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

Authors


Address correspondence and reprint requests to Santosh R. D’Mello, Department of Molecular and Cell Biology, University of Texas at Dallas, 2601N. Floyd Road, Richardson, TX 75083, USA.
E-mail: dmello@utdallas.edu

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 apoptosis of cerebellar granule neurons, we report here that the protective action of GW5074 is mediated through the activation 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 cultures. All four compounds also inhibited ATF-3 expression. Taken together our results indicate that all four indolones mediate neuroprotection by a common mechanism which involves B-Raf activation, and that a downstream target of B-Raf is ATF-3.

Abbreviations 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 protein kinase inhibitor

SAR

structure activity relationship

SDS

sodium dodecyl sulfate

TUNEL

terminal transferase dUTP nick end labeling

Neurodegenerative diseases disrupt the quality of the lives of patients and often lead to their death prematurely. A common feature of these pathologies is the abnormal degeneration of neurons, which results from an inappropriate activation of apoptosis. Drugs that inhibit neuronal apoptosis could thus be candidates for therapeutic intervention in neurodegenerative disorders. Moreover, identifying the molecular targets of such neuroprotective drugs and understanding the signal transduction pathways that are utilized in their action would lead to the development of more effective therapeutic strategies. In 2004, we reported the identification of a pharmacological compound, GW5074 that completely inhibits neuronal cell death in vitro (Chin et al. 2004). GW5074 also prevents striatal degeneration and improves behavioral performance in mice administered with 3-nitropropionic acid (3-NP), a commonly used in vivo paradigm of Huntington’s disease. GW5074 could therefore have therapeutic value against neurodegenerative pathologies and understanding how 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, however, the treatment of cultured neurons with GW leads to c-Raf activation (Chin et al. 2004). The phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated protein kinase (ERK), downstream effectors of Raf kinases, is also stimulated by GW5074. Such a paradoxical activation of c-Raf has also been reported to occur in cell lines treated with other structurally distinct c-Raf inhibitors (Hall-Jackson et al. 1999a,b). Although the molecular basis for this paradoxical activation is not understood, it is likely that cells respond to GW5074 treatment by triggering compensatory mechanisms, such as the accumulation of activating modifications on c-Raf, which are revealed in assays performed in the absence of the inhibitor.

GW5074 is a 3′ substituted indolone. We recently showed that several other commercially available 3′ substituted indolones designed to inhibit a variety of other protein kinases are also capable of preventing neuronal apoptosis (Johnson et al. 2005). In this study, we extended our investigation to include another commercially available 3′ substituted indolone sold as an inhibitor against RNA-dependent protein kinase (PKR) designated in this report as PKRi (Jammi et al. 2003). To identify chemical groups within 3′ substituted indolones that contribute to neuroprotective efficacy we initiated a preliminary structure activity relationship (SAR) analysis using GW5074 as the parent compound. This analysis led to the identification of two other compounds SK4 and SK6.

Here, we report that PKRi, SK4, and SK6 are all neuroprotective. Like GW5074, these compounds inhibit c-Raf in vitro. We show that all four compounds also activate B-Raf and that their neuroprotective action is mediated by B-Raf. Finally, we report that neuroprotection by B-Raf does not involve the well-described MEK–ERK signaling pathway but acts through the suppression of activating transcription factor-3 (ATF-3) of the ATF/cAMP response element binding protein family.

Materials and methods

Materials

Unless 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 Biotechnology. 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 promoter-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-TARGET™ vector and PCR master mix were from Promega.

Neuronal culture, treatments, and viability assay

Cerebellar granule neurons were obtained from dissociation of 7- to 8-day-old rats as described previously (D’Mello et al. 1993). The cells were plated in basal minimal Eagle’s medium, supplemented with 10% fetal bovine serum (FBS), 25 mM KCl, 2 μM 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 μM) 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 homocysteic acid (HCA) for 24 h to induce oxidative stress 1 day after plating or 5 μM camptothecin for 6 h 3 days after plating. HCA was made as 150 mM stock solution that was adjusted to pH 7.5.

Western blotting

Equivalent 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 membrane. 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. Immunoreactivity was developed by enhanced chemiluminescence and visualized by autoradiography.

ATF-3 cloning, transfection, and immunocytochemistry

Flag-ATF-3 was cloned to p-TARGET™ 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 μL of 2x HEPES-buffered saline buffer, pH 7.05, was combined with 15 μL of a 0.25 M CaCl2 solution containing 3 μg 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 immunocytochemistry 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 assay

Two hundred and fifty micrograms of protein was incubated with 1.0 μg of primary antibody and 12 μL 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 μM ATP 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–polyacrylamide gel electrophoresis and subjected to western blotting. The level of kinase activity was detected by a phospho-MEK antibody.

GeneChip microarray analysis

Total RNA was extracted from cultures treated with or without 1 μM GW 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-PCR

RNA was 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′-CCTGCTCACCACCTTCTTG-3′; Rat β-Adducin, forward 5′-GGCGATGCAGATACCAAAGAT-3′ and reverse 5′-ATCTCCTCTTGCCCTCCTTG-3′; Rat insulin-related protein-2, forward 5′-GTGTGGTTTCAGAACAAGCGTT-3′ and reverse 5′-TTACAAGGGCCGGAGATTTT-3′; Rat SH3/ankyrin domain gene 3, forward 5′-TAAGCTCCATCCACGTCACTC-3′and reverse 5′-ATCTCTGTGGGGTCTATCACAA-3′; Rat Jun, forward 5′-TGAAGCAGAGCATGACCTTG-3′ and reverse 5′-GACACTGGGCAGCGTATTCT-3′; Rat ATF-3, forward 5′-ATGCTTCAACATCCAGGCCA-3′ and reverse, 5′-CGCCTCCTTTTTCTCTCATCT-3′; Rat stathmin-like 4, forward 5′-GGCTGCAAGAGAAGGACAA-3′ and reverse 5′-TGACAATGAACAGACGCACA-3′; Rat tumor necrosis factor (ligand) superfamily member 11, forward 5′-TATGATGGAAGGTTCGTGGCT-3′ and reverse 5′-AGTACGTCGCATCTTGATCCG-3′.

In vivo 3-nitropropionic acid treatment

3-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 assay

The 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 μg/mL streptomycin. N1E-115 cells were plated into 24-well tissue culture dishes at 5 × 104 per well 1 day before 6-hydroxydopamine (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 assay

N1E-115 cells were cultured in antibiotic-free DMEM media containing 10% FBS 1 day before transfection. Transient transfection 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 in cultured cerebellar granule neurons

GW5074 inhibits c-Raf when tested in vitro on purified enzyme (Lackey et al. 2000; Chin et al. 2004). As we have previously reported, however, when cultured cerebellar granule neurons induced to undergo apoptosis are treated with GW5074, the activity of endogenous c-Raf is robustly activated (Chin et al. 2004; Fig. 1a). This paradoxical activation of c-Raf has also been observed by other investigators with other structurally distinct c-Raf inhibitors (Hall-Jackson et al. 1999a,b; Chin et al. 2004). Not surprisingly, when activated c-Raf that is immunoprecipitated from GW5074-treated neuronal cultures is exposed to GW5074 readded in vitro, the kinase activity of c-Raf is again inhibited (Chin et al. 2004; Fig. 1a). In addition to c-Raf, B-Raf activity is stimulated by GW5074 in neuronal cultures switched to LK-treatment (Fig. 1b). To examine how quickly c-Raf and B-Raf activation occurs, we immunoprecipitated these enzymes from LK-treated neuronal cultures exposed to GW5074 for 10 min, 20 min, 1 h, and 2 h, and evaluated their activation status in vitro. As shown in Fig. 1c and d, activation of both c-Raf and B-Raf is clearly discernible at 10 min. The rapidity at which c-Raf and B-Raf activation occurs suggests a more direct effect of GW5074 on enzyme activity as opposed to a feedback-loop mechanism activated as a consequence of enzyme inhibition.

Figure 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 μM 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 demonstrate 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 μM GW5074 and a kinase assay performed. The 3rd lane (GW + GW) 100 nM GW5074 was added before kinase assay performed. (b) B-Raf was pulled down from 3 h treated LK or LK medium containing 1 μM GW5074 and a kinase assay performed. (c) Cerebellar granule neuron cultures were treated for different times in LK or LK medium with 1 μM GW5074 (GW). B-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). (d) Cerebellar granule neuron cultures were treated for different times in LK or LK medium with 1 μM 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).

B-Raf activity is required for GW5074-mediated neuroprotection

As the activation of B-Raf and c-Raf is an early response to GW5074 treatment, we hypothesized that either B-Raf or c-Raf activation is important for GW5074-mediated neuroprotection. To test this hypothesis we performed over-expression experiments in which active or kinase-dead forms of the two kinases were introduced into neurons which were then switched to HK or LK medium. Preliminary studies indicated that 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 of B-Raf on the other hand, blocked GW5074-mediated neuroprotection (Fig. 2a). Furthermore, the over-expression of either of two different constitutively active forms of B-Raf prevented LK-induced neuronal death even in the absence of GW5074 (Fig. 2b). These results suggest that B-Raf activity is necessary for GW5074-mediated neuroprotection.

Figure 2.

 B-Raf activity is required for GW5074-mediated neuroprotection. 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 condensed 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 μM GW5074 (GW) or 1 μM 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 < 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.

ATF-3 is a downstream mediator of neuroprotection by GW5074 and B-Raf

Although Raf kinases typically act through MEK and ERK, this pathway is not necessary for GW5074-mediated neuroprotection (Chin et al. 2004). To gain insight into downstream regulators of GW5074, we performed GeneChip microarray analyses. These experiments identified 12 mRNAs that were up-regulated in neurons by GW5074 treatment and 13 mRNAs that were down-regulated (Table 1). Altered expression of a subset of these mRNAs was validated by RT-PCR analyses (Fig. 3). Among the genes whose expression was confirmed to be down-regulated by GW5074 is the one encoding ATF-3. ATF-3 has been implicated in the promotion of neuronal death in some experimental paradigms (Hai et al. 1999; Vlug et al. 2005). In cerebellar granule neuron cultures, ATF-3 is induced after switching to LK media within 3 h (Fig. 4a and b). Previous studies have demonstrated that in this paradigm, neurons are irreversibly 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 committed to dying. More long-term analysis of ATF-3 indicates that its expression remains elevated for at least 12 h after LK treatment before declining to normal levels (Fig. 4c). A similar profile of expression is observed with c-Jun. In comparison, caspase 3 activation, as judged by generation of the 17 kDa proteolytic product, begins later and is more sustained (Fig. 4c). It is noteworthy that ATF-3 has been shown to play an essential role in stress-induced caspase 3 activation (Lu et al., 2007).

Table 1.   Genes that are regulated by GW5074 treatment of cerebellar granule neurons
Gene titleGene symbolFold change
  1. Three independent sets of RNA were prepared from CGNs treated with LK or LK plus 1 μM 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.

Beta-1 adducinβ-adducin2.636
Glycine receptor, alpha 1 subunitGlra12.13
Insulin related protein 2Isl22.093
Calcium channel, voltage-dependent, beta 1 subunitCacnb11.762
Apoptotic protease activating factor 1Apaf11.694
Glutamate receptor, metabotropic 4Grm41.673
W307 proteinW3071.59
Ribonuclease, RNase A family 4RNase41.555
Transporter-like proteinCtl11.537
SH3/ankyrin domain gene 3Shank31.531
Nuerabin 2Neb21.531
CTD-binding SR-like rA1LOC560811.529
Hepcidin antimicrobial peptideHamp0.634
v-jun sarcoma virus 17 oncogene homolog (avian)jun0.586
Protein kinase (cAMP dependent, catalytic) inhibitor betapkib0.583
Protein tyrosine phosphatase, non-receptor type 2Ptpn20.578
Netrin 1Ntn10.524
Epithelial calcium channel 1Ecac10.512
Protein tyrosine phosphatase, non-receptor type 16Ptpn160.509
CaveolinCav0.477
Phosphatidylinositol 3-kinase, regulatory subunit, polypeptide 1Pik3r10.467
Activating transcription factor 3 (Atf3)Atf30.46
Calpain 8Capn80.404
Stathmin-like 4Stmn40.383
Tumor necrosis factor (ligand) superfamily, member 11Tnfsf110.252
Figure 3.

 Validation of microarray analysis. RNA was prepared from cerebellar granule neurons treated with LK or LK with 1 μM 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.

Figure 4.

 ATF-3 is up-regulated during neuronal apoptosis. (a) Cerebellar 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.

To examine if the stimulation of ATF-3 played a causal role in LK-mediated neuronal death, we over-expressed it in neurons. As shown in Fig. 5a, the over-expression of ATF-3 reduces neuronal survival in HK and blocks the ability of GW5074 to neuroprotect in LK medium. The pro-apoptotic effect of ATF-3 on HK and GW5074-treated neurons was confirmed by TUNEL staining (Supplementary Fig. S2). This finding suggests that ATF-3 promotes apoptosis in LK and that GW5074 and HK maintain neuronal survival by inhibiting the increase in ATF-3 expression. To verify this conclusion, we knocked down ATF-3 expression using siRNA. As shown in Fig. 5b, the forced down-regulation of ATF-3 expression reduces neuronal death induced by LK medium.

Figure 5.

 ATF-3 expression regulates apoptosis in neurons. (a) Cerebellar 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 μM GW5074 (GW) or 1 μM PKRi. The proportion of positively transfected neurons that were viable was quantified following immunocytochemistry with an antibody against Flag or GFP and 4′6′-diamidino-2-phenylindole hydrochloride staining. Neurons with condensed and/or fragmented nuclei were scored as apoptotic. *Indicates value of < 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 blotting 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 deviation 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 μM 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 expression 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 demonstrate equal amount of protein was loaded.

We looked at ATF-3 expression in two other well-established paradigms of neurodegeneration. Camptothecin is a DNA damaging agent which induces apoptosis in cultured cortical neurons (Morris and Geller 1996). In contrast to LK-induced death which is mediated in a p53-independent mechanism, neuronal death by camptothecin is p53-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 camptothecin treatment. HCA treatment of cortical neurons is also used widely as a paradigm to study the mechanisms underlying neuronal death. By causing glutathione depletion, HCA induces oxidative stress leading to apoptosis (Murphy et al. 1990; Ratan et al. 1994a,b). As shown in Fig. 5d, HCA treatment induces ATF-3 expression in cortical neurons. We extended our studies to an in vivo paradigm of neurodegeneration. Administration of 3-NP to mice causes selective striatal neurodegeneration and is widely used as an experimental model of Huntington’s disease. As shown in Fig. 5e, ATF-3 expression is stimulated in the degenerating striatum of 3-NP administered mice (also see Supplementary Fig. S3 for evidence of striatal degeneration). In contrast, the expression of ATF-3 is not increased in the non-striatal brain tissue, which does not display significant neurodegeneration. These results suggest that ATF-3 stimulation contributes to cell death in multiple neuronal types and in response to other apoptotic stimuli.

We next proceeded to examine whether the increased expression of ATF-3 was transcriptionally mediated or mediated post-transcriptionally. As primary neurons are poorly transfected, we utilized the dopaminergic N1E-115 cell line to investigate this issue. Treatment of N1E-115 with 6-OHDA induces apoptosis (Fig. 6a and b). As observed in primary neurons, ATF-3 expression is up-regulated by 6-OHDA (Fig. 6c). Using a promoter construct containing 514 bp of upstream sequence of the ATF-3 gene fused to a luciferase reporter we find that 6-OHDA results in a small but significant increase in transcriptional activity (Fig. 6d).

Figure 6.

 6-Hydroxydopamine (6-OHDA) induced ATF-3 promoter activity in N1E-115 cell line. (a) N1E-115 cells were treated with 50 μM 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-Luciferase 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 μM 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.

To investigate whether the suppression of ATF-3 by GW5074 (Fig. 7a) was mediated by B-Raf, we over-expressed B-Raf in neuronal cultures. The over-expression of an active form of B-Raf reduced the apoptosis-associated stimulation of ATF-3 expression even in the absence of GW5074 (Fig. 7b). Another molecule that is known to play a pivotal role in neuronal apoptosis is the c-Jun transcription factor. The need for c-Jun activation in the induction of LK-mediated death of cerebellar granule neurons as well as other 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, the expression of active B-Raf also blocks c-Jun induction by LK.

Figure 7.

 GW5074 and active B-Raf inhibit ATF-3 expression. (a) Cerebellar granule neurons were treated with HK, LK media or LK media with 1 μM 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.

Other neuronal protective compounds also utilize similar mechanisms while mediating neuronal survival

In a previous study, we reported the identification of other 3′ substituted indolones which were also capable of preventing neuronal apoptosis (Johnson et al. 2005). In this study, we investigated PKRi, an inhibitor of PKR first described by Jammi et al. (2003) and that has a 3′ substituted indolone core. As shown in Fig. 8, treatment with PKRi prevented the death of cerebellar granule neurons induced by LK. In addition to PKRi, two other 3′ substituted indolones, SK4 and SK6, synthesized as part of an ongoing SAR analysis were also found to be neuroprotective (Fig. 8). In contrast to SK4 and SK6, a closely related 3′ substituted indolone designated as SK9, fails to block LK-induced neuronal death at any of the concentrations tested (Fig. 8). A description of the SAR analysis along with other compounds analyzed will be described elsewhere.

Figure 8.

 PKRi, SK4, and SK6 protect cerebellar 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 hydrochloride 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 comparing with LK.

Given their structural similarity, it was possible that PKRi, SK4, and SK6 exerted their neuroprotective action by a common mechanism. We first examined whether the three compounds inhibited c-Raf in vitro. As shown in Fig. 9a, all three neuroprotective compounds (SK4, SK6, and PKRi) inhibit the activity of c-Raf that had been immunoprecipitated from neurons. In comparison, SK9 is less effective. While SK6 and SK4 also produced the paradoxical activation of c-Raf in intact neuronal cultures, PKRi did not. This finding suggested that the in vivo activation of c-Raf is not an essential feature for neuroprotection by 3′ substituted indolones (Fig. 9b). In contrast, PKRi, SK4, and SK6 all activated B-Raf kinase activity while treated in neuronal culture (Fig. 9c). PKRi, SK4, and SK6 also inhibited the apoptosis-associated stimulation of ATF-3 and c-Jun expression that is triggered in cerebellar granule neurons (Fig. 9d and e).

Figure 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 μM 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 concentration 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 blotting 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 treatment (GW = 1.0). (b) Neuronal cultures were treated for 3 h with HK, LK, or LK medium containing 1 μM GW5074, 5 μM PKRi, 1 μM SK4, 2 μM SK6, or 1 μM 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 μM GW5074, 5 μM PKRi, 1 μM SK4, 2 μM SK6, or 1 μM SK9. B-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 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 μM GW5074, 5 μM PKRi, 1 μM SK4, 2 μM SK6, or 1 μM 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 cultures were treated for 3 h with HK, LK, or LK medium containing 1 μM GW5074, 5 μM PKRi, 1 μM SK4, 2 μM SK6, or 1 μM 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 antibody against tubulin, which serves as a loading control.

Since its discovery in 2003, PKRi has been used in a number of studies and a variety of biological effects produced by this compound have been attributed to its ability to inhibit PKR (Page et al. 2006; Shimazawa and Hara 2006; Ingrand et al. 2007). As described above, PKRi does not activate c-Raf in response to GW5074 treatment. PKRi also inhibits c-Jun and ATF-3 expression much more substantially that GW5074, SK4, and SK6 (Fig. 9). It was therefore conceivable that although sharing structural similarity with the other three indolones, PKRi mediates neuroprotection via a distinct mechanism. To address this issue, we examined more rigorously whether B-Raf is necessary for neuroprotection by PKRi. As shown in Fig. 2a, over-expression of the kinase-dead form of B-Raf blocks neuroprotection by PKRi. Over-expression of ATF-3 also inhibits PKRi-mediated neuroprotection (Fig. 5a) as was observed with GW5074.

Discussion

Our results indicate that GW5074 exerts its neuroprotective action by activating B-Raf. Indeed, the over-expression of a kinase-dead form of B-Raf blocks neuroprotection whereas expression of an active form of B-Raf promotes neuronal survival even in the absence of GW5074.

Our conclusion that B-Raf has neuroprotective effects is consistent with the finding that sensory neurons and motor neurons cultured from B-Raf-deficient embryos (but not from c-Raf or A-Raf deficient embryos) fail to survive in response to neurotrophic factors (Wiese et al. 2001). In response to growth factor stimulation, activated B-Raf directly phosphorylates MEK, a serine–threonine kinase that then phosphorylates and activates ERK (Baccarini 2002; Hindley and Kolch 2002). Not unexpectedly, we find that MEK and ERK are stimulated by all four neuroprotective compounds (Supplementary Fig. S4a). Surprisingly, the inhibition of MEK–ERK signaling has no effect on neuroprotection (Supplementary Fig. S4b). It is noteworthy that while MEK remains the most widely accepted substrate, recent studies have identified a number of other proteins including Bad, p53, and Rb that can be directly phosphorylated by Raf (Bonni et al. 1999; Baccarini 2002; Hindley and Kolch 2002). Moreover, Raf is able to prevent apoptosis by interacting with the anti-apoptotic molecule, Bag-1(Rudolf Götz et al. 2005), or sequestering the pro-apoptotic molecule apoptosis signal-regulating kinase-1 (Chen et al. 2001) through a MEK and ERK independent pathway. Although further work is needed to clarify the mechanism by which it occurs, we find that neuroprotection by B-Raf involves the inhibition of the ATF-3 transcription factor whose expression is induced in neurons primed to undergo apoptosis. Over-expression of active B-Raf blocks the apoptosis-induced increase in ATF-3 expression. A relationship between B-Raf and ATF-3 has been described in actively proliferating cell lines (Nilsson et al. 1997). In these cells, B-Raf has been described to actually stimulate ATF-3 expression. Whether ATF-3 is induced or suppressed by B-Raf may therefore be cell and context specific.

Activating transcription factor-3 is a member of the ATF/cAMP response element binding protein family of basic leucine zipper-type transcription factors which can form both homo- and heterodimers with other basic leucine zipper-type transcription factors (Hai et al. 1999). While ATF-3 homodimers are believed to inhibit gene transcription, heterodimers can activate transcription. The most commonly described dimerization partners for ATF-3 are members of the Jun family (c-Jun, Jun B, and Jun D). ATF-3 expression is generally induced by cellular damage and a variety of stress stimuli in different tissue types (Hai et al. 1999). In the nervous system, ATF-3 has been implicated in neuronal degeneration following spinal cord damage (Tsujino et al. 2000; Averill et al. 2004; Huang et al. 2006), cerebral artery occlusion (Ohba et al. 2004), and neurotoxin-induced oxidative stress (Holtz et al. 2006). Elevated ATF-3 expression also precedes the death of spinal motor neurons in a transgenic mouse model of amyotrophic lateral sclerosis (Vlug et al. 2005) and of retinal ganglion cells after crushing of the optic nerve (Takeda et al. 2000). In addition to cerebellar granule neurons, we find that ATF-3 expression is increased in cortical neurons induced to die by oxidative stress and DNA damage, and in the degenerating striatum of 3-NP administered mice. In some cases however, ATF-3 has been reported to promote neuronal survival. For example, ATF-3 over-expression protects hippocampal neurons from kianic acid-induced degeneration (Francis et al. 2004) and sympathetic neurons from nerve growth factor deprivation-induced apoptosis (Nakagomi et al. 2003). Whether ATF-3 kills or promotes neuronal survival may be dependent on whether it acts as a homo- or heterodimer or on other proteins with which it associates within the specific cell type. Other mechanisms such as differential post-translational modifications on ATF-3 may also contribute to the different roles that ATF-3 displays. Initial indications based on the utilization of an ATF-3 promoter-luciferase reporter construct is that the increase in ATF-3 expression during apoptosis is mediated at least in part at the transcriptional level. The small increase in promoter activity we observe in 6-OHDA-treated N1E-115 cells does not, however, explain the substantial increase in ATF-3 protein expression. It is possible that the 514 bp of 5′ genomic sequence that we have utilized does not contain all the transcriptional elements necessary for full induction. Alternatively, post-transcriptional mechanisms such as increased mRNA stability might contribute to increased expression. Post-transcriptional regulation of ATF-3 expression has been suggested in other studies (Lu et al., 2007).

In cerebellar granule neurons, ATF-3 induction is causally involved in promoting apoptosis. Indeed, the over-expression of ATF-3 in healthy neurons is sufficient to induce apoptosis whereas the forced suppression of ATF-3 reduces the extent of LK-induced neuronal death. The partial protection is likely to reflect the incomplete suppression of ATF-3 expression by the siRNA used in this study. It is also possible that suppression of ATF-3 alone is not sufficient for complete protection and that other molecules cooperate with ATF-3 to promote neuronal death. One such molecule is c-Jun. Several different 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; Crocker et al. 2001). Interestingly, the apoptosis-associated stimulation of c-Jun expression is also inhibited by active B-Raf.

Another 3′ substituted indolone utilized in this study is PKRi, a compound identified based on its ability to inhibit PKR activity in vitro (Jammi et al. 2003). We have discovered that PKRi protects against LK-induced death of cerebellar granule neurons. While this study was in progress other investigators also reported neuroprotective actions of PKRi 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 PKR activity is elevated in certain tissue culture models of neurodegeneration and in the brains of patients with Alzheimer’s diseases raising the possibility that activation of PKR may 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 its neuroprotective effect in cerebellar granule neurons is dependent, at least in part, on B-Raf. Furthermore, PKRi also blocks the apoptosis-associated induction of ATF-3. As observed with GW5074, over-expression of ATF-3 blocks the ability of PKRi to be neuroprotective. At the present time we do not know whether activation of PKR is necessary for apoptosis in the paradigms we have investigated comprising cerebellar granule neurons or cortical neurons. Preliminary work in cerebellar granule neurons indicates that PKRi treatment does not inhibit the phosphorylation of eIF2α, which is a downstream target of PKR (Chen and D’Mello, unpublished observation). Further studies are necessary to determine whether PKR inhibition is involved in the neuroprotective action of PKRi, or whether this compound acts through separate effects on B-Raf activity. In addition to PKRi, we have studied two other 3′ substituted indolones which were identified in a SAR screen. Both SK4 and SK6 activate B-Raf and suppress ATF-3 expression.

In summary, we identify B-Raf and ATF-3 as important players in the regulation of neuronal survival. These proteins can serve as molecular targets in the development of neuroprotective therapeutic strategies. Furthermore, our studies identify 3′ substituted indolones as a core chemical structure that can be used as a starting point for the development of therapeutic drugs against neurodegenerative disorders. At the present time we do not know whether activation of PKR is necessary for apoptosis in the paradigms we have investigated comprising cerebellar granule neurons or cortical neurons. Perliminary work in cerebellar granule neurons indicates that PKRi treatment does not inhibit the phosphorylation of eIF2α, 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).

Ancillary