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

Production andβRegulates Alzheimer's AThe Spleen Tyrosine Kinase (syk)Molecular Bases of Disease:

published online October 20, 2014J. Biol. Chem. 

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Syk controls Alzheimer's disease pathological lesions

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The spleen tyrosine kinase (syk) regulates Alzheimer's A production and tau hyperphosphorylation*

Daniel Paris1, Ghania Ait-Ghezala, Corbin Bachmeier, Gary Laco, David Beaulieu-Abdelahad,

Yong Lin, Chao Jin, Fiona Crawford, Michael Mullan

From the Roskamp Institute, Sarasota, FL 34243

*Running title: Syk controls Alzheimer's disease pathological lesions

1 To whom correspondence should be addressed: Daniel Paris, The Roskamp Institute, 2040 Whitfield

Avenue, Sarasota, FL 34243, USA, Tel.: (941) 752-2949; Fax: (941) 752-2948; Email: dparis@rfdn.org

Keywords: Alzheimer disease; amyloid (A); glycogen synthase kinase 3 (GSK-3); secretase; Tau

protein (Tau); spleen tyrosine kinase (syk)

Background: Spleen tyrosine kinase (syk)

mediates microglial activation and neurotoxicity

elicited by Alzheimer's Aβ peptides.

Results: Syk regulates Aβ production via NFκB

dependent mechanisms and tau phosphorylation

by controlling the activation of glycogen synthase

3β.

Conclusion: Inhibition of syk can interrupt the

neuroinflammation, pathological Aβ accumulation

and tau hyperphosphorylation in Alzheimer's.

Significance: Syk represents a therapeutic target

for the key pathological lesions that define

Alzheimer's.

ABSTRACT

We have previously shown that the L-

type calcium channel (LCC) antagonist

nilvadipine reduces brain A accumulation by

affecting both A production and A clearance

across the blood-brain barrier (BBB).

Nilvadipine consists of a mixture of two

enantiomers, (+)-nilvadipine and (-)-

nilvadipine, in equal proportion. (+)-

nilvadipine is the active enantiomer responsible

for the inhibition of LCC whereas (-)-

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 (Tg

Tau P301S) with (-)-nilvadipine reduces tau

hyperphosphorylation at several AD pertinent

epitopes. A search for the mechanism of action

of (-)-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 downregulation of syk expression

reduces A production and increases the

clearance of A across the BBB mimicking (-)-

nilvadipine effects. Moreover, treatment of

transgenic mice overexpressing A and Tg 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 Ser9 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.

Alzheimer's disease (AD) is the most

prevalent form of dementia in the elderly. AD

pathology is characterized by the presence of

extracellular deposits of A peptides and by the

accumulation of intraneuronal tangles made of

hyperphosphorylated tau. Although the amount of

http://www.jbc.org/cgi/doi/10.1074/jbc.M114.608091The latest version is at JBC Papers in Press. Published on October 20, 2014 as Manuscript M114.608091

Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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Syk controls Alzheimer's disease pathological lesions

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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

aiming at detecting A in the brain of subjects

with mild cognitive impairment (MCI), AD and

normal controls revealed that elevated brain A

levels at baseline are associated with greater

cognitive worsening in both AD, MCI 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 A38, A40 and

A42. Soluble A oligomers are now widely

recognized as pathogenic since they have been

shown to inhibit synaptic function, to cause

synaptic degeneration, to induce tau

hyperphosphorylation and to impair memory (8-

11).

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 disease-associated 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

(DHP) display some A lowering properties by

affecting both the processing of APP and the

clearance of A across the blood-brain barrier

(BBB) (18,19), properties which are unrelated to

the inhibition of calcium channels or to their anti-

hypertensive activity (18). Among the DHP CCB

tested, we identified nilvadipine for its ability to

readily cross the BBB, to lower brain A levels, to

improve cognition and 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 to 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 since we have shown previously

that racemic nilvadipine stimulates the BBB

clearance of A (18). Additionally, we evaluated

the A lowering properties of (+) and (-)-

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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.

EXPERIMENTAL PROCEDURES

Chemicals and reagents

Racemic nilvadipine, (-)-nilvadipine and

(+)-nilvadipine were synthesized as previously

described (26) and were obtained from Archer

Pharmaceuticals (FL, USA). BAY61-3606,

phorbol 12-myristate 13-acetate (PMA), human

recombinant TNF-, dimethyl sulfoxide (DMSO),

2-mercaptoethanol, imidazole, sodium chloride

and phenylmethylsulfony fluoride (PMSF) were

purchased from Sigma-Aldrich (MO, USA).

KT5270 was obtained from R&D systems (MN,

USA). All antibiotics, fungizone, PBS, culture

media and fetal bovine serum were purchased

from Life Technologies (CA, USA). Fluorescein-

A1–42 was purchased from rPeptide (Bogart, GA).

The MPER reagent and the protease/phosphatase

inhibitors cocktail were purchased from Thermo

Fisher Scientific (IL, USA). Guanidine

hydrochloride was obtained from EMD Millipore

(MA, USA).

In vitro assays for A production and sAPP

secretion.

7W CHO (29) cells stably transfected with

human APP751 were maintained in DMEM

medium containing 10% fetal bovine serum, 1X

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 hours with a dose range of BAY61-

3606 (0.5, 1, 5 and 10µM), a dose range of (-)-

nilvadipine (1, 5, 10, and 20 µM), (+)-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, Germany) and

no significant toxicity was observed for the

different treatments (data not shown). Following

the treatments with nilvadipine enantiomers, A40

and A42 were analyzed in the culture medium by

using commercially available sandwich ELISAs

(Life Technologies, CA, USA) according to

recommendations of the manufacturer. Following

the treatments with a dose range of BAY61-3606,

A38, A40 and A42 were quantified by

electrochemoluminescence using multiplex A

assays according to the manufacturer’s

recommendations (Meso Scale Discovery, MD,

USA). All experiments were performed at least in

quadruplicate for each treatment dose.

Additionally, sAPP was detected by Western-

blot in the culture medium surrounding 7 W CHO

cells using the antibody 6E10 (Signet Laboratories

Inc., MA, USA) 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 previously described (30).

In vitro blood-brain barrier model

The in vitro model of the blood-brain

barrier (BBB) consisting of a polarized human

brain microvascular endothelial cells monolayer

grown on cell culture inserts that separates into

apical ("blood") and basolateral ("brain")

compartments, was established as previously

described by our group (18,19,38-41). A

exchange dynamics across the BBB model were

examined using a fluorometric A42 assay as we

previously described (18,19,31-34). Briefly, the

apical (receiver) side of the membrane was

exposed to various concentrations of racemic

nilvadipine, (-)-nilvadipine, (+)-nilvadipine or

BAY61-3606. The donor compartment was

sampled at time 0 to establish the initial

concentration of fluorescein labeled A1-42 (FlA1-

42) in each group. Following exposure of the insert

to the well containing FlA1-42, samples were

collected from the apical compartment at various

time points up to 90 minutes to assess the

movement of FlA1-42 across the HBMEC

monolayer (basolateral-to-apical). The samples

were analyzed (λex = 485 nm and λem = 516 nm)

for FlA1-42 using a BioTek Synergy HT multi-

detection microplate reader (Winooski, VT, USA).

The apparent permeability (Papp) of FlA1-42 was

determined using the equation Papp = 1/AC0 *

(dQ/dt), where A represents the surface area of the

membrane, C0 is the initial concentration of FlA1-

42 in the basolateral compartment, and dQ/dt is the

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amount of FlA1-42 appearing in the apical

compartment in the given time period. The Papp

of FlAβ1-42 in the presence of drug was compared

to 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 in the

Association for Assessment and Accreditation of

Laboratory Animal Care International (AAALAC)

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 one hour after

the last injection, animals were humanely

euthanatized, their brains (without cerebella) were

snap frozen in liquid nitrogen until being analyzed

for A40 and A42 levels using commercially

available ELISA kits (Life Technologies, CA,

USA). Briefly, brains were homogenized at 4°C in

MPER reagent (Thermo Fisher Scientific Inc., IL,

USA) containing 1 mM PMSF and 1X

protease/phosphatase inhibitors cocktail (Thermo

Fisher Scientific Inc., IL, USA) and centrifuged at

15,000 g for 30 min at 4°C. The supernatant

containing detergent solubilized A was collected

and denatured in 5M guanidine for one hour

before being diluted with the sample diluent

provided in the A ELISA kits and assessed for

A40 and A42 according to the manufacturer's

protocol. The pellet containing detergent insoluble

material was resuspended in 5M 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 (Life

Technologies, CA, USA) and results of brain A40

and A42 were calculated in pg/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. BAY61-3606 was

dissolved in sterile PBS. Vehicle-treated Tg

PS1/APPsw mice received an intraperitoneal

injection of PBS for 4 days. Mice were humanely

euthanatized 30 minutes after the last injection,

their brains (without cerebella) were snap frozen

in liquid nitrogen until analysis for A38, A40 and

A42 levels by electrochemoluminescence using

multiplex A assays according to the

manufacturer’s recommendations (Meso Scale

Discovery, MD, USA). Brain homogenates for the

quantification of A peptides were prepared as

described above.

Silencing of the SYK gene by shRNA in NFB

luciferase reporter cells and in 7W CHO APP

overexpressing cells

Short hairpin RNAs (shRNAs) were used

to stably knock-down SYK gene expression in

HEK-NFB luciferase reporter cells (Panomics,

CA, USA). SYK shRNAs cloned into the GIPZ

lentiviral vector were purchased from Origene

(Rockville, MD, USA). Five different SYK

shRNAs and a scrambled control shRNA were

used separately for transfection. Approximately

one million HEK-NFB luciferase reporter cells

detached with TripLE (Invitrogen, IL, USA) were

mixed together with 10μg shRNA in Bio-Rad

(Hercules, CA, USA) genepulser cuvettes (0.4 cm)

and electroporated using a square wave protocol:

110V, one pulse length 25ms using a Bio-Rad

(Hercules, CA, USA) gene pulser and were seeded

in 6-well plates. 48 hours later, the cell culture

media were replaced by selective media containing

6mg/ml of 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

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including the control shRNA (control) to confirm

silencing of the SYK gene using the anti-SYK

antibody from Santa Cruz Biotechnology (CA,

USA) (1:500 dilution). The clones (83D04, 38G10

and 69F04) with the most profound silencing were

chosen to evaluate the effect of SYK deficiency on

NFB luciferase activity. Briefly, NFB activation

was triggered with 10ng/ml of PMA for 24 hours

and NFB luciferase activity quantified by

chemiluminescence using the Luc-Screen

Extended-Glow from Applied Biosystems (CA,

USA) with a Synergy HT chemiluminescence

reader (Biotek, VT, USA) as we previously

described (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 (Ser338) 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 Inc., MA, USA).

Expression and purification of human biotinylated

syk

The plasmid pDW445 (38) containing the

birA biotin-protein ligase gene was obtained from

Addgene (MA, USA) and digested with Bam HI

and Hind III restriction enzymes to remove the

birA gene. The birA gene was then ligated into

pBac-1 (EMD Millipore, MA, USA) 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 Bam HI

restriction site followed by the ATG start codon

and to the 3' end codons in the following order for

a Gly(4)Ser linker, an AviTag (Avidity)

GLNDIFEAQKIEWHE which is biotinylated on

the Lys by the BirA enzyme (39) and His(6)

followed by a stop codon and an Eco RI restriction

site (GenScript, NJ, USA). The synthesized gene

(sykA6H) was blunt end cloned into pUC57 using

an Eco RV site to give pSykAvi6H, the gene

sequence was verified by DNA sequencing

(GenScript, NJ, USA). The pSykAvi6H plasmid

was digested with Bam HI and Eco RI and the

sykAvi6H gene was ligated into pBac-1 which had

been digested with the same restriction enzymes to

give pBacSykAvi6H. Both pBacBirA and

pBacSykAvi6H were transfected into Sf9 insect

cells as previously described (39) with the

following modification: the flashBAC plasmid

(Oxford Expression Technologies, Inc., CA, USA)

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 (Life

Technologies, CA, USA) supplemented with 100

M biotin.

The fBAC-BirA and fBAC-SykAvi6H co-

infected insect cells were harvested after 60-70 hr

and centrifuged at 2,000g. The pellet was then

resuspended in ice-cold lysis buffer (50 mM

sodium phosphate pH 7.4, 100 mM NaCl, 0.5%

NP40, 5 mM 2-mercaptoethanol and cOmplete

EDTA-free protease inhibitor cocktail (Roche,

Life Technologies, CA, USA) and then incubated

on ice for 15 min with gentle mixing every 3 min

to keep cells suspended. The sample was then

centrifuged at 6,000g for 10 min at 5°C, the

supernatant was removed and placed in a new tube

and centrifuged at 15,000g 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 Life Sciences,

PA, USA) with inversion for 1 hr at 5oC, 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 a

FPLC column. The column was then 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 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 full-length (data not shown). The

SykAvi6H was >95% biotinylated as determined

using Streptavidin Agarose resin (Thermo Fisher

Scientific Inc., IL, USA) in a pull-down 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 (Life Technologies, CA, USA).

Enzymatic reactions were carried out at 30°C

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Syk controls Alzheimer's disease pathological lesions

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using a Biotek Synergy HT plate reader (VT,

USA). Fluorescence intensity readings

(ex=360nm/em=485) were collected every 39

seconds for 40 minutes.

Biolayer interferometry (BLI)

A fortéBio Octet Red platform (fortéBio

Corp., CA, USA) 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 (Greiger Bio-One, NC, USA) 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 superstreptavidin (SSA)

biosensors (fortéBio Corp., CA, USA). Syk

biosensors were washed several times in kinetic

buffer prior to performing the binding

experiments. Reference SSA biosensors loaded

with biotinylated-PEG4 (Thermo Fisher Scientific

Inc., IL, USA) were used to correct for non

specific binding of (-)-nilvadipine and BAY61-

3606 to the biosensors. For (-)-nilvadipine and

BAY61-3606, the association step was performed

for 120 seconds, the dissociation step for 180

seconds followed by an additional wash step of 30

seconds 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 10M). Data analysis and curve fitting

were done using the Octet software version 6.4

(fortéBio Corp., CA, USA). Steady-state kinetic

analyses were performed for every data set to

calculate the KD using the estimated response at

the equilibrium for each concentration of (-)-

nilvadipine and BAY61-3606. An average KD (±

S.E.M) representative of 6 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 (ME,

USA). (-)-nilvadipine was administered

intraperitoneally at a dosage of 2mg/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, USA) and PHF-1 (41) as

we previously described (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 previously described (42) using

PHF-1, RZ3, CP13 (48) and 9G3 (MédiMabs,

Canada) antibodies.

SHSY-5Y cell culture experiments

SH-SY5Y cells were purchased from

American Type Culture Collection (VA, USA).

SH-SY5Y cells were grown in DMEM/F12

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 while BACE-

1 protein expression was analyzed by western-

blots as we previously described (36,37). SHSY-

5Y were treated with a dose range of BAY61-3606

for 30 minutes and 24 hours to assess the impact

of syk inhibition on AKT phosphorylation and

GSK3 Ser9 phosphorylation by western-blots

using phospho-selective Ser9 GSK3 and

phospho-Ser473 AKT antibodies (Cell signaling

Technology Inc., MA, USA) at 1:1000 dilutions.

The effects of BAY61-3606 on tau

phosphorylation at multiple AD relevant epitopes

(9G3 (phospho-Tyr18), PHF-1, RZ3, CP13) were

studied by western-blot as we previously described

(42). The involvement of PKA in mediating the

stimulation of Ser9 phosphorylation of GSK3

was tested by using the selective PKA inhibitor

KT5270 (Tocris, CA, USA). SHSY-5Y cells were

pretreated with KT5270 for 5 minutes prior to be

challenged with 10µM of BAY61-3606 for a

period of 30 minutes. The impact of these

treatments on Ser9 GSK3 phosphorylation and

on CREB phosphorylation (Ser133) were followed

by western-blots using phospho-selective

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antibodies from Cell signaling Technology (MA,

USA) at 1:1000 dilutions.

Statistical analyses Results are expressed as mean ± SEM.

Statistical analyses were performed using SPSS

V12.0.1 for Windows. Data were examined for

assumption of normality using the Shapiro-Wilk

statistic and for homogeneity of variance 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.

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, (+)-nilvadipine and of 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 hours of treatment 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

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 neuronal like SHSY-5Y

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 blood-brain barrier (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 A42 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 to (+)-nilvadipine or racemic

nilvadipine and 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 observed

that both (-)-nilvadipine and (+)-nilvadipine

acutely reduced brain A levels with similar

potency (Fig. 2C&D). We also evaluated the

impact of (-)-nilvadipine on the clearance of

human A42 across the blood-brain barrier using

wild-type mice as we previously described (18).

Wild-type mice were intracranially injected with

human A42 and the appearance of human A42

was followed in the blood of the animals. Data

show that (-)-nilvadipine stimulated the clearance

of human A42 across the BBB as more human

A42 was detected in the plasma of (-)-nilvadipine

treated mice than control animals (Fig. 2B).

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We explored the possible impact of (-)-

nilvadipine on tau phosphorylation using Tg Tau

P301S mice as 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 at AT8 (phosphorylated

Ser199/Ser202/Thr205) and PHF-1

(phosphorylated Ser396/Ser404) epitopes (Fig. 3).

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 LCC 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. Since (-)-nilvadipine and

racemic nilvadipine inhibit BACE-1 transcription,

we evaluated whether (-)-nilvadipine was

impacting NFB activation as NFB 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 NFB activation in response to TNF

(Fig. 4A) or phorbol 12-myristate 13-acetate

(PMA) (Fig. 4B) using an NFB-luciferase

reporter cell line to monitor NFB activation

suggesting a possible mechanism responsible for

the inhibition of BACE-1 transcription following

nilvadipine treatment. The signaling pathways

responsible for the activation of NFB 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 NFB 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 NFB

activation induced by (-)-nilvadipine, 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 which

ultimately induces NFB activation (46-48), hence

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. 4C&D)

suggesting that (-)-nilvadipine is impacting a

target upstream of RAF. We tested the possible

effect of (-)-nilvadipine of RAS activity, and

found that (-)-nilvadipine is unable to affect RAS

activity in a cell free assay ruling out RAS as a

possible target of (-)-nilvadipine (data not shown).

Tyrosine kinases including the spleen tyrosine

kinase (syk) and the Bruton's tyrosine kinase

(BTK) are activated following PMA treatment

(49,50), act upstream of RAS/RAF (51,52) and

mediate the activation of the NFB pathway (53).

We tested a selective BTK inhibitor (BTK

inhibitor III, 1-(3-(4-Amino-3-(4-

phenyloxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-

1-yl)piperidin-1-yl)prop-2-en-1-one, N-Acryloyl-

(3-(4-Amino-3-(4-phenyloxyphenyl)-1H-

pyrazolo[3,4-d]pyrimidin-1-yl)piperidine) on A

production and 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 dose dependent inhibition

of syk activity was observed with BAY61-3606 as

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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 µM (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 0.4µM was obtained

for BAY61-3606. In addition, we verified that

pharmacological inhibition of syk with BAY61-

3606 results in a blockade of NFB activation and

that genetic downregulation of syk using shRNA

also prevents NFB activation (data not shown)

highlighting syk as a key player of NFB

activation. The magnitude of the NFB inhibition

following (-)-nilvadipine treatment was also

reduced in clones of HEK293 NFB luciferase

reporter cells in which syk expression has been

silenced (data not shown) further suggesting that

syk is required to mediate the inhibition of NFB

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-(1-Methyl-

1H-indol-3-yl-methylene)-2-oxo-2,3-dihydro-1H-

indole-5-sulfonamide and 2-(2-

Aminoethylamino)-4-(3-trifluoromethylanilino)-

pyrimidine-5-carboxamide) and 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 suppressed 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 to 7W CHO cells (Fig. 6C)

was significantly reduced, further demonstrating

the involvement of syk in the regulation of A

production.

Since 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.

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 A38,

A40 and A42 levels in Tg PS1/APPsw mice (Fig.

7C&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 A42 in mice

treated with the syk inhibitor compared to vehicle

treated mice following the intracranial injection of

human A42 (Fig. 7B).

Effects of syk inhibition on tau phosphorylation

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Syk has been shown to mediate the

phosphorylation of tau at Tyr18 (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

Tyr18 and also investigated whether other tau

phosphoepitopes were affected by the treatment.

We observed a reduction in tau phosphorylation at

the Tyr18 epitope as expected (Fig. 8) in BAY61-

3606 treated mice. Interestingly, we also detected

a reduction in tau phosphorylation at PHF-1

(pSer396/pSer404) and CP13 (pSer202) epitopes

following treatment of Tg Tau P301S mice with

BAY61-3606 whereas the RZ3 (pThr231) 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 neuronal like SHSY-5Y to delineate a

possible mechanism of action responsible for the

reduction of tau phosphorylation observed at

multiple tau epitopes following syk inhibition.

Since we observed a reduction of tau

phosphorylation at the characteristic GSK3 motif

(pSer396/pSer404 (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 Ser9 which is

known to inactivate the enzyme (57). We observed

that pharmacological inhibition of syk with

BAY61-3606 stimulates Ser9 phosphorylation of

GSK3 in SHSY-5Y cells (Fig. 9A&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 SHSY-5Y cells

with BAY61-3606 whereas tau phosphorylation at

the RZ3 site was not significantly impacted in

SHSY-5Y cells (Fig. 9C). GSK3 phosphorylation

at Ser9 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

SHSY-5Y cells with BAY61-3606 inhibits AKT

phosphorylation (Fig. 9A&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 Ser9 phosphorylation induced

by syk inhibition in SHSY-5Y cells. We then

explored whether a selective PKA inhibitor

(KT5270) was able to mitigate GSK3 Ser9

phosphorylation induced by treatment of SHSY-

5Y cells with BAY61-3606. We found that

KT5270 effectively suppressed GSK3

phosphorylation at Ser9 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 Tyr330

of the PKA catalytic subunit (60) further

supporting our observation. To verify that syk

inhibition with BAY61-3606 was resulting 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 while that event is inhibited in

presence of a selective PKA inhibitor (Fig.

10A&B) further showing that syk inhibition

results in PKA activation.

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

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. Chronic treatment with

nilvadipine has been shown to prevent the

conversion of MCI 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 the present 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

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activity of the compound (24-26). We confirmed

ex vivo in rat aortae that (-)-nilvadipine was

approximately 1000 time less potent than (+)-

nilvadipine for opposing the vasoconstriction

induced by a selective L-type calcium channel

agonist (FPL64176) showing 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 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). We therefore 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 show

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 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 NFB

activation suggesting a possible mechanism for the

reduction of BACE-1 transcription observed

following syk inhibition as NFB has been shown

previously to play an important role in the

regulation of BACE-1 transcription and expression

(36,37,44,64). Since NFB 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 Tyr18 residue (56),

which is considered to be an early event in the

pathophysiology of AD (56,71). We show that

treatment of SHSY-5Y cells with the selective syk

inhibitor BAY61-3606 results in a partial

reduction of tau phosphorylation at Tyr18 but also

in an almost complete inhibition of tau

phosphorylation at Ser202 (CP13) and

Ser396/Ser404 (PHF-1). The partial inhibition of

tau phosphorylation at Tyr18 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

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the selective syk inhibitor BAY61-3606 results in

a reduction of tau Tyr18 phosphorylation as

expected but also in a significant decrease in tau

phosphorylation at other epitopes including

Ser202 (CP13) and Ser396/Ser404 (PHF-1) which

are phosphorylated by GSK3 (13). Since syk

inhibition results in decreased tau phosphorylation

at typical GSK3 sites in Tg P301S mice and

SHSY-5Y cells, we investigated whether

pharmacological inactivation of syk could affect

GSK3. We found that inhibition of syk in SHSY-

5Y cells stimulates Ser9 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 Ser202 and Ser396/Ser404

epitopes phosphorylated by GSK3. Previous data

have revealed that syk phosphorylates Tyr330 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 Ser9 phosphorylation induced

by syk inhibition. Our data show that PKA

inactivation with KT5270 opposes the increased

Ser9 phosphorylation of GSK3 induced by syk

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 NFB

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 NFB 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 as

stimulation of CREB phosphorylation by PKA is

known to confer neuroprotection (73-75). Also,

PKA and CREB phosphorylation are

downregulated 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 NFB 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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ACKNOWLEGEMENTS-We are grateful to Dr. Peter Davies (Albert Einstein College of Medicine,

Bronx, NY, USA) for the gift of the PHF-1, CP13 and RZ3 antibodies. We thank Dr. Michael Wolf

(Harvard Medical School, Boston, MA, USA) for providing 7W CHO APP overexpressing cells. We

thank the Roskamp Foundation for providing support for this work.

FOOTNOTES 1 To whom correspondence may be addressed: The Roskamp Institute, 2040 Whitfield Avenue, Sarasota,

FL 34243, USA, Tel.: (941) 752-2949; Fax: (941) 752-2948; Email: dparis@rfdn.org

2 The abbreviations used are: shRNA, small hairpin ribonucleic acid; PKA, protein kinase A; NFB,

nuclear factor kappa-light-chain-enhancer of activated B cells; syk, spleen tyrosine kinase; APP, amyloid

precursor protein; BACE-1, beta-site amyloid precursor protein cleaving enzyme 1; GSK3, glycogen

synthase 3; sAPP, secreted amyloid precursor protein; RT-PCR, reverse transcription polymerase chain

reaction; PMA, phorbol 12-myristate 13-acetate; TNF, tumor necrosis factor-; LCC, L-type calcium

channel; AKT, protein kinase B; PKA, protein kinase A; RAF kinase, Rapidly Accelerated Fibrosarcoma

kinase; CREB, cyclic adenosine monophosphate response element-binding protein; FU, fluorescence

units; CCB, calcium channel blocker; DHP, dihydropyridine

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FIGURE LEGENDS

FIGURE 1. Effects of nilvadipine enantiomers on A production, sAPP secretion and BACE-1

expression. A) Dose dependent inhibition of A40 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 A40 production. Post-hoc comparisons show significant differences in

A40 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 1µM. No significant

difference in A40 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 A40 to the same extent. B) Representative western-blots showing

that (-)-nilvadipine reduces the secretion of sAPP in 7W CHO cells overexpressing APP showing 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 neuronal like

cells SHSY-5Y. SHSY-5Y 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 SHSY-5Y cells co-treated with

TNF and (-)-nilvadipine (P<0.01) and in cells co-treated with TNF and racemic nilvadipine (P<0.001)

compared to the TNF treatment conditions for all the time points tested showing that (-)-nilvadipine and

racemic nilvadipine antagonize BACE-1 transcription induced by TNF. The symbol "*" denotes

statistically significant differences compared to the control conditions whereas the "$" sign indicates

statistically significant differences compared to the TNF treatment conditions. D) Effects of racemic

nilvadipine and (-)-nilvadipine on basal BACE-1 protein expression. HEK293 cells were treated for 24

hours with 20M of 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.

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 A1-42 in an in

vitro BBB model employing human brain microvascular endothelial cells. The histogram represents the

apparent permeability of A1-42 calculated for a period of 90 minutes in response to a dose range of (+)-

nilvadipine and (-)-nilvadipine depicting the transport of A1-42 from the basolateral ("brain") to the apical

("blood") compartment of the BBB model. Results are expressed as the percentage of A1-42 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 A1-42 levels transported to the apical of the

BBB model. Post-hoc comparisons reveal statistically significant differences in the amount of A1-42

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 A1-42 across the BBB in this in vitro

model. B) Effect of (-)-nilvadipine on the clearance of human A1-42 across the BBB in vivo. The

histogram represents the amount of human A1-42 quantified in the plasma of vehicle and (-)-nilvadipine

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treated wild-type mice that received an intracranial injection of human A1-42. A significant increase in

human A1-42 was detected in the plasma of (-)-nilvadipine treated mice compared to the mice that

received the vehicle used to dissolve (-)-nilvadipine (T-test, P<0.001) showing that (-)-nilvadipine

stimulates the clearance of human A1-42 across the BBB in vivo. C) Impact of (-)-nilvadipine and (+)-

nilvadipine on brain A accumulation in Tg PS1/APPsw mice. Tg PS1/APPsw (26 week-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 pg per 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 A40 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

4mg/Kg. D) The histogram represents the amount of detergent soluble and insoluble A42 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 4mg/Kg. ANOVA show a significant main effect of (-)-nilvadipine (P<0.001) and

of (+)-nilvadipine (P<0.001) for detergent soluble A40 , detergent soluble A42, detergent insoluble A40

and detergent insoluble A42. Post-hoc comparisons reveal statistically significant differences in brain

detergent soluble A40 levels between vehicle and (-)-nilvadipine 2mg/Kg (P<0.007), vehicle and (-)-

nilvadipine 4 mg/Kg (P<0.003), vehicle and (+)-nilvadipine 2mg/Kg (P<0.02) and vehicle and (+)-

nilvadipine 4mg/Kg (P<0.008). Post-hoc comparisons show statistically significant differences in brain

detergent soluble A42 levels between vehicle and (-)-nilvadipine 2mg/Kg (P<0.03), vehicle and (-)-

nilvadipine 4 mg/Kg (P<0.02) and vehicle and (+)-nilvadipine 4mg/Kg (P<0.05). Post-hoc comparisons

reveal statistically significant differences in brain detergent insoluble soluble A40 levels between vehicle

and (-)-nilvadipine 2mg/Kg (P<0.005), vehicle and (-)-nilvadipine 4 mg/Kg (P<0.01), vehicle and (+)-

nilvadipine 2mg/Kg (P<0.05) and vehicle and (+)-nilvadipine 4mg/Kg (P<0.01). Post-hoc comparisons

show also statistically significant differences in brain detergent insoluble soluble A42 levels between

vehicle and (-)-nilvadipine 2mg/Kg (P<0.001), vehicle and (-)-nilvadipine 4 mg/Kg (P<0.001), vehicle

and (+)-nilvadipine 2mg/Kg (P<0.001) and vehicle and (+)-nilvadipine 4mg/Kg (P<0.001). The symbol

"*" on the histograms indicates statistically significant differences compared to 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 (Ser396/Ser404) and AT8 (Ser202/Thr205) 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 hyperphopshorylation in Tg

Tau P301S mice. The symbol "*" on the histogram indicates statistically significant differences compared

to vehicle treated Tg Tau P301S mice.

FIGURE 4. Effect of (-)-nilvadipine on NFB activation. NFB activation was measured by quantifying

intracellular luciferase activity using an NFB luciferase reporter HEK293 cell line. A) Histogram

showing that (-)-nilvadipine (200nM) inhibits NFB activation induced by TNF (3 hours treatment).

ANOVA shows a significant main effect of TNF (P<0.001) and (-)-nilvadipine on NFB 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 NFB luciferase

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activity showing that (-)-nilvadipine suppresses NFB activity induced by TNF. B) Histogram showing

that (-)-nilvadipine antagonizes PMA induced NFB activation (24 hours treatment). ANOVA shows a

significant main effect of PMA (P<0.001) and (-)-nilvadipine on NFB 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 NFB luciferase activity showing that (-)-

nilvadipine suppresses NFB activity induced by PMA. C) Western-blot depicting the impact of (-)-

nilvadipine on RAF phosphorylation (Ser338) induced by PMA. HELA cells were challenged with 100

ng/ml of PMA with or without (-)-nilvadipine for 15 minutes. 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), 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 (Ser338) 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), 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.

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) which 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 reveals significant differences between control and (-)-nilvadipine treatments for

all the doses tested (P<0.001) showing that (-)-nilvadipine inhibits syk activity. The symbol "*" indicates

statistically significant differences compared to 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 reveals significant differences between control and BAY61-3606

treatments for all the doses greater than 0.3125µM (P<0.001). The symbol "*" indicates statistically

significant differences compared to 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 6 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 6 independent experiments and generated an average KD = 0.42 ± 0.1 M.

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FIGURE 6. Effect of pharmacological inhibition of syk and downregulation of syk expression on A

production. A) Dose dependent inhibition of A38, A40 and A42 production by 7W CHO overexpressing

APP treated with the syk inhibitor BAY61-3606. ANOVA shows a significant main effect of BAY61-

3606 doses on A38, A40 and A42 production (P<0.001). Post-hoc comparisons reveal significant

differences between the A38, A40 and A42 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. The symbol "*" indicates statistically significant differences compared to 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 downregulation 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 to the control conditions. No statistically significant difference in RAF

phosphorylation levels were observed between SYK shRNA 7W CHO cells untreated and treated with

PMA (P=0.304) showing that downregulation 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 A38, A40 and A42 production in control 7W CHO cells and in 7W

CHO stably transfected with SYK shRNA. T-tests reveal significant differences for A38, A40 and A42

(P<0.001) produced by 7W CHO cells and 7W CHO cells transfected with SYK shRNA showing that

downregulation of syk expression inhibits A production.

FIGURE 7. Effects of pharacological 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 A1-42 in an in vitro BBB

model employing human brain microvascular endothelial cells. The histogram represents the apparent

permeability of A1-42 calculated for a period of 90 minutes in response to a dose range of BAY61-3606

and depicts the transport of A1-42 from the basolateral ("brain") to the apical ("blood") compartment of

the BBB model. Results are expressed as the percentage of A1-42 apparent permeability observed in the

control conditions. ANOVA show a significant main effect of BAY61-3606 (P<0.05) on A1-42 levels

transported to the apical side of the BBB model. Post-hoc comparisons reveal statistically significant

differences in the amount of A1-42 transported from the basolateral (brain side) to the apical side (blood

side) for human brain microvascular endothelial cells treated with 5 M of BAY61-3606 (P<0.05)

showing that pharmacological inhibition of syk activity stimulates the transport of A1-42 across the BBB

in this in vitro model. The symbol "*" indicates statistically significant differences compared to the

control conditions. B) BAY61-3606 increases the clearance of human A1-42 across the BBB in vivo.

ANOVA shows a significant main effect of BAY61-3606 on plasma human A1-42 (P<0.03) detected

following an intracranial injection of the peptide. Post-hoc comparisons reveal statistically significant

difference in plasma human A1-42 between vehicle treated mice and mice treated with 4 mg/Kg of

BAY61-3606 (P<0.05) showing that at this dose BAY61-3606 stimulates the clearance of A1-42 across

the BBB in vivo. The symbol "*" indicates statistically significant differences compared to the vehicle

treatment conditions. C) Effects of pharmacological inhibition of syk with BAY61-3606 on brain A38,

A40 and A42 levels in Tg PS1/APPsw mice. Histogram representing the amount of detergent soluble

A38, A40 and A42 detected in the brains of vehicle and BAY61-3606 treated Tg PS1/APPsw mice. A

significant reduction in brain detergent soluble A38 (P<0.01), A40 (P<0.01) and A42 (P<0.01) levels

was observed in BAY61-3606 treated Tg PS1/APPsw compared to vehicle treated mice (placebo). D)

Histogram representing the amount of detergent insoluble A38, A40 and A42 detected in the brains of

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vehicle (placebo) and BAY61-3606 treated Tg PS1/APPsw mice. A significant reduction in brain

detergent insoluble A38 (P<0.01), A40 (P<0.01) and A42 (P<0.05) levels was observed in BAY61-3606

treated Tg PS1/APPsw compared to vehicle treated mice (placebo).

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 Tyr18 (9G3), pSer396/pSer404 (PHF-1), pSer202 (CP13) and pThr231(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) phospho-epitopes but not at the RZ3 epitope

(P=0.382). The symbol "*" indicates statistically significant differences compared to the vehicle

treatment.

FIGURE 9. Effect of pharmacological inhibition of syk with BAY61-3606 on GSK3 Ser9

phosphorylation, AKT phosphorylation in SHSY-5Y cells and tau phosphorylation. A) Representative

western-blot depicting the impact of a 30 minutes treatment with a dose range of BAY61-3606 on

GSK3 Ser9 phosphorylation and on AKT phosphorylation. GAPDH was used as a reference protein.

The histogram represents the average chemiluminescent signals quantified for GSK3 Ser9

phosphorylation/GAPDH and AKT Ser473 phosphorylation/GAPDH. ANOVA shows a significant main

effect of BAY61-3606 on GSK3 Ser9 phosphorylation/GAPDH (P<0.001) and AKT Ser473

phosphorylation/GAPDH (P<0.001). Post-hoc comparisons reveal statistically significant differences for

GSK3 Ser9 phosphorylation/GAPDH (P<0.001) and AKT Ser473 phosphorylation/GAPDH at all dose

of BAY61-3606 tested (P<0.02) showing that BAY61-3606 treatment stimulates GSK3 Ser9

phosphorylation and inhibits AKT phosphorylation. The symbol "*" indicates statistically significant

differences compared to the control conditions. B) Representative western-blot depicting the impact of a

24 hour treatment with a dose range of BAY61-3606 on GSK3 Ser9 phosphorylation and on AKT

phosphorylation. GAPDH was used as a reference protein. The histogram represents the average

chemiluminescent signals quantified for GSK3 Ser9 phosphorylation/GAPDH and AKT Ser473

phosphorylation/GAPDH. ANOVA shows a significant main effect of BAY61-3606 on GSK3 Ser9

phosphorylation/GAPDH (P<0.001) and AKT Ser473 phosphorylation/GAPDH (P<0.001). Post-hoc

comparisons reveal statistically significant differences for GSK3 Ser9 phosphorylation/GAPDH

(P<0.015) and AKT Ser473 phosphorylation/GAPDH at all dose of BAY61-3606 tested (P<0.001)

showing that BAY61-3606 treatment stimulates GSK3 Ser9 phosphorylation and inhibits AKT

phosphorylation. The symbol "*" indicates statistically significant differences compared to the control

conditions. C) Impact of a 24 hour treatment with 10µM of the syk inhibitor BAY61-3606 on tau

phosphorylation in SHSY-5Y cells. Western-blots showing the impact of BAY61-3606 on tau

phosphorylation at Tyr18, Ser396/Ser404, Thr231 and Ser202. 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 SHSY-5Y 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 10M of BAY61-3606. The symbol "*" indicates statistically

significant differences compared to the control conditions.

FIGURE 10. PKA mediates GSK3 Ser9 phosphorylation induced by pharmacological inhibition of syk

activity with BAY61-3606 in SHSY-5Y cells. A) Western-blot depicting the effect of a 24 hour treatment

with BAY61-3606 on CREB phosphorylation in SHSY-5Y cells. GAPDH was used as a reference

protein. The histogram represents the average CREB Ser133 phosphorylation/GAPDH chemiluminescent

signals observed in control and BAY61-3606 treated SHSY-5Y cells. A significant induction of CREB

phosphorylation was observed following treatment with BAY61-3606 (P<0.004). The symbol "*"

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indicates statistically significant differences compared to the control conditions. B) Representative

western-blot depicting the effect of a 30 minute treatment with BAY61-3606 on GSK3 Ser9

phosphorylation with or without cotreatment with the selective PKA inhibitor KT5270. GAPDH was used

as a reference protein. The histogram represents the average GSK3 Ser9 phosphorylation/GAPDH and

CREB Ser133 phosphorylation/GAPDH chemiluminescent signals observed for the different treatment

conditions. ANOVA shows a significant main effect of BAY61-3606 on GSK3 Ser9 phosphorylation

(P<0.001) and CREB phosphorylation (P<0.007) and a significant main effect of KT5270 on GSK3

Ser9 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 Ser9

phosphorylation (P<0.001) and CREB phosphorylation (P<0.007) showing that BAY61-3606 stimulates

GSK3 Ser9 phosphorylation and CREB phosphorylation. Compared to the BAY61-3606 treatment, a

significant reduction in GSK3 Ser9 phosphorylation (P<0.001) and CREB phosphorylation (P<0.002)

was observed in SHSY-5Y cells cotreated with KT5270 and BAY61-3606 showing that PKA inhibition

prevents GSK3 Ser9 phosphorylation and CREB phosphorylation induced by BAY61-3606.

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 NFB

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 Tyr18 (63) which is considered to be an early event in the pathophysiology of AD.

Our data illustrates 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 which phosphorylates the inhibitory Ser9 of GSK3 resulting in GSK3 inactivation and a

reduction of tau hyperphosphorylation at multiple GSK3dependent epitopes. Activated PKA also

phosphorylates CREB which is expected to be neuroprotective and to improve cognition. In addition, syk

inhibition prevents NFB 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.

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Figure 10

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Figure 11

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