FIBROBLAST GROWTH FACTOR 1 REGULATES SIGNALING VIA THE

GSK3 PATHWAY: IMPLICATIONS FOR NEUROPROTECTION

Makoto Hashimoto1, Yutaka Sagara1, Dianne Langford2, Ian P. Everall3, Margaret Mallory1,

Analisa Everson1, Murat Digicaylioglu4, and Eliezer Masliah1,2

Departments of Neurosciences1 and Pathology2

University of California, San Diego

La Jolla, CA, USA

and

Section of Experimental Neuropathology and Psychiatry3

Institute of Psychiatry,

London, United Kingdom

and

The Burnham Institute4

Center for Neuroscience and Aging

La Jolla, CA, USA

Running title: FGF1 neuroprotection and GSK3β regulation

Correspondence and reprint requests should be addressed to: Dr. E. Masliah, Department of

Neurosciences, University of California San Diego, La Jolla, CA 92093-0624

Phone (858) 534-8992, Fax (858) 534-6232, email address: emasliah@ucsd.edu

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on July 2, 2002 as Manuscript M202803200 by guest on January 21, 2020

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SUMMARY

We hypothesize that in neurodegenerative disorders such as Alzheimer's Disease and HIV

encephalitis, the neuroprotective activity of fibroblast growth factor 1 (FGF1) against several

neurotoxic agents might involve regulation of glycogen synthase kinase-β (GSK3-β), a pathway

important in determining cell fate. In primary rat neuronal and HT22 cells, FGF1 promoted a

time-dependent inactivation of GSK3-β by phosphorylation at serine 9. Blocking FGF1

receptors with heparinase reduced this effect. The effects of FGF1 on GSK3-β were dependent

on phosphatidylinositol-3 kinase (PI3K) /protein kinase B (Akt) because inhibitors of this

pathway or infection with dominant negative Akt adenovirus blocked inactivation. Furthermore,

treatment of neuronal cells with FGF1 resulted in ERK-independent Akt phosphorylation and β-

catenin translocation into the nucleus. On the other hand, infection with wildtype GSK3-β

recombinant adenovirus associated-virus increased activity of GSK3-β and cell death, both of

which were reduced by FGF1 treatment. Moreover, FGF1 protection against glutamate toxicity

was dependent on GSK3-β inactivation by the PI3K-Akt, but was independent of ERK. Taken

together, these results suggest that neuroprotective effects of FGF1 might involve inactivation of

GSK3-β by a pathway involving activation of the PI3K-Akt cascades.

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INTRODUCTION

Neurotrophic factors are capable of maintaining particular neuronal populations during

cellular stress. While some factors, such as nerve growth factor (NGF), support a narrowly

defined neuronal population (e.g. cholinergic neurons), other factors such as fibroblast growth

factor (FGF) support more diverse populations (1). Among the more than 20 members of the

FGF family (2) (3), FGF1 (or acidic FGF) is abundant in sensory and motor neurons and FGF2

(or basic FGF) is primarily produced by astrocytes, although it can be taken up by neurons and

translocated to the nucleus (4). Of the four FGF receptors (FGFR), three are found in the brain:

FGFR1 is mainly expressed on neurons, while FGFR2 and FGFR3 are found on glial cells (1) (5)

(6) (7). Binding of FGF leads to dimerization of FGFR, followed by tyrosine kinase activation

(5). FGF2 promotes survival of cortical and hippocampal neurons (8) (9) and is also capable of

rescuing neurons from denervation and injury (1). Similarly, FGF1 protects selective neuronal

populations against the neurotoxic effects of molecules involved in the pathogenesis of

neurodegenerative disorders such as Alzheimer’s Disease (AD) (10) (11) and HIV encephalitis

(HIVE) (12).

FGF1 and 2 are potent regulators of the CNS development (13) (14) and maintenance

after neuronal injury (1). However, there is no consensus as to the signal transduction pathways

initiated by FGF during neuronal differentiation or during neuroprotection. Some studies

suggest that during FGF2-induced neuronal differentiation: (i) activation of the mitogen-

activated protein kinase (MAPK), such as extracellular signal regulated kinases (ERK1 and

ERK2) is neither necessary nor sufficient; (ii) activation of Src kinases is necessary but not

sufficient; and (iii) FGF2 requires at least two signaling pathways activated by Ras and Src (15)

(16) (17). These results indicate that the neuroactive effects of FGF depends on signaling

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pathways other than ERK. Most studies have focused on the neurotrophic effects of FGF2,

while fewer studies have investigated the effects of FGF1. We hypothesize that the neurotrophic

effects of FGF1 might involve regulation of other signaling cascades, such as the glycogen

synthase kinase-β (GSK3β) pathway, which is important in determining cell fate (18) (19).

Supporting this possibility, a recent study showed that FGF2-mediated tau hyperphosphorylation

was inhibited by lithium, an inhibitor of GSK3β, but not by inhibitors of ERK or the cyclin-

dependent kinases (20). For the present study, we investigated the effects of FGF1 on the

GSK3β pathway in rat primary neuronal and HT22 cells. Our results suggest that the

neuroprotective properties of FGF1 might involve phosphorylation-mediated inactivation of

GSK3β via the phosphatidylinositol-3 kinase (PI3K)-Akt signaling pathway.

EXPERIMENTAL PROCEDURES

Cell culture and treatments. All experiments were performed with rat primary cortical neurons

prepared from embryonic day 17 Sprague Dawley rats, as described previously (21) and with

HT22 cells (22), a mouse hippocampal cells derived from the HT4 cell line (23) . Briefly,

primary cortical neurons were dissociated from the cortex and maintained in tissue culture dishes

coated with 100µg/ml poly-D lysine in MEM supplemented with 30mM glucose, 2mM

glutamine, 1mM pyruvate, and 10% FBS. Cultures were used within one week after preparation.

HT22 cells were maintained at no greater than 70% confluence in 10% fetal bovine serum (FBS)

in Dulbecco's Modified Eagle Medium (DMEM; high glucose, Irvine Scientific, Irvine, CA),

with 1 mM L-glutamine, 1 mM sodium pyruvate, and 1% penicillin/streptomycin (GIBCO/BRL,

Bethesda, MD).

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To analyze the effects of FGF1 on neurons, cells were placed in N2-supplemented serum-

free medium (GIBCO-BRL, Grand Island, NY) 24 hr before treatment. Neurons were then

exposed to FGF1 (10ng/ml, Sigma Chemical Co., St. Louis, MO) for 0, 1, 2, 5, 10, 30 and 60

min, and analyzed by immunoblot for levels of GSK3β, Akt, and ERK1/2. To confirm that

FGF1 effects involve signaling through the FGFR pathway, cells were treated with heparinase

since FGF signal transduction involves binding both at the high affinity FGF receptor and the

low-affinity heparin sulfate receptor. Because heparin optimizes the effects of FGF (24) (25),

additional experiments were performed where heparin (2.5 U/ml, Sigma) and heparinase (1U/ml,

Sigma) were added 1 hr prior to FGF1 treatment (11).

To investigate the intracellular signaling pathways involved in mediating FGF1 effects,

cells were pretreated for 30 min with: (i) ERK inhibitors U0126 and PD98059 (10µM,

Calbiochem, San Diego, CA) or (ii) PI3K inhibitor LY294002 (10µM, Calbiochem). All

experiments were performed at least three times.

Adenoviral and recombinant adenoviral associated vector constructs and transfection.

Replication-defective adenovirus vectors expressing mouse Akt protein fused in-frame to the

FLAG epitope under the control of the cytomegalovirus (CMV) promoter were constructed as

described previously (kind gifts from Dr. Kenneth Walsh, Tufts University, Boston, MA) (26)

(27). The dominant-negative mutant Akt (S473A) protein cannot be activated by

phosphorylation (28) and functions in a inactive fashion (29). The constitutively active Akt

construct has the c-src myristoylation sequence fused in-frame to the N terminus of the FLAG-

Akt (wild-type) coding sequence (27). Adenoviral Akt constructs were amplified in 293A cells

and purified by ultracentrifugation through a CsCl gradient (30).

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For infections, HT22 neuronal cells were plated at approximately 50% confluence in

serum free media and cultured for 24 hr at 37oC, 5% CO2. Half of the conditioned serum-free

media was removed from each sample, pooled and at stored at 37oC, 5% CO2. Cells were

infected at a multiplicity of infection of 50 in pre-conditioned serum free media for 4 hr. Cells

were rinsed three times in warm PBS and media was replaced with pooled pre-conditioned

serum-free media, followed by a 48 hr incubation at 37oC, 5% CO2. After 48 hr, cells were

treated with FGF1 (10ng/ml) for 10 min, harvested in lysis buffer, stored at -20oC, and later used

for the Akt and GSK3β kinase activity assays and Western analyses. For immunocytochemistry,

cells were cultured on coverslips and treated as described above, fixed in 4% paraformaldehyde,

and blocked overnight at 4oC in 10% horse serum and 5% BSA. Cells on coverslips were then

labeled overnight at 4oC with primary anti-FLAG (1:50) (Sigma) followed by incubation with

secondary biotinylated IgG (Vector Laboratories, Inc., Burlingame, CA) (1:200) for 1 hr at room

temperature. FLAG proteins were detected with 3, 3’ diaminobenzidine (DAB) (Sigma) and

visualized by light microscopy to access FLAG production and transduction efficiency (data not

shown). This assay confirmed that transfection efficiency was up to 90%. Experiments were

conducted at least three times to ensure reproducibility.

Expression of exogenous GSK3β was performed using recombinant adenoviral

associated-virus (rAAV) Helper-Free System (Stratagene, San Diego, CA) according to the

manufacturer’s instructions with minor modifications. Briefly, pXT7 plasmids bearing wildtype

GSK3β cDNA (kind gifts from Dr. Zhengui Xia, University of Washington, Seattle, WA and Dr.

Isabel Dominguez, Harvard University, Cambridge, MA) were digested with XbaI, followed by

ligation of the fragments into the XbaI site of the pCMV-MCS expression vector (Stratagene).

The resulting pCMV-MCS-GSK3β plasmids were then cut with NotI, followed by ligation of

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GSK3β containing fragments to the NotI fragments of pAAV-LacZ (Stratagene), to yield the

pAAV-GSK3β.

For generation of the rAAV, sub-confluent 293T cells were transfected without or with

the pAAV-GSK3β, together with pAAV-RC and pHelper plasmid (Stratagene) using Superfect

(Qiagen,Valencia, CA). After incubation for 72 hr, cells were harvested and lysed by four

freeze-thaw cycles. The lysates were centrifuged at 10,000 x g for 10 min to remove cell debris,

and frozen at -80oC until use. As a positive control, pAAV-GFP (Stratagene) was used to

confirm the transfection efficiency.

Sub-confluent neuronal cells were pre-incubated with hydroxyurea (40mM) and sodium

butylate (1mM) for 6 hr. After washing with PBS, cells were incubated with the pAAV lysates

(1/10 dilution) in DMEM with 2% FCS for 24 hr. The cells were then washed with PBS and

incubated in DMEM with 2% FCS for an additional 36 hr. Finally, cells treated with or without

FGF1 were harvested and further analyzed, as described above. As a positive control, HT1080

human fibrosarcoma cells were used to confirm the transfection efficiency. Experiments were

conducted at least three times to ensure reproducibility.

Western blot analysis. Briefly, as previously described (11) (31), pellets of whole neuronal cell

homogenates treated as previously described, were sonicated for 30 sec in HEPES

homogenization buffer (1mM HEPES, 5mM benzamidine, 2mM 2-mercaptoethanol, 3mM

EDTA, 0.5mM magnesium sulfate, 0.05% sodium azide, 1mM sodium orthovanadate and

leupeptin (0.01mg/ml). Protein concentrations were determined by the method of Lowry and 10-

15 µg of protein per well were loaded onto 10% Tris-Glycine ready gels (Bio-RAD, Hercules,

CA). Samples were electroblotted onto Immobilon-P membranes (Millipore, Bedford, MA), and

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immunolabeled with primary antibodies against total GSK3β (mouse monoclonal, 1:2500,

Transduction Laboratories, Lexington, KY), phosphoGSK3β (Ser 9, rabbit polyclonal, 1:2500,

NEB, Beverly, MA), total ERK1/2 (mouse monoclonal, 1:2500, NEB), phosphoERK1/2

(Thr202/Tyr204, mouse monoclonal, 1:2500, NEB), total Akt (rabbit polyclonal, 1:2500, NEB)

and phosphoAkt (Thr308, rabbit polyclonal, 1:2500, NEB). Membranes were incubated with the

HRP-tagged secondary antibody (1:5000) and exposed to the ECL reagent (DuPont NEN,

Boston, MA), followed by autoradiography. Western blots were performed at least three times

to ensure reproducibility.

Immunocomplex kinase assay for GSK3 and Akt. Assays were performed essentially as

previously described with some modifications (18). Briefly, for the GSK3β assay, cells were

rinsed twice with cold PBS and incubated for 20 min on ice in lysis buffer (1% Triton X-100, 10

% glycerol, 50mM HEPES, pH 7.4, 140mM NaCl, 1mM EDTA, 1mM Na3VO4, 1mM phenyl-

methylsulfonylfluoride, 1mM dithiothreitol, aprotinin (5µg/ml) and leupeptin (5µg/ml). The cell

lysates were then centrifuged for 10 min at 14,000 rpm and protein concentration was

determined using the BCA reagent (Pierce, Rockford, IL). Two hundred micrograms of the

supernatant were pre-absorbed with a protein G-sepharose (Amersham Pharmacia Biotech,

Uppsala, Sweden) for 1 hr and the pre-cleared lysates were incubated with anti-GSK3β

monoclonal antibody (1:50, Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C,

followed by incubation with protein G-sepharose for 2 hr at 4°C. The immune complexes were

then washed twice with the lysis buffer and twice with kinase buffer (20mM HEPES, pH 7.2,

0.1mM Na3VO4, 10mM glycerophosphate, 10mM MgCl2, 1mM dithiothreitol, 0.1mM EGTA).

Finally, the immune complexes were incubated in 30µl of the kinase buffer containing either

2.5µg of phosphoglycogen synthase-2 peptides or the Ala21 mutant peptides (Upstate

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Biotechnology, Lake Placid NY) and 10µCi of [γ-32P] ATP (6000 Ci/mmol, PerkinElmer,

Boston, MA) for 20 min at 30 °C. Reactions were terminated by the addition of 5µl of 500mM

EDTA and 5mM ATP. Samples were then spotted onto Whatman P81 phosphocellulose filter

paper. The filters were washed with 180mM phosphoric acid, dried with acetone, and analyzed

by scintillation counting.

Immune complex kinase assays for Akt were performed as described for the GSK3β

assay with minor modifications. Briefly, the pre-cleared lysates were incubated with polyclonal

anti-human Akt antibody (anti-PKB 88-100) (1µg/sample) (Calbiochem, San Diego, CA),

followed by incubation in protein G-sepharose. After washing, immune complex assays were

performed in the presence of 1.0µg of GSK3β fusion protein (Cell Signaling, Beverly, MA) as

substrate. Reactions were terminated by an addition of the SDS sample buffer. The samples

were then subjected to SDS-PAGE (15%) analysis, followed by autoradiography. Quantification

was performed with the PhorphorImager using the Image Quant software (Molecular Dynamics,

Sunnyvale, CA).

Immunocytochemical analysis of -catenin by laser scanning confocal microscopy (LSCM).

Further analysis of catalytically active GSK3β was assayed in neuronal cultures by

immunocytochemical visualization of the cellular localization of β-catenin. Using this method,

inactivation of GSK3β is associated with nuclear translocation of β-catenin (32) (33). Briefly,

cells were plated on poly-L lysine-coated coverslips, exposed to FGF1 in the presence or absence

of inhibitors as described above, and fixed for 10 min with 4% paraformaldehyde in PBS. Cells

were then immunolabeled with the mouse monoclonal antibody against β-catenin (1:1000,

Transduction Laboratories), followed by incubation with FITC-conjugated horse anti-mouse IgG

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(1:75) (Vector Laboratories). Cells on coverslips were analyzed with the LSCM (BioRad MRC

1024). For each experimental condition, a total of 20 cells were analyzed in duplicate. For each

condition, cellular distribution of β-catenin was recorded and computer assisted image analysis

was performed to estimate the percentage of cells displaying predominantly nuclear versus

cytoplasmic distribution of β-catenin.

Analysis of neuronal cell viability and cell death. To determine if the effects of FGF1 on the

PI3K-Akt, GSK3β and ERK pathways influenced cell viability, neuronal cells treated with

glutamate (5mM, Sigma) were analyzed for DNA fragmentation and by the MTT assay and

Trypan blue exclusion.

FACS analysis for the DNA fragmentation was conducted using the flow cytometric

method of Nicoletti et al., as previously described (34). This assay is highly correlated (R2 =

0.82) with the percentage of cells undergoing apoptosis as determined by annexin-V staining.

Briefly, cell nuclei were stained with 50µg/ml of propidium iodide in a hypotonic lysis buffer at

4°C for 2 hr. Samples were then run on a FACScan (Becton Dickinson) to quantify DNA

fragmentation on the FL3 channel (propidium iodide). Cell debris was excluded from nuclei by

an empirical setting of FSC and SSC threshold levels. Apoptotic nuclear bodies appeared as a

broad hypo-diploid peak easily discernable from the narrow peak of cells with normal diploid

DNA content.

For the MTT assay, 2.5 x 103 cells/well were plated in 100µl DMEM with 10% FBS in

96-well microtiter plates. On the following day, after replacing the growth medium with the N2-

supplemented serum-free medium, cells were treated with inhibitors or activators

(pharmacological, adenoviral or adenoviral associated constructs) of the PI3K-Akt or GSK3β

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pathways followed by a 24 hr treatment with FGF1 (10ng/ml). The next day, cells were treated

with 5mM glutamate for an additional 24 hr, followed by cell survival assays. For the MTT

assay (35), 10µl of MTT solution (2.5mg/ml) was added to each well, incubated at 37ºC for 4-8

hr, followed by the addition of 100µl solubilization solution (50% dimetylformamide and 20%

SDS, pH 4.8). The next day, absorption values were read at 570nm. Experiments were done in

triplicate and results averaged.

For Trypan blue exclusion, after treating as described, cells were stained with Trypan

blue (Sigma) and 100 cells/sample were visualized and counted as either dead or alive with an

Axiovert inverted microscope. Cells staining blue, indicating cell membrane compromise, were

counted as dead.

RESULTS

FGF1 promotes Akt, GSK3 , and ERK phosphorylation in neuronal cells. To investigate

the effects of FGF1 on Akt, GSK3ß, and ERK, we first determined the time-course for

phosphorylation of these protein kinases in rat cortical neurons and HT22 cells. Western blot

analysis showed that stimulation with FGF1 resulted in maximum Akt, GSK3β and ERK

phosphorylation at 10-15 min, followed by a progressive decrease reaching non-detectable levels

at 60 min (Fig. 1). Since FGF1 signal transduction involves binding both at the high affinity

FGF receptor and the low-affinity heparin sulfate receptor, optimizing the effects of FGF (24),

experiments were performed with heparin and heparinase. While pretreatment with heparinase

blocked primarily Akt and GSK3β phosphorylation and, to a lesser extent reduced ERK

phosphorylation (Fig.2), heparin enhanced FGF1-mediated phosphorylation of all three kinases

(not shown).

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FGF1 regulation of GSK3 activity is dependent on the PI3K-Akt pathway. Since time-

course experiments (Fig. 1) suggested that the effects of FGF1 on GSK3β might be mediated via

either the PI3K-Akt or the ERK signaling pathways, further analysis of the molecular events was

conducted by treating cells with the PI3K inhibitor, LY294002 or the ERK inhibitors U0126 and

PD98059 prior to FGF1 stimulation. Inhibition of PI3K, which is upstream of Akt (18), resulted

in decreased phosphorylation of Akt and GSK3β, but had no effect on ERK phosphorylation

(Fig. 3). In contrast, inhibitors of the ERK pathway completely blocked FGF1-mediated ERK

phosphorylation with only slight effects on Akt and GSK3β phosphorylation (Fig. 3). Taken

together, these results indicate that phosphorylation of GSK3β by FGF1 stimulation is mediated

via the PI3K-Akt pathway, but is independent of ERK.

To further confirm that FGF1-mediated phosphorylation of GSK3β via PI3K-Akt

resulted in inactivation of GSK3β, immunocomplex kinase assays were performed. FGF1

treatment decreased GSK3β activity by 40% while inhibition of the PI3K-Akt pathway with

LY294002 re-established GSK3β activity even in the presence of FGF1 (Fig. 4A). Inhibition of

the ERK pathway with U0126 did not interfere with the FGF1-mediated effect on

GSK3β activity (Fig.4A). Moreover, while transfection of neuronal cells with dominant

negative Akt re-established GSK3β activity and blocked FGF1 effects on GSK3β (Fig. 4B),

constitutively active Akt reduced GSK3β activity in a similar fashion to FGF1 (Fig. 4B).

Immunocomplex activity assays for Akt confirmed that dominant negative Akt blocked FGF1

effects on Akt (Fig. 4C) and constitutively active Akt increased basal Akt activity levels (Fig.

4C). Consistent with these results, Western blot analysis showed that dominant negative Akt

resulted in reduced Akt and GSK3β phosphorylation, while constitutively active Akt increased

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Akt and GSK3β phosphorylation (Fig. 4D). Furthermore, these effects were enhanced by FGF1

(Fig. 4D).

Neuronal cells were also infected with a rAAV expressing wildtype GSK3β or with a

control vector (AdV-GFP). In cells transfected with rAAV wildtype GSK3β, the activity of this

enzyme (Fig. 5A) as well as cell death (not shown) increased over basal levels compared to

vector control. In contrast, pretreatment with FGF1 decreased GSK3β activity in neuronal cells

infected with rAAV wildtype GSK3β (Fig. 5A). Western blot analysis using antibodies against

phosphorylated and total GSK3β (Fig. 5B) confirmed these findings. Taken together, these

results support the notion that FGF1 might regulate GSK3β activity via PI3K-Akt activation.

Inactivation (phosphorylation) of GSK3 by FGF1 signaling is associated with -catenin

translocation to the nucleus. Since previous studies show that inactivation of GSK3β by

phosphorylation at serine 9 results in translocation of β-catenin to the nucleus (33),

immunocytochemical analyses with β-catenin antibodies were performed in neurons treated with

FGF1 with or without pharmacological inhibitors. Laser scanning confocal microscopy showed

that under basal conditions, β-catenin immunoreactivity was primarily in the cytoplasm and, to a

lesser extent, in the nucleus (Fig. 6A, B). After stimulation with FGF1, β-catenin labeling in the

nucleus was significantly increased (Fig. 6C, D). Consistent with Western blot studies (Fig. 3)

and kinase assays (Fig. 4), the effects of FGF1 on β-catenin nuclear localization were blocked by

LY294002 (PI3K inhibitor) (Fig.6E), but not by the ERK inhibitors (PD98059) (Fig. 6F) and

U0126 (Fig.6G), supporting the idea that FGF1 blocks GSK3β activation leading to β-catenin

degradation.

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Neuroprotective effects of FGF1 against glutamate are mediated by GSK3 . To investigate

the physiological relevance of FGF1 on the PI3K-Akt, GSK3β and ERK pathways, cell viability

was measured by DNA fragmentation, the MTT assay and Trypan blue exclusion in cells

pretreated with FGF1 (10 ng/ml, 24 hrs) and challenged with glutamate (5mM, 24 hrs) in the

presence or absence of specific inhibitors. Consistent with results from the MTT assay and

Trypan blue exclusion (not shown), DNA fragmentation studies showed that FGF1 protected

neurons against neurotoxic effects of glutamate and that these effects were blocked by

LY294002 but not by U0126 (Fig. 7). Analysis of DNA fragmentation by FACS showed that

treatment of the neuronal cells with glutamate resulted in the formation of apoptotic bodies (Fig.

7) whereas pretreatment with FGF1 reduced the formation of apoptotic bodies in the cells.

Treatment with LY294002 (PI3K inhibitor) (Fig. 7A), but not U0126 (Fig. 7B), blocked the

neuroprotective effects of FGF1 against glutamate. Similarly, dominant negative Akt blocked

the neuroprotective effects of FGF1, while constitutively active Akt was protective against

glutamate toxicity in the absence of FGF1 (Fig. 7C).

DISCUSSION

The present study shows that the neurotrophic effects of FGF1 involve signaling via the

PI3K-Akt and GSK3β pathways. The FGFR tyrosine kinase is linked to the G-protein Ras,

which stimulates the ERK signal transduction cascade (15) (25) (Fig. 8). In this context, since

ERK plays a central role in mediating cellular responses to a variety of signaling molecules (36)

(37) most studies in both neuronal and non-neuronal cells have concentrated on characterizing

the effects of FGF2 (rather than FGF1) on the ERK pathway (16) (17) (25) (38) (36) (39) (40)

(41) (42).

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Although ERK plays an important role in regulating the trophic effects of FGF, other

pathways may also be involved (Fig. 8). For example, the neurotrophic activity of FGF1 is

dependent on endogenous FGF1 expression but independent of ERK (43). In agreement with

this finding, we showed that stimulation of neuronal cells with FGF1 resulted in GSK3β

inactivation and β-catenin translocation to the nucleus independent of ERK. Trophic factors

such as insulin also promote GSK3β inactivation by phosphorylation at serine 9 (44), facilitating

β-catenin nuclear localization (45). While inactivation of GSK3β correlates with cell survival,

activation of GSK3β results in cell death (18). These effects are important for understanding the

neurotrophic activity of FGF1 because the GSK3β signaling pathway has been shown to play an

important role in regulating CNS development (32) (46) and cell fate (18) (19). Phosphorylation

of GSK3β serine 9 by the dishevelled signal (Wnt) through the frizzled receptor and activated

through the Notch receptor results in its inactivation (32) with subsequent ß-catenin translocation

to the nucleus (45). Alterations of this pathway are also currently being recognized as important

in the pathogenesis of neurodegenerative disorders such as AD (47) (48) and HIVE (49). For

example, activation of GSK3β might facilitate HIV-mediated neurotoxicity since recent studies

have shown that the HIV protein, Tat, may activate GSK3β (49). In contrast, the neuroprotective

effects of FGF1 against neurotoxins such as HIV-derived proteins and the amyloid β protein of

AD might be associated with its ability to block GSK3β. In support of this possibility, the

present study shows that protection against glutamate toxicity is associated with inactivation of

GSK3β. Furthermore, the neurotoxic effects of gp120, (12) and amyloid β (10) are blocked by

inhibitors of GSK3β such as LiCl2 (50) (51) (52). In addition, in individuals with HIVE high

levels of neuronal FGF1 expression correlate with improved cognitive performance and

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preservation of the dendritic integrity (12). Similarly, neurons that express high levels of FGF1,

such as motor neurons, are resistant to β−amyloid toxicity (11).

As to the potential mechanisms mediating the effects of FGF1 on GSK3β, the present

study shows that inhibitors of the PI3K-Akt pathway block the effects of FGF1 on GSK3β, while

ERK inhibitors have no effect. Further confirming the involvement of the PI3K pathway, FGF1

treatment of neuronal cells resulted in Akt phosphorylation independent of ERK activation. This

is consistent with previous studies showing that the PI3K-Akt signaling inactivates GSK3β,

which is important for cell survival (53) (54). Both PI3K and Akt are activated by other growth

factors including platelet-derived growth factor (55), insulin (56) and brain derived neurotrophic

factor (57). Growth factor-induced cell survival is dependent on the activation of PI3K and its

downstream effector, Akt (18). Akt phosphorylates several intracellular substrates, thus

affecting cell survival and programmed cell death (18) (58). GSK3β has been previously

identified as one of the main substrates for the PI3K-Akt pathway (18) (54). Overexpression of

catalytically active GSK3β in neuronal cell lines results in apoptosis, while dominant negative

GSK3β prevents cell death following the phosphorylation-mediated inhibition by the PI3K-Akt

cascade (18). Similarly, recent studies have shown that in primary neuronal cultures, apoptosis

induced by withdrawal of trophic factors or by PI3K inhibition is dependent on GSK3β

activation (57). These studies also show that both the expression of an inhibitory GSK3β

binding protein or a dominant interfering-form of GSK3β reduced neuronal apoptosis (57). In

contrast, expression of a mutant β-catenin, not affected by GSK3β activity, did not protect

against apoptosis. Thus, although stabilization of β-catenin as a result of GSK3β inactivation is

an important physiological effect, several other pathways downstream of GSK3β might regulate

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cell fate (57). Taken together, these results suggest that the neuroprotective effects of FGF1

involve a PI3K-Akt-mediated inactivation of GSK3β.

In conclusion, FGF1 neuroprotective effects might involve inactivation of GSK3β by a

pathway involving activation of PI3K-Akt cascades (Fig. 8). To the best of our knowledge, this

is the first study showing that FGF1 is capable of acting on these intracellular pathways to

enhance cell viability. This finding is significant because alterations in FGF1 and GSK3β are

being recognized as important in the pathogenesis of neurodegenerative disorders such as AD

and HIVE.

ACKNOWLEDGEMENTS

We wish to thank Dr. Kenneth Walsh (Akt), and Dr. Zhengui Xia and Dr. Isabel Dominguez

(GSK3β) for their generous gifts of the adenoviral constructs. This work was supported by NIH

Grants MH 62962, MH59745, MH45294, MH58164, and DA12065 (to EM), AG01029 (to YS),

and the Medical Research Council, Great Britain (to IPE).

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FIGURE LEGENDS

Figure 1. Time course of FGF1 effects on GSK3β, Akt and ERK phosphorylation in primary

cortical neurons. Western blot demonstrates the inducible expression of phospho-Akt, -GSK3β

and -ERK and constitutive expression of total Akt, GSK3β and ERK by exposure to FGF1.

Figure 2. Effects of heparinase on FGF1-mediated Akt, GSK3β and ERK phosphorylation in

primary cortical neurons. Western blot analysis showed that pre-treatment of neuronal cells with

heparinase (1U/ml, 1 hr) reduced the effects of FGF1 in promoting phosphorylation of Akt, and

GSK3β, but had no effects on ERK phosphotylation.

Figure 3. Effects of inhibitors on FGF1-mediated phosphorylation on Akt, GSK3β, and ERK in

primary cortical neurons. Inhibitors of PI3K-Akt (LY294002) abolished expression of phospho-

Akt and phospho-GSK3β, while neither of the ERK inhibitors (U0126, PD98059) suppressed

expression of phospho-Akt nor phospho-GSK3β.

Figure 4. Effects of inhibitors on FGF1-mediated activation of Akt and GSK3β in HT22 cells.

(A) Analysis of GSK3β activity by immunocomplex assay in the presence of inhibitors of PI3K

(LY294002) and ERK (U0126). FGF1 reduced GSK3β activity. This effect was LY294002 but

not by U0126. (B) Analysis of GSK3β activity by immunocomplex assay in the presence of

FGF1 and dominant negative and constitutively active Akt adenoviral infection. FGF1 alone or

constitutively active Akt reduced GSK3β activity, while dominant negative Akt re-established

GSK3β activity. (C) Analysis of Akt activity by immunocomplex assay showed that FGF1 alone

or in the presence of constitutively active Akt induced an increase Akt activity, while dominant

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negative Akt reduced Akt activity. (D) Western blot analysis confirmed that dominant negative

Akt reduced Akt and GSK3β phosphorylation while constitutively active Akt increased Akt and

GSK3β phosphorylation. These effects were enhanced by FGF1.

Figure 5. Effects of rAAV-GSK3β on kinase activity and cell survival in HT22 cells. (A)

Analysis of GSK3β activity by immunocomplex assay showed that transfection of neuronal cells

with rAAV-wild type GSK3β increased activity. This effect was reduced by FGF1. (B) Western

blot analysis confirmed that treatment of neuronal cells with FGF1 and rAAV-wild type GSK3β

resulted in increased GSK3β phosphorylation.

Figure 6. Effects of FGF1 on β-catenin subcellular localization in a series of confocal

microscopic images of HT22 neurons. (A) and (B) are low (x630) and high power (x900) views

of control neurons, respectively in the absence of FGF1 demonstrating cytoplasmic localization

of β-catenin with no labeling in the nucleus. In the low (C) and high power (D) images of

neurons treated with FGF1, β-catenin is present in the nucleus. In neurons treated with FGF1,

pre-exposure to the PI3K-Akt inhibitor (LY294002) (E) prevented nuclear localization of β-

catenin, while pre-exposure to the ERK inhibitors (PD98059 or U0126) (F-G) resulted in β-

catenin localization to the nucleus. (H) No immunoreactivity was observed in the abscense of the

primary antibody.

Figure 7. FGF1 neuroprotective effects against glutamate are mediated by PI3K-Akt. Cell death

in HT22 cells was analyzed by the DNA fragmentation assay. Neuroprotection against glutamate

(5mM) was blocked in the presence of inhibitors of ; (A) PI3K-Akt (LY294002), but not by (B)

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ERK inhibitor (U0126). (C) Dominant negative Akt blocked the neuroprotective effects of FGF1

against glutamate, while constitutively active Akt was protective against glutamate toxicity in the

absence of FGF1.

Figure 8. Graphic representation of the intracellular signaling events regulated by FGF1. It is

proposed that based on observations from this study with various inhibitor proteins, FGF1

regulation of GSK3β is mediated by the PI3K-Akt pathway.

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Analisa Everson, Murat Digicaylioglu and Eliezer MasliahMakoto Hashimoto, Yutaka Sagara, Dianne Langford, Ian P. Everall, Margaret Mallory,

for neuroprotection pathway: implicationsβFibroblast growth factor 1 regulates signaling via the GSK3

published online July 2, 2002J. Biol. Chem. 

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