statistically significant differences in the amount of A␤(1– 42) transported from the basolateral (brain side) to the apical side (blood side) for doses of (⫺)-nilvadipine greater than 5 ␮M (p ⬍ 0.05) and for doses of (⫹)-nilvadipine greater than 1 ␮M (p ⬍ 0.05), showing that both (⫺)-nilvadipine and (⫹)-nilvadipine stimulate the transport of A␤(1– 42) across the BBB in this in vitro model. B, effect of (⫺)-nilvadipine on the clearance of human A␤(1– 42) across the BBB in vivo. The histogram represents the amount of human A␤(1– 42) quantified in the plasma of vehicle and (⫺)-nilvadipine-treated wild-type mice that received an intracranial injection of human A␤(1– 42). A significant increase in human A␤(1– 42) was detected in the plasma of (⫺)-nilvadipine-treated mice compared with the mice that received the vehicle used to dissolve (⫺)nilvadipine (t test, p ⬍ 0.001) showing that (⫺)-nilvadipine stimulates the clearance of human A␤(1– 42) across the BBB in vivo. C, impact of (⫺)-nilvadipine and (⫹)-nilvadipine on brain A␤ accumulation in Tg PS1/APPsw mice. Tg PS1/APPsw mice (26 weeks old) were treated intraperitoneally daily for 4 consecutive days with (⫺)-nilvadipine, (⫹)-nilvadipine, or the vehicle used to dissolve nilvadipine. Brain detergent-soluble and -insoluble A␤ levels were quantified by ELISAs and calculated in picograms/mg of total protein. A␤ values were standardized to the vehicle control conditions and expressed as a percentage of the A␤ levels measured in vehicle-treated mice. The histogram represents the amount of detergent-soluble and -insoluble A␤40 quantified in the brains of Tg PS1/APPsw following treatment with the vehicle, (⫹)-nilvadipine at 2 and 4 mg/kg, and (⫺)-nilvadipine at 2 and 4 mg/kg. D, histogram represents the amount of detergent-soluble and -insoluble A␤42 quantified in the brains of Tg PS1/APPsw following treatment with the vehicle, (⫹)-nilvadipine at 2 and 4 mg/kg, and (⫺)-nilvadipine at 2 and 4 mg/kg. ANOVA shows a significant main effect of (⫺)-nilvadipine (p ⬍ 0.001) and of (⫹)-nilvadipine (p ⬍ 0.001) for detergent-soluble A␤40, detergent-soluble A␤42, detergent-insoluble A␤40, and detergent-insoluble A␤42. Post hoc comparisons revealed statistically significant differences in brain detergent-soluble A␤40 levels between vehicle and (⫺)-nilvadipine 2 mg/kg (p ⬍ 0.007), vehicle and (⫺)-nilvadipine 4 mg/kg (p ⬍ 0.003), vehicle and (⫹)-nilvadipine 2 mg/kg (p ⬍ 0.02), and vehicle and (⫹)-nilvadipine 4 mg/kg (p ⬍ 0.008). Post hoc comparisons show statistically significant differences in brain detergent-soluble A␤42 levels between vehicle and (⫺)-nilvadipine 2 mg/kg (p ⬍ 0.03), vehicle and (⫺)-nilvadipine 4 mg/kg (p ⬍ 0.02), and vehicle and (⫹)-nilvadipine 4 mg/kg (p ⬍ 0.05). Post hoc comparisons reveal statistically significant differences in brain detergent-insoluble and -soluble A␤40 levels between vehicle and (⫺)-nilvadipine 2 mg/kg (p ⬍ 0.005), vehicle and (⫺)-nilvadipine 4 mg/kg (p ⬍ 0.01), vehicle and (⫹)-nilvadipine 2 mg/kg (p ⬍ 0.05), and vehicle and (⫹)-nilvadipine 4 mg/kg (p ⬍ 0.01). Post hoc comparisons also show statistically significant differences in brain detergent-insoluble and -soluble A␤42 levels between vehicle and (⫺)-nilvadipine 2 mg/kg (p ⬍ 0.001), vehicle, and (-)-nilvadipine 4 mg/kg (p ⬍ 0.001), vehicle and (⫹)-nilvadipine 2 mg/kg (p ⬍ 0.001), and vehicle and (⫹)-nilvadipine 4 mg/kg (p ⬍ 0.001). * on the histograms indicates statistically significant differences compared with the vehicle-treated mice.



FIGURE 3. Impact of (⫺)-nilvadipine on Tau hyperphosphorylation in a transgenic mouse model of tauopathy (Tg Tau P301S mice). Representative Western blots depicting Tau hyperphosphorylation at PHF-1 (Ser-396/ Ser-404) and AT8 (Ser-202/Thr-205) Tau epitopes in brain homogenates from wild-type, Tg Tau P301S vehicle-treated and Tg Tau P301S mice treated with (⫺)-nilvadipine are shown. Actin was used as a reference protein. The histogram represents the quantification of Tau hyperphosphorylation at PHF-1 and AT8 epitopes in brain homogenates from wild-type, Tg Tau P301S vehicle-treated, and Tg Tau P301S mice treated with (⫺)-nilvadipine. ANOVA reveals a significant main effect of treatment (p ⬍ 0.001) and genotype (p ⬍ 0.001) for AT8 and PHF-1 levels. Post hoc comparisons show statistically significant differences between wild-type and Tg Tau P301S for PHF-1 (p ⬍ 0.001) and AT8 levels (p ⬍ 0.001) and between Tg Tau P301S vehicle and Tg Tau P301S treated with (⫺)-nilvadipine for PHF-1 (p ⬍ 0.05) and AT8 levels (p ⬍ 0.05) showing that (⫺)-nilvadipine significantly reduces Tau hyperphosphorylation in Tg Tau P301S mice. * on the histogram indicates statistically significant differences compared with vehicle-treated Tg Tau P301S mice.

Mechanism of Action of Nilvadipine—Overall, our data show that the A␤-lowering properties of (⫺)-nilvadipine are similar to racemic nilvadipine and (⫹)-nilvadipine revealing that the A␤ inhibition observed following nilvadipine treatment is not due to an inhibition of the L-type calcium channel and suggesting that another target is responsible for the A␤-lowering properties of nilvadipine. This prompted us to explore in further detail the mechanisms responsible for the A␤-lowering properties of nilvadipine and to identify a possible molecular target controlling both A␤ production, A␤ clearance across the BBB, and Tau hyperphosphorylation. As (⫺)-nilvadipine and racemic nilvadipine inhibit BACE-1 transcription, we evaluated whether (⫺)-nilvadipine was impacting NF␬B activation because NF␬B has been shown to play an important role in the regulation of BACE-1 transcription and expression (36, 37, 43, 44). We observed that (⫺)-nilvadipine inhibits NF␬B activation in response to TNF␣ (Fig. 4A) or phorbol 12-myristate 13-aceJOURNAL OF BIOLOGICAL CHEMISTRY

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reduces sAPP␤ secretion (18). Analyses of sAPP␤ secretion in the culture media surrounding 7W CHO cells reveal that (⫺)nilvadipine inhibits sAPP␤ secretion suggesting an inhibition of the ␤-cleavage of APP (Fig. 1B). We found that (⫺)-nilvadipine was, however, unable to directly affect BACE-1 activity using a cell-free assay (data not shown). We therefore evaluated the possible impact of racemic nilvadipine and (⫺)-nilvadipine on BACE-1 expression. Tumor necrosis factor-␣ (TNF␣) has been shown to induce BACE-1 expression and to contribute to brain accumulation of A␤ peptides (43). We therefore tested the possible impact of (⫺)-nilvadipine and racemic nilvadipine on TNF␣-induced BACE-1 transcription in human neuron-like SH-SY5Y cells. We found that both (⫺)-nilvadipine and racemic nilvadipine reduce BACE-1 mRNA expression (Fig. 1C) induced by TNF␣. In addition, a reduction in BACE-1 protein levels was observed following treatment of HEK293 cells with (⫺)-nilvadipine or racemic nilvadipine (Fig. 1D) further suggesting that the inhibition of A␤ production observed following nilvadipine treatment is mediated in part by a reduction of BACE-1 expression. Next, we assessed the impact of a dose range of nilvadipine enantiomers on A␤ transcytosis across the BBB using an in vitro model employing human brain microvascular endothelial cells that we have abundantly characterized (18, 19, 31–34) since we have shown previously that racemic nilvadipine increases the clearance of A␤ across the BBB (18). We found that both the (⫹)- and (⫺)-nilvadipine enantiomers enhance A␤42 clearance from the brain to the peripheral side of the in vitro BBB model (Fig. 2A). Altogether, these data show that (⫺)-nilvadipine retains similar A␤-lowering properties compared with (⫹)-nilvadipine or racemic nilvadipine, and we further demonstrate that the A␤-lowering properties of nilvadipine are not related to a blockade of L-type calcium channels. In Vivo Effects of (⫺)-Nilvadipine on Brain A␤ Levels, A␤ Transport across the BBB, and Tau Phosphorylation in Tg Tau P301S Mice—We tested the effect of an acute treatment with (⫺)-nilvadipine or (⫹)-nilvadipine on brain A␤ levels using Tg PS1/APPsw mice, and we observed that both (⫺)-nilvadipine and (⫹)-nilvadipine acutely reduced brain A␤ levels with similar potency (Fig. 2, C and D). We also evaluated the impact of (⫺)-nilvadipine on the clearance of human A␤42 across the blood-brain barrier using wild-type mice as we described previously (18). Wild-type mice were intracranially injected with human A␤42, and the appearance of human A␤42 was followed in the blood of the animals. Data show that (⫺)-nilvadipine stimulated the clearance of human A␤42 across the BBB as more human A␤42 was detected in the plasma of (⫺)-nilvadipine-treated mice than control animals (Fig. 2B). We explored the possible impact of (⫺)-nilvadipine on Tau phosphorylation using Tg Tau P301S mice because this model of Tauopathy displays Tau hyperphosphorylation at multiple AD relevant epitopes (40, 42). Tg Tau P301S mice were treated for 10 days with a daily intraperitoneal injection of (⫺)-nilvadipine (2 mg/kg) or a vehicle. Western blot analyses of brain homogenates show that (⫺)-nilvadipine significantly reduces Tau phosphorylation in AT8 (phosphorylated Ser-199/Ser-202/Thr-205) and PHF-1 (phosphorylated Ser-396/Ser-404) epitopes (Fig. 3).


tate (PMA) (Fig. 4B) by using an NF␬B-luciferase reporter cell line to monitor NF␬B activation, thus suggesting a possible mechanism responsible for the inhibition of BACE-1 transcription following nilvadipine treatment. The signaling pathways responsible for the activation of NF␬B by TNF␣ or PMA have been described in the literature, and we explored whether elements of these pathways were impacted by (⫺)-nilvadipine. For instance, the small GTPase Rho and its downstream effector


Rho-associated coiled-coil containing protein kinase (ROCK) have been shown to contribute to TNF␣ induction of NF␬B activation (45). We found that nilvadipine was ineffective at inhibiting ROCK activity in a cell-free assay excluding ROCK as possible target of nilvadipine (data not shown). In addition, we observed that Rho inhibition with C3 cell-permeable transferase (CT04) and ROCK inhibition with Y27632 were unable to prevent the inhibition of NF␬B activation induced by (⫺)-nilVOLUME 289 • NUMBER 49 • DECEMBER 5, 2014


FIGURE 4. Effect of (⫺)-nilvadipine on NF␬B activation. NF␬B activation was measured by quantifying intracellular luciferase activity using an NF␬B luciferase reporter HEK293 cell line. A, histogram showing that (⫺)-nilvadipine (200 nM) inhibits NF␬B activation induced by TNF␣ (3-h treatment). ANOVA shows a significant main effect of TNF␣ (p ⬍ 0.001) and (⫺)-nilvadipine on NF␬B luciferase activity. Post hoc comparisons reveal statistically significant differences between control and TNF␣ (p ⬍ 0.001) and between TNF␣ and TNF␣ ⫹ (⫺)-nilvadipine treatments (p ⬍ 0.001) for NF␬B luciferase activity showing that (⫺)-nilvadipine suppresses NF␬B activity induced by TNF␣. B, histogram showing that (⫺)-nilvadipine antagonizes PMA-induced NF␬B activation (24-h treatment). ANOVA shows a significant main effect of PMA (p ⬍ 0.001) and (⫺)-nilvadipine on NF␬B luciferase activity. Post hoc comparisons reveal statistically significant differences between control and PMA (p ⬍ 0.001) and between PMA and PMA ⫹ (⫺)-nilvadipine treatments (p ⬍ 0.001) for NF␬B luciferase activity showing that (⫺)-nilvadipine suppresses NF␬B activity induced by PMA. C, Western blot depicting the impact of (⫺)-nilvadipine on RAF phosphorylation (Ser-338) induced by PMA. HeLa cells were challenged with 100 ng/ml PMA with or without (⫺)-nilvadipine for 15 min. The histogram represents the amount of phosphorylated RAF over actin (used as a reference protein) quantified for the different treatment conditions. ANOVA shows a significant main effect of (⫺)-nilvadipine (p ⬍ 0.002) and PMA (p ⬍ 0.001) on RAF phosphorylation levels as well as an interactive term between them (p ⬍ 0.05). Post hoc analyses reveal statistically significant differences between the level of phosphorylated RAF observed in the control conditions and the PMA treatment (p ⬍ 0.001) and the PMA treatment and the PMA ⫹ (⫺)-nilvadipine treatment conditions (p ⬍ 0.002) showing that PMA stimulates RAF phosphorylation, whereas (⫺)-nilvadipine reduces RAF phosphorylation induced by PMA. D, Western blot showing the impact of (⫺)-nilvadipine on RAF phosphorylation (Ser-338) induced by PMA in HEK293 cells. The histogram represents the amount of phosphorylated RAF over actin (used as a reference protein) quantified for the different treatment conditions. ANOVA shows a significant main effect of (⫺)-nilvadipine (p ⬍ 0.005) and PMA (p ⬍ 0.001) on RAF phosphorylation levels. Post hoc analyses reveal statistically significant differences between the level of phosphorylated RAF observed in the control conditions and the PMA treatment (p ⬍ 0.001) and the PMA treatment and the PMA ⫹ (⫺)-nilvadipine treatment conditions (p ⬍ 0.005) showing that PMA stimulates RAF phosphorylation, whereas (⫺)-nilvadipine reduces RAF phosphorylation induced by PMA in HEK293 cells.


vadipine and did not significantly impact BACE-1 expression ruling out the possible involvement of Rho/ROCK for mediating the A␤-lowering properties of (⫺)-nilvadipine (data not shown). PMA is a known agonist of PKC, which leads to the activation of the PKC/RAS/RAF/MEK/MAPK pathway that ultimately induces NF␬B activation (46 – 48). Therefore, we evaluated whether nilvadipine could inhibit some members of this signaling pathway. In particular, we monitored RAF phosphorylation following treatment with (⫺)-nilvadipine and observed that (⫺)-nilvadipine prevents RAF phosphorylation induced by PMA (Fig. 4, C and D) suggesting that (⫺)-nilvadipine is impacting a target upstream of RAF. We tested the possible effect of (⫺)-nilvadipine on RAS activity and found that (⫺)-nilvadipine was unable to affect RAS activity in a cellfree assay ruling out RAS as a possible target of (⫺)-nilvadipine (data not shown). Tyrosine kinases, including Syk and Bruton’s DECEMBER 5, 2014 • VOLUME 289 • NUMBER 49

tyrosine kinase (BTK), are activated following PMA treatment (49, 50), act upstream of RAS/RAF (51, 52), and mediate the activation of the NF␬B pathway (53). We tested a selective BTK inhibitor (BTK inhibitor III, 1-(3-(4-amino-3-(4-phenyloxy phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop2-en-1-one, N-acryloyl-(3-(4-amino-3-(4-phenyloxyphenyl)1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine) on A␤ production, and we found that this compound was unable to significantly inhibit A␤ production (data not shown) suggesting that the A␤-lowering properties of nilvadipine are not mediated via an inhibition of BTK. We explored whether Syk activity was impacted by (⫺)-nilvadipine as nilvadipine reduces RAF phosphorylation. Using a cell-free assay using human recombinant Syk, we observed that (⫺)-nilvadipine dose-dependently inhibits Syk activity (Fig. 5A). The Syk inhibitor BAY61-3606 was used as a positive control in the Syk activity assay, and a JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 5. (⫺)-Nilvadipine binds to human Syk and inhibits Syk enzymatic activity. A, Syk enzymatic kinetics with (⫺)-nilvadipine. The graph depicts the amount of Syk product formed (mFU) in function of time in response to a dose range of (⫺)-nilvadipine. Phosphorylation of the peptide substrate by Syk results in an increased fluorescence intensity (mFU) that is directly proportional to the amount of peptide phosphorylation. ANOVA shows a significant main effect of time (p ⬍ 0.001), (⫺)-nilvadipine dose (p ⬍ 0.001) as well as an interactive term between them (p ⬍ 0.001) for the amount of Syk product formed. Post hoc comparisons reveal significant differences between control and (⫺)-nilvadipine treatments for all the doses tested (p ⬍ 0.001) showing that (⫺)-nilvadipine inhibits Syk activity. * indicates statistically significant differences compared with the control conditions. B, Syk enzymatic kinetics with BAY61-3606. The graph depicts the amount of Syk product formed (mFU) in function of time in response to a dose range of BAY61-3606. ANOVA shows a significant main effect of time (p ⬍ 0.001), BAY61-3606 dose (p ⬍ 0.001), as well as an interactive term between them (p ⬍ 0.001) for the amount of Syk product formed. Post hoc comparisons reveal significant differences between control and BAY61-3606 treatments for all the doses greater than 0.3125 ␮M (p ⬍ 0.001). * indicates statistically significant differences compared with the control conditions. C, interferometry studies of the interaction between Syk and (⫺)-nilvadipine. Experimental binding data showing the association and dissociation of (⫺)-nilvadipine to human recombinant Syk are shown for a single experiment using a dose range of (⫺)-nilvadipine. For the determination of the equilibrium dissociation constants (KD), steady-state analyses of the binding data were performed for six independent experiments and generated an average KD ⫽ 2.1 ⫾ 0.3 ␮M. D, interferometry studies of the interaction between Syk and BAY61-3606. Experimental binding data showing the association and dissociation of BAY61-3606 to human recombinant Syk are shown for a single experiment using a dose range of BAY61-3606. Steady-state analyses of the binding data were performed for six independent experiments and generated an average KD ⫽ 0.42 ⫾ 0.1 ␮M.


FIGURE 6. Effect of pharmacological inhibition of Syk and down-regulation of syk expression on A␤ production. A, dose-dependent inhibition of A␤38, A␤40, and A␤42 production by 7W CHO overexpressing APP treated with the Syk inhibitor BAY61-3606. ANOVA shows a significant main effect of BAY61-3606 doses on A␤38, A␤40, and A␤42 production (p ⬍ 0.001). Post hoc


comparisons reveal significant differences between the A␤38, A␤40, and A␤42 values observed in the control conditions and after treatment for all doses of BAY61-3606 tested (p ⬍ 0.001) showing that BAY61-3606 inhibits the production of A␤ peptides in 7W CHO cells. * indicates statistically significant differences compared with the control conditions. B, effects of an inhibition of syk expression with shRNA on RAF activation and A␤ production by 7W CHO cells overexpressing APP. Western blot depicting the level of RAF phosphorylation observed after PMA treatment in 7W CHO cells overexpressing APP and in 7W CHO cells stably transfected with syk shRNA. ANOVA shows a significant main effect of PMA (p ⬍ 0.001) and syk shRNA (p ⬍ 0.001) transfection on RAF phosphorylation as well as an interactive term between them (p ⬍ 0.001). Post hoc comparisons reveal statistically significant differences between basal RAF phosphorylation levels in 7W CHO cells and 7W CHO cells transfected with syk shRNA (p ⬍ 0.02) showing that down-regulation of syk expression lowers basal RAF phosphorylation. A significant elevation of RAF phosphorylation was observed following treatment of 7W CHO cells with PMA (p ⬍ 0.001) compared with the control conditions. No statistically significant difference in RAF phosphorylation levels was observed between syk shRNA 7W CHO cells untreated and treated with PMA (p ⫽ 0.304) showing that down-regulation of syk expression abolishes RAF phosphorylation induced by PMA confirming the absence of active Syk in syk shRNA-transfected cells. C, histogram representing the quantification of A␤38, A␤40, and A␤42 production in control 7W CHO cells and in 7W CHO stably transfected with syk shRNA. t tests reveal significant differences for A␤38, A␤40, and A␤42 (p ⬍ 0.001) produced by 7W CHO cells and 7W CHO cells transfected with syk shRNA showing that down-regulation of Syk expression inhibits A␤ production.

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dose-dependent inhibition of Syk activity was observed with BAY61-3606 as expected (Fig. 5B). To ensure that the reduction in Syk activity observed was not due to an interaction of the peptide substrate with (⫺)-nilvadipine, we also verified that (⫺)-nilvadipine was able to directly bind to Syk. We measured the binding affinity of (⫺)-nilvadipine for Syk using biolayer interferometry and confirmed that (⫺)-nilvadipine binds to human recombinant Syk with a binding dissociation constant (KD) of 2.1 ␮M (Fig. 5C), further suggesting that Syk is the possible target impacted by nilvadipine. Binding kinetics were also performed using BAY61-3606 as a positive control (Fig. 5D), and a KD of 0.4 ␮M was obtained for BAY61-3606. In addition, we verified that pharmacological inhibition of Syk with BAY61-3606 resulted in a blockade of NF␬B activation and that genetic down-regulation of SYK using shRNA also prevented NF␬B activation (data not shown) thus highlighting Syk as a key player of NF␬B activation. The magnitude of the NF␬B inhibition following (⫺)-nilvadipine treatment was also reduced in clones of HEK293 NF␬B luciferase reporter cells in which Syk expression had been silenced (data not shown) further suggesting that Syk is required to mediate the inhibition of NF␬B activity induced by (⫺)-nilvadipine. As (⫺)-nilvadipine affects both A␤ production and A␤ clearance across the BBB, we investigated whether pharmacological inhibition of Syk could also affect these two processes. We found that Syk inhibition with the selective Syk inhibitor BAY61-3606 suppresses A␤ production in 7W CHO cells overexpressing APP (Fig. 6A). We also tested other Syk inhibitors with a chemical structure distinct from BAY61-3606 (3-(1methyl-1H-indol-3-yl-methylene)-2-oxo-2,3-dihydro-1H-indole5-sulfonamide and 2-(2-aminoethylamino)-4-(3-trifluoromethylanilino)-pyrimidine-5-carboxamide), and we observed that these compounds inhibited A␤ production in vitro (data not shown). We found that, like (⫺)-nilvadipine, Syk inhibition with the selective Syk inhibitor BAY61-3606 resulted in decreased sAPP␤ secretion, BACE-1 mRNA, and BACE-1 protein expression (data not shown). To further demonstrate the involvement of Syk in the regulation of A␤ production, we sup-


pressed syk expression in 7W CHO cells overexpressing A␤ using shRNA. To verify that Syk activity has been functionally suppressed in 7W CHO cells transfected with SYK shRNA, we monitored RAF phosphorylation. As expected, RAF phosphorylation induced by PMA as well as basal RAF phosphorylation were reduced in 7W CHO cells transfected with SYK shRNA confirming a reduction in Syk activity (Fig. 6B). Interestingly, A␤ production in 7W CHO cells transfected with SYK shRNA compared with 7W CHO cells (Fig. 6C) was significantly reduced, further demonstrating the involvement of Syk in the regulation of A␤ production. DECEMBER 5, 2014 • VOLUME 289 • NUMBER 49

Because we have shown that (⫺)-nilvadipine improves the clearance of A␤ across the BBB, we also explored whether Syk inhibition could affect the transport of A␤ across the BBB. We show that the selective Syk inhibitor BAY61-3606 stimulates the transport of A␤ across the BBB in vitro mimicking the biological activity of (⫺)-nilvadipine in this model (Fig. 7A). We tested other Syk inhibitors of unrelated chemical structures in our in vitro BBB model and observed that all these Syk inhibitors stimulated the clearance of A␤ from the basolateral to apical side of the BBB (data not shown) further demonstrating the involvement of Syk in the clearance of A␤ across the BBB. JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 7. Effects of pharmacological inhibition of Syk on A␤ clearance across the BBB and on brain A␤ levels in Tg PS1/APPsw mice. A, BAY61-3606 stimulates the transport of A␤(1– 42) in an in vitro BBB model employing human brain microvascular endothelial cells. The histogram represents the apparent permeability of A␤(1– 42) calculated for a period of 90 min in response to a dose range of BAY61-3606 and depicts the transport of A␤(1– 42) from the basolateral (brain) to the apical (blood) compartment of the BBB model. Results are expressed as the percentage of A␤(1– 42) apparent permeability observed in the control conditions. ANOVA show a significant main effect of BAY61-3606 (p ⬍ 0.05) on A␤(1– 42) levels transported to the apical side of the BBB model. Post hoc comparisons reveal statistically significant differences in the amount of A␤(1– 42) transported from the basolateral (brain side) to the apical side (blood side) for human brain microvascular endothelial cells treated with 5 ␮M BAY61-3606 (p ⬍ 0.05) showing that pharmacological inhibition of Syk activity stimulates the transport of A␤(1– 42) across the BBB in this in vitro model. * indicates statistically significant differences compared with the control conditions. B, BAY61-3606 increases the clearance of human A␤(1– 42) across the BBB in vivo. ANOVA shows a significant main effect of BAY61-3606 on plasma human A␤(1– 42) (p ⬍ 0.03) detected following an intracranial injection of the peptide. Post hoc comparisons reveal a statistically significant difference in plasma human A␤(1– 42) between vehicle-treated mice and mice treated with 4 mg/kg BAY61-3606 (p ⬍ 0.05) showing that at this dose BAY61-3606 stimulates the clearance of A␤(1– 42) across the BBB in vivo. * indicates statistically significant differences compared with the vehicle treatment conditions. C, effects of pharmacological inhibition of Syk with BAY61-3606 on brain A␤38, A␤40, and A␤42 levels in Tg PS1/APPsw mice. Histogram representing the amount of detergent-soluble A␤38, A␤40, and A␤42 detected in the brains of vehicle and BAY61-3606-treated Tg PS1/APPsw mice. A significant reduction in brain detergent-soluble A␤38 (p ⬍ 0.01), A␤40 (p ⬍ 0.01), and A␤42 (p ⬍ 0.01) levels was observed in BAY61-3606-treated Tg PS1/APPsw compared with vehicletreated mice (placebo). D, histogram representing the amount of detergent-insoluble A␤38, A␤40, and A␤42 detected in the brains of vehicle (placebo) and BAY61-3606-treated Tg PS1/APPsw mice. A significant reduction in brain detergent-insoluble A␤38 (p ⬍ 0.01), A␤40 (p ⬍ 0.01), and A␤42 (p ⬍ 0.05) levels was observed in BAY61-3606-treated Tg PS1/APPsw compared with vehicle-treated mice (placebo).


In Vivo Effects of the Selective Syk Inhibitor BAY61-3606 on Brain A␤ Levels and A␤ Transport across the BBB—To further validate Syk as a potential target for regulating brain A␤ levels and A␤ clearance across the BBB, we investigated the impact of the selective Syk inhibitor BAY61-3606 on these different processes in vivo. BAY61-3606 was selected over other Syk inhibitors because it has been shown previously to inhibit Syk activity in vivo in rodents (54, 55). We observed that BAY61-3606 significantly reduces brain A␤38, A␤40, and A␤42 levels in Tg PS1/ APPsw mice (Fig. 7, C and D). In addition, we found that BAY61-3606 stimulates the clearance of A␤ across the BBB in wild-type mice as demonstrated by increased circulating plasma levels of human A␤42 in mice treated with the Syk inhibitor compared with vehicle-treated mice following the intracranial injection of human A␤42 (Fig. 7B). Effects of Syk Inhibition on Tau Phosphorylation—Syk has been shown to mediate the phosphorylation of Tau at Tyr-18 (56). We evaluated the possible effects of the selective Syk inhibitor BAY61-3606 on Tau phosphorylation in Tg Tau P301S mice using a dot-blot approach that we have validated for monitoring Tau phosphorylation in P301S mice (42). We tested the impact of BAY61-3606 on Tau phosphorylation at Tyr-18 and also investigated whether other Tau phosphoepitopes were affected by the treatment. We observed a reduction in Tau phosphorylation at the Tyr-18 epitope as expected (Fig. 8) in BAY61-3606-treated mice. Interestingly, we also detected a reduction in Tau phosphorylation at PHF-1 (Ser(P)396/Ser(P)-404) and CP13 (Ser(P)-202) in epitopes following treatment of Tg Tau P301S mice with BAY61-3606, whereas the RZ3 (Thr(P)-231) Tau epitope was not significantly impacted (Fig. 8) suggesting that Syk inhibition may also control the activity of other downstream kinases involved in Tau hyperphosphorylation. We conducted some mechanistic studies in human neuronlike SH-SY5Y cells to delineate a possible mechanism of action responsible for the reduction of Tau phosphorylation observed at multiple Tau epitopes following Syk inhibition. Because we observed a reduction of Tau phosphorylation at the character-


istic GSK3␤ motif (Ser(P)-396/Ser(P)-404 (PHF-1)) following treatment of Tg Tau P301S mice with BAY61-3606, we investigated whether pharmacological inhibition of Syk could affect GSK3␤ by monitoring the phosphorylation of GSK3␤ at Ser-9, which is known to inactivate the enzyme (57). We observed that pharmacological inhibition of Syk with BAY61-3606 stimulates Ser-9 phosphorylation of GSK3␤ in SH-SY5Y cells (Fig. 9, A and B) suggesting that blocking Syk activity results in GSK3␤ inhibition. We further confirmed that possibility by showing that Tau phosphorylation at the typical GSK3␤ sites (PHF-1 and CP13) is reduced following treatment of SH-SY5Y cells with BAY61-3606, whereas Tau phosphorylation at the RZ3 site was not significantly impacted in SH-SY5Y cells (Fig. 9C). GSK3␤ phosphorylation at Ser-9 has been shown to be mediated by PKA and AKT (protein kinase B) (57, 58). We therefore examined whether Syk inhibition was affecting AKT and PKA. We found that treatment of SH-SY5Y cells with BAY61-3606 inhibits AKT phosphorylation (Fig. 9, A and B), which is consistent with previous studies (59) investigating the impact of Syk inhibition on AKT activation. Our data therefore show that AKT cannot mediate the increased GSK3␤ Ser-9 phosphorylation induced by Syk inhibition in SH-SY5Y cells. We then explored whether a selective PKA inhibitor (KT5270) was able to mitigate GSK3␤ Ser-9 phosphorylation induced by treatment of SH-SY5Y cells with BAY61-3606. We found that KT5270 effectively suppressed GSK3␤ phosphorylation at Ser-9 induced by BAY61-3606 (Fig. 10B) suggesting that this event is mediated by an activation of PKA. PKA is a known substrate of Syk, and it has been shown that Syk inhibits PKA activity by phosphorylating Tyr-330 of the PKA catalytic subunit (60), further supporting our observation. To verify that Syk inhibition with BAY61-3606 resulted in PKA activation, we evaluated the phosphorylation of CREB, a direct PKA substrate, following pharmacological inhibition of Syk. We found that Syk inhibition with BAY61-3606 induced CREB phosphorylation, although that event is inhibited in the presence of a selective PKA inhibitor (Fig. 10, A and B) further showing that Syk inhibition results in PKA activation. VOLUME 289 • NUMBER 49 • DECEMBER 5, 2014


FIGURE 8. Effects of pharmacological inhibition of Syk with BAY61-3606 on Tau hyperphosphorylation in Tg Tau P301S mice. Tau hyperphosphorylation at multiple AD pertinent epitopes was analyzed by dot blots using brain homogenates from vehicle (placebo) and BAY61-3606-treated Tg Tau P301S mice (left panel). GAPDH was used as a reference protein. The histogram represents the quantification of Tau phosphorylation at Tyr-18 (9G3), Ser(P)-396/Ser(P)-404 (PHF-1), Ser(P)-202 (CP13), and Thr(P)-231(RZ3). A significant reduction in Tau hyperphosphorylation was observed in BAY61-3606-treated mice at the 9G3 (p ⬍ 0.025), PHF-1 (p ⬍ 0.006), and CP13 (p ⬍ 0.004) phosphoepitopes but not at the RZ3 epitope (p ⫽ 0.382). * indicates statistically significant differences compared with the vehicle treatment.


DISCUSSION Hypertension in mid-life has been associated with an increased risk of dementia, including AD in the elderly (61). Interestingly, the double-blind, placebo-controlled systolic DECEMBER 5, 2014 • VOLUME 289 • NUMBER 49

hypertension study (Syst-Eur) has revealed that the long term use of a dihydropyridine CCB (nitrendipine) reduces the risk of dementia by 55%, including AD (62, 63), suggesting that antihypertensive therapies may be beneficial against dementia. JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 9. Effect of pharmacological inhibition of Syk with BAY61-3606 on GSK3␤ Ser-9 phosphorylation, AKT phosphorylation in SH-SY5Y cells, and Tau phosphorylation. A, representative Western blot depicting the impact of a 30-min treatment with a dose range of BAY61-3606 on GSK3␤ Ser-9 phosphorylation and on AKT phosphorylation. GAPDH was used as a reference protein. The histogram represents the average chemiluminescent signals quantified for GSK3␤ Ser-9 phosphorylation/GAPDH and AKT Ser-473 phosphorylation/GAPDH. ANOVA shows a significant main effect of BAY61-3606 on GSK3␤ Ser-9 phosphorylation/GAPDH (p ⬍ 0.001) and AKT Ser-473 phosphorylation/GAPDH (p ⬍ 0.001). Post hoc comparisons reveal statistically significant differences for GSK3␤ Ser-9 phosphorylation/GAPDH (p ⬍ 0.001) and AKT Ser-473 phosphorylation/GAPDH at all doses of BAY61-3606 tested (p ⬍ 0.02) showing that BAY61-3606 treatment stimulates GSK3␤ Ser-9 phosphorylation and inhibits AKT phosphorylation. * indicates statistically significant differences compared with the control conditions. B, representative Western blot depicting the impact of a 24-h treatment with a dose range of BAY61-3606 on GSK3␤ Ser-9 phosphorylation and on AKT phosphorylation. GAPDH was used as a reference protein. The histogram represents the average chemiluminescent signals quantified for GSK3␤ Ser-9 phosphorylation/GAPDH and AKT Ser-473 phosphorylation/GAPDH. ANOVA shows a significant main effect of BAY61-3606 on GSK3␤ Ser-9 phosphorylation/GAPDH (p ⬍ 0.001) and AKT Ser-473 phosphorylation/GAPDH (p ⬍ 0.001). Post hoc comparisons reveal statistically significant differences for GSK3␤ Ser-9 phosphorylation/GAPDH (p ⬍ 0.015) and AKT Ser-473 phosphorylation/GAPDH at all doses of BAY61-3606 tested (p ⬍ 0.001) showing that BAY61-3606 treatment stimulates GSK3␤ Ser-9 phosphorylation and inhibits AKT phosphorylation. * indicates statistically significant differences compared with the control conditions. C, impact of a 24-h treatment with 10 ␮M of the Syk inhibitor BAY61-3606 on Tau phosphorylation in SH-SY5Y cells. Western blots showing the impact of BAY61-3606 on Tau phosphorylation at Tyr-18, Ser-396/Ser-404, Thr-231, and Ser-202. GAPDH was used as a reference protein. The histogram represents the average amount of Tau phosphorylation detected at 9G3, PHF-1, CP13, and RZ3 phospho-Tau epitopes in control and BAY61-3606-treated SH-SY5Y cells. Statistically significant reductions (t test) in Tau phosphorylation at the PHF-1 (p ⬍ 0.001) and CP13 epitopes (p ⬍ 0.001) were observed following treatment with 10 ␮M BAY61-3606. * indicates statistically significant differences compared with the control conditions.

Syk Controls Alzheimer Disease Pathological Lesions Chronic treatment with nilvadipine has been shown to prevent the conversion of mild cognitive impairment to AD (23) suggesting that nilvadipine may have disease-modifying properties. In addition, a short treatment duration with nilvadipine in AD patients has been shown to reduce cognitive decline (21, 22).

In this study, we investigated the A␤-lowering properties of nilvadipine enantiomers, (⫹)-nilvadipine and (⫺)-nilvadipine. (⫹)-Nilvadipine is the enantiomer of racemic nilvadipine responsible for the anti-hypertensive activity of the compound (24 –26). We confirmed ex vivo in rat aortae that (⫺)-nilvadipine was ⬃1,000 time less potent than (⫹)-nilvadipine for




Syk Controls Alzheimer Disease Pathological Lesions identify Syk as an important regulator of A␤ production both in vitro and in vivo. We show additionally that (⫺)-nilvadipine, as well as a selective Syk inhibitor, or a reduction of syk expression using shRNA all prevent NF␬B activation suggesting a possible mechanism for the reduction of BACE-1 transcription observed following Syk inhibition as NF␬B has been shown previously to play an important role in the regulation of BACE-1 transcription and expression (36, 37, 44, 64). Because NF␬B is involved in the regulation of neuroinflammation and cytokine production, these data also suggest that Syk may serve as an important target for regulating neuroinflammation. Pharmacological inhibitors of Syk have been developed and represent potential immunomodulatory agents for the treatment of autoimmune and inflammatory conditions. Interestingly, A␤ has been shown to stimulate Syk activity resulting in microglial activation and neurotoxicity (65–70). Syk has also been shown to phosphorylate Tau in vitro, primarily at the Tyr-18 residue (56), which is considered to be an early event in the pathophysiology of AD (56, 71). We show that treatment of SH-SY5Y cells with the selective Syk inhibitor BAY61-3606 results in a partial reduction of Tau phosphorylation at Tyr-18 but also an almost complete inhibition of Tau phosphorylation at Ser-202 (CP13) and Ser-396/Ser-404 (PHF1). The partial inhibition of Tau phosphorylation at Tyr-18 following Syk inhibition may be explained by the fact that other tyrosine kinases are also known to phosphorylate Tau at this particular residue (71). Additionally, we show that treatment of Tg Tau P301S mice with the selective Syk inhibitor BAY613606 results in a reduction of Tau Tyr-18 phosphorylation as expected but also in a significant decrease in Tau phosphorylation at other epitopes, including Ser-202 (CP13) and Ser-396/ Ser-404 (PHF-1) that are phosphorylated by GSK3␤ (13). Because Syk inhibition results in decreased Tau phosphorylation at typical GSK3␤ sites in Tg P301S mice and SH-SY5Y cells, we investigated whether pharmacological inactivation of Syk could affect GSK3␤. We found that inhibition of Syk in SH-SY5Y cells stimulates Ser-9 phosphorylation of GSK3␤, which is known to inactivate the enzyme (57). We hypothesized that by this mechanism, Syk inhibition results in a reduction of Tau phosphorylation at Ser-202 and Ser-396/Ser-404 epitopes phosphorylated by GSK3␤. Previous data have revealed that Syk phosphorylates Tyr-330 in the catalytic subunit of PKA resulting in the inactivation of PKA (60). We therefore tested the possible involvement of PKA for mediating the increased GSK3␤ Ser-9 phosphorylation induced by Syk inhibition. Our data show that PKA inactivation with KT5270 opposes the increased Ser-9 phosphorylation of GSK3␤ induced by Syk

FIGURE 10. PKA mediates GSK3␤ Ser-9 phosphorylation induced by pharmacological inhibition of Syk activity with BAY61-3606 in SH-SY5Y cells. A, Western blot depicting the effect of a 24-h treatment with BAY61-3606 on CREB phosphorylation in SH-SY5Y cells. GAPDH was used as a reference protein. The histogram represents the average CREB Ser-133 phosphorylation/GAPDH chemiluminescent signals observed in control and BAY61-3606-treated SH-SY5Y cells. A significant induction of CREB phosphorylation was observed following treatment with BAY61-3606 (p ⬍ 0.004). * indicates statistically significant differences compared with the control conditions. B, representative Western blot depicting the effect of a 30-min treatment with BAY61-3606 on GSK3␤ Ser-9 phosphorylation with or without co-treatment with the selective PKA inhibitor KT5270. GAPDH was used as a reference protein. The histogram represents the average GSK3␤ Ser-9 phosphorylation/GAPDH and CREB Ser-133 phosphorylation/GAPDH chemiluminescent signals observed for the different treatment conditions. ANOVA shows a significant main effect of BAY61-3606 on GSK3␤ Ser-9 phosphorylation (p ⬍ 0.001) and CREB phosphorylation (p ⬍ 0.007) and a significant main effect of KT5270 on GSK3␤ Ser-9 phosphorylation (p ⬍ 0.001) and CREB phosphorylation (p ⬍ 0.002). Post hoc comparisons reveal statistically significant differences between control and BAY61-3606-treated cells for GSK3␤ Ser-9 phosphorylation (p ⬍ 0.001) and CREB phosphorylation (p ⬍ 0.007) showing that BAY61-3606 stimulates GSK3␤ Ser-9 phosphorylation and CREB phosphorylation. Compared with the BAY61-3606 treatment, a significant reduction in GSK3␤ Ser-9 phosphorylation (p ⬍ 0.001) and CREB phosphorylation (p ⬍ 0.002) was observed in SH-SY5Y cells co-treated with KT5270 and BAY61-3606 showing that PKA inhibition prevents GSK3␤ Ser-9 phosphorylation and CREB phosphorylation induced by BAY61-3606.



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opposing the vasoconstriction induced by a selective L-type calcium channel agonist (FPL64176) showing that it is a much weaker L-type calcium channel antagonist than (⫹)-nilvadipine (data not shown). Interestingly, we observed that both enantiomers are equipotent at lowering A␤ production in a cell line overexpressing A␤ thus showing that the A␤-lowering properties of nilvadipine are not related to L-type calcium channel inhibition. We have shown previously that a subset of CCB dihydropyridines prevent brain A␤ accumulation by opposing A␤ production and by stimulating the clearance of A␤ across the BBB (18). Therefore, we also investigated the impact of nilvadipine enantiomers on the transport of A␤ across the BBB to determine whether the two enantiomers also retain the ability to increase the clearance of A␤ across the BBB. Our data show that (⫺)-nilvadipine also stimulates the clearance of A␤ across the BBB both in vitro and in vivo duplicating the properties of the racemic mixture and further substantiating the fact that the A␤-lowering properties of nilvadipine are not related to L-type calcium channel inhibition. As (⫺)-nilvadipine retains the anti-amyloidogenic properties of the racemic mixture but is deprived of anti-hypertensive activity, (⫺)-nilvadipine may represent a new lead compound for the treatment of AD. Interestingly, we found that (⫺)-nilvadipine also reduces Tau phosphorylation at AD-pertinent epitopes (AT8 and PHF-1) in a mouse model of tauopathy (Tg Tau P301S mice) suggesting that (⫺)-nilvadipine may also be beneficial for modulating Tau phosphorylation in AD. The fact that (⫺)-nilvadipine affects pathological Tau phosphorylation and A␤ accumulation prompted us to further explore the mechanism of action of (⫺)-nilvadipine. Our search for the mechanism responsible for the A␤-lowering properties of nilvadipine led to the identification of Syk as a possible target responsible for mediating the effects of (⫺)-nilvadipine on A␤ and Tau phosphorylation. We show that nilvadipine enantiomers inhibit Syk activity in a cell-free assay, whereas analysis of binding kinetics by biolayer interferometry shows that nilvadipine enantiomers bind to full-length human Syk with relatively high affinity (KD 2.1 ␮M) identifying Syk as a possible nilvadipine target responsible for its A␤-lowering properties. We further confirmed this possibility by showing that pharmacological inhibition of Syk in vitro and in vivo as well as downregulation of syk expression result in a reduction of A␤ production. Moreover, Syk inhibition reduces sAPP␤ production and BACE-1 transcription mimicking the effect of (⫺)-nilvadipine. In addition, we show that Syk inhibition increases the clearance of A␤ across the BBB recapitulating to the full extent the A␤-lowering properties of (⫺)-nilvadipine. Our data therefore


inhibition suggesting that Syk inhibition favors PKA activation providing a mechanism explaining the inactivation of GSK3␤ following Syk inactivation. This is also supported by the fact that phosphorylation of CREB (a substrate of PKA) is increased following Syk inhibition, whereas PKA inhibition decreases CREB phosphorylation induced by Syk inhibition. Altogether these data suggest that PKA activation following Syk inhibition plays an important role in the regulation of pathological Tau phosphorylation at GSK3␤ sites. Recent data have also emphasized this possibility by showing that PKA stimulation using a cAMP phosphodiesterase inhibitor leads to an inhibition of GSK3␤ activity and attenuates A␤-induced tauopathy (72). GSK3␤ has also been shown to regulate A␤ production in vivo by controlling BACE-1 gene expression in a NF␬B-dependent manner (15); therefore, the inhibition of GSK3␤ activity following Syk inhibition may also contribute to the inhibition of A␤ and BACE-1 expression observed following Syk inhibition. Alternatively, Syk inhibition may also result in a suppression of NF␬B activation via an inhibition of RAF phosphorylation and AKT phosphorylation, which consequently is expected to lower BACE-1 expression and A␤ production. Interestingly, we show that Syk inhibition results in a stimulation of CREB phosphorylation via PKA activation suggesting that Syk inhibition could also have neuroprotective properties because stimulation of CREB phosphorylation by PKA is known to confer neuroprotection (73–75). Also, PKA and CREB phosphorylation are


Acknowledgments—We are grateful to Dr. Peter Davies (Albert Einstein College of Medicine, Bronx, NY) for the gift of the PHF-1, CP13, and RZ3 antibodies. We thank Dr. Michael Wolf (Harvard Medical School, Boston) for providing 7W CHO APP-overexpressing cells.

REFERENCES 1. Nelson, P. T., Alafuzoff, I., Bigio, E. H., Bouras, C., Braak, H., Cairns, N. J., Castellani, R. J., Crain, B. J., Davies, P., Del Tredici, K., Duyckaerts, C., Frosch, M. P., Haroutunian, V., Hof, P. R., Hulette, C. M., Hyman, B. T., Iwatsubo, T., Jellinger, K. A., Jicha, G. A., Kövari, E., Kukull, W. A., Leverenz, J. B., Love, S., Mackenzie, I. R., Mann, D. M., Masliah, E., McKee, A. C., Montine, T. J., Morris, J. C., Schneider, J. A., Sonnen, J. A., Thal, D. R., Trojanowski, J. Q., Troncoso, J. C., Wisniewski, T., Woltjer, R. L., and Beach, T. G. (2012) Correlation of Alzheimer disease neuropathologic changes with cognitive status: a review of the literature. J. Neuropathol. Exp. Neurol. 71, 362–381 2. Santos, A. N., Ewers, M., Minthon, L., Simm, A., Silber, R. E., Blennow, K., Prvulovic, D., Hansson, O., and Hampel, H. (2012) Amyloid-␤ oligomers in cerebrospinal fluid are associated with cognitive decline in patients with Alzheimer’s disease. J. Alzheimers Dis. 29, 171–176 3. Doraiswamy, P. M., Sperling, R. A., Coleman, R. E., Johnson, K. A., Reiman, E. M., Davis, M. D., Grundman, M., Sabbagh, M. N., Sadowsky, C. H., Fleisher, A. S., Carpenter, A., Clark, C. M., Joshi, A. D., Mintun, M. A., Skovronsky, D. M., Pontecorvo, M. J., and AV45-A11 Study Group (2012) Amyloid-␤ assessed by florbetapir F 18 PET and 18-month cognitive decline: a multicenter study. Neurology 79, 1636 –1644 4. Piaceri, I., Nacmias, B., and Sorbi, S. (2013) Genetics of familial and sporadic Alzheimer’s disease. Front. Biosci. 5, 167–177 5. Walsh, D. M., and Teplow, D. B. (2012) Alzheimer’s disease and the amyloid ␤-protein. Prog. Mol. Biol. Transl. Sci. 107, 101–124 6. Jonsson, T., Atwal, J. K., Steinberg, S., Snaedal, J., Jonsson, P. V., Bjornsson, S., Stefansson, H., Sulem, P., Gudbjartsson, D., Maloney, J., Hoyte, K., Gustafson, A., Liu, Y., Lu, Y., Bhangale, T., Graham, R. R., Huttenlocher, J., Bjornsdottir, G., Andreassen, O. A., Jönsson, E. G., Palotie, A., Behrens, T. W., Magnusson, O. T., Kong, A., Thorsteinsdottir, U., Watts, R. J., and Stefansson, K. (2012) A mutation in APP protects against Alzheimer’s disease and age-related cognitive decline. Nature 488, 96 –99 7. Kero, M., PaeTau, A., Polvikoski, T., Tanskanen, M., Sulkava, R., Jansson, L., Myllykangas, L., and Tienari, P. J. (2013) Amyloid precursor protein (APP) A673T mutation in the elderly Finnish population. Neurobiol. Aging 34, 1518.e1–3 8. Kumar-Singh, S., Theuns, J., Van Broeck, B., Pirici, D., Vennekens, K., Corsmit, E., Cruts, M., Dermaut, B., Wang, R., and Van Broeckhoven, C. (2006) Mean age-of-onset of familial Alzheimer disease caused by presenilin mutations correlates with both increased A␤42 and decreased A␤40. Hum. Mutat. 27, 686 – 695



FIGURE 11. Schematic representation of the signaling pathways affected by Syk that drive A␤ production, neuroinflammation, and Tau hyperphosphorylation. Our data show that Syk regulates NF␬B activity, which besides its well known role in neuroinflammatory processes also regulates BACE-1 expression, the rate-limiting enzyme responsible for A␤ production. A␤ has been shown to stimulate Syk activity resulting in microglial activation and neurotoxicity (74 –79). In addition, Syk phosphorylates Tau directly at the residue Tyr-18 (63), which is considered to be an early event in the pathophysiology of AD. Our data illustrate that Syk also plays an important role in the regulation of GSK3␤, one of the main kinases involved in pathological Tau phosphorylation. When Syk is inhibited, this induces an activation of PKA that phosphorylates the inhibitory Ser-9 of GSK3␤ resulting in GSK3␤ inactivation and a reduction of Tau hyperphosphorylation at multiple GSK3␤-dependent epitopes. Activated PKA also phosphorylates CREB, which is expected to be neuroprotective and to improve cognition. In addition, Syk inhibition prevents NF␬B pathway activation lowering neuroinflammation and BACE-1 expression, resulting in a reduction of A␤ accumulation. Neuroinflammatory events with the ensuing gliosis, Tau hyperphosphorylation, and A␤ accumulation seem therefore intertwined and have the potential for feed-forward effects that can create a self-propagating pathological cascade in AD, which can be interrupted by inhibiting Syk activity.

down-regulated in AD brains (76) and in transgenic mouse models of AD overexpressing A␤, whereas stimulation of PKA/ CREB phosphorylation with various compounds improves memory in AD mouse models (77– 80) suggesting that Syk inhibition has the potential to reduce memory impairments by stimulating PKA/CREB signaling. We have summarized the Syk signaling events that regulate AD-related processes highlighting the potential therapeutic application of Syk inhibitors in AD (Fig. 11). Altogether our data show that Syk plays a key role in the regulation of A␤ production, Tau hyperphosphorylation, and NF␬B activation, suggesting that Syk inhibition has the potential to oppose the three main pathologies found in AD brain (A␤ deposition, Tau hyperphosphorylation, and neuroinflammation) and may therefore represent a particularly attractive target for the treatment of AD.



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Getting Syk: spleen tyrosine kinase as a therapeutic target.

Phenyl carboxamide analogues as spleen tyrosine kinase (syk) inhibitors.

Zinc binding directly regulates tau toxicity independent of tau hyperphosphorylation.

From CLL to Multiple Myeloma - Spleen Tyrosine Kinase (SYK) influences multiple myeloma cell survival and migration.

Suppression of LPS-induced tau hyperphosphorylation by serum amyloid A.

Alzheimer disease therapy--moving from amyloid-β to tau.

Defined neurofilament, tau, and beta-amyloid precursor protein epitopes distinguish Alzheimer from non-Alzheimer senile plaques.

Human secreted tau increases amyloid-beta production.

Cytoplasmic retention of protein phosphatase 2A inhibitor 2 (I2PP2A) induces Alzheimer-like abnormal hyperphosphorylation of Tau.

Spleen Tyrosine Kinase (Syk) Mediates IL-1β Induction by Primary Human Monocytes during Antibody-enhanced Dengue Virus Infection.

Spleen tyrosine kinase inhibition attenuates autoantibody production and reverses experimental autoimmune GN.

Alternative splicing of spleen tyrosine kinase differentially regulates colorectal cancer progression.

Amyloid-β and tau: the trigger and bullet in Alzheimer disease pathogenesis.

Alzheimer's disease pathological lesions activate the spleen tyrosine kinase.

Targeting spleen tyrosine kinase-Bruton's tyrosine kinase axis for immunologically mediated glomerulonephritis.

GSk3β pathway in the mouse hippocampus.

Phosphorylation of Alzheimer amyloid precursor protein by protein kinase C.

Evaluation of Tau Imaging in Staging Alzheimer Disease and Revealing Interactions Between β-Amyloid and Tauopathy.

DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk.

Isorhynchophylline treatment improves the amyloid-β-induced cognitive impairment in rats via inhibition of neuronal apoptosis and tau protein hyperphosphorylation.

Golgin-84-associated Golgi fragmentation triggers tau hyperphosphorylation by activation of cyclin-dependent kinase-5 and extracellular signal-regulated kinase.

Cortical laminar binding of PET amyloid and tau tracers in Alzheimer disease.

Spatially distinct atrophy is linked to β-amyloid and tau in preclinical Alzheimer disease.

Cerebrospinal fluid β-amyloid and phospho-tau biomarker interactions affecting brain structure in preclinical Alzheimer disease.