PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci. Author manuscript; available in PMC Apr 30, 2013.
Published in final edited form as:
PMCID: PMC3504487
NIHMSID: NIHMS418878
Loss of STAT3 signaling during elevated activity causes vulnerability in hippocampal neurons
Sachiko Murase,1 Eunyoung Kim,2 Lin Lin,2 Dax A. Hoffman,2 and Ronald D. McKay1,3
1Laboratory of Molecular Biology, National Institute of Neurological Disorder and Stroke, National Institutes of Health, Bethesda, MD 20892
2Molecular Neurophysiology and Biophysics Unit, Program in Developmental Neuroscience, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
3Lieber Institute for Brain Development, 855 North Wolfe St., Baltimore, MD 21205
Correspondence should be addressed to S. M. (sachikom/at/ninds.nih.gov)
Chronically altered levels of network activity lead to changes in the morphology and functions of neurons. However, little is known of how changes in neuronal activity alter the intracellular signaling pathways mediating neuronal survival. Here we use primary cultures of rat hippocampal neurons to show that elevated neuronal activity impairs phosphorylation of the serine/threonine kinase, Erk1/2 and the activation of signal transducer and activator of transcription 3 (STAT3) by phosphorylation of Serine 727. Chronically stimulated neurons go through apoptosis when they fail to activate another serine/threonine kinase, Akt. Gain and loss of function experiments show that STAT3 plays the key role directly downstream from Erk1/2 as the alternative survival pathway. Elevated neuronal activity resulted in increased expression of a tumor suppressor, p53 and its target gene, Bax. These changes are observed in Kv4.2 knockout mouse hippocampal neurons, which are also sensitive to the blockade of TrkB signaling, confirming that the alteration occurs in vivo. Thus, this study provides new insight to a mechanism by which chronic elevation of activity may cause neurodegeneration.
Neurons can detect global changes in input levels and can adjust their own levels of excitability to maintain an appropriate range of spiking activity (Davis, 2006; Turrigiano, 2007). Although the molecular details of synaptic scaling mechanisms that ensure this homeostatic plasticity have been studied extensively (Beattie et al., 2002; Shepherd et al., 2006; Cingolani et al., 2008; Seeburg et al., 2008; Cohen et al., 2011), little is known of the impact these mechanisms exert on the survival signaling of neurons.
Recently, we reported that neurons in newborn rodent hippocampus, while they are under selection through a process of developmental death, depend on neurotrophin for survival. Neurotrophins activate signaling pathways through the tropomyosin receptor kinase (Trk) (Zweifel et al., 2005). Interestingly, we found that the direct activation of these pathways by TrkB was not sufficient to promote survival of neurons. Rather, brain-derived neurotrophic factor (BDNF) recruits neurons into active networks, and this activity, together with integrin signals, mediates sustained Akt activation and promotes neuronal survival (Murase et al., 2011a). Whether or not this sustained Akt activation remains critical for the survival of mature neurons has not been tested.
STAT proteins are transcription factors that respond to cytokines such as interleukins (ILs) (Levy and Darnell, 2002). STAT3-mediated cytokine signaling regulates gliogenesis as well as neurogenesis during brain development (Deverman and Patterson, 2009). Although neuroprotective roles of STAT3 have been reported in the context of injury (Schweizer et al., 2002; Dziennis et al., 2007; Jung et al., 2009), roles of STAT3 in the survival of neurons under healthy conditions have not been explored.
Here we report that after the vulnerable period, neurons lose their dependence on neurotrophins unless they are challenged by elevated activity. In this more mature state, elevation of network activity restores a dependence on neurotrophin induced survival signals. Gain and loss of function experiments identify STAT3 as a key mediator of the second survival pathway that mature neurons acquire. This study shows chronic elevation of activity may cause neurodegeneration through a mechanism similar to developmental death.
Reagents
LY294002, PD98059, rapamycin and roscovitine were purchased from Calbiochem (La Jolla, CA). Fluo-4 AM was purchased from Invitrogen (Eugene, OR). 4-Aminopyridine (4-AP), tetrodotoxin (TTX), picrotoxin (PTX) 4′,6-diamidino-2-phenylindole (DAPI), K252a were purchased from Sigma-Aldrich (St. Louis, MO). BDNF was purchased from R&D Systems (Minneapolis, MN).
Antibodies
Antibodies were used at the following dilutions: polyclonal rabbit anti-phospho-Ser727 STAT3 antibody, anti-phospho-Tyr705 STAT3 antibody, anti-STAT3 antibody, anti-p53 antibody and anti-Bax antibody (Santa Cruz Biotechnology, Santa Cruz, CA), 1:500; anti-Erk1/2 antibody, anti-phospho-Erk1/2 antibody, anti-phospho-Ser-743 Akt antibody, cleaved caspase3 (c-cas3) antibody, and monoclonal mouse anti-STAT3 antibody (Cell Signaling Technology, Danvers, MA), 1: 500; monoclonal mouse anti-cytochrome c antibody and NeuN antibody (BD Biosciences Pharmingen, San Diego, CA), 1:1000; monoclonal mouse anti-β-actin antibody, anti-MAP2 antibody (Sigma-Aldrich), 1:10000 and 1:1000, respectively. Polyclonal goat anti-TrkB antibody (R&D Systems) was used at various concentrations for function blocking experiments.
Dissociated primary hippocampal culture
Culture was prepared as described previously (Murase and McKay, 2006). Hippocampi from embryonic day 18 (E18) Sprague Dawley rat embryos of either sex were used for both astrocyte (plated at a density of 80,000 cells/ml) and neuron (density: 200,000 cells/ml) cultures. Astrocytes were cultured in Neurobasal (Invitrogen) with 5% fetal bovine serum (FBS) in 5% CO2 at 37°C for 14 days. Medium was changed completely twice weekly. Neurons were plated on confluent astrocyte beds and cultured in Neurobasal and B27 in 5% CO2 at 37°C. Half of the medium was changed every 2 days. Experiments were performed using 14 days in vitro (DIV14) neurons.
Calcium imaging
Cultures were incubated with 2 μM fluo4-AM for 15 min. Images were obtained with a BX51W1 microscope (Olympus America, Center Valley, PA) equipped with a 10x objective lens. The cultures were imaged in Hepes buffered saline (HBS: 110 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 10 mM D-glucose, 10 mM Hepes-NaOH (pH 7.4), 290 mOsm) at 37°C using a CCD camera (QImaging, Blaine, WA) at 20 Hz for 40 sec. Typically, 20 neurons in one experiment were used for analyses.
Immuno-precipitation
Rat hippocampal culture was incubated with lysis buffer (150 mM NaCl, 1% NP-40, 50 μM Tris-HCl, pH8.0) containing a protease inhibitor cocktail (Roche) for 20 min on ice (60 μl per one 24-well culture dish). Lysate was centrifuged at 12,000 rpm for 5 min at 4°C. The supernatant was pre-absorbed with 10% (v/v) of protein A Sepharose beads (Pierce, Rockford, IL) for 1 hr, and incubated with 3 μg/ml of anti-STAT3 antibody for 2 hr followed by incubation with 10 % (v/v) of protein A Sepharose beads for 1 hr at 4°C. The beads were then rinsed 3 times with lysis buffer with protease inhibitor at 4°C prior to elution with an equal volume of 1XSDS loading buffer by boiling for 5 min.
Western blot
Samples from dissociated culture were collected with 1XSDS loading buffer (60 μl per one 24-well culture dish). Hippocampi from wild-type C57BL/6 mice and Kv4.2−/− mice were homogenized in 300% (v/w) lysis buffer with protease inhibitor on ice. The homogenates were diluted with 2XSDS loading buffer. The samples were boiled for 5 min, then applied to a 4–10% gradient SDS gel (BioRad, Hercules, CA). The proteins were transferred to a nitrocellulose membrane. The membranes were blocked with 4% skim milk in phosphate buffered saline (PBS) for 30 min. Incubation with antibodies was performed in the blocking solution. Membranes were washed with Tris-buffered saline with 0.05% Tween 20. The proteins were visualized with SuperSignal West Pico System (Pierce), and detected and analyzed with a BioChemi System (UVP BioImaging Systems, Upland, CA). Mean±SEM are plotted.
Immunocytochemistry
Cultures were fixed with 4% paraformaldehyde, permeabilized in 0.5% Triton X-100, and blocked with PBS containing 5% normal goat serum (NGS, Vector Laboratories, CA). Primary and secondary antibodies were diluted with the blocking solution. Samples were incubated for 2 hr with antibodies.
For the in vivo injection analyses, C57BL/6 and Kv4.2−/− mice were perfused with 4% PFA two days after the injection. Consecutive coronal slices of 50 μm thickness were made by a Leica VT100S vibrating microtome (Leica, Allendale, NJ) and were immunostained with a neuronal marker, NeuN, and the apoptotic marker, c-cas3. Slices were compared with respect to distance from the injection site. Four consecutive slices per animal and three animals per condition were combined for the analyses. The analysis was done blind with respect to the content of the injections.
Cell quantification
Neurons were visualized by immunostaining against neuron specific microtubule associated protein 2 (MAP2; (Izant and McIntosh, 1980)). Fluorescent images were taken with a Zeiss confocal microscope (LSM-510) equipped with 10x lens or a 25x lens. Z-stacked images from eight sections (1 μm intervals) were used for the analyses. All experiments were repeated in at least 3 independent culture preparations. Image analyses were done using ImageJ. Images were taken from 5 fields; one from the center of the coverslip, and two vertically and two horizontally 400–3000 μm from the center. Because the densities of neurons were higher in the rim of coverslips than in other regions, we avoided sampling the edge of coverslips. Each coverslip was defined as an individual culture. Numbers represent mean±SEM. All analyses were done blind.
Transfection
Transfection was performed using Lipofectamine 2000 (Invitrogen). Cells were transfected with 1.6 μg/ml of pEGFPC1 vector (Clontech, Mountain View, CA), and/or 8 μg/ml activated Akt1/pUSE vector (Millipore, Bedford, MA), or 30 pM rat STAT3 siRNA (Santa Cruz) or wild-type and mutant STAT3 IRES EGFP/pMX plasmids (gift from Dr. Y. Gotoh, Tokyo, Japan) in OPTI-MEM (Invitrogen) for 30 min, then the medium was replaced with NeuroBasal Medium. Transfection was performed 4 days prior to the experiments.
Reverse transcription (RT)-PCR
Hippocampi from C57BL/6 and Kv4.2−/− mice were homogenized in 300% (v/w) lysis buffer on ice. Rat hippocampal culture was incubated with lysis buffer with protease inhibitor cocktail for 20 min on ice (60 μl per one 24-well culture dish). RNA was isolated from the homogenates using TriPure Isolation Reagent (Roche, Welwyn Garden City, UK). RT-PCR was performed using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Using 5 μg of total RNA, first-strand cDNA synthesis reaction by reverse transcriptase was done using Oligo(dT)12–18 as primers. PCR was performed using Taq polymerase (Roche). The sequences of the primers are the following: 5′-CCACACTTTCTACAATGAGC-3′ and 5′-CCGTCAGGATCTTCATGAGG-3′ for rat β-actin, 5′-CTACTAAGGTCGTGAGACGCTGCC-3′ and 5′-TCAGCATACAGGTTTCCTTCCACC-3′ for rat p53, 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′ for mouse GAPDH, 5′-GATGGTGATGGCCTGGCTCC-3′ and 5′-GGTCGGCGGTTCATGCCCCC-3′ for mouse p53. Conditions for PCR reactions are: 44 cycles of 95°C (15 sec), 60°C (20 sec), 72°C (15 sec) for rat p53; and 35 cycles of 95°C (30 sec), 62°C (30 sec), 72°C (30 sec) for rat β-actin, mouse p53 and GAPDH. The PCR products were separated in 2% agarose gel.
Chromatin immunoprecipitation (ChIP)
Chromatin immuno-precipitation assays were performed as described by Ballas et al. (Ballas et al., 2001). C57BL/6 and Kv4.2−/− mice were perfused with 4% PFA. Hippocampi were homogenized with cell lysis buffer (CLB; 5 mM Hepes pH 8, 85 mM KCl, and 0.5% Triton X-100) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) using a glass tissue grinder on ice. The homogenate was centrifuged at 3000 rpm for 2 min at 4°C, and the pellet was resuspended in CLB with PMSF and centrifuged at 3000 rpm for 2 min at 4°C two times. The pellet was then resuspended in nuclear lysis buffer (NLB; 50 mM Tris-HCl pH8, 10 mM EDTA, 1% SDS) with 1 mM PMSF and was sonicated to yield 100 bp to 1000 bp DNA on ice, and was centrifuged at 12000 rpm for 15 min at at 4°C. The nuclear lysate was pre-absorbed with recombinant protein G agarose (rProtein G agarose; Life Technologies, Grand Island, NY) pre-incubated with 200 μg/ml yeast tRNA and 200 μg/ml salmon sperm (Invitrogen) for 1 hr at 4°C. The chromatin suspension was diluted with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8, 167 mM NaCl), then immuno-precipitated with 5 μg/ml of monoclonal mouse anti-STAT3 overnight at 4°C. The chromatin suspension was incubated with rProtein G agarose pre-treated with 3% BSA and yeast tRNA and salmon sperm for 4 hr at 4°C. Agarose beads were washed with series of solutions as following at room temperature: ChIP dilution buffer, dialysis buffer (2 mM EDTA, 50 mM Tris-HCl pH 8, 0.2% sarkosyl), TSE-500 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8, 500 mM NaCl), LiCl detergent (100 mM Tris pH 8, 500 mM LiCl, 1% Triton X-100, 1% deoxycholic acid), and TE (10 mM Tris-HCl pH 8, 1 mM EDTA). To change the solution, the beads were centrifuged at 3000 rpm for 1 min and the supernatant was aspirated. The samples were eluted from the beads with 300 μl of elution buffer (50 mM NaHCO3, 1% SDS). Samples were incubated overnight at 65°C to reverse PFA cross links following the addition of 20 μl of 5 M NaCl. DNA was then purified from the eluted samples using Qiagen PCR purification kit (Qiagen, Valencia CA). PCR was performed to analyze the STAT3 binding site in the p53 promoter using the following DNA primers: 5′-GGGCCCGTFTTGGTTCATCC-3′ and 5′-CCGCGAGACTCCTGGCACAA-3′. Conditions for PCR reactions were: 30 cycles of 94°C (30 sec), 60°C (30 sec), 72°C (1 min). The PCR products were separated in 1.5% agarose gel.
In vivo injection
The C57BL/6 and Kv4.2KO mice (8 to 9 weeks old) of either sex were anesthetized by ketamine/xylazine cocktail (3.33ml/kg) via intraperitoneal injection, before the surgery. The delivery of reagents to CA1 was done using the following stereotaxic coordinates from bregma: anteroposterior, 2.4 mm; mediolateral, ±2.28 mm; ventrodorsal, 1.6 mm. Reagents (0.5 μl) were delivered at a rate of 0.12 μl/min using a Hamilton needle and syringe attached with a microsyringe pump controller (World Precision Instruments (Sarasota, FL). Anti-TrkB (1.0 mg/ml) and vehicle (PBS) alone were injected, one to each side of the brain. The incision was closed using a polyglycolic acid suture (CP Medical, Portland, OR). Animals were allowed to recover at 37°C for 1–2 hr.
Statistical analyses
Statistical significance between two groups was determined with a two-tailed paired Student’s t test. For multiple groups, statistical comparisons were made by ANOVA followed by individual group tests with the Bonferroni correction made for multiple comparisons.
Hippocampal neurons become BDNF-dependent during elevated activity
As we reported previously, spontaneous network activity develops during the first 12 days in our primary cultures of hippocampal neurons (Murase et al., 2011a). After the conclusion of this period, on day 14 in vitro (DIV14), we elevated the activity of the network by increasing the excitability of neurons by application of 4-aminopyridine (4AP), a drug that blocks the current mediated by A-type voltage-gated potassium channels (Zagotta et al., 1988). We monitored spontaneous network activity with the calcium indicator Fluo-4 AM (Fig. 1A) and found treatment with 5 mM 4AP increased the mean frequency of calcium responses (>100%, Fig. 1A).
Fig. 1
Fig. 1
Neurons become TrkB-dependent when network activity is elevated
To test whether these neurons required neurotrophin for survival, we first showed that in neurons (at DIV14) robust phosphorylation of Trk receptor followed exogenous application of BDNF. This effect was blocked by pre-incubation with a function-blocking anti-TrkB antibody (data not shown). Using this antibody, we found that blocking BDNF-TrkB signaling alone did not decrease the number of surviving neurons (Fig. 1B and C). This is strikingly different from the dramatic loss of immature neurons caused by this treatment during the developmental death period (Murase et al., 2011a). In mature neurons, elevation of network activity with 4AP alone also did not affect the number of surviving neurons (Fig. 1B and C). However, elevating activity in the presence of the anti-TrkB antibody resulted in a significant decrease in neuron number (Fig. 1B and C). Blocking neuronal activity by applying the voltage-gated sodium channel blocker, tetrodotoxin (TTX) attenuated 4AP-induced TrkB dependency (Fig. 1C). Blocking phosphorylation of Trk receptors with 200 nM K252a, the specific inhibitor of Trk (Berg et al., 1992), also induced a loss of neurons but only when 4AP was co-applied with the inhibitor (K252a alone; 100.5±3.4% of control, K252a and 4AP; 65.1±7.2% of control, n=6, p<0.05). Elevating network activity by applying picrotoxin (PTX), the γ-aminobutyric acid receptor A (GABAA receptor) antagonist, also induced TrkB dependency (Fig. 1C). These results suggest that elevation of activity led to changes in intracellular signaling, causing the neurons to become, once again, dependent on BDNF-TrkB signaling.
Akt is necessary and sufficient for survival of chronically stimulated neurons
Activation of PI3K and recruitment of the serine-threonine kinase Akt to the plasma membrane initiates cell survival responses in many types of cells (Toker and Newton, 2000; Jacinto et al., 2006). Moreover, we recently reported that BDNF-triggered Akt activation is critical for the survival of immature neurons during the death period (Murase et al., 2011a). To determine if Akt activation was sensitive to Trk activation we exposed mature neurons to the function-blocking anti-TrkB antibody. The results show levels of phospho-Ser 473 Akt in neurons at DIV14 were severely reduced by the function blocking anti-TrkB antibody (Fig. 2A), suggesting BDNF continues to play a major role in activating Akt signaling in these neurons. However, our new data suggest that the survival of these mature cells is not sensitive to neurotrophin reduced Akt activation.
Fig. 2
Fig. 2
Neurons become dependent on Akt signaling when treated with 4AP
Other serine/threonine kinases, Erk1/2, are activated by many receptor tyrosine kinases including TrkB, and are also often implicated in survival responses (Huang and Reichardt, 2003). Incubation with anti-TrkB antibody, however, did not affect levels of phospho-Erk1/2 without affecting Akt levels (Fig. 2A). Addition of TTX attenuated the effect of 4AP, confirming that impairment of Erk1/2 signaling was due to the elevation of spiking activity (Fig. 2A). When neurons were treated with 4AP and anti-TrkB antibody, both Akt and Erk1/2 signaling pathways were impaired (Fig. 2A).
To test whether blocking Akt signals can have the same effect as applying anti-TrkB antibody, we then added LY294002, the PI3K inhibitor. When treated with both 4AP and LY294002, many neurons showed: (1) activation of caspase 3 (c-cas3), a protein that plays a central role in apoptosis (Fernandes-Alnemri et al., 1994); (2) a diffuse pattern of cytochrome c, a protein that initiates apoptosis (Liu et al., 1996); and (3) fragmented nuclei revealed by DAPI staining (Fig. 2B). These results suggest that, in over-activated neurons, Akt signals mediate survival. Consistent with these observations, treatment with 4AP and LY294002 decreased the number of neurons, whereas LY294002 alone did not affect the number of neurons (Fig. 2C). Moreover, exogenous BDNF did not rescue these neurons from death caused by co-application of 4AP and LY294002 (Fig. 2C), suggesting Akt is critical for BDNF-mediated neuronal survival.
Phosphorylation triggers Akt to translocate to the plasma membrane, a step critical for its activation. Akt tagged with a myristoylation site is constitutively active (Kohn et al., 1996). When neurons were transfected with a control plasmid expressing only green fluorescent protein (GFP), numbers of transfected neurons decreased upon co-application of 4AP and anti-TrkB, whereas neurons co-transfected with a plasmid encoding myristoylated Akt were not affected by the treatment (Fig. 2D). Thus, constitutively active Akt is sufficient to block the induction of death caused by the 4AP and anti-TrkB treatment. Taken together, these results suggest Erk1/2 signal is impaired during elevated neural activity, causing neurons to become dependent on Akt signals mediated mainly by TrkB activation.
STAT3 provides the alternative survival signal
When studying potential mediators of the Erk1/2 survival signal, we found that levels of phospho Ser-727 STAT3 were severely impaired by treatment with 4AP (Fig. 3A). This treatment caused no change in levels of phospho Tyr-705 STAT3 (data not shown). Blocking network activity by applying TTX completely abolished this effect of 4AP (Fig. 3A). Inhibiting Erk1/2 with PD98059 effectively suppressed the phosphorylation of Ser-727 STAT3 and occluded the effect of 4AP (Fig. 3A). Neither the treatments with anti-TrkB nor LY294002 affected the phosphorylation (Fig. 3A). Inhibition of mTOR by rapamycin (Brown et al., 1994), Cyclin-dependent kinase 5 (Cdk5) by roscovitine (Meijer et al., 1997), on the other hand, resulted in a minor reduction of phosphorylation (Fig. 3B). Next, we analyzed the interaction between Erk1/2 and STAT3 with an immuno-coprecipitation experiment. When the lysate from hippocampal culture was precipitated with anti-STAT3, we observed it co-precipitated Erk1/2, suggesting that there is an interaction between Erk1/2 and STAT3 (Fig. 3C). These results suggest that STAT3 may be phosphorylated at Ser-727 by Erk1/2.
Fig. 3
Fig. 3
Phosphorylation of STAT3 is impaired following treatment with 4AP
To directly test whether phosphorylation of STAT3 plays a role in the survival of neurons, we over-expressed the phospho-mimetic Ser727Glu STAT3 (SE-STAT3). Neurons expressing only GFP declined significantly in number after treatment with 4AP in the presence of the anti-TrkB antibody or LY294002, but the survival of neurons expressing Ser727Glu STAT3 was not affected by these treatments (Fig. 4A and B). These results suggest that STAT3 signaling mediates neuronal survival and the role of TrkB in neuronal survival can only be assessed when the Erk-STAT3 pathway is inhibited.
Fig. 4
Fig. 4
Ser727Glu STAT3 prevents neurons from becoming TrkB and Akt dependent during elevated activity
Consistent with a central role for STAT3 in regulating the alternate survival pathway, neurons expressing Ser727Ala STAT3, which cannot be phosphorylated, were vulnerable to anti-TrkB or LY294002 treatment, which does not change activity of Erk phosphorylation (Fig. 5). Neurons expressing GFP only or co-expressing wild-type STAT3 were not affected by these treatments (Fig. 5). These results show that serine phosphorylation of STAT3 is necessary and sufficient for the survival of mature hippocampal neurons.
Fig. 5
Fig. 5
Ser727Ala STAT3 induces TrkB- and Akt-dependency in neurons
Finally, to confirm that loss of STAT3 signaling is responsible for TrkB-and Akt-dependency, we examined the effect of knocking down endogenous STAT3 expression. We co-transfected neurons after the death period (DIV9) with STAT3 siRNA and GFP vector. Application of STAT3 siRNA significantly down-regulated STAT3 levels, and up-regulated a tumor suppressor, p53, and Bax, a proapoptotic target of p53 (Deckwerth et al., 1996) (Fig. 6A). Numbers of siRNA-containing neurons dramatically decreased upon treatment with anti-TrkB or LY294002 (Fig. 6A and B). Application of BDNF did not rescue the effect of LY294002 on STAT3 knockdown neurons, indicating BDNF provides its survival effect mainly through Akt. These results are consistent with our observation that endogenous STAT3 is required for resistance against neurotrophin deprivation.
Fig. 6
Fig. 6
STAT3 blockade by siRNA induces TrkB- and Akt-dependency in neurons
We reported previously that young neurons go through a well-characterized period of developmental death, and during this period, neurons are dependent upon Akt signaling for their survival (Murase et al., 2011a). Our new findings reported here prompted us to investigate levels of STAT3 during these distinct phases of neuronal vulnerability. We found that the total level of STAT3 protein was much lower in young neurons (DIV7) than in mature neurons (DIV14). The levels of PSer STAT3 followed the same proportions (Fig. 7A). We also observed slightly lower levels of pErk1/2. In the neonatal hippocampus, p53 is a key regulator for the survival of neonatal neurons (Murase et al., 2011b). We found levels of p53 and Bax declined significantly after the developmental death period (Fig. 7A). Neurons co-transfected with GFP and wild-type STAT3-expressing plasmids showed lower levels of p53 and Bax than those transfected with GFP plasmid only (Fig. 7B). Further, co-transfection with GFP and wild-type STAT3-expressing plasmids rescued the young neurons from death induced by anti-TrkB or LY294002, whereas S727A STAT3 co-expression failed to rescue the neurons (Fig. 7C). These results suggest immature neurons are vulnerable because they contain low levels of STAT3. Taken together, our results show levels of STAT3 signaling are regulated developmentally, effectively controlling the vulnerability of neurons.
Fig. 7
Fig. 7
STAT3 is responsible for the change in vulnerability during development
Kv4.2 knockout mouse hippocampus shows impairment of Erk1/2-STAT3 signaling
The finding that levels of p53 declined significantly after the developmental death period prompted us to check whether p53 levels increased in neurons challenged by elevated activity. Western blot analyses showed that the treatment of hippocampal neurons (DIV14) with 4AP resulted in the up-regulation of p53 (Fig. 8A). Bax also increased in the treated neurons (Fig. 8A). RT-PCR showed the amount of p53 mRNA had increased (Fig. 8A), suggesting that the transcription of p53 is stimulated by the treatment. These results suggest the increase in a proapoptotic protein, Bax contributes to induction of vulnerability.
Fig. 8
Fig. 8
Elevated excitability increases levels of p53 and Bax in vitro and in vivo
We then checked whether these alterations observed in vitro also occur in vivo by comparing the hippocampi of Kv4.2 knockout mice with those of wild-type mice. Kv4.2 is an A-type voltage-gated potassium channel subunit expressed in hippocampal neurons (Sheng et al., 1992) and the loss of Kv4.2 function results in dendritic excitability (Kim et al., 2007). Western blot analyses showed that genetic deletion of Kv4.2 resulted in loss of Erk1/2-STAT3 signaling, and up-regulation of p53 and Bax (Fig. 8B). RT-PCR showed that the knockout hippocampus contained higher levels of p53 mRNA than the wild type. To check if loss of STAT3 signaling directly affects p53 expression, we performed a chromatin immuno-precipitation (ChIP) assay on wild type and Kv4.2KO hippocampal neurons. Immuno-precipitation of STAT3 showed the binding of STAT3 to p53 promoter, which was impaired in Kv4.2KO neurons (Fig. 8B), suggesting that the change in STAT3 signaling directly affects p53 expression. To confirm that elevated excitability causes vulnerability in vivo, we then made bilateral injections to the wild-type and Kv4.2KO CA1 in adult mice; the function-blocking anti-TrkB antibody was delivered to one side of the brain, and vehicle control was delivered to the other side. Neither the antibody nor vehicle injections caused notable neuronal death in the wild type hippocampi (Fig. 8D). However, we observed an area of significantly increased numbers of apoptotic neurons centered around the antibody-injected region of Kv4.2KO hippocampi, but no increase in neuronal death on the contra-lateral side where vehicle alone had been injected (Fig. 8C and D), suggesting that Kv4.2KO hippocampal neurons were indeed sensitive to the blockade of TrkB signaling. Together, these results suggest that elevated excitability in vitro and in vivo inhibits the Erk1/2-STAT3 survival pathway that controls the survival of neurons through transcription of p53 (Fig. 9).
Fig. 9
Fig. 9
Elevation of activity impairs Erk1/2-STAT3 signaling to control p53 expression
It remains unclear how neurons integrate signals from different extracellular cues to initiate events that support survival. Previously we reported that during the period of developmental neuronal death, neurons require neurotrophin-induced Akt signaling for their survival (Murase et al., 2011a). Neurons that survive this vulnerable period, the subjects of the current study, no longer depend on Akt signaling for continued viability. Here we show that elevation of network activity induces neurotrophin dependency in neurons by inhibiting a survival-signaling pathway that inhibits activation of STAT3. This inhibition is caused by the impairment of Erk1/2 and, as a consequence, these neurons become dependent on Trk mediated Akt activation. Erk1/2 and Akt can be simultaneously activated by many tyrosine receptor kinases including Trk (Huang and Reichardt, 2003), and these kinases share common substrates; one is cAMP response element-binding protein (Tan et al., 1996; Du and Montminy, 1998), which plays an important role in the survival of neurons (Finkbeiner et al., 1997). The response to elevated activity, a severe reduction in levels of PErk1/2 with little change in the levels of PAkt, suggests that Erk1/2 and Akt are activated independently.
A variety of serine/threonine kinases have been reported to phosphorylate STAT3 at Ser-727 (Chung et al., 1997; Oh et al., 1998; Jain et al., 1999; Lim and Cao, 2001; Fu et al., 2004). However, as we show here, in hippocampal neurons, Ser phosphorylation of STAT3 largely depends on Erk1/2. Over-expression of the phospho-mimetic mutant Ser727Glu STAT3 completely attenuated neurotrophin-dependency in chronically activated neurons, suggesting STAT3 is the key molecule downstream from the Erk1/2 survival signal. Ser727Ala STAT3, on the other hand, acts as a dominant negative species. Studies have shown different effects of Ser phosphorylation on STAT3 activity (Decker and Kovarik, 2000): Originally, the dominant role was assigned to PTyr-705, and PSer-727 was reported to be required for maximum activity (Wen et al., 1995; Decker and Kovarik, 2000). Others observed transcriptional activity in the absence of detectable PTyr-705 (Ceresa and Pessin, 1996)(Ng et al., 2006). In some cases, PSer-727 inhibited PTyr-705 and STAT3 activity (Chung et al., 1997)(Jain et al., 1998)(Lim and Cao, 1999). Only low levels of PTyr-705 were detected in our study, and the elevation of network activity in vivo and in vitro did not affect the levels of Tyr phosphorylation. These contradictory findings may reflect differences of cell type and cellular conditions.
Previously, we reported that young neurons, during the developmental death period, require neurotrophin and Akt signaling for their survival (Murase et al., 2011a). Notably, neurons that survive this period of vulnerability have outgrown their dependency on Akt signaling for continued viability. Similar changes in vulnerability to death have been observed in developing sympathetic neurons (Easton et al., 1997). Several lines of evidence suggest STAT3 signaling controls the vulnerability of neurons: 1) during development, STAT3 levels increase dramatically as neurons become resistant to neurotrophin deprivation; 2) over-expression of STAT3 in immature neurons attenuates death caused by the blockade of Trk-Akt signaling; 3) expression of dominant-negative (Ser727Ala) STAT3 as well as knock-down of endogenous STAT3 in older neurons (DIV14) induces Trk-Akt dependency. Following chronic elevation of network activity, these mature neurons once again became dependent on Trk-Akt signaling caused by the loss of STAT3 signaling. Thus, these mature neurons revert to features characteristic of immature neurons.
Our new results indicate that chronically activated neurons in vitro and in vivo express higher levels of p53 and its proapoptotic target, Bax. We found that this change in p53 expression occurs at its transcription level. These results are consistent with a previous study that demonstrated p53 transcription is negatively regulated by STAT3 (Niu et al., 2005). During the developmental death period, p53 serves as a key regulator to control survival of hippocampal neurons (Murase et al., 2011b). The increased p53 expression in chronically activated neurons reflects this feature of immature neurons.
The identification of STAT3 phospho-serine modification as a critical link between neuronal activity and survival raises many further questions. Amongst the most interesting is how STAT3 activation is quantitatively linked to the appropriate level of activity. This mechanism is of potential clinical interest as an altered balance of excitation/inhibition (E/I) is thought to contribute to many diseases (Heinemann, 2004; Belforte et al., 2009; Rubenstein, 2010; Yizhar et al., 2011). In these pathological conditions, neurons are subject to chronically elevated activity. Although neurons can use compensatory synaptic scaling mechanisms to adjust their net input (Davis, 2006; Turrigiano, 2007), our results suggest neurons can undergo dramatic changes in vulnerability that may contribute to the degeneration of dendritic morphology and neuronal death observed in neurological diseases characterized by altered E/I balance (Paul et al., 1981; Garey et al., 1998; Boda et al., 2010). As STAT3 has been reported to modulate the expression of synaptic proteins (Lund et al., 2008), it would be interesting to determine whether STAT3 also plays a role in homeostatic plasticity.
Acknowledgments
We thank Dr. Yukiko Gotoh for wild-type and mutant STAT3 IRES EGFP/pMX plasmids. This research was supported by the Intramural Research Program of the NIH, NINDS.
Footnotes
No conflict of interest.
  • Ballas N, Battaglioli E, Atouf F, Andres ME, Chenoweth J, Anderson ME, Burger C, Moniwa M, Davie JR, Bowers WJ, Federoff HJ, Rose DW, Rosenfeld MG, Brehm P, Mandel G. Regulation of neuronal traits by a novel transcriptional complex. Neuron. 2001;31:353–365. [PubMed]
  • Beattie EC, Stellwagen D, Morishita W, Bresnahan JC, Ha BK, Von Zastrow M, Beattie MS, Malenka RC. Control of synaptic strength by glial TNFalpha. Science. 2002;295:2282–2285. [PubMed]
  • Belforte JE, Zsiros V, Sklar ER, Jiang Z, Yu G, Li Y, Quinlan EM, Nakazawa K. Postnatal NMDA receptor ablation in corticolimbic interneurons confers schizophrenia-like phenotypes. Nat Neurosci. 2009;13:76–83. [PMC free article] [PubMed]
  • Berg MM, Sternberg DW, Parada LF, Chao MV. K-252a inhibits nerve growth factor-induced trk proto-oncogene tyrosine phosphorylation and kinase activity. J Biol Chem. 1992;267:13–16. [PubMed]
  • Boda B, Dubos A, Muller D. Signaling mechanisms regulating synapse formation and function in mental retardation. Curr Opin Neurobiol. 2010;20:519–527. [PubMed]
  • Brown EJ, Albers MW, Shin TB, Ichikawa K, Keith CT, Lane WS, Schreiber SL. A mammalianprotein targeted by G1-arresting rapamycin-receptor complex. Nature. 1994;369:756–758. [PubMed]
  • Ceresa BP, Pessin JE. Insulin stimulates the serine phosphorylation of the signal transducer and activator of transcription (STAT3) isoform. J Biol Chem. 1996;271:12121–12124. [PubMed]
  • Chung J, Uchida E, Grammer TC, Blenis J. STAT3 serine phosphorylation by ERK-dependent and -independent pathways negatively modulates its tyrosine phosphorylation. Mol Cell Biol. 1997;17:6508–6516. [PMC free article] [PubMed]
  • Cingolani LA, Thalhammer A, Yu LM, Catalano M, Ramos T, Colicos MA, Goda Y. Activity-dependent regulation of synaptic AMPA receptor composition and abundance by beta3 integrins. Neuron. 2008;58:749–762. [PMC free article] [PubMed]
  • Cohen JE, Lee PR, Chen S, Li W, Fields RD. MicroRNA regulation of homeostatic synaptic plasticity. ProcNatl Acad Sci U S A. 2011;108:11650–11655. [PubMed]
  • Davis GW. Homeostatic control of neural activity: from phenomenology to molecular design. Annu Rev Neurosci. 2006;29:307–323. [PubMed]
  • Decker T, Kovarik P. Serine phosphorylation of STATs. Oncogene. 2000;19:2628–2637. [PubMed]
  • Deckwerth TL, Elliott JL, Knudson CM, Johnson EM, Jr, Snider WD, Korsmeyer SJ. BAX is required for neuronal death after trophic factor deprivation and during development. Neuron. 1996;17:401–411. [PubMed]
  • Deverman BE, Patterson PH. Cytokines and CNS development. Neuron. 2009;64:61–78. [PubMed]
  • Du K, Montminy M. CREB is a regulatory target for the protein kinase Akt/PKB. J Biol Chem. 1998;273:32377–32379. [PubMed]
  • Dziennis S, Jia T, Ronnekleiv OK, Hurn PD, Alkayed NJ. Role of signal transducer and activator of transcription-3 in estradiol-mediated neuroprotection. J Neurosci. 2007;27:7268–7274. [PMC free article] [PubMed]
  • Easton RM, Deckwerth TL, Parsadanian AS, Johnson EM., Jr Analysis of the mechanism of loss of trophic factor dependence associated with neuronal maturation: a phenotype indistinguishable from Baxdeletion. J Neurosci. 1997;17:9656–9666. [PubMed]
  • Fernandes-Alnemri T, Litwack G, Alnemri ES. CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 beta-converting enzyme. J Biol Chem. 1994;269:30761–30764. [PubMed]
  • Finkbeiner S, Tavazoie SF, Maloratsky A, Jacobs KM, Harris KM, Greenberg ME. CREB: a major mediator of neuronal neurotrophin responses. Neuron. 1997;19:1031–1047. [PubMed]
  • Fu AK, Fu WY, Ng AK, Chien WW, Ng YP, Wang JH, Ip NY. Cyclin-dependent kinase 5 phosphorylates signal transducer and activator of transcription 3 and regulates its transcriptional activity. Proc Natl Acad Sci U S A. 2004;101:6728–6733. [PubMed]
  • Garey LJ, Ong WY, Patel TS, Kanani M, Davis A, Mortimer AM, Barnes TR, Hirsch SR. Reduced dendritic spine density on cerebral cortical pyramidal neurons in schizophrenia. J Neurol Neurosurg Psychiatry. 1998;65:446–453. [PMC free article] [PubMed]
  • Heinemann U. Basic mechanisms of partial epilepsies. Curr Opin Neurol. 2004;17:155–159. [PubMed]
  • Huang EJ, Reichardt LF. Trk receptors: roles in neuronal signal transduction. Annu Rev Biochem. 2003;72:609–642. [PubMed]
  • Izant JG, McIntosh JR. Microtubule-associated proteins: a monoclonal antibody to MAP2 binds to differentiated neurons. Proc Natl Acad Sci U S A. 1980;77:4741–4745. [PubMed]
  • Jacinto E, Facchinetti V, Liu D, Soto N, Wei S, Jung SY, Huang Q, Qin J, Su B. SIN1/MIP1 maintains rictor-mTOR complex integrity and regulates Akt phosphorylation and substrate specificity. Cell. 2006;127:125–137. [PubMed]
  • Jain N, Zhang T, Fong SL, Lim CP, Cao X. Repression of Stat3 activity by activation of mitogen-activated protein kinase (MAPK) Oncogene. 1998;17:3157–3167. [PubMed]
  • Jain N, Zhang T, Kee WH, Li W, Cao X. Protein kinase C delta associates with and phosphorylates Stat3 in an interleukin-6-dependent manner. J Biol Chem. 1999;274:24392–24400. [PubMed]
  • Jung JE, Kim GS, Narasimhan P, Song YS, Chan PH. Regulation of Mn-superoxide dismutase activity and neuroprotection by STAT3 in mice after cerebral ischemia. J Neurosci. 2009;29:7003–7014. [PMC free article] [PubMed]
  • Kim J, Jung SC, Clemens AM, Petralia RS, Hoffman DA. Regulation of dendritic excitability by activity-dependent trafficking of the A-type K+ channel subunit Kv4.2 in hippocampal neurons. Neuron. 2007;54:933–947. [PMC free article] [PubMed]
  • Kohn AD, Takeuchi F, Roth RA. Akt, a pleckstrin homology domain containing kinase, is activated primarily by phosphorylation. J Biol Chem. 1996;271:21920–21926. [PubMed]
  • Levy DE, Darnell JE., Jr Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol. 2002;3:651–662. [PubMed]
  • Lim CP, Cao X. Serine phosphorylation and negative regulation of Stat3 by JNK. J Biol Chem. 1999;274:31055–31061. [PubMed]
  • Lim CP, Cao X. Regulation of Stat3 activation by MEK kinase 1. J Biol Chem. 2001;276:21004–21011. [PubMed]
  • Liu X, Kim CN, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell. 1996;86:147–157. [PubMed]
  • Lund IV, Hu Y, Raol YH, Benham RS, Faris R, Russek SJ, Brooks-Kayal AR. BDNF selectively regulates GABAA receptor transcription by activation of the JAK/STAT pathway. Sci Signal. 2008;1:ra9. [PMC free article] [PubMed]
  • Meijer L, Borgne A, Mulner O, Chong JP, Blow JJ, Inagaki N, Inagaki M, Delcros JG, Moulinoux JP. Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur J Biochem. 1997;243:527–536. [PubMed]
  • Murase S, McKay RD. A specific survival response in dopamine neurons at most risk in Parkinson’s disease. J Neurosci. 2006;26:9750–9760. [PubMed]
  • Murase S, Owens DF, McKay RD. In the newborn hippocampus, neurotrophin-dependent survival requires spontaneous activity and integrin signaling. J Neurosci. 2011a;31:7791–7800. [PMC free article] [PubMed]
  • Murase S, Poser SW, Joseph J, McKay RD. p53 controls neuronal death in the CA3 region of the newborn mouse hippocampus. Eur J Neurosci. 2011b;34:374–381. [PubMed]
  • Ng YP, Cheung ZH, Ip NY. STAT3 as a downstream mediator ofTrk signaling and functions. J Biol Chem. 2006;281:15636–15644. [PubMed]
  • Niu G, Wright KL, Ma Y, Wright GM, Huang M, Irby R, Briggs J, Karras J, Cress WD, Pardoll D, Jove R, Chen J, Yu H. Role of Stat3 in regulating p53 expression and function. Mol Cell Biol. 2005;25:7432–7440. [PMC free article] [PubMed]
  • Oh H, Fujio Y, Kunisada K, Hirota H, Matsui H, Kishimoto T, Yamauchi-Takihara K. Activation of phosphatidylinositol 3-kinase through glycoprotein 130 induces protein kinase B and p70 S6 kinase phosphorylation in cardiac myocytes. J Biol Chem. 1998;273:9703–9710. [PubMed]
  • Paul LA, Fried I, Watanabe K, Forsythe AB, Scheibel AB. Structural correlates of seizure behavior in the mongolian gerbil. Science. 1981;213:924–926. [PubMed]
  • Rubenstein JL. Three hypotheses for developmental defects that may underlie some forms of autism spectrum disorder. Curr Opin Neurol. 2010;23:118–123. [PubMed]
  • Schweizer U, Gunnersen J, Karch C, Wiese S, Holtmann B, Takeda K, Akira S, Sendtner M. Conditional gene ablation of Stat3 reveals differential signaling requirements for survival of motoneurons during development and after nerve injury in the adult. J Cell Biol. 2002;156:287–297. [PMC free article] [PubMed]
  • Seeburg DP, Feliu-Mojer M, Gaiottino J, Pak DT, Sheng M. Critical role of CDK5 and Polo-like kinase 2 in homeostatic synaptic plasticity during elevated activity. Neuron. 2008;58:571–583. [PMC free article] [PubMed]
  • Sheng M, Tsaur ML, Jan YN, Jan LY. Subcellular segregation of two A-type K+ channel proteins in rat central neurons. Neuron. 1992;9:271–284. [PubMed]
  • Shepherd JD, Rumbaugh G, Wu J, Chowdhury S, Plath N, Kuhl D, Huganir RL, Worley PF. Arc/Arg3.1 mediates homeostatic synaptic scaling of AMPA receptors. Neuron. 2006;52:475–484. [PMC free article] [PubMed]
  • Tan Y, Rouse J, Zhang A, Cariati S, Cohen P, Comb MJ. FGF and stress regulate CREB and ATF-1 via a pathway involving p38 MAP kinase and MAPKAP kinase-2. Embo J. 1996;15:4629–4642. [PubMed]
  • Toker A, Newton AC. Cellular signaling: pivoting around PDK-1. Cell. 2000;103:185–188. [PubMed]
  • Turrigiano G. Homeostatic signaling: the positive side of negative feedback. Curr Opin Neurobiol. 2007;17:318–324. [PubMed]
  • Wen Z, Zhong Z, Darnell JE., Jr Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell. 1995;82:241–250. [PubMed]
  • Yizhar O, Fenno LE, Prigge M, Schneider F, Davidson TJ, O’Shea DJ, Sohal VS, Goshen I, Finkelstein J, Paz JT, Stehfest K, Fudim R, Ramakrishnan C, Huguenard JR, Hegemann P, Deisseroth K. Neocortical excitation/inhibition balance in information processing and social dysfunction. Nature. 2011;477:171–178. [PubMed]
  • Zagotta WN, Brainard MS, Aldrich RW. Single-channel analysis of four distinct classes of potassium channels in Drosophila muscle. J Neurosci. 1988;8:4765–4779. [PubMed]