Inhibition of ATF-3 expression by B-Raf mediates the neuroprotective action of GW5074

Inhibition of ATF-3 expression by B-Raf mediates theneuroprotective action of GW5074

Hsin-Mei Chen, Lulu Wang and Santosh R. D’Mello

Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX, USA

Neurodegenerative diseases disrupt the quality of the lives ofpatients and often lead to their death prematurely. A commonfeature of these pathologies is the abnormal degeneration ofneurons, which results from an inappropriate activation ofapoptosis. Drugs that inhibit neuronal apoptosis could thusbe candidates for therapeutic intervention in neurodegener-ative disorders. Moreover, identifying the molecular targetsof such neuroprotective drugs and understanding the signaltransduction pathways that are utilized in their action wouldlead to the development of more effective therapeuticstrategies. In 2004, we reported the identification of apharmacological compound, GW5074 that completely inhib-its neuronal cell death in vitro (Chin et al. 2004). GW5074also prevents striatal degeneration and improves behavioralperformance in mice administered with 3-nitropropionic acid(3-NP), a commonly used in vivo paradigm of Huntington’sdisease. GW5074 could therefore have therapeutic valueagainst neurodegenerative pathologies and understandinghow it acts is therefore of significance.

GW5074 is a potent inhibitor of c-Raf when tested in vitro(Lackey et al. 2000; Chin et al. 2004). Surprisingly, how-ever, the treatment of cultured neurons with GW leads toc-Raf activation (Chin et al. 2004). The phosphorylation ofmitogen-activated protein kinase kinase (MEK) and extra-cellular signal-regulated protein kinase (ERK), downstream

Received October 12, 2007; revised manuscript received December 21,2007; accepted January 8, 2008.Address correspondence and reprint requests to Santosh R. D’Mello,

Department of Molecular and Cell Biology, University of Texas atDallas, 2601N. Floyd Road, Richardson, TX 75083, USA.E-mail: [email protected] used: 3-NP, 3-nitropropionic acid; 6-OHDA, 6-

hydroxydopamine; ATF-3, activating transcription factor-3; DMEM,Dulbecco’s modified Eagle’s medium; ERK, extracellular signal-regulated protein kinase; HCA, homocysteic acid; HK, high potassium;LK, low potassium; MEK, mitogen-activated protein kinase kinase;PKR, RNA-dependent protein kinase; PKRi, RNA-dependent proteinkinase inhibitor; SAR, structure activity relationship; SDS, sodiumdodecyl sulfate; TUNEL, terminal transferase dUTP nick end labeling.

Abstract

GW5074 a brain-permeable 3¢ substituted indolone, protects

neurons from death in culture and in an in vivo paradigm of

neurodegeneration. Using low potassium (LK) induced apop-

tosis of cerebellar granule neurons, we report here that the

protective action of GW5074 is mediated through the activa-

tion of B-Raf. Over-expression of a kinase-dead form of B-Raf

blocks the ability of GW5074 to neuroprotect, whereas over-

expression of active forms of B-Raf protect even in the

absence of GW5074. Although mitogen-activated protein

kinase kinase (MEK) and extracellular signal-regulated protein

kinase (ERK) are activated by GW5074, pharmacological

inhibition of MEK-ERK signaling by U0126 or PD98059 does

not reduce neuroprotection suggesting that B-Raf signals

through a non-canonical signaling pathway. GeneChip

microarray analyses identified activating transcription factor-3

(ATF-3) as a gene whose expression is induced by LK but

that is negatively regulated by GW5074. Forced inhibition of

ATF-3 expression using siRNA protects neurons against LK-

induced apoptosis, whereas the over-expression of ATF-3

blocks GW5074-mediated neuroprotection. Not unexpectedly,

expression of active B-Raf inhibits the apoptosis-associated

increase in ATF-3 expression. We extended our work to

include three other 3¢ substituted indolones – a commercially

available inhibitor of RNA-dependent protein kinase and two

novel compounds designated as SK4 and SK6. Like GW5074,

RNA-dependent protein kinase inhibitor, SK4, and SK6 all

inhibited c-Raf in vitro but activated B-Raf in neuronal cul-

tures. All four compounds also inhibited ATF-3 expression.

Taken together our results indicate that all four indolones

mediate neuroprotection by a common mechanism which in-

volves B-Raf activation, and that a downstream target of B-

Raf is ATF-3.

Keywords: activating transcription factor-3, apoptosis, B-

Raf, cerebellar granule neurons, c-Raf, neuroprotection.

J. Neurochem. (2008) 105, 1300–1312.

d JOURNAL OF NEUROCHEMISTRY | 2008 | 105 | 1300–1312 doi: 10.1111/j.1471-4159.2008.05226.x

1300 Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1300–1312� 2008 The Authors

effectors of Raf kinases, is also stimulated by GW5074. Sucha paradoxical activation of c-Raf has also been reportedto occur in cell lines treated with other structurally distinctc-Raf inhibitors (Hall-Jackson et al. 1999a,b). Although themolecular basis for this paradoxical activation is notunderstood, it is likely that cells respond to GW5074treatment by triggering compensatory mechanisms, such asthe accumulation of activating modifications on c-Raf, whichare revealed in assays performed in the absence of theinhibitor.

GW5074 is a 3¢ substituted indolone. We recently showedthat several other commercially available 3¢ substitutedindolones designed to inhibit a variety of other proteinkinases are also capable of preventing neuronal apoptosis(Johnson et al. 2005). In this study, we extended ourinvestigation to include another commercially available 3¢substituted indolone sold as an inhibitor against RNA-dependent protein kinase (PKR) designated in this report asPKRi (Jammi et al. 2003). To identify chemical groupswithin 3¢ substituted indolones that contribute to neuropro-tective efficacy we initiated a preliminary structure activityrelationship (SAR) analysis using GW5074 as the parentcompound. This analysis led to the identification of two othercompounds SK4 and SK6.

Here, we report that PKRi, SK4, and SK6 are allneuroprotective. Like GW5074, these compounds inhibitc-Raf in vitro. We show that all four compounds also activateB-Raf and that their neuroprotective action is mediated byB-Raf. Finally, we report that neuroprotection by B-Raf doesnot involve the well-described MEK–ERK signaling path-way but acts through the suppression of activating transcrip-tion factor-3 (ATF-3) of the ATF/cAMP response elementbinding protein family.

Materials and methods

MaterialsUnless specified otherwise, all chemicals, including GW5074,

were purchased from Sigma Chemicals (St Louis, MO, USA).

PKRi, PD98059, and U0126 were purchased from Calbiochem

(La Jolla, CA, USA). SK4, SK6, and SK9 were synthesized by

Edward Biehl (Department of Chemistry, Southern Methodist

University, Dallas, TX, USA). Enhanced polyvinylidene difluoride

membrane was from Bio-Rad (Hercules, CA, USA). Enhanced

chemiluminescence was from GE Healthcare (Piscataway, NJ,

USA). Phospho-MEK, MEK, and Phospho-ERK antibodies were

purchased from Cell Signaling Technology (Beverly, MA, USA);

c-Raf, B-Raf, c-Jun, and ATF-3 antibodies were purchased from

Santa Cruz Biotechnology (Santa Cruz, CA, USA); c-myc, Flag,

and tubulin were purchased from Sigma (St Louis, MO, USA).

A/G PLUS-Agarose was also obtained from Santa Cruz Biotech-

nology. All cell culture media used in these experiments were

purchased from Invitrogen (Carlsbad, CA, USA). Trizol and

Thermoscript reverse transcriptase PCR (RT-PCR) system were

also from Invitrogen. Terminal transferase dUTP nick end

labeling (TUNEL)-staining was performed using the DeadEnd

Fluorometric TUNEL system from Promega (Madison, WI, USA).

The B-Raf constructs, myc-B-Raf KD, myc-B-Raf CAT, and myc-

B-Raf V600E, are kind gifts from Michael White’s lab at the

University of Texas-Southwestern Medical Center (Dallas, TX,

USA). The ability of these constructs to increase or block B-Raf

activity has been tested in 293T (Supplementary Fig. S1). The

expression plasmid encoding kinase-dead MEK (GST-MEK1

K97M) was also a kind gift from Melanie Cobb, the University

of Texas-Southwestern Medical Center. The ATF-3 gene pro-

moter-luciferase reporter plasmid (pGL3-ATF3-Luc) was provided

by Seung Baek, University of Tennessee (Knoxville, TN, USA).

ATF-3 siRNA was purchased from Ambion (Austin, TX, USA).

p-TARGETTM vector and PCR master mix were from Promega.

Neuronal culture, treatments, and viability assayCerebellar granule neurons were obtained from dissociation of 7- to

8-day-old rats as described previously (D’Mello et al. 1993). Thecells were plated in basal minimal Eagle’s medium, supplemented

with 10% fetal bovine serum (FBS), 25 mM KCl, 2 lM glutamine,

and 0.2% gentamycin (D’Mello et al. 1993), in 24-well dishes

(1 · 106 cells/well for viability assay), or 60-mm dishes (12 · 106

cells/dish for immunoprecipitation and western blotting). Cytosine

arabinoforanoside (10 lM) was added to the culture medium

18–22 h after plating to prevent replication of non-neuronal cells.

Unless indicated otherwise, treatments were performed 6–7 days

after plating. For treatments, the cultures were switched to serum-

free basal minimal Eagle’s medium in the absence [low potassium

(LK) medium] or presence of 25 mM KCl [high potassium (HK)

medium]. Purchased or synthesized compounds (GW5074, PKRi,

SK4, SK6, and SK9) were added to LK medium at the time the

medium was switched. After 24 h treatment, cells were fixed and

stained with DNA fluorescent dye, 4¢6¢-diamidino-2-phenylindole

hydrochloride. Cell viability was quantified by visualizing the

morphology of the nuclei. Apoptotic cell death is revealed by nuclei

condensation and/or fragmentation.

Cortical cultures were obtained from the cerebral cortex of

Wistar rats (day 17 of gestation) as described previously (Murphy

et al. 1990). Cortical neurons were treated with 1 mM homocy-

steic acid (HCA) for 24 h to induce oxidative stress 1 day after

plating or 5 lM camptothecin for 6 h 3 days after plating.

HCA was made as 150 mM stock solution that was adjusted to

pH 7.5.

Western blottingEquivalent amounts of protein were mixed with 6x sodium dodecyl

sulfate (SDS) sample buffer (375 mM Tris–HCl, pH 6.8 at 25�C,12% SDS, 60% glycerol, 300 mM dithiothreitol, and 0.012%

bromophenol blue). This was followed by heating at 95�C for

5 min, subjected to SDS–polyacrylamide gel electrophoresis, and

transferred electrophoretically to polyvinylidene difluoride mem-

brane. After staining with Ponceau S to verify uniformity of protein

loads/transfer, the membranes were blocked. Incubation with

primary antibodies was performed overnight at 4�C or for 1 h at

25�C followed by secondary antibodies for 1 h at 25�C. Immuno-

reactivity was developed by enhanced chemiluminescence and

visualized by autoradiography.

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1300–1312

Neuroprotection by GW5074 | 1301

ATF-3 cloning, transfection, and immunocytochemistryFlag-ATF-3 was cloned to p-TARGETTM vector with reverse primer

5¢-TAAGCTCTGCAATGTTCCTTCTT-3¢ and forward primer 5¢-ATGATGCTTCAACACCCAGG-3¢ from an embryonic human

brain cDNA library. Plasmids were purified by performing CsCl

equilibrium centrifugation. Cerebellar granule neurons are able to be

transfected after 4–5 days following plating by using a well-

established calcium–phosphate protocol (Dudek et al. 1997).

Briefly, cell media was replaced with serum-free Dulbecco’s

modified Eagle’s medium (DMEM) for 1 h. Original conditioned

media was saved at 37�C in a CO2 incubator. For each well a

mixture was prepared as follows: 15 lL of 2x HEPES-buffered

saline buffer, pH 7.05, was combined with 15 lL of a 0.25 M CaCl2solution containing 3 lg of plasmid DNA or 40–80 pmole of ATF-3

siRNA. The mixtures were vortexed and incubated at 25�C for

30 min. The mixture was added to the cells drop-wise and allowed

to incubate for another 45 min. After washing cells three times with

serum-free DMEM, original medium was added back to cultures.

One day following transfection, cerebellar granule neurons were

treated with HK, LK, or LK plus drugs for 24 h for immunocyto-

chemistry or 3 h for western blotting. Immunocytochemistry was

performed by fixing cells for 20 min with 4% formaldehyde. After

blocking in 0.1 M phosphate-buffered saline containing 5% bovine

serum albumin and 5% goat serum for 30 min, immunostaining was

performed by incubation with the primary antibody for 90 min and

the secondary antibody for 45 min at 25�C. During washing of the

secondary antibody, nuclei were stained with 4¢6¢-diamidino-2-

phenylindole hydrochloride for 10 min at 25�C.

Immunoprecipitation and kinase assayTwo hundred and fifty micrograms of protein was incubated with

1.0 lg of primary antibody and 12 lL of Protein A/G PLUS-

Agarose beads overnight. Immunoprecipitates were collected by

centrifugation at 3000 g for 30 s and washed twice with lysis buffer,

twice with lysis buffer supplemented with 350 mM NaCl, and twice

with kinase buffer (25 mM HEPES, pH 7.4, and 10 mM MgCl2).

Purified recombinant GST-MEK1 K97M protein was added as a

substrate in kinase buffer supplemented with 85 lMATP for 35 min

at 30�C. For in vitro kinase assays, drugs were added in kinase

buffer and incubated for 5 min at 30�C prior to kinase reaction. The

kinase reactions were stopped by addition of 6x SDS sample buffer

and boiled for 5 min. Proteins were resolved by SDS–polyacryl-

amide gel electrophoresis and subjected to western blotting. The

level of kinase activity was detected by a phospho-MEK antibody.

GeneChip microarray analysisTotal RNAwas extracted from cultures treated with or without 1 lMGW using Trizol according to the manufacturer’s instructions.

Integrity of the RNA was confirmed by gel electrophoresis. To

reduce false-positives resulting from technical variance and potential

biological variation between samples, three independent sets of

RNA samples were prepared from three different sets of cerebellar

granule neuron cultures and separately subjected to microarray

analysis. Probe preparation and hybridizations were performed

through the NIH MicroArray Consortium at the Translational

Genomic Research Institute (TGRI, Tempe, AZ, USA) on a fee-for-

service basis using the Affymetrix GeneChip Rat Expression 230

2.0 Array (Affymetrix, Santa Clara, CA, USA), which permits

analysis of the expression of over 31 000 transcripts and variants

from over 28 000 well-substantiated rat genes. The QVALUE program

from GeneSpring R-integration package (Agilent Technologies,

Santa Clara, CA, USA) was used for data analysis.

RNA preparation and semi-quantitative RT-PCRRNAwas extracted from cultured neurons by using Trizol according

to the manufacturer’s instructions. Three micrograms of total RNA

from each sample was reverse transcribed by Thermoscript reverse

transcriptase PCR (RT-PCR) system (Invitrogen) according to the

manufacturer’s instructions. PCR was performed with PCR master

mix (Promega). The primers used for PCR amplification were as

follows: Rat glyceraldehyde 3-phosphate dehydrogenase, forward

5¢-CCATCACCATCTTCCAGGAG-3¢ and reverse 5¢-CCTGCTC-ACCACCTTCTTG-3¢; Rat b-Adducin, forward 5¢-GGCGATGCA-GATACCAAAGAT-3¢ and reverse 5¢-ATCTCCTCTTGCCCTCCT-TG-3¢; Rat insulin-related protein-2, forward 5¢-GTGTGGTTT-CAGAACAAGCGTT-3¢ and reverse 5¢-TTACAAGGGCCGGA-GATTTT-3¢; Rat SH3/ankyrin domain gene 3, forward 5¢-TAAG-CTCCATCCACGTCACTC-3¢and reverse 5¢-ATCTCTGTGGGG-TCTATCACAA-3¢; Rat Jun, forward 5¢-TGAAGCAGAGCATGA-CCTTG-3¢ and reverse 5¢-GACACTGGGCAGCGTATTCT-3¢; RatATF-3, forward 5¢-ATGCTTCAACATCCAGGCCA-3¢ and reverse,

5¢-CGCCTCCTTTTTCTCTCATCT-3¢; Rat stathmin-like 4, forward

5¢-GGCTGCAAGAGAAGGACAA-3¢ and reverse 5¢-TGACAAT-GAACAGACGCACA-3¢; Rat tumor necrosis factor (ligand) super-

family member 11, forward 5¢-TATGATGGAAGGTTCGTGGCT-3¢and reverse 5¢-AGTACGTCGCATCTTGATCCG-3¢.

In vivo 3-nitropropionic acid treatment3-Nitropropionic acid administration and analysis of brain sections

was performed as previously described (Chin et al. 2004). Briefly,mice were injected intraperioneally with 3-NP at 50–55 mg/kg twice

a day for 4 days. The following day, the mice were deeply

anesthetized, brains removed, washed in phosphate-buffered saline,

and rapidly frozen in Cryo-Stat embedding medium (StatLab

Medical Products, Lewisville, TX, USA). Fifty micron coronal

sections were cut on a cryostat and stained for Nissl substance with

cresyl violet (Chin et al. 2004). For western blotting analysis,

striatum from control or 3-NP injected animal were homogenized in

RIPA buffer. Then, protein was extracted after centrifugation for

15 min at 15 000 g. Protein was also extracted from the rest of the

brain (lacking striatum) and used for western blotting.

N1E-115 cell culture and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assayThe N1E-115 cell line was purchased from ATCC (Manassas, VA,

USA) and cultured in DMEM with 4.5 g/L glucose (without sodium

pyruvate) supplemented with 10% FBS, 100 U/mL penicillin, and

100 lg/mL streptomycin. N1E-115 cells were plated into 24-well

tissue culture dishes at 5 · 104 per well 1 day before 6-hydroxy-

dopamine (6-OHDA) treatment. Cell death was determined 24 h

later by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide assay as previously described (Koulich et al. 2001).

Luciferase assayN1E-115 cells were cultured in antibiotic-free DMEM media

containing 10% FBS 1 day before transfection. Transient transfec-

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1300–1312� 2008 The Authors

1302 | H.-M. Chen et al.

tion was performed using Lipofectamine 2000 (Invitrogen).

Cultured/transfected media was switched to regular N1E-115 culture

media 2 h after transfection. The next morning, cells were treated

with or without 6-OHDA for 6 h. Cells then were harvested and

luciferase activity determined by using BD Monolight Enhanced

Luciferase Assay Kit (BD Biosciences, San Jose, CA, USA)

following the manufacturer’s instructions.

Results

GW5074 activates both B-Raf and c-Raf kinase activity incultured cerebellar granule neuronsGW5074 inhibits c-Raf when tested in vitro on purifiedenzyme (Lackey et al. 2000; Chin et al. 2004). As we havepreviously reported, however, when cultured cerebellar

granule neurons induced to undergo apoptosis are treatedwith GW5074, the activity of endogenous c-Raf is robustlyactivated (Chin et al. 2004; Fig. 1a). This paradoxicalactivation of c-Raf has also been observed by otherinvestigators with other structurally distinct c-Raf inhibitors(Hall-Jackson et al. 1999a,b; Chin et al. 2004). Not surpris-ingly, when activated c-Raf that is immunoprecipitated fromGW5074-treated neuronal cultures is exposed to GW5074readded in vitro, the kinase activity of c-Raf is againinhibited (Chin et al. 2004; Fig. 1a). In addition to c-Raf, B-Raf activity is stimulated by GW5074 in neuronal culturesswitched to LK-treatment (Fig. 1b). To examine how quicklyc-Raf and B-Raf activation occurs, we immunoprecipitatedthese enzymes from LK-treated neuronal cultures exposed toGW5074 for 10 min, 20 min, 1 h, and 2 h, and evaluated

(a)

(c)

(d)

(b)

Fig. 1 GW5074 activates both B-Raf and c-Raf kinase activity when

treated in cerebellar granule neuronal cultures. Cerebellar granule

neuron cultures were treated with LK or LK medium with 1 lM

GW5074 (GW) (as described in Chin et al. 2004). Lysates were made

and B-Raf or c-Raf was immunoprecipitated. GST-MEK was used as

substrate in the kinase assay. The kinase activity was determined by

western blotting using a phospho-MEK antibody. The same blot was

reprobed with antibodies to B-Raf or c-Raf and MEK to demonstrate

that comparable amounts of kinase and substrate were used in each

reaction. An aliquot of the lysates prior to immunoprecipitation was

also analyzed by western blotting using tubulin antibody to demon-

strate that similar quantities of the lysates were used for pull-down. (a)

c-Raf was pulled down from 3 h treated LK or LK medium containing

1 lM GW5074 and a kinase assay performed. The 3rd lane

(GW + GW) 100 nM GW5074 was added before kinase assay per-

formed. (b) B-Raf was pulled down from 3 h treated LK or LK medium

containing 1 lM GW5074 and a kinase assay performed. (c) Cere-

bellar granule neuron cultures were treated for different times in LK or

LK medium with 1 lM GW5074 (GW). B-Raf was pulled down and a

kinase assay performed. Kinase activity was quantified by densito-

metric analysis of the bands and normalized to LK at 10¢ treatment (LK

10¢ = 1.0). (d) Cerebellar granule neuron cultures were treated for

different times in LK or LK medium with 1 lM GW5074 (GW). c-Raf

was pulled down and a kinase assay performed. Kinase activity was

quantified by densitometric analysis of the bands and normalized to LK

at 10¢ treatment (LK 10¢ = 1.0).



their activation status in vitro. As shown in Fig. 1c and d,activation of both c-Raf and B-Raf is clearly discernible at10 min. The rapidity at which c-Raf and B-Raf activationoccurs suggests a more direct effect of GW5074 on enzymeactivity as opposed to a feedback-loop mechanism activatedas a consequence of enzyme inhibition.

B-Raf activity is required for GW5074-mediatedneuroprotectionAs the activation of B-Raf and c-Raf is an early response toGW5074 treatment, we hypothesized that either B-Raf or c-Raf activation is important for GW5074-mediated neuropro-tection. To test this hypothesis we performed over-expressionexperiments in which active or kinase-dead forms of the twokinases were introduced into neurons which were thenswitched to HK or LK medium. Preliminary studies indicatedthat the over-expression of c-Raf constructs do not have a

substantial effect on the ability of GW5074 to neuroprotect(data not shown). Over-expression of a kinase-dead form ofB-Raf on the other hand, blocked GW5074-mediated neu-roprotection (Fig. 2a). Furthermore, the over-expression ofeither of two different constitutively active forms of B-Rafprevented LK-induced neuronal death even in the absence ofGW5074 (Fig. 2b). These results suggest that B-Raf activityis necessary for GW5074-mediated neuroprotection.

ATF-3 is a downstream mediator of neuroprotection byGW5074 and B-RafAlthough Raf kinases typically act through MEK and ERK,this pathway is not necessary for GW5074-mediated neuro-protection (Chin et al. 2004). To gain insight into down-stream regulators of GW5074, we performed GeneChipmicroarray analyses. These experiments identified 12mRNAs that were up-regulated in neurons by GW5074

(a)

(b)

Fig. 2 B-Raf activity is required for GW5074-mediated neuroprotec-

tion. Cultured cerebellar granule neurons were transfected with an

expression plasmid encoding myc-tagged kinase-dead B-Raf or two

separate plasmids encoding myc-tagged constitutively active B-Raf.

Control cultures were transfected with a GFP-expressing plasmid. The

proportion of positively transfected neurons that were viable was

quantified following immunocytochemistry with an antibody against c-

myc or GFP. 4¢6¢-diamidino-2-phenylindole hydrochloride (DAPI)

staining was performed to visualize chromatin. Neurons with con-

densed and/or fragmented nuclei were scored as apoptotic. Panels on

the left show representative fields after immunocytochemistry, while

panels on the right show quantification of viability. The results show

mean and standard deviation from three independent experiments.

(a) Neurons were transfected with plasmids expressing GFP or

kinase-dead B-Raf (B-Raf KD) and then switched to HK, LK, or LK

medium containing 1 lM GW5074 (GW) or 1 lM PKRi. Viability was

quantified 24 h after the switch. C-myc immunoreactivity was detected

using a Texas Red (TX)-conjugated secondary antibody. *Indicates

value of p < 0.05 using the Student’s t-test comparing values with

GFP transfected neurons in the same treatment. (b) Neurons were

transfected with two plasmids expressing myc-tagged forms of active

B-Raf (B-Raf CAT and B-Raf V600E) or GFP and then switched to HK

or LK medium. Viability was quantified 24 h after the switch. C-myc

immunoreactivity was detected using a Texas Red (TX)-conjugated

secondary antibody. **Indicates value of p < 0.01 using the Student’s

t-test compared with GFP transfected neurons.



treatment and 13 mRNAs that were down-regulated(Table 1). Altered expression of a subset of these mRNAswas validated by RT-PCR analyses (Fig. 3). Among thegenes whose expression was confirmed to be down-regulatedby GW5074 is the one encoding ATF-3. ATF-3 has beenimplicated in the promotion of neuronal death in someexperimental paradigms (Hai et al. 1999; Vlug et al. 2005).In cerebellar granule neuron cultures, ATF-3 is induced afterswitching to LK media within 3 h (Fig. 4a and b). Previousstudies have demonstrated that in this paradigm, neurons areirreversibly committed to death within 4–6 h of LK treatment(Galli et al. 1995; Schulz et al. 1996; Nardi et al. 1997;Borodezt and D’Mello 1998). Thus, ATF-3 expression

occurs prior to the point at which the neurons are committedto dying. More long-term analysis of ATF-3 indicates that itsexpression remains elevated for at least 12 h after LK

Table 1 Genes that are regulated by GW5074 treatment of cerebellar

granule neurons

Gene title

Gene

symbol

Fold

change

Beta-1 adducin b-adducin 2.636

Glycine receptor, alpha 1 subunit Glra1 2.13

Insulin related protein 2 Isl2 2.093

Calcium channel, voltage-dependent,

beta 1 subunit

Cacnb1 1.762

Apoptotic protease activating factor 1 Apaf1 1.694

Glutamate receptor, metabotropic 4 Grm4 1.673

W307 protein W307 1.59

Ribonuclease, RNase A family 4 RNase4 1.555

Transporter-like protein Ctl1 1.537

SH3/ankyrin domain gene 3 Shank3 1.531

Nuerabin 2 Neb2 1.531

CTD-binding SR-like rA1 LOC56081 1.529

Hepcidin antimicrobial peptide Hamp 0.634

v-jun sarcoma virus 17 oncogene

homolog (avian)

jun 0.586

Protein kinase (cAMP dependent, catalytic)

inhibitor beta

pkib 0.583

Protein tyrosine phosphatase, non-receptor

type 2

Ptpn2 0.578

Netrin 1 Ntn1 0.524

Epithelial calcium channel 1 Ecac1 0.512

Protein tyrosine phosphatase, non-receptor

type 16

Ptpn16 0.509

Caveolin Cav 0.477

Phosphatidylinositol 3-kinase, regulatory

subunit, polypeptide 1

Pik3r1 0.467

Activating transcription factor 3 (Atf3) Atf3 0.46

Calpain 8 Capn8 0.404

Stathmin-like 4 Stmn4 0.383

Tumor necrosis factor (ligand) superfamily,

member 11

Tnfsf11 0.252

Three independent sets of RNA were prepared from CGNs treated

with LK or LK plus 1 lM GW5074 for microarray analysis. Candidate

genes were identified as those exhibiting 1.5-fold or greater change

with GW5074 treatment compared with LK alone. A list of genes was

created with q < 0.05 using Student’s t-test.

Fig. 3 Validation of microarray analysis. RNA was prepared from

cerebellar granule neurons treated with LK or LK with 1 lM GW5074

(GW) for 3 h and subjected to RT-PCR analysis to detect expression

of a subset of genes that were previously identified in microarray

analysis. GAPDH was amplified as a loading control.

(a)

(b)

(c)

Fig. 4 ATF-3 is up-regulated during neuronal apoptosis. (a) Cere-

bellar granule neurons were treated with LK medium for different

times. RNA was prepared and RT-PCR analysis was performed. Actin

was amplified as a loading control. (b) Cerebellar granule neurons

were treated with HK or LK media for different times and lysates from

these cultures subjected to western blotting analysis using an ATF-3

antibody. The same membrane was reprobed with tubulin to ensure

equal loading. (c) Cerebellar granule neurons were treated with HK or

LK media for 9, 12, 18, and 24 h and lysates subjected to western

blotting analysis using an ATF-3 antibody. The same membrane was

reprobed with tubulin to ensure equal loading. The same membrane

was reprobed with antibodies to c-Jun and cleaved caspase 3.



treatment before declining to normal levels (Fig. 4c). Asimilar profile of expression is observed with c-Jun. Incomparison, caspase 3 activation, as judged by generation ofthe 17 kDa proteolytic product, begins later and is moresustained (Fig. 4c). It is noteworthy that ATF-3 has beenshown to play an essential role in stress-induced caspase 3activation (Lu et al., 2007).

To examine if the stimulation of ATF-3 played a causalrole in LK-mediated neuronal death, we over-expressed it in

neurons. As shown in Fig. 5a, the over-expression of ATF-3reduces neuronal survival in HK and blocks the ability ofGW5074 to neuroprotect in LK medium. The pro-apoptoticeffect of ATF-3 on HK and GW5074-treated neurons wasconfirmed by TUNEL staining (Supplementary Fig. S2).This finding suggests that ATF-3 promotes apoptosis in LKand that GW5074 and HK maintain neuronal survival byinhibiting the increase in ATF-3 expression. To verify thisconclusion, we knocked down ATF-3 expression using

(a)

(c) (d)

(e)

(b)

Fig. 5 ATF-3 expression regulates apoptosis in neurons. (a) Cere-

bellar granule neurons were transfected with an expression plasmid

encoding Flag-tagged ATF-3, or GFP, and then switched to HK, LK, or

LK medium containing 1 lM GW5074 (GW) or 1 lM PKRi. The pro-

portion of positively transfected neurons that were viable was quanti-

fied following immunocytochemistry with an antibody against Flag or

GFP and 4¢6¢-diamidino-2-phenylindole hydrochloride staining. Neu-

rons with condensed and/or fragmented nuclei were scored as apop-

totic. *Indicates value of p < 0.05 using the Student’s t-test comparing

with GFP transfected neurons in the same treatment. (b) Cultured

cerebellar granule neurons were transfected with ATF-3 siRNA or a

control siRNA and then switched to HK or LK medium. Western blot-

ting analysis was performed 24 h after transfection using ATF-3 and c-

Jun antibodies. The same membrane was reprobed with tubulin to

show equal amount of protein loaded. Viability was also quantified

24 h after the switch. Data represents the mean plus standard devi-

ation from six independent experiments. *Indicates value of p < 0.05

from the Student’s t-test compared with control. (c) Cortical neurons

were treated with 5 lM camptothecin (CPT) for 4 h. Untreated (Un)

and CPT treated lysates were harvested and subjected to western

blotting analysis. ATF-3 expression level was determined using an

ATF-3 antibody. The same membrane was reprobed with tubulin to

indicate equal sample loading. (d) Cortical neurons were treated with

1 mM HCA for 6 h. Untreated (Un) and HCA treated neurons were

harvested and subjected to western blotting analysis. ATF-3 expres-

sion level was determined using an ATF-3 antibody. The same

membrane was reprobed with tubulin to indicate equal amount of

protein was loaded. (e) Mice were injected with 3-NP or saline (Ctrl)

twice a day for 4 days as described in Materials and methods. After

the last day of injection, whole brains were removed. The striatum was

dissected out and lysates were prepared from it and from the

remaining extra striatal brain tissue (whole brain without striatum). The

lysates were subjected to western blotting analysis using an ATF-3

antibody. The same membrane was reprobed with tubulin to demon-

strate equal amount of protein was loaded.



siRNA. As shown in Fig. 5b, the forced down-regulation ofATF-3 expression reduces neuronal death induced by LKmedium.

We looked at ATF-3 expression in two other well-established paradigms of neurodegeneration. Camptothecinis a DNA damaging agent which induces apoptosis incultured cortical neurons (Morris and Geller 1996). Incontrast to LK-induced death which is mediated in a p53-independent mechanism, neuronal death by camptothecin isp53-dependent (Enokido et al. 1996; Xiang et al. 1998;Morris et al. 2001). As shown in Fig. 5c, ATF-3 is also up-regulated in cortical neurons primed to die by camptothecintreatment. HCA treatment of cortical neurons is also usedwidely as a paradigm to study the mechanisms underlyingneuronal death. By causing glutathione depletion, HCAinduces oxidative stress leading to apoptosis (Murphy et al.1990; Ratan et al. 1994a,b). As shown in Fig. 5d, HCAtreatment induces ATF-3 expression in cortical neurons. Weextended our studies to an in vivo paradigm of neurode-generation. Administration of 3-NP to mice causes selectivestriatal neurodegeneration and is widely used as an exper-imental model of Huntington’s disease. As shown in Fig. 5e,ATF-3 expression is stimulated in the degenerating striatumof 3-NP administered mice (also see Supplementary Fig. S3for evidence of striatal degeneration). In contrast, theexpression of ATF-3 is not increased in the non-striatalbrain tissue, which does not display significant neurodegen-eration. These results suggest that ATF-3 stimulationcontributes to cell death in multiple neuronal types and inresponse to other apoptotic stimuli.

We next proceeded to examine whether the increasedexpression of ATF-3 was transcriptionally mediated ormediated post-transcriptionally. As primary neurons arepoorly transfected, we utilized the dopaminergic N1E-115cell line to investigate this issue. Treatment of N1E-115with 6-OHDA induces apoptosis (Fig. 6a and b). Asobserved in primary neurons, ATF-3 expression is up-regulated by 6-OHDA (Fig. 6c). Using a promoter constructcontaining 514 bp of upstream sequence of the ATF-3 genefused to a luciferase reporter we find that 6-OHDA resultsin a small but significant increase in transcriptional activity(Fig. 6d).

To investigate whether the suppression of ATF-3 byGW5074 (Fig. 7a) was mediated by B-Raf, we over-expressed B-Raf in neuronal cultures. The over-expressionof an active form of B-Raf reduced the apoptosis-associatedstimulation of ATF-3 expression even in the absence ofGW5074 (Fig. 7b). Another molecule that is known to play apivotal role in neuronal apoptosis is the c-Jun transcriptionfactor. The need for c-Jun activation in the induction ofLK-mediated death of cerebellar granule neurons as well asother paradigms of neurodegeneration is well-established(Estus et al. 1994; Ham et al. 1995; Watson et al. 1998).GW5074 inhibits the apoptosis-associated stimulation of

c-Jun expression (Fig. 7a). As shown in Fig. 7b, theexpression of active B-Raf also blocks c-Jun induction byLK.

Other neuronal protective compounds also utilize similarmechanisms while mediating neuronal survivalIn a previous study, we reported the identification of other 3¢substituted indolones which were also capable of preventingneuronal apoptosis (Johnson et al. 2005). In this study, weinvestigated PKRi, an inhibitor of PKR first described byJammi et al. (2003) and that has a 3¢ substituted indolonecore. As shown in Fig. 8, treatment with PKRi prevented thedeath of cerebellar granule neurons induced by LK. Inaddition to PKRi, two other 3¢ substituted indolones, SK4and SK6, synthesized as part of an ongoing SAR analysis

(a) (b)

(c) (d)

Fig. 6 6-Hydroxydopamine (6-OHDA) induced ATF-3 promoter

activity in N1E-115 cell line. (a) N1E-115 cells were treated with 50 lM

6-OHDA for 24 h. The morphology of untreated or 6-OHDA treated

cells was observed under phase contrast microscope. (b) Viability of

untreated or 6-OHDA treated N1E-115 cells was determined by the 3-

(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The

survival rate of 6-OHDA treated N1E-115 cells was normalized to

untreated cells. *Indicates value of p < 0.05 using the Student’s t-test.

(c) Whole cell lysates prepared from untreated or 3 h 6-OHDA-treated

N1E-115 cells were subjected to western blotting analysis using an

ATF-3 antibody. The same membrane was reprobed with tubulin

antibody to show equal amount of protein loaded. (d) ATF-3-Lucifer-

ase reporter vector containing 514 bp of upstream sequence (pGL3-

ATF3-Luc) and mock vector (lacking the promoter) were transfected

into N1E-115. The next day, the cultures were treated with 50 lM 6-

OHDA for 6 h. The percentage of ATF-3 promoter activity induced by

6-OHDA was normalized to the promoter activity of untreated cells.

The data indicates the mean of three independent experiments.

*Indicates value of p < 0.05 using the Student’s t-test.



were also found to be neuroprotective (Fig. 8). In contrast toSK4 and SK6, a closely related 3¢ substituted indolonedesignated as SK9, fails to block LK-induced neuronal deathat any of the concentrations tested (Fig. 8). A description ofthe SAR analysis along with other compounds analyzed willbe described elsewhere.

Given their structural similarity, it was possible that PKRi,SK4, and SK6 exerted their neuroprotective action by acommon mechanism. We first examined whether the three

compounds inhibited c-Raf in vitro. As shown in Fig. 9a, allthree neuroprotective compounds (SK4, SK6, and PKRi)inhibit the activity of c-Raf that had been immunoprecipi-tated from neurons. In comparison, SK9 is less effective.While SK6 and SK4 also produced the paradoxical activationof c-Raf in intact neuronal cultures, PKRi did not. Thisfinding suggested that the in vivo activation of c-Raf is notan essential feature for neuroprotection by 3¢ substitutedindolones (Fig. 9b). In contrast, PKRi, SK4, and SK6 allactivated B-Raf kinase activity while treated in neuronalculture (Fig. 9c). PKRi, SK4, and SK6 also inhibitedthe apoptosis-associated stimulation of ATF-3 and c-Junexpression that is triggered in cerebellar granule neurons(Fig. 9d and e).

Since its discovery in 2003, PKRi has been used in anumber of studies and a variety of biological effectsproduced by this compound have been attributed to itsability to inhibit PKR (Page et al. 2006; Shimazawa andHara 2006; Ingrand et al. 2007). As described above, PKRidoes not activate c-Raf in response to GW5074 treatment.PKRi also inhibits c-Jun and ATF-3 expression much moresubstantially that GW5074, SK4, and SK6 (Fig. 9). It wastherefore conceivable that although sharing structural simi-larity with the other three indolones, PKRi mediatesneuroprotection via a distinct mechanism. To address thisissue, we examined more rigorously whether B-Raf isnecessary for neuroprotection by PKRi. As shown in Fig. 2a,over-expression of the kinase-dead form of B-Raf blocksneuroprotection by PKRi. Over-expression of ATF-3 alsoinhibits PKRi-mediated neuroprotection (Fig. 5a) as wasobserved with GW5074.

(a)

(b)

Fig. 7 GW5074 and active B-Raf inhibit ATF-3 expression. (a) Cere-

bellar granule neurons were treated with HK, LK media or LK media

with 1 lM GW5074 for 3 h and subjected to western blotting analysis

in which the same blot was probed sequentially with ATF-3 and c-Jun

antibodies. Tubulin was used as a loading control. (b) Cerebellar

granule neurons were transfected with plasmids expressing GFP or

B-Raf V600E for 48 h followed by treatment with HK or LK for 3 h.

Lysates were prepared and subjected to western blotting analysis in

which the same blot was probed sequentially with ATF-3, B-Raf, and

c-Jun antibodies. Actin serves to demonstrate similar loading.

Fig. 8 PKRi, SK4, and SK6 protect cere-

bellar granule neurons against LK-mediated

apoptosis; 6–7 days after plating, cerebellar

granule neurons were switched to HK

medium (control) or LK medium containing

various concentrations of PKRi, SK4, SK6,

or SK9. Viability was quantified after 24 h

using 4¢6¢-diamidino-2-phenylindole hydro-

chloride staining. Percentage of survival

was normalized to HK treatment. The data

represents the mean values of viability with

standard deviation from three independent

experiments. *Indicates the value of

p < 0.05 using the Student’s t-test com-

paring with LK.



Discussion

Our results indicate that GW5074 exerts its neuroprotectiveaction by activating B-Raf. Indeed, the over-expression of akinase-dead form of B-Raf blocks neuroprotection whereasexpression of an active form of B-Raf promotes neuronalsurvival even in the absence of GW5074.

Our conclusion that B-Raf has neuroprotective effects isconsistent with the finding that sensory neurons and motorneurons cultured from B-Raf-deficient embryos (but not from

c-Raf or A-Raf deficient embryos) fail to survive in responseto neurotrophic factors (Wiese et al. 2001). In response togrowth factor stimulation, activated B-Raf directly phospho-rylates MEK, a serine–threonine kinase that then phospho-rylates and activates ERK (Baccarini 2002; Hindley andKolch 2002). Not unexpectedly, we find that MEK and ERKare stimulated by all four neuroprotective compounds(Supplementary Fig. S4a). Surprisingly, the inhibition ofMEK–ERK signaling has no effect on neuroprotection(Supplementary Fig. S4b). It is noteworthy that while

(a) (b)

(c) (d)

(e)

Fig. 9 Different neuroprotective indolones act through a common

mechanism. (a) Cerebellar granule neuron cultures were treated for

3 h in LK or LK medium with 1 lM GW5074 (GW) (as described in

Chin et al. 2004). C-Raf was immunoprecipitated from the cultures.

The lysates from LK + GW treated cultures were subjected to in vitro

kinase assays with no additives ()) or the addition of GW5074

100 nM, PKRi 500 nM, SK4 100 nM, SK6 200 nM, or SK9 100 nM into

the kinase reaction mixture. All drugs were added at 1/10 the con-

centration at which they were found to be most neuroprotective in

culture. GST-MEK was used as substrate in the kinase assay. The

reaction was terminated and the products subjected to western blot-

ting using a phopho-MEK antibody. The same blot was reprobed with

antibodies to c-Raf and MEK to demonstrate that comparable amounts

of c-Raf kinase and substrate were used in each reaction. An aliquot of

the lysates prior to immunoprecipitation was also analyzed by western

blotting using tubulin antibody to demonstrate that similar quantities of

lysates were used for c-Raf pull-down. Kinase activity was quantified

by densitometric analysis of the bands and normalized to GW treat-

ment (GW = 1.0). (b) Neuronal cultures were treated for 3 h with HK,

LK, or LK medium containing 1 lM GW5074, 5 lM PKRi, 1 lM SK4,

2 lM SK6, or 1 lM SK9. C-Raf was immunoprecipitated down from

treated lysates and kinase assay performed. Western blotting analysis

of the reaction product was performed with a phospho-MEK antibody.

The same blot was reprobed with c-Raf and with MEK antibodies to

show equal pull-down and substrate amount in each lane. Western

blot of an aliquot of pre-immunoprecipitated cell lysates with a tubulin

antibody serves to show equal amount of input lysate used. Kinase

activity was quantified by densitometric analysis of the bands and

normalized to LK (LK = 1.0). (c) Neuronal cultures were treated for 3 h

with HK, LK, or LK medium containing 1 lM GW5074, 5 lM PKRi,

1 lM SK4, 2 lM SK6, or 1 lM SK9. B-Raf was immunoprecipitated

down from treated lysates and kinase assay performed. Western

blotting analysis of the reaction product was performed with a phos-

pho-MEK antibody. The same blot was reprobed with B-Raf and with

MEK antibodies to show equal pull-down and substrate amount in

each lane. Western blot of an aliquot of pre-immunoprecipitated cell

lysates with a tubulin antibody serves to show equal amount of input

lysate used. Kinase activity was quantified by densitometric analysis of

the bands and normalized to LK (LK = 1.0). (d) Neuronal cultures were

treated for 3 h with HK, LK, or LK medium containing 1 lM GW5074,

5 lM PKRi, 1 lM SK4, 2 lM SK6, or 1 lM SK9. Whole cell lysates

from treated cells were prepared and used in western blots to analyze

ATF-3 expression. The same blot was reprobed with an antibody

against tubulin, which serves as a loading control. (e) Neuronal cul-

tures were treated for 3 h with HK, LK, or LK medium containing 1 lM

GW5074, 5 lM PKRi, 1 lM SK4, 2 lM SK6, or 1 lM SK9. Whole cell

lysates from treated cells were prepared and used in western blots to

analyze c-Jun expression. The same blot was reprobed with an anti-

body against tubulin, which serves as a loading control.



MEK remains the most widely accepted substrate, recentstudies have identified a number of other proteins includingBad, p53, and Rb that can be directly phosphorylated by Raf(Bonni et al. 1999; Baccarini 2002; Hindley and Kolch2002). Moreover, Raf is able to prevent apoptosis byinteracting with the anti-apoptotic molecule, Bag-1(RudolfGotz et al. 2005), or sequestering the pro-apoptotic moleculeapoptosis signal-regulating kinase-1 (Chen et al. 2001)through a MEK and ERK independent pathway. Althoughfurther work is needed to clarify the mechanism by which itoccurs, we find that neuroprotection by B-Raf involves theinhibition of the ATF-3 transcription factor whose expressionis induced in neurons primed to undergo apoptosis. Over-expression of active B-Raf blocks the apoptosis-inducedincrease in ATF-3 expression. A relationship between B-Rafand ATF-3 has been described in actively proliferating celllines (Nilsson et al. 1997). In these cells, B-Raf has beendescribed to actually stimulate ATF-3 expression. WhetherATF-3 is induced or suppressed by B-Raf may therefore becell and context specific.

Activating transcription factor-3 is a member of the ATF/cAMP response element binding protein family of basicleucine zipper-type transcription factors which can form bothhomo- and heterodimers with other basic leucine zipper-typetranscription factors (Hai et al. 1999). While ATF-3 homod-imers are believed to inhibit gene transcription, heterodimerscan activate transcription. The most commonly describeddimerization partners for ATF-3 are members of the Junfamily (c-Jun, Jun B, and Jun D). ATF-3 expression isgenerally induced by cellular damage and a variety of stressstimuli in different tissue types (Hai et al. 1999). In thenervous system, ATF-3 has been implicated in neuronaldegeneration following spinal cord damage (Tsujino et al.2000; Averill et al. 2004; Huang et al. 2006), cerebral arteryocclusion (Ohba et al. 2004), and neurotoxin-induced oxi-dative stress (Holtz et al. 2006). Elevated ATF-3 expressionalso precedes the death of spinal motor neurons in atransgenic mouse model of amyotrophic lateral sclerosis(Vlug et al. 2005) and of retinal ganglion cells after crushingof the optic nerve (Takeda et al. 2000). In addition tocerebellar granule neurons, we find that ATF-3 expression isincreased in cortical neurons induced to die by oxidativestress and DNA damage, and in the degenerating striatum of3-NP administered mice. In some cases however, ATF-3 hasbeen reported to promote neuronal survival. For example,ATF-3 over-expression protects hippocampal neurons fromkianic acid-induced degeneration (Francis et al. 2004) andsympathetic neurons from nerve growth factor deprivation-induced apoptosis (Nakagomi et al. 2003). Whether ATF-3kills or promotes neuronal survival may be dependent onwhether it acts as a homo- or heterodimer or on other proteinswith which it associates within the specific cell type. Othermechanisms such as differential post-translational modifica-tions on ATF-3 may also contribute to the different roles that

ATF-3 displays. Initial indications based on the utilization ofan ATF-3 promoter-luciferase reporter construct is that theincrease in ATF-3 expression during apoptosis is mediated atleast in part at the transcriptional level. The small increase inpromoter activity we observe in 6-OHDA-treated N1E-115cells does not, however, explain the substantial increase inATF-3 protein expression. It is possible that the 514 bp of 5¢genomic sequence that we have utilized does not contain allthe transcriptional elements necessary for full induction.Alternatively, post-transcriptional mechanisms such as in-creased mRNA stability might contribute to increasedexpression. Post-transcriptional regulation of ATF-3 expres-sion has been suggested in other studies (Lu et al., 2007).

In cerebellar granule neurons, ATF-3 induction iscausally involved in promoting apoptosis. Indeed, the over-expression of ATF-3 in healthy neurons is sufficient to induceapoptosis whereas the forced suppression of ATF-3 reducesthe extent of LK-induced neuronal death. The partial protec-tion is likely to reflect the incomplete suppression of ATF-3expression by the siRNA used in this study. It is also possiblethat suppression of ATF-3 alone is not sufficient for completeprotection and that other molecules cooperate with ATF-3 topromote neuronal death. One such molecule is c-Jun. Severaldifferent investigators have established the importance of c-Jun in promoting neuronal death (Estus et al. 1994; Ham et al.1995; Herdegen et al. 1998; Watson et al. 1998; Crockeret al. 2001). Interestingly, the apoptosis-associated stimula-tion of c-Jun expression is also inhibited by active B-Raf.

Another 3¢ substituted indolone utilized in this study isPKRi, a compound identified based on its ability to inhibitPKR activity in vitro (Jammi et al. 2003). We have discov-ered that PKRi protects against LK-induced death ofcerebellar granule neurons. While this study was in progressother investigators also reported neuroprotective actions ofPKRi in other experimental paradigms of neuronal death(Page et al. 2006; Shimazawa and Hara 2006; Ingrand et al.2007). Some investigators have also reported that PKRactivity is elevated in certain tissue culture models ofneurodegeneration and in the brains of patients with Alzhei-mer’s diseases raising the possibility that activation of PKRmay contribute to neurodegeneration (Bando et al. 2005;Page et al. 2006; Eley et al. 2007; Shimazawa et al. 2007).We find that PKRi stimulates B-Raf activity and that itsneuroprotective effect in cerebellar granule neurons isdependent, at least in part, on B-Raf. Furthermore, PKRialso blocks the apoptosis-associated induction of ATF-3. Asobserved with GW5074, over-expression of ATF-3 blocks theability of PKRi to be neuroprotective. At the present time wedo not know whether activation of PKR is necessary forapoptosis in the paradigms we have investigated comprisingcerebellar granule neurons or cortical neurons. Preliminarywork in cerebellar granule neurons indicates that PKRitreatment does not inhibit the phosphorylation of eIF2a,which is a downstream target of PKR (Chen and D’Mello,



unpublished observation). Further studies are necessary todetermine whether PKR inhibition is involved in the neuro-protective action of PKRi, or whether this compound actsthrough separate effects on B-Raf activity. In addition toPKRi, we have studied two other 3¢ substituted indoloneswhich were identified in a SAR screen. Both SK4 and SK6activate B-Raf and suppress ATF-3 expression.

In summary, we identify B-Raf and ATF-3 as importantplayers in the regulation of neuronal survival. These proteinscan serve as molecular targets in the development of neuro-protective therapeutic strategies. Furthermore, our studiesidentify 3¢ substituted indolones as a core chemical structurethat can be used as a starting point for the development oftherapeutic drugs against neurodegenerative disorders. At thepresent time we do not know whether activation of PKR isnecessary for apoptosis in the paradigms we have investi-gated comprising cerebellar granule neurons or corticalneurons. Perliminary work in cerebellar granule neuronsindicates that PKRi treatment does not inhibit the phosphor-ylation of eIF2a, which is a downstream target of PKR.

Acknowledgements

This work was supported by a grant from the National Institute of

Neurological Diseases and Stroke (NS047201) to SRD. We

acknowledge the help of Edward Biehl and Kamila Sukanta

(Southern Methodist University, Dallas, TX, USA) who synthesized

some of the compounds used in this study. We thank Michael White

(University of Texas Southwestern Medical Center, Dallas, TX,

USA) for the c-Raf and B-Raf expression constructs and

Seung Baek (University of Tennessee, Knoxville, TN, USA) for

the ATF-3 gene promoter-luciferase reporter plasmid (pGL3-

ATF3-Luc).

Supplementary material

The following supplementary material is available for this article

online:

Fig. S1 Raf constructs exhibit appropriate kinase activity.

Fig. S2 ATF-3 induced neuronal apoptosis is confirmed by

TUNEL staining.

Fig. S3 3-NP induces striatal degeneration.

Fig. S4 MEK inhibitors do not block neuroprotective com-

pound-mediated neuronal survival.

This material is available as part of the online article from http://

www.blackwell-synergy.com.

Please note: Blackwell Publishing are not responsible for the

content or functionality of any supplementary materials supplied by

the authors. Any queries (other than missing material) should be

directed to the corresponding author for the article.

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Inhibition of ATF-3 expression by B-Raf mediates the neuroprotective action of GW5074

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