Biochemical and Biophysical Research Communications 319 (2004) 993–1000

BBRCwww.elsevier.com/locate/ybbrc

The retinoic acid and brain-derived neurotrophic factordifferentiated SH-SY5Y cell line as a model for Alzheimer’s

disease-like tau phosphorylation

Anne J€ams€a,a,b Kristina Hasslund,b Richard F. Cowburn,a

Anders B€ackstr€om,b and Mervi Vas€angeb,*

a Karolinska Institutet, Neurotec Department, Division of Experimental Geriatrics, Novum, S-141 86 Huddinge, Swedenb AstraZeneca R&D, Forskargatan 20, Building 212, S-151 85 S€odert€alje, Sweden

Received 6 May 2004

Available online

Abstract

The paired helical filaments of highly phosphorylated tau protein are the main components of neurofibrillary tangles (NFT) in

Alzheimer’s disease (AD). Protein kinases including glycogen synthase kinase 3 beta (GSK3b), cyclin-dependent kinase 5 (Cdk5),

and c-Jun N-terminal kinase (JNK) have been implicated in NFT formation making the use of selective kinase inhibitors an at-

tractive treatment possibility in AD. When sequentially treated with retinoic acid (RA) and brain-derived neurotrophic factor

(BDNF), the human neuroblastoma SH-SY5Y differentiates to neuron-like cells. We found that coincident with morphologically

evident neurite outgrowth, both the content and phosphorylation state of tau increased in RA-BDNF differentiated SH-SY5Y cells.

Tau phosphorylation increased at all the examined sites ser-199, ser-202, thr-205, ser-396, and ser-404, all of which are hyper-

phosphorylated in AD brain. We also investigated whether GSK3b, Cdk5 or JNK was involved in tau phosphorylation in the

differentiated SH-SY5Y cells. We found that GSK3b contributed most and that Cdk5 made a minor contribution. JNK was not

involved in tau phosphorylation in this system. The GSK3b-inhibitor, lithium, inhibited tau phosphorylation in a concentration-

dependent manner and with good reproducibility, which enables ranking of substances in this cell model. RA-BDNF differentiated

SH-SY5Y cells could serve as a suitable model for studying the mechanisms of tau phosphorylation and for screening potential

GSK3b inhibitors.

� 2004 Elsevier Inc. All rights reserved.

Keywords: SH-SY5Y cells; GSK3b; Lithium; Cdk5; JNK

Neurofibrillary tangles (NFT) and amyloid plaques

are the pivotal lesions in Alzheimer’s disease (AD). The

major components of NFTs are paired helical filaments

(PHF) of highly phosphorylated microtubule-associated

protein tau [1,2]. Unlike normal tau that maintains the

structure of microtubules, hyperphosphorylated tau se-

questers normal tau and other microtubule-associated

proteins leading to microtubule disassembly and desta-bilization of neuronal cytoskeleton [3].

Many kinases including glycogen synthase kinase 3

beta (GSK3b), cyclin-dependent kinase 5 (Cdk5), and

* Corresponding author. Fax: +46-8-553-23-840.

E-mail address: mervi.vasange@astrazeneca.com (M. Vas€ange).

0006-291X/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2004.05.075

c-Jun N-terminal kinase (JNK) are implicated in AD

NFT formation. Furthermore, there is evidence of either

increased or deregulated activity of these three kinases in

AD brain. Increased levels of GSK3b have been found

in AD brain as compared to non-diseased controls and

immunohistochemical studies have co-localized GSK3bwith NFT in AD brains [4,5]. Additionally, active

GSK3b was found to accumulate in pretangle neurons[6]. Elevated Cdk5 enzyme activity in post mortem AD

brain has been reported in two independent studies [7,8]

and there is also evidence for accumulation of p25, a

cleavage product of the Cdk5 activator p35, in AD

brains [7,9]. P25 has been reported to cause prolonged

and mislocalized activation of Cdk5 [7]. Increased Cdk5

994 A. J€ams€a et al. / Biochemical and Biophysical Research Communications 319 (2004) 993–1000

immunoreactivity was also found colocalized to NFTsin AD brain [4,10]. The activated phospho-JNK

(p-JNK) in turn was detected in NFTs in hippocampal

and cortical regions in the brains from individuals with

severe AD [11].

Specific tau phosphorylation sites have been shown

to correlate with the severity of AD pathology [12]

with the AT8 and PHF-1 epitopes showing intense

staining in advanced AD cases. The AT8 epitopecomprises phosphorylated ser-199, ser-202, and thr-

205, whereas PHF-1 recognizes tau phosphorylated at

ser-396 and ser-404 sites. GSK3b and Cdk5 are able to

phosphorylate tau at all these sites in vitro [13] whereas

stress activated protein kinase (SAPK)/JNK has

been reported to phosphorylate ser-202, thr-205, and

ser-396 [14].

This involvement of protein kinases in tau hyper-phosphorylation makes the use of selective kinase in-

hibitors an attractive treatment possibility in AD

(reviewed in [15]). To enable screening for potential ki-

nase inhibitors, a well-characterized cell model is nee-

ded. SH-SY5Y is a human neuroblastoma cell line that

upon sequential treatment with retinoic acid (RA) and

brain-derived neurotrophic factor (BDNF) differentiates

to neuron-like cells [16]. The RA-treatment inducesexpression of functional TrkB-receptors making cells

responsive to BDNF [17]. BDNF, in turn, activates

phosphatidylinositol 3-kinase (PI 3-K) and extracellular

regulated kinase (ERK) pathways that mediate survival

and neuritogenesis, respectively [18]. RA-BDNF differ-

entiated SH-SY5Y cells express neuronal markers

including neuron-specific enolase (NSE) and growth-

associated protein-43 (GAP-43), and neuronal polaritymarkers such as microtubule-associated protein 2

(MAP2) and tau [16]. Tau immunoreactivity was pre-

viously reported to increase dramatically in RA-predif-

ferentiated SH-SY5Y cells that were exposed to 7-day

treatment with BDNF as compared to undifferentiated

control cells [16].

We wanted to investigate whether differentiated SH-

SY5Y cells could serve as a model for testing selectiveinhibitors targeting kinases proposed to be involved in

aberrant tau phosphorylation in AD. First, we tested

whether a shorter differentiation protocol would induce

appearance of tau in SH-SY5Y cells. We found that

48 h BDNF treatment of RA-predifferentiated SH-

SY5Y cells was enough to increase tau content in the

cells. In addition, tau phosphorylation increased at all

the examined sites ser-199, ser-202, thr-205, ser-396,and ser-404. We also investigated whether GSK3b,Cdk5 or JNK was involved in tau phosphorylation

and found that GSK3b contributed most to the tau

phosphorylation in the differentiated SH-SY5Y cells

whereas the contribution by Cdk5 was minor. JNK

was not involved in tau phosphorylation in this

system.

Materials and methods

Cell cultures and treatments. The human neuroblastoma SH-SY5Y

cell line from European collection of cell cultures (ECACC) was used

for these experiments. SH-SY5Y cells were cultured in medium with

equal amount of minimum essential medium (MEM, Gibco) and

Nutrient Mixture Ham’s F-12 (Gibco), supplemented with 1% non-

essential amino acids (Gibco) and 0–10% heat-inactivated fetal calf

serum (FCS) (HyClone). No antibiotics were added to the medium.

Cells were plated at a density of 5.0� 103 cells/cm2 in 60mm-diameter

culture dishes (Corning) using cell medium with 10% FCS. From day 1

after plating, cells were differentiated in the presence of 10 lM all-

trans-retinoic acid (RA, Sigma–Aldrich) for 6 days in the cell medium

containing 1% FCS. Thereafter, cells were switched to serum-free

medium supplemented with 2 nM brain-derived neurotrophic factor

(BDNF, Sigma–Aldrich) in which they were grown for 48 h. Cells were

maintained at 37 �C in a humidified atmosphere containing 95% air

and 5% CO2.

Cell cultures were treated with 0.5, 1, 2, 10 or 20mM lithium

(Sigma–Aldrich), 1, 10 or 20lM Roscovitine (Biomol) or 1, 10 or

50 lM SP600125 (synthesized in-house under the name AR-

CO82659YY, AstraZeneca) for 2 h before the cells were lysed. Lithium

was diluted in distilled water as a 1M stock solution, whereas Ros-

covitine and SP600125 were prepared as 10mM and 25mM stock

solutions, respectively, in dimethyl sulfoxide (DMSO, Sigma–Aldrich).

All cultures treated with Roscovitine or SP600125 received equal

volumes of DMSO including the control cells, the final concentration

being 0.2%. UV-treatment was carried out for 20 s in UV Crosslinker

(Hoefer, UVC 500) 30min before lysis of the cells. UV light intensity

was 254 nm and the energy was 8mJ/cm2.

Western blot. Cells were lysed in buffer containing 50mM Tris–

HCl, pH 8.0, 150mM NaCl, 1% Triton X-100, 1mM EDTA, 10mM

NaF, 1mM Na3VO4, and one complete protease inhibitor cocktail

tablet (Roche Diagnostics)/10ml buffer. Cell lysates were centrifuged

at 14,000 rpm (Eppendorf 5417R) for 15min. The protein content in

the supernatants was measured using BCA Protein Assay kit (Pierce).

Samples containing 35 lg protein were resolved in 10% NuPage Bis–

Tris gels (Invitrogen). To detect p-JNK 70 lg protein was loaded in

each well. The proteins were transfered to Hybond nitrocellulose

membranes (Amersham Biosciences) using a TRANS-BLOT Semi-dry

Transfer Cell (Bio-Rad). Membranes were blocked in PBS with 0.05%

Tween 20 containing 5% non-fat dried milk for 1 h at room tempera-

ture (RT). Primary antibodies were diluted in either 5% BSA or 5%

milk and incubations were carried out at 4 �C overnight. Primary an-

tibodies were used at the following dilutions: GSK3a=b [pY279=216]

(Biosource International) 1:1500, GSK3b (Santa Cruz, H-76) 1:1500,

Cdk5 (Santa Cruz, C-8) 1:2000, p35 (Sigma) 1:1000, p-SAPK/JNK

(Cell Signaling) 1:1000, SAPK/JNK (Cell Signaling) 1:1000, Tau-5

(RDI) 1:200, Tau[pS199] 1:3000, Tau[pS202] 1:1000, Tau[pT205] 1:500,

Tau[pS396]1:4000, and Tau[pS404] 1:1000. All anti-pTau antibodies

were from Biosource International. Horseradish peroxidase (HRP)

conjugated secondary antibodies (Amersham Biosciences) were incu-

bated for 1 h at RT in 5% milk at the dilution of 1:5000 for anti-rabbit

and 1:10000 for anti-mouse antibody. Blots were developed using the

enhanced chemiluminescence (ECL) Western blotting detection system

(Amersham Biosciences). When needed, membranes were stripped with

Restore Western blot stripping buffer (Pierce) for 30min at 50 �C.Average density of the bands was measured in Fluor-S MultiIm-

ager (Bio-Rad) by using Quantity One software. Density of pTau at

the different residues was normalized to total tau levels detected with

the phosphorylation-independent Tau-5 antibody. When using phos-

phorylation-dependent and site-specific tau antibodies Tau[pS199],

Tau[pS202], and Tau[pT205] two tau species were detected, the lower one

of which was interpreted as non-phosphorylated tau since it over-

lapped with the tau band from the Tau-5 antibody. Our interpretation

is that although the phospho-antibodies are reported to be specific,

A. J€ams€a et al. / Biochemical and Biophysical Research Communications 319 (2004) 993–1000 995

they might not be specific enough for a total discrimination of p-tau

and unphosphorylated tau. Tau[pS396] and Tau[pS404] antibodies de-

tected only one tau band. The degree of phosphorylation was ex-

pressed as a percent of untreated control. All the results were from

three to four separate experiments and the data presented as mean-

s� SEM. The program Graph Pad Prism was used to calculate IC50

values and for statistical analyses. Statistical significance was deter-

mined using one way ANOVA with Dunnett’s test.

Fig. 1. Effect of differentiation on morphological appearance of SH-

SY5Y cells. (A) Undifferentiated SH-SY5Y cells exhibited roundish

features with few short processes. (B) RA alone induced modest degree

of neuronal differentiation resulting in cells that acquired a more

neuron-like phenotype with loss of the round morphology and signs of

neurite outgrowth. (C) Sequential treatment with RA and BDNF gave

rise to cells with mature neuronal morphology with neurite extensions

that occasionally connected the cells.

Results

BDNF enhances the differentiating effects of RA

Undifferentiated SH-SY5Y cells exhibited roundish

features (Fig. 1A) with few short processes. Treatment

with 10 lM RA for 6 days induced a modest degree of

neuronal differentiation resulting in cells that acquired a

more neuron-like phenotype with loss of the round

morphology and signs of neurite outgrowth (Fig. 1B).When treated sequentially with 10 lM RA for 6 days

and 2 nM BDNF for 2 days the cells displayed a mature

neuronal morphology with neurite extensions that

occasionally connected the cells (Fig. 1C).

The content and phosphorylation state of tau are

increased in RA-BDNF differentiated SH-SY5Y cells

The tau isoform detected in undifferentiated SH-

SY5Y cells was of approximately 48 kDa, which

corresponds to the molecular weight of fetal tau [19].

Treatment with either RA or RA-BDNF did not gen-

erate any additional tau isoforms (data not shown).Differentiation with RA or RA-BDNF increased tau

content in the cells although the increase was significant

only in the RA-BDNF treated cells (p < 0:05) (Fig. 2A,

n ¼ 4). Tau phosphorylation occurred only at low levels

in undifferentiated cells. A slight increase was seen in cells

treated with RA, whereas RA differentiation followed by

48 h BDNF treatment robustly increased tau phosphor-

ylation (Figs. 2B, and C). Twenty-four hour BDNFtreatment after RA differentiation was also tested but

showed similar results to RA alone (data not shown). RA

increased tau phosphorylation at all examined sites by

20–30% as compared to undifferentiated control (Fig. 2B,

n ¼ 4). A more prominent increase was seen in the

RA-BDNF differentiated cells, the increase in tau phos-

phorylation being statistically significant (p < 0:05) forser-199, ser-202, ser-396, and ser-404 (Fig. 2B,n ¼ 4).Thr-205 did not quite reach statistical significance (p ¼ 0:08).

Levels of GSK3b and p35/Cdk5 but not of JNK are

increased in RA and RA-BDNF differentiated SH-SY5Y

cells

Levels of total GSK3b and GSK3b Y216 phosphory-

lation that denotes active enzyme increased when the

cells were differentiated with RA but no additional in-

crease was seen after treatment with RA+BDNF

(Fig. 3A, n ¼ 3). Levels of p35, a neuron-specific acti-vator of Cdk5, were increased in RA differentiated cells

and an additional slight increase was seen after sub-

sequent treatment with BDNF (Fig. 3B, n ¼ 3). Levels

of Cdk5 were slightly higher in differentiated cells as

compared to undifferentiated controls (Fig. 3B, n ¼ 3).

There was, however, no difference in Cdk5 levels

Fig. 2. Differentiation of SH-SY5Y cells with RA or RA-BDNF in-

creased the content and the phosphorylation state of tau. (A) Differ-

entiation with RA increased tau with 32% whereas sequential

treatment with RA and BDNF increased tau content in the cells with

69% (p < 0:05) as compared to undifferentiated control cells. (B) RA

increased tau phosphorylation at all the examined sites by 15–25%.

More prominent increase in tau phosphorylation was seen when RA

predifferentiation was followed by 48 h BDNF treatment. Increase in

tau phosphorylation was statistically significant (p < 0:05) at ser-199,

ser-202, ser-396, and ser-404 whereas thr-205 did not quite reach sta-

tistical significance (p ¼ 0:08). (C) Representative Western blots

showing phosphorylation at ser-199, ser-202, thr-205, ser-396, and ser-

404 and total tau as assessed with Tau-5 antibody in undifferentiated,

RA and RA-BDNF differentiated SH-SY5Y cells. Lane 1, undiffer-

entiated; lane 2, RA; and lane 3 RA+BDNF. *p < 0:05.

Fig. 3. Levels of GSK3b and p35/Cdk5 but not of JNK were in-

creased in RA and RA-BDNF differentiated SH-SY5Y cells. (A)

Levels of total GSK3b and activation-associated GSK3b Y216 phos-

phorylation increased when the cells were differentiated with RA but

no additional increase was seen after subsequent treatment with

BDNF. (B) Levels of p35, a neuron-specific activator of Cdk5, were

increased in RA differentiated cells and an additional slight increase

was seen after subsequent treatment with BDNF. Similar levels of

Cdk5 were detected in RA and RA-BDNF differentiated cells and

were only slightly higher than in undifferentiated control cells. (C)

Levels of JNK were unaffected by differentiation. p-JNK was unde-

tectable in undifferentiated cells and was barely above the detection

level in RA and RA-BDNF differentiated cells as compared to UV

treated cells that showed a strong p-JNK signal. Lane 1, undiffer-

entiated; lane 2, RA; lane 3, RA+BDNF; and lane 4, RA+

BDNF+UV.

996 A. J€ams€a et al. / Biochemical and Biophysical Research Communications 319 (2004) 993–1000

between RA and RA-BDNF differentiated cells. JNK

levels were unaffected by differentiation (Fig. 3C, n ¼ 3).

P-JNK was undetectable in undifferentiated cells and

was barely above the detection level in RA andRA-BDNF differentiated cells. Cells treated with UV as

a positive control showed a strong p-JNK signal

(Fig. 3C).

GSK3b contributes most to the tau phosphorylation in

RA-BDNF differentiated SH-SY5Y cells

To pharmacologically assess the role of GSK3b,Cdk5, and JNK in tau phosphorylation in the RA-

BDNF differentiated SH-SY5Y cells, the cell cultures

were treated with kinase inhibitors: lithium for GSK3b,Roscovitine for Cdk5, and SP600125 for JNK. Initially

the cell cultures were treated for 2 and 4 h. However, 4 htreatment was discontinued since it resulted either in

unchanged (lithium) or decreased (Roscovitine and

SP600125) inhibition of tau phosphorylation as com-

pared to 2 h treatment.

Lithium at 10 and 20mM induced prominent inhi-

bition of tau phosphorylation whereas inhibition was

modest at the lowest lithium concentrations 0.5, 1, and

2mM. Inhibition of tau phosphorylation by lithium wasstatistically significant for ser-199 at the concentration

of 20mM (p < 0:001), for ser-202 at 10 and 20mM

(p < 0:001), for thr-205 at 10 and 20mM (p < 0:001),and for ser-396 at 10mM (p < 0:05) and 20mM

(p < 0:001) (Fig. 4, n ¼ 3). Lithium did not inhibit

phosphorylation significantly at ser-404, the maximal

inhibition being about 20%. The IC50 values for lithium

Fig. 4. Lithium concentration-dependently inhibited tau phosphorylation in RA-BDNF differentiated SH-SY5Y cells at ser-199, ser-396, ser-202, and

thr-205 in this order of potency but not at ser-404. Inhibition of tau phosphorylation by lithium was statistically significant for ser-199 at the

concentration of 20mM (p < 0:001), for ser-202 at 10 and 20mM (p < 0:001), for thr-205 at 10 and 20mM (p < 0:001), and for ser-396 at 10mM

(p < 0:05) and 20mM (p < 0:001). Density of pTau at the different residues was normalized to total tau levels detected with the phosphorylation-

independent Tau-5 antibody. Ser-199, ser-202, and thr-205 antibodies detected two bands, the upper one being the pTau band. Lane 1, untreated

control; lanes 2–6, treated with 0.5, 1, 2, 10, and 20mM lithium. ***p < 0:001, *p < 0:05.

A. J€ams€a et al. / Biochemical and Biophysical Research Communications 319 (2004) 993–1000 997

were 2mM for ser-199, 2.5mM for ser-396, 3.1mM for

thr-205, and 4mM for ser-202.Roscovitine significantly inhibited tau phosphoryla-

tion only at the ser-404 residue and at the concentrations

10 and 20 lM (p < 0:05) (Fig. 5, n ¼ 3).

SP600125 did not significantly inhibit tau phosphor-

ylation at any of the sites examined either in UV-treated

or non-UV-treated cells (data not shown). However,

SP600125 had a tendency to reduce Tau-5 immunore-

activity at concentrations of 10 and 50 lM indicating adecreased tau content in the cells although this decrease

was not statistically significant.

Discussion

In SH-SY5Y cells, treatment with RA induces

expression of TrkB-receptors thus making the cells re-

sponsive to BDNF [18]. The ability of BDNF to acti-

vate various intracellular pathways can then be used to

induce neuronal differentiation. Maximal TrkB receptor

expression in SH-SY5Y cells has been reported to

occur after 5–6 days RA treatment [16,20]. Therefore,

we decided to conduct experiments after 6 days pre-exposure to RA to obtain optimal BDNF-induced

response.

Fig. 5. Roscovitine inhibited tau phosphorylation significantly only at

the ser-404 residue of the all examined sites at the concentrations 10

and 20 lM (p < 0:05). Density of pTau was normalized to total tau

levels detected with Tau-5 antibody. *p < 0:05.

998 A. J€ams€a et al. / Biochemical and Biophysical Research Communications 319 (2004) 993–1000

Differentiation with RA increased both the tau con-

tent and tau phosphorylation and these effects were even

more prominent in the RA-BDNF differentiated cells.Induction of tau promoter activity by ERK in PC12

cells has been demonstrated by Sadot et al. [21]. Since

BDNF treatment is known to activate ERK [18], this

may explain the observed increase in tau content in the

RA-BDNF differentiated SH-SY5Y cells. The present

differentiation protocol did not generate any additional

tau isoforms other than fetal tau. Recently, Paccioretti

et al. [22] showed that 7-day BDNF treatment of RA-predifferentiated SH-SY5Y cells resulted in the expres-

sion of all six tau isoforms. Thus, our treatment with

BDNF for 2 days could be too short for induction of

mature tau isoforms. However, the reported 7-day

treatment with BDNF generated tau that was in a low

phosphorylated state, which is in contrast to the high

level of tau phosphorylation seen with our protocol.

Since the aim of this study was to identify a simple andfast protocol for generation of highly phosphorylated

tau, we chose the shorter protocol of 2 day BDNF

treatment of RA-predifferentiated SH-SY5Y cells that

increased tau phosphorylation at all the examined sites.

In the present study, highly phosphorylated tau in

RA-BDNF differentiated SH-SY5Y cells coincided with

morphologically evident neurite outgrowth. Previously,

Brion et al. [23] demonstrated that high levels of phos-phorylated tau are present in neurons during the period

of intense neurite outgrowth. In fact, the transient hy-

perphosphorylation of tau that occurs in developing

neurons has been found to be very similar to the path-

ological phosphorylation of tau in NFT [23,24].

We found that differentiation with RA and RA-

BDNF increased levels of total GSK3b as well as the

activation-associated phosphorylation at Y216. Thus,RA alone was enough to induce active GSK3b. BDNF

has been reported to induce p35 expression and thereby

increase Cdk5 activity [25]. We were able to detect ele-

vated p35 levels although this increase was already ob-

served in cells treated with RA alone and the additional

increase by subsequent treatment with BDNF was mi-

nor. Levels of Cdk5 were only slightly higher in RA and

RA-BDNF differentiated cells than in undifferentiatedcontrols. The enzyme levels of JNK were not altered by

the differentiation protocol used. Furthermore, the ac-

tive phosphorylated form of JNK (p-JNK) was not in-

creased by either RA or RA-BDNF differentiation. UV

treatment of the cells as a positive control gave detect-

able levels of the phosphorylated enzyme. In response to

UV radiation, mitogen-activated protein kinase

(MAPK) kinase kinase is activated, which then activatesMAPK kinase that in turn elevates JNK activity by

phosphorylating it [26]. UV is obviously not a relevant

stress in AD but it is known to be an effective inducer of

the p-JNK in SH-SY5Y cells [27]. It could thus be used

for screening purposes since the same intracellular

pathways are activated regardless of which stress the

cells are exposed to [26].

Our differentiation protocol increased tau phosphor-ylation at all the examined sites, i.e., ser-199, ser-202,

thr-205, ser-396, and ser-404. All these sites are reported

to be hyperphosphorylated in PHF tau in AD brain [28],

which increases the relevance of using this model for

screening compounds that can reduce aberrant tau

phosphorylation in AD. To pharmacologically validate

the assay and to elucidate the contribution of the three

examined kinases to tau phosphorylation we used threedocumented reference compounds. Lithium is a selective

non-competitive inhibitor of GSK3b with in vitro IC50

value of 2mM [29]. Roscovitine is reported to be a se-

lective inhibitor of Cdk5, Cdk2, and Cdc2 [30]. It is an

ATP competitor with in vitro IC50 values ranging from

0.2 to 0.7 lM for these three cyclin-dependent kinases.

The JNK inhibitor, SP600125, also exerts its inhibitory

action via ATP competition [31]. SP600125 inhibits allJNK isoforms (JNK1, 2, and 3) with similar potency

and IC50 values ranging from 0.04 to 0.09 lM in in vitro

assays.

In the RA-BDNF differentiated SH-SY5Y cells,

lithium inhibited concentration-dependent tau phos-

phorylation at ser-199, ser-396, thr-205, and ser-202 in

this order of potency but not at ser-404. Similar results

have been obtained in rat hippocampal cultures wherelithium was able to inhibit tau phosphorylation at the

ser-199, ser-396, and AT8 epitopes but not at ser-404

[32]. IC50 values of lithium for ser-199, ser-202, thr-205,

and ser-396 ranged from 2 to 4mM in our cell model

being somewhat higher than the reported in vitro IC50

value of 2mM [29]. The inhibitory effect of lithium was

reproducible in the concentration range 0.5–20mM

A. J€ams€a et al. / Biochemical and Biophysical Research Communications 319 (2004) 993–1000 999

lithium, which indicates that this model can be used torank substances.

Roscovitine was able to significantly reduce tau

phosphorylation only at ser-404, the maximal inhibition

being about 20%. This indicates that Cdk5 plays a minor

role in tau phosphorylation in the described system, at

least at the sites examined here. Despite the elevated

p-JNK levels in UV-treated cells, the JNK inhibitor

SP600125 did not have any significant effect on tauphosphorylation. Neither could we see any effect on tau

phosphorylation in non-UV-treated cells. As a conclu-

sion, JNK is either not involved in tau phosphorylation

in our cell system or does not phosphorylate the sites

examined here.

Although RA alone was able to increase the levels of

active GSK3b, we chose a protocol with RA and BDNF

because this treatment gave highly phosphorylated tauand thus a better signal to noise ratio for screening

purposes than RA alone. However, the prominent in-

crease in tau phosphorylation after BDNF treatment

cannot solely be attributed to GSK3b, since BDNF per

se did not increase the levels of GSK3b. In addition to

the three kinases we studied, other proline directed Ser/

Thr kinases could contribute to tau phosphorylation in

our system. For instance, ERK1/2 that is activated byBDNF [16] has been shown to be capable of phos-

phorylating tau in human neuroblastoma SK-N-SH

cells [33]. However, this enzyme was not within the

scope of the present investigation.

In conclusion, the present differentiation protocol

promotes neuron-like cell morphology and increases tau

phosphorylation at relevant epitopes for AD. The model

was thus investigated for involvement of GSK3b, Cdk5,and JNK, kinases implicated in AD tau phosphoryla-

tion. GSK3b was shown to be the only one of these

kinases with a clear involvement in tau phosphorylation.

Lithium, a GSK3b-inhibitor, was able to inhibit tau

phosphorylation in a concentration-dependent manner

with good reproducibility, which would enable ranking

of substances in this cell model. RA-BDNF differenti-

ated SH-SY5Y cells could thus serve as a suitable modelfor studying the mechanisms of tau phosphorylation

and for screening potential GSK3b inhibitors.

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