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Keywords:

  • cytokine;
  • hypothalamus;
  • inflammation;
  • interleukin;
  • neuron;
  • tyrosine kinase

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The proinflammatory cytokine interleukin1β (IL-1β), acting at IL-1R1 receptors, affects neuronal signaling under both physiological and pathophysiological conditions. The molecular mechanism of the rapid synaptic actions of IL-1β in neurons is not known. We show here that within minutes of IL-1β exposure, the firing rate of anterior hypothalamic (AH) neurons in culture was inhibited. This effect was prevented by pre-exposure of the cells to the Src family inhibitor, PP2, suggesting the involvement of Src in the hyperpolarizing effects of IL-1β. The IL-1β stimulation of neurons induced a rapid increase in the phosphorylation of the tyrosine kinase Src and kinase suppressor of Ras (ceramide activated protein kinase (CAPK)/KSR) in neurons grown on glia from IL-1RI(–/–) mice. These effects of IL-1β were dependent on the association of the cytosolic adaptor protein, MyD88, to the IL-1 receptor, and on the activation of the neutral sphingomyelinase, leading to production of ceramide. A cell-permeable analog of ceramide mimicked the effects of IL-1β on the cultured AH neurons. These results suggest that ceramide may be the second messenger of the fast IL-1β actions in AH neurons, and that this IL-1β/ceramide pathway may underlie the fast non-transcription-dependent, electrophysiological effects of IL-1β observed in AH neurons in vivo.

Abbreviations used
AH

anterior hypothalamic

COX2

cyclooxygenase 2

FAN

factor associated with N-SMase

IL-1β

interleukin-1β

IL-6

interleukin-6

IL-1RI

interleukin-1 type-I receptor

IL-1RacP

interleukin-1 type-I receptor-associated protein

IRAK

interleukin-1 receptor-associated kinase

KSR

kinase suppressor of Ras

LTP

long-term potentiation

MAPK

mitogen-activated protein kinase

MyD88

myeloid differentiation primary-response protein 88

NF-κB

nuclear factor kappa B

N-Smase

neutral sphingomyelinase

PBS

phosphate-buffered saline

PGE2

prostaglandin E2

PI3-kinase

phosphoinositide-3-kinase

POA

preoptic area

TIR

Toll/Interleukin-1 receptor

Interleukin-1β (IL-1β) is synthesized in many cells of the peripheral and central nervous system (Schultzberg et al. 1987; Dinarello and Bunn 1997). Healthy neurons in the hippocampus, pituitary and hypothalamus synthesize IL-1β and express the interleukin-1 type-I receptor (IL-1RI) and the receptor-associated protein (IL-1RAcP) as a heterodimeric receptor complex (Bristulf et al. 1991; Boutin et al. 2003). The pro-inflammatory actions of IL-1β at the IL-1R/IL-1RAcP receptor complex in the brain, occurring during trauma, neuroinflammation and neurodegeneration, are slow in onset (45 min to 3 h) and chronic in duration, involving the transcription-dependent synthesis of inflammatory mediators such as prostaglandin E2 (PGE2) and nitric oxide (NO) (Dinarello and Bunn 1997; Lucas et al. 2006). The time for the transcription-dependent formation of these mediators is too long to explain the rapid IL-1-mediated affects that we and others have observed in neurons.

The pyrogenic effects of IL-1β in hypothalamic neurons can be observed within minutes of cytokine exposure, and do not require cyclooxygenase 2 (COX2) induction and PGE2 synthesis and release (Kakucska et al. 1993), while the sustained phase of fever is dependent on PGE2 action (Dinarello 2004). Direct application of IL-1β was shown to produce a rapid reduction of the firing rate of hypothalamic neurons in vivo (Hori et al. 1988a) and in vitro (Vasilenko et al. 2000). Rapid neuronal actions of IL-1β were also observed in the hippocampus, the locus coeruleus and the dorsal raphe nucleus (DRN) (Takao et al. 1990; Borsody and Weiss 2002; Manfridi et al. 2003). IL-1β can suppress long-term potentiation (LTP) induction, and rapid effects of IL-1β on synaptic transmission were observed in hippocampal slices (Bellinger et al. 1993; Schneider et al. 1998; Vereker et al. 2000). In the DRN, IL-1β exposure induced a rapid inhibition of serotoninergic neurons that led to increased non-rapid eye movement (REM) sleep (De Sarro et al. 1997; Manfridi et al. 2003).

These electrophysiological observations do not provide a molecular mechanism that explains how the binding of IL-1β to the IL-1RI/RAcP receptor complex leads to rapid changes in the activity of ion channels, membrane potential and neuronal activation. One possible mechanism for the fast actions of IL-1β is the activation of signaling kinases, such as the protein kinase, Src, that rapidly phosphorylate ion channels (see Salter and Kalia 2004 for review). Furthermore, IL-1β exposure was shown to induce the activation of Src and enhance NMDA receptor function in hippocampal neurons (Viviani et al. 2003).

IL-1β was previously shown to induce the activation of neutral sphingomyelinase (N-SMase), resulting in a rapid ceramide production in non-neuronal cells (Kolesnick and Golde 1994) and in mouse neuronal synaptosomes from wild-type, but not from IL-1R1(–/–), mice (Nalivaeva et al. 2000). Zumbansen and Stoffel (2002) have further shown that neutral sphingomyelinase 2 (N-SMase2) is responsible for most of the production of ceramide in the brains of mice. Therefore, when we refer to N-SMase activity in these centrally derived neurons, we are referring to the activity of N-SMase2.

Here, we examined the possibility that IL-1β-mediated activation of neuronal N-SMase and subsequent ceramide production is the second messenger system activated by IL-1β that leads to the rapid activation of Src and to the subsequent effect on neuronal activity in anterior hypothalamic (AH) neurons. Ceramide formation has been shown in non-neuronal cell types to activate the kinase suppressor of Ras (KSR) and to be mimicked by the cell-penetrating analog C2-ceramide, but not by the non-penetrating dihydroceramide (Kolesnick and Golde 1994). We have used these readouts of N-SMase activation and ceramide production together with an inhibitor of N-SMase (Arenz and Giannis 2000). We have previously shown that hypothalamic neurons in culture, grown on glia derived from wild-type and from IL-1R1(–/–) mice, exhibit the same morphological and electrophysiological properties as neurons in tissue slices from the anterior hypothalamus (Tabarean et al. 2005). Therefore, these cultures serve as useful tools for the study of the second messenger mechanisms mediating the rapid neuronal effects of IL-1β.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Anterior hypothalamic cultures

Wild-type mixed AH cultures (containing neurons and glia) and mosaic cultures were prepared from anterior hypothalami from Swiss Webster (Taconic, Hudson, NY, USA) mice. Dissociated anterior hypothalami from fetal mice at 13–14 days of gestation were plated at a density of 1–2 anterior hypothalami/mL onto either poly d-lysine-coated coverslips (for confocal microscopy), or onto pre-established astrocyte monolayers obtained essentially as described (Rose et al. 1993) from 1–3-day-old wild-type or IL-1RI(–/–) pups when preparing mosaic cultures. The IL-1RI(–/–) mouse strain was a kind gift from Dr Mark Labow, Hoffman La Roche. Cultures were kept in Eagle's minimal essential medium (MS: with the addition of 20 mm glucose and 26.2 mm NaHCO3) supplemented with 5% fetal bovine serum, 5% horse serum and 2 mm glutamine. To prevent non-neuronal cell proliferation, 10 µm cytosine-arabinoside was added to mixed AH cultures 5–7 days after plating, or to mosaic cultures 24 h after plating. Cultures were then fed every 4 days with MS + glutamine containing 10% horse serum. Cultures were maintained in a 37°C humidified incubator in a 5% CO2 atmosphere and used at 30–45 days in vitro (DIV) for biochemical determinations and immunocytochemistry.

IL-1β exposures, immunoprecipitation and western blots

Plates containing neurons and glia were washed in serum-free medium (HCSS) as described (Heidinger et al. 2002) and equilibrated in this medium for 3 h at 37°C to allow recovery. Murine recombinant IL-1β (expressed in Escherichia coli) was purchased from R & D systems (Minneapolis, MN, USA). The bioactivity of murine recombinant IL-1β has been shown to be 5–10 pg/mL as measured by its effects on murine T-cell proliferation (Symons et al. 1987). IL-1β (10–12 nm) or C2-ceramide (5–10 µm) was applied, for the times indicated, in the absence or presence of the myeloid differentiation primary-response protein 88 (MyD88) mimic, AS-1 (50–100 µm) (Bartfai et al. 2003), the Src family inhibitor, PP2 (2 µm; Calbiochem, San Diego, CA, USA), or the N-SMase inhibitor, spiroexpoxide (10 µm; Calbiochem) (Arenz and Giannis 2000), or IL-1β (1.2 nm) in the presence of the IL-1Ra Kineret (0.5–100 nm; Amgen, Thousand Oaks, CA, USA), after which cells were washed and proteins extracted. Whole-cell extracts were separated in 8% (immunoprecipitations) or 10% (phospho-Src westerns) sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels and transferred to nitrocellulose membranes as described (Heidinger et al. 2002). For the determination of Src-Tyr416 phosphorylation, membranes were incubated in a 1 : 1000 dilution of anti-phospho-Src antibody (Biosource, Camarillo, CA, USA and/or Cell Signaling Technology, Beverly, MA, USA) for 2–4 h at room temperature (25°C) or overnight at 4°C. Blots were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Vector Laboratories, Burlingame, CA, USA) followed by chemiluminiscense (Pierce, Rockford, IL, USA). Blots were then stripped of antibodies and total Src protein was determined with the use of the pan-Src monoclonal antibody (Upstate Biotechnology, Lake Placid, NY, USA) at 1 : 1000 dilution for 2 h at room temperature (25°C). The ratios of phospho-Src/Src were obtained by densitometric analysis of the bands in the films and expressed as percentage of control (no treatment). Phosphorylation of KSR was determined by immunoprecipitation of KSR from 500–800 µg whole-cell extracts using 2 µg rat monoclonal anti-KSR antibody, clone C3H7D2 (Sigma, St. Louis, MO, USA). Lysates were pre-cleared with protein G plus agarose (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) for 2–3 h, and incubation with antibody was performed overnight, with rotation, at 4°C. Protein–antibody complexes were incubated for 1 h with protein G plus agarose and washed three times with lysis buffer. Immunoprecipitates were resolved in 8% SDS–PAGE gels and transferred as before. For serine phosphorylation detection, membranes were blocked in milk-free buffer (Vector Laboratories) and incubated with antibodies against phospho-serine (1 : 1000 dilution). Following detection, the membranes were stripped and re-incubated in anti-KSR (rabbit polyclonal 1 µg/mL, Santa Cruz Biotechnologies). The ratios of phospho-KSR/KSR were obtained as for Src and expressed similarly as percentage of control (no treatment).

Immunocytochemistry

Coverslips were washed in phosphate-buffered saline (PBS), fixed in ice-cold 4% paraformaldehyde for 30 min, and then incubated for 10 min at room temperature (25°C) in PBS containing 0.25% Triton X-100. Non-specific sites were blocked by incubation in PBS containing 10% normal goat/horse serum. For double immunostaining, the coverslips were incubated in 2% normal goat serum containing antibody against IL-1RI (4 µg/mL, rat polyclonal; R & D Systems), antibodies against glial fibrillary acidic protein (GFAP) (rabbit polyclonal, 1 : 750 dilution; Cell Signaling) or antibodies against MAP-2 (mouse monoclonal, 1 : 750 dilution; Calbiochem), for 2 h at 37°C. Specific binding was detected using secondary antibodies conjugated to AlexaFluor dyes (594, red; 488, green: Molecular Probes, Eugene, OR, USA). Images were collected on a Delta Vision Optical Sectioning Microscope (Applied Precision, Issaquah, WA, USA) consisting of an Olympus IX-70 microscope (Olympus, Melville, NY, USA) equipped with a mercury arc lamp. A photometrics CH 350 cooled charge coupled device (CCD) camera (Roper Scientific, Tuscon, AZ, USA) and a high precision motorized XYZ stage were used to acquire multiple consecutive optical sections at a 0.2 µm interval for each of the fluorescent probes using a 60× oil objective. Three independent experiments were performed on different sets of DIV 30 AH neurons.

Electrophysiology

Standard tight seal recordings in current-clamp mode (I-clamp fast) were performed with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA). The external recording solution was (in mm): 155 NaCl, 3.5 KCl, 2 CaCl2, 1.5 MgSO4, 10 glucose, 10 HEPES (pH 7.4). The osmolarity was 300–305 mOsm. Indomethacin (10 µm) was added to all the extracellular solutions in order to block COX1 and COX2, and to prevent prostaglandin E2 production. The pipette solution was (in mm): 130 K-gluconate, 10 KCl, 10 HEPES, 2 MgCl2, 0.5 EGTA, 2 ATP, 1 GTP (pH 7.4). Glass micropipettes were pulled with a horizontal puller (P-87; Sutter Instruments, Novato, CA, USA) using borosilicate glass. The electrode resistance after back-filling was 2–6 MΩ. All voltage measurements were corrected for the liquid junction potential (approximately 12 mV). The recording chamber was perfused at 3 mL/min, and its content was fully changed within 1 min. The temperature of the external solution was controlled with an HCC-100A heating/cooling bath temperature controller (Dagan Corporation, Minneapolis, MN, USA). The temperature during the recordings was usually 36–37°C. To prevent changes induced in the electrode reference potential, the ground electrode was thermally isolated in a separate bath connected to the recording bath by a filter paper bridge.

Data acquisition and analysis

Recordings were digitized using a Digidata 1320A interface and the Pclamp8 (Molecular Devices) software package. The mean firing rate was determined for stretches of 2–4 min of recording (number of events/duration) before and after treatment. Statistical calculations were performed with InStat (GraphPad Software, San Diego, CA, USA). For spontaneously-firing neurons, an apparent resting membrane potential was determined by averaging the voltage with spikes removed over a period of 300 ms (as described in Tabarean et al. 2004, 2005). Data are presented as means ± SD.

Results

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

IL-1β exposure has been shown to decrease the firing rate of AH neurons both in vivo and in vitro (Hori et al. 1988b; Vasilenko et al. 2000). Here, we tested whether these effects are also observed in AH neurons developed in culture. Neurons in this culture system develop to maturity in a similar way to neurons in hypothalamic slices, as we have recently characterized using morphological and electrophysiological techniques (Tabarean et al. 2005). IL-1β (300 pm) hyperpolarized six out of 25 AH neurons studied. The effect developed with a delay of approximately 1 min (Fig. 1a), reached a plateau within 3 min and persisted for 2–4 min after removal of the cytokine. The resting membrane potential was − 56.3 ± 5.2 mV (n = 6) in the control and − 62.5 ± 6.3 mV (n = 6) after IL-1β (300 pm) application, i.e. a hyperpolarization of 6.2 ± 1.4 mV (n = 6). This hyperpolarization was associated with a reduction in the firing rate from 5.9 ± 2.2 (n = 6) in the control to 1.2 ± 1.2 (n = 6) after IL-1β. At 100 pm, the effects of IL-1β are similar, i.e. IL-1 hyperpolarizes preoptic area (POA) neurons by 4.1 ± 2.9 mV (n = 6). At lower concentrations (10 pm or less), no effect was observed (data not shown). We examined whether the IL-1β-dependent activation of the protein tyrosine kinase, Src, was involved in these rapid effects on the AH neurons. Figure 1(b) indicates that neurons pre-treated with the Src family inhibitor, PP2 (1 µm), for 30 min did not respond to subsequent treatment with 300 pm IL-1β (n = 19). These results suggest that Src-kinase activation is required for the hyperpolarizing effect of IL-1β in AH neurons in culture.

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Figure 1.  IL-1β hyperpolarizes and inhibits the firing of anterior hypothalamic neurons in culture. This effect is prevented by the Src kinase inhibitor, PP2. (a) IL-1β hyperpolarizes and inhibits the firing of a subpopulation of AH neurons. Current-clamp recording of the response of an AH neuron to a 90 s application of IL-1β (300 pm). (b) The neuron hyperpolarized by about 6 mV and spontaneous firing was abolished. Incubation with 1 µm PP2 blocks the hyperpolarizing effect of 300 pm IL-1β.

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IL-1RI receptors are expressed in both neurons and glia in the anterior hypothalamus and in AH cultures (Figs 2a and b). To characterize biochemically the neuronal effects of IL-RI activation in isolation, we cultured the AH neurons on top of a pre-formed glial bed obtained from IL-1RI(–/–) mice. The entire population of IL-1RI-expressing cells is purely neuronal and responses to IL-1β are in the neurons alone. Neurons in this mosaic culture system continued to express the neuronal marker, microtubule associated protein 2 (MAP-2), and IL-1RI, but no expression of these receptors was found in the otherwise GFAP-positive underlying glial bed (Figs 2c and d). Electrophysiological studies showed that IL-1RI-mediated responses were indistinguishable from neurons in a wild-type culture (data not shown). The mosaic cultures enabled biochemical characterization of the neuronal effects of IL-RI/MyD88 interactions in isolation from IL-1β-induced events in glia.

image

Figure 2.  Expression of IL-1RI in wild-type and mosaic anterior hypothalamic cultures. Wild-type neurons dissected from E13 mouse embryos were seeded onto either a wild-type glial monolayer (a, b) or a IL-1RI(–/–) glial monolayer (c, d) and allowed to develop for 30–45 days in vitro. At this time point, the coverslips containing the co-cultures were fixed and processed for immunocytochemistry for the detection of IL-1RI (green fluorescence in a–d), GFAP (red fluorescence in a and c) and MAP-2 (red fluorescence in b and d).

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We used this mosaic culture system to analyze the IL-1β-mediated changes in the phosphorylation state of Src by Western blot, using phospho-specific antibodies developed against the activated form of Src phosphorylated at Tyr416. Exposure to IL-1β induced a rapid concentration and time-dependent phosphorylation of Src in AH neurons (Figs 3a and b). To determine the specificity of the actions of IL-1β on cultured AH neurons, we tested the ability of the cytokine to induce the phosphorylation of Src in the presence of the IL-1 receptor antagonist (IL-1Ra; Kineret, Amgen, Thousand Oaks, CA, USA). The IL-1β-induced activation of Src was blocked by the addition of 50 or 100 nm IL-1Ra in the AH neurons (Fig. 3c). To examine whether IL-1β-mediated Src activation requires the recruitment of the cytosolic adaptor protein, MyD88 (involved in mediating signaling from multiple Toll receptors including the agonist occupied IL-1RI), we used the Toll/Interleukin-1 receptor (TIR)-domain mimic, AS-1, which was previously shown by us to disrupt IL-1RI-mediated signaling leading to mitogen-activated protein kinase (MAPK) activation in vitro and in vivo (Bartfai et al. 2003). In addition, we examined the possibility that IL-1β-mediated activation of N-SMase and subsequent ceramide production is the rapid second messenger system activated by IL-1β that leads to the rapid activation of Src.

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Figure 3.  IL-1β induces a dose- and time-dependent activation of the protein kinase Src. Anterior hypothalamic cultures were exposed to (a) varying concentrations of IL-1β or (b) IL-1β (10 nm) for the indicated periods of time. Protein extracts were separated on SDS–PAGE gels, transferred, and processed for western blot determination of the phosphorylation state of Tyr416 of Src, using a phospho-specific antibody, and re-detected with an antibody against Src. The ratios of pSrc-Tyr416/Src are expressed as percentage of control conditions (untreated in a and t0 in b). Values are means ± SEM for three independent experiments. *Statistically significant as compared with control conditions at p < 0.05 by anova. (c) Increasing concentrations of the IL-1 receptor antagonist (IL-1Ra) Kineret (Amgen) block IL-1β (1.2 nm, 10 min)-induced activation of Src. Representative western blot is shown from two independent experiments.

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We have taken advantage of a specific inhibitor of N-SMase, described by Arenz and Giannis (2000), which was shown to inhibit enzyme activity specifically and selectively by more than 85% within 30 min. Sphingomyelin is converted to ceramide by N-SMase, and inhibition of this enzyme blocks the production of ceramide in the cell. Figure 4 indicates that the enhanced Src phosphorylation was prevented by pre-incubation of the mosaic cultures with spiroepoxide (10 µm, applied 30 min prior to IL-1β exposure) and the MyD88 mimic, AS-1 (100 µm, 15 min pre-exposure). As expected, pre-incubation with the specific Src family inhibitor, PP2, blocked the increase in Src phosphorylation (2 µm, 15 min pre-exposure). In addition, PP2 reduced Src phosphorylation significantly below basal levels of activation. The Src family inhibitor, PP2, was toxic to the neurons in culture at concentrations higher than 10 µm. We have chosen a concentration of the inhibitor that is non-toxic and still provides inhibition of Src phosphorylation. The use of PP2 as an inhibitor of the Src family of kinases does not allow us to distinguish between the effects of specific Src family members, such as Fyn and Lck, in the IL-1-mediated hyperpolarization of AH/POA neurons.

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Figure 4.  IL-1β-mediated activation of the protein kinase Src depends on MyD88/IL-1RI interaction and N-SMase activation. (a) Anterior hypothalamic cultures were treated with IL-1β (10 nm) for 10 min following at least a 15 min pre-incubation of the MyD88 mimic AS-1 (100 µm) or a 30 min pre-incubation of the N-SMase inhibitor spiroepoxide (10 µm), and processed for western blot determination of the phosphorylation state of Tyr416-Src as in Fig. 3. Values are means ± SEM for seven independent experiments. *Statistically significant as compared with control (no treatment) conditions at p < 0.05 by anova; **statistically significant as compared with IL-1β treatment conditions at p < 0.05 by anova. (b) Pre-treatment with the MyD88/TIR domain mimic, hydrocinnamoyl-l-valyl pyrrolidine (AS-1), 100 µm, for about 10 min completely blocked the effects of IL-1β (n = 10).

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To confirm the effects of AS-1 blocking Src phosphorylation, we tested the ability of IL-1β to activate Src in MyD88(–/–) AH neurons. These MyD88 knockout neurons were developed on a bed of IL-1RI(–/–) glia and stimulated with IL-1β. Similar to the results obtained with the TIR mimic, AS-1, on neurons from wild-type mice, no stimulation of Src was detected in the MyD88(–/–) AH neurons (Davis et al. 2006).

In addition to blocking the phosphorylation of Src, AS-1 and spiroepoxide block IL-1β-induced hyperpolarization of POA neurons in culture. Pre-treatment with the MyD88/TIR domain mimic, AS-1 (100 µm for approximately 10 min) completely blocked the hyperpolarizing effects of IL-1β (300 pm) (n = 10) (Fig. 4b). Similar to the results with AS-1, incubation with the N-SMase inhibitor (50 µm for 30 min) prevented the hyperpolarization of POA neurons by IL-1β (300 pm) in all neurons tested (n = 15) (data not shown).

The activation of CAPK/KSR following IL-1β stimulation serves as a rapid and sensitive readout of ceramide production, as has been shown in non-excitable cells (Mathias et al. 1993; Kolesnick and Golde 1994). Exposure of AH neurons to IL-1β induced a rapid (within 10 min) serine phosphorylation of the ceramide-activated adaptor protein, CAPK/KSR (Fig. 5). Similar to the observation regarding Src phosphorylation, the IL-1β-mediated increase in CAPK/KSR phosphorylation was also prevented by pre-exposure of the cultures to the MyD88 mimic, AS-1, and the N-SMase inhibitor, spiroepoxide. These data suggest that Src and CAPK/KSR phosphorylation occurs downstream of MyD88 binding to IL-1RI, and that it requires N-SMase activation and thus, ceramide production.

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Figure 5.  IL-1β-mediated phosphorylation of CAPK/KSR depends on MyD88/IL-1RI interaction and N-SMase activation in anterior hypothalamic neurons. DIV 30-45 cultures were exposed to IL-1β (10 nm) for 10 min following at least a 15 min pre-incubation of the MyD88 mimic AS-1 (100 µm), or a 30 min pre-incubation of the N-SMase inhibitor, spiroepoxide (10 µm), and processed for immunoprecipitation using an antibody directed against CAPK/KSR. Immunoprecipitates were then separated on SDS–PAGE gels and transferred for western blot detection of serine phosphorylation. The ratios of pSer/KSR are expressed as percentage of control conditions (no treatment). Values are means ± SEM for five experiments per condition. *Statistically significant as compared with control condition at p < 0.05 by anova; **statistically significant as compared with IL-1β treatment conditions at p < 0.05 by anova.

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Src activation is involved in the rapid hyperpolarizing effects of IL-1β. We next examined the cell-permeable analog of ceramide, C2-ceramide, for its ability to induce Src phosphorylation in the AH cultures. Similar to the effects of IL-1β exposure, C2-ceramide induced a concentration- and time-dependent phosphorylation of Src (Figs 6a and b). To determine whether ceramide can reproduce IL-1β electrophysiological affects on AH neurons, C2-ceramide (10 µm) was applied to the bath in a way similar to IL-1β in Fig. 1. C2-ceramide hyperpolarized 32% of the neurons tested (eight out of 25) (Fig. 6c). The hyperpolarization averaged 9.5 ± 5.2 mV (mean ± SD, n = 8) and silenced the neurons that initially fired action potentials. The responses developed gradually, similar to the responses to IL-1β. However, the recovery during wash-out was slower (> 5 min) and the effect was only partially reversible in all experiments (n = 8). Taking advantage of the slow recovery after C2-ceramide treatment, we tested whether the effects of IL-1β (300 pm) were additive by applying it immediately after ceramide. In three AH neurons that were hyperpolarized by C2-ceramide (10 µm), IL-1β (300 pm) did not have further effect (data not shown). Also, in 10 other AH neurons that did not respond to C2-ceramide, IL-1β was also without effect. We tested the ability of a membrane-impermeable analog of ceramide (dihydroceramide) to reproduce the effects of the C2-ceramide. Dihydroceramide was unable to stimulate the phosphorylation of Src or hyperpolarize the cells in AH neuronal cultures within 60 min of incubation (data not shown). Ceramide can be rapidly converted to ceramide-1P or sphingosine-1P, both emerging as important signaling molecules (Chalfant and Spiegel 2005). Therefore, we investigated whether they could mimic the effects of IL-1 stimulation and C2-ceramide exposure, but found no electrophysiological effects in the short 0–30 min time frame studied (supplementary data). We thus believe that most of the rapid neuronal effects of IL-1 are accounted for by the fast production of ceramide and by the ceramide activation of Src.

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Figure 6.  C2-ceramide induces the hyperpolarization and activation of Src in anterior hypothalamic neurons. Anterior hypothalamic cultures were exposed to varying concentrations of C2-ceramide (a), or to C2-ceramide (10 µm) for the indicated periods of time (b). Protein extracts were separated and pSrc-Tyr416 detected as in Fig. 3. Values are means ± SEM for three independent experiments. *Statistically significant as compared with control (untreated in a and t0 in b) conditions at p < 0.05 by anova. (c) Application of C2-ceramide (10 µm) for 2 min hyperpolarizes and abolishes the firing of an AH neuron (b).

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One possible mechanism for the fast actions of IL-1β is the activation of signaling kinases, such as Src, that rapidly phosphorylate ion channels (see Salter and Kalia 2004 for review). Indeed, IL-1β exposure was shown to induce the activation of Src and enhance NMDA receptor function in hippocampal neurons (Viviani et al. 2003). Because it is possible for Src to modulate NMDA receptors in neurons, we tested whether NMDA receptor activation was involved in the IL-1β-induced hyperpolarization of POA neurons in culture. The IL-1-induced hyperpolarization was not affected by the presence of the NMDA receptor antagonists AP-5 (n = 3; (Fig. 7a) or MK-801 (n = 3, not shown) in the bath solution, thus suggesting that NMDA receptors were not involved in this response.

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Figure 7.  Rapid modulation of NMDA receptors are not involved in IL-1-induced hyperpolarization of POA neurons. (a) The IL-1-induced hypepolarization was not affected by the presence of the NMDA receptor antagonist, AP-5 (n = 3). (b) In the three bursting POA neurons studied here, IL-1 was without effect on the burst frequency or duration. In contrast, MK-801 shortened the burst duration, an effect similar to that of AP-5 (Tabarean et al. 2005).

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In a previous study, we showed that NMDA receptors play a role during bursting activity of POA neurons (Tabarean et al. 2005). Such activity is present only in about 5% of POA neurons. In the three bursting POA neurons studied here, IL-1 was without effect on the burst frequency or duration (Fig. 7b). In contrast, MK-801 shortened the burst duration (Fig. 7b), an effect similar to the one produced by AP-5 (Tabarean et al. 2005). These data suggest that modulation of NMDA receptors by IL-1β stimulation is not playing a role in the hypothalamic neuronal actions of IL-1 β.

Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The effects of the cytokine IL-1β on hypothalamic neurons are assumed to underlie its actions as a pyrogen (Dinarello 2004). The fever response to IL-1β occurs over several hours, of which the period beyond the first 30–45 min is dependent on the IL-1β-mediated, nuclear factor kappa B (NF-κB)-dependent transcription of COX2 and production of PGE2. This phase of IL-1β effects can be inhibited by indomethacin (Dinarello 1997). However, faster neuronal effects of IL-1β, such as the inhibition of warm-sensitive neurons, have also been shown in the hypothalamus (Hori et al. 1988a; Vasilenko et al. 2000). Our results confirm that IL-1β induces a rapid hyperpolarization in AH neurons in culture, and show that this effect is inhibited in the presence of the Src family inhibitor, PP2. These data suggest that Src activation is an initial step in IL-IRI-mediated rapid signaling in AH neurons. The neuronal effects occurred within minutes of IL-1β application and have been observed in the presence of the COX2 inhibitor, indomethacin. This suggests that the effects occur independently of IL-1β-induced transcription of COX2 leading to production of PGE2. A primary goal of this study was to identify the rapidly-activated signaling mechanisms in the anterior hypothalamus that might explain the fast actions of IL-1β.

We characterized the rapid neuronal effects of IL-1β using a mosaic primary hypothalamic culture system that permitted the study of the neuronal effects of IL-1β independent of its massive effects on glia. This was achieved by developing AH neurons from wild-type mice that express IL-1RI over a bed of glia from IL-1RI(–/–) mice. The neurons in these mosaic cultures developed indistinguishably from wild-type cultures regarding expression of neuronal markers (Conti et al. 2004; Tabarean et al. 2005), IL-1RI (Fig. 2) and ion channels (data not shown).

We demonstrated that brief exposure (2–10 min) to IL-1β in subnanomolar to nanomolar concentrations induces a swift activation of the protein kinase Src, and the Ser-phosphorylation of CAPK/KSR in neurons (Figs 3 and 5). The subnanomolar concentration range of IL-1β is similar to that required for the rapid hyperpolarizing electrophysiological affects in the hypothalamus and AH neurons. It is also within the same range as concentrations used earlier to demonstrate rapid neuronal effects in the hippocampus on LTP induction, and on the modification of NMDA receptor currents (Katsuki et al. 1990; Bellinger et al. 1993; Viviani et al. 2003). The earliest we could detect increases in Src phosphorylation was 30 s. However, IL-1β did not increase phospho-Src significantly above control until 2 min. The 300 pm concentration of IL-1β used to hyperpolarize the neurons did not stimulate phospho-Src significantly above control although, as in the time-course experiments, a slight increase was detected biochemically when examining Src phosphorylation. These biochemical data match the electrophysiological effects seen in the AH neuronal cultures, suggesting that Src could play a role in the speedy electrophysiological changes in AH neurons. It should be acknowledged that in the biochemical study, we sampled a population of neurons and in the electrophysiological study, we examined a single neuron. Taking this into consideration, the concentration and time dependence of IL-1-mediated Src phosphorylation and of IL-1-mediated hyperpolarization, are in agreement. In addition, we tested the IL-1β-mediated modulation of NMDA receptor activation with specific NMDA receptor antagonists to determine whether these receptors were playing a role in the IL-1-induced hyperpolarization of POA neurons. Our data suggest that rapid Src modulation of the NMDA receptor is not involved in these responses, unlike the case of hippocamapal neurons where IL-1 enhanced NMDA-mediated depolarization (Viviani et al. 2003).

The Toll family of receptors, of which the IL-1RI/RAcP receptor complex is a member, requires the recruitment of cytosolic adaptor proteins, such as MyD88, Trip or Tollip, to transduce their signal (O'Neill 2000). In the case of IL-1β signaling, MyD88 is the intracellular adaptor molecule that functions to recruit IL-1R-associated kinases to the IL-1R complex following stimulation with the cytokine. We have previously shown that the TIR domain/MyD88 mimic, AS-1, can inhibit the association of IL-1RI and MyD88, thus blunting IL-1β-mediated MAPK signaling in vitro and IL-1β-mediated fever induction in vivo (Bartfai et al. 2003). The inhibitor, AS-1, did not block lipopolysaccharide (LPS)-induced signaling in lymphocytes and it did not block the association of MyD88 with TLR4, showing its specificity for the IL−1R1/MyD88 interaction. In the present study, we used AS-1 to show that IL-1β-mediated phosphorylation of Src and KSR requires the interaction between IL-1RI and the cytosolic adaptor protein, MyD88, which is probably required for the activation of the N-SMase.

Previous experiments using IL-1RI(–/–) mice had shown that this receptor subtype is responsible for the IL-1β-mediated activation of N-SMase in mouse brain (Nalivaeva et al. 2000). Two forms of N-SMase have been identified and cloned (Tomiuk et al. 1998; Hofmann et al. 2000). Staining for N-SMase2 was shown to co-localize with neuronal cells (Hofmann et al. 2000). Furthermore, Zumbansen and Stoffel (2002) demonstrated that sphingomyelinase activity remained in the brains of N-SMase1 knockout mice, suggesting that the majority of sphingomyelinase activity in the brain is represented by N-SMase2. Here, we used an inhibitor of N-SMase (which inhibits N-SMase from the brain, i.e. N-SMase2), spiroepoxide, to show that IL-1β-mediated activation of Src and KSR phosphorylation were both dependent on N-SMase activation. The IL-1β action on hypothalamic neurons suggests that a putative bifurcation of the fast (ceramide-mediated) and slow (NF-κB-mediated) IL-1β signaling (Sanchez-Alavez et al. 2006) occurs downstream from the IL-1RI receptor/MyD88 interaction in hypothalamic neurons involving the activation of N-SMase.

Ceramide is one of the products of N-Smase, and it was previously suggested to act as a rapid intracellular second messenger transducing the actions of the pro-inflammatory cytokine, tumor necrosis factor α (TNFα) (Dressler et al. 1992; Mathias et al. 1998; Brann et al. 1999). In the nervous system, it was shown that ceramide may exert different actions on cell survival depending on the developmental stage of the tissue under analysis, and whether it is a primary culture system or tumor cell line (Goodman and Mattson 1996; Irie and Hirabayashi 1998; Mitoma et al. 1998; Taniwaki et al. 1999; Blazquez et al. 2000; Birbes et al. 2001). Ceramide and its phosphorylated metabolite, ceramide-1-phosphate, have been shown to regulate the activity of several ion channels, such as calcium release activated calcium, and tetrodotoxin-resistant Na+ channels, K+ channels and L-Ca2+ channels (Hida et al. 1998; Chik et al. 1999; Lepple-Wienhues et al. 1999; Wu et al. 2001; Zhang et al. 2002; Bock et al. 2003; Tornquist et al. 2004). Several of these ion channels have been also proposed as substrates of the Src family of kinases (Hu et al. 1998; Cook and Fadool 2002; Nitabach et al. 2002; Tiran et al. 2003).

There is increasing evidence pointing to the role of factor associated with N-SMase (FAN) as a linker of cytokine receptors to N-SMase. Originally, FAN was shown to link N-SMase to the p55 TNF receptor (Adam-Klages et al. 1996). It was further shown that there was impaired TNFα-induced activation of N-SMase in FAN knockout mice, but FAN was not necessary for the TNFα-mediated activation of extra cellular related kinase (ERK) (Kreder et al. 1999; Luschen et al. 2000). Our results indicate that activation of Src by IL-1β was dependent both on the activation of N-SMase and ceramide production. Recently, we have shown that activation of Src is abolished in neurons from mice lacking the expression of MyD88 (Davis et al. 2006). These data suggest that the activation of Src occurs downstream of N-SMase activity, leading to ceramide production, and is completely dependent on MyD88 and not other proteins such as FAN. However, an interaction of MyD88 or other adaptor proteins with FAN cannot be ruled out in these studies. Distinct sphingolipid- and cholesterol-enriched membrane domain rafts exist in the plasma membrane. These rafts appear to merge into large membrane domains upon the generation of ceramide within the rafts (for review see Bollinger et al. 2005). These rafts have been shown to couple sphingomyelinase to the IL-1R and are involved in IL-1-induced signaling (Mathias et al. 1993). It is possible that the rafts recruit the Src kinases to the IL-1 signaling complex in neurons and provide a mechanism by which Src could regulate other membrane-bound proteins, including ion channels.

These data, combined with the proposed effects of Src, suggest that IL-1β binding to its receptor can lead to the regulation of ion channels, either pre- or post-synaptically, via rapid non-transcription-dependent phosphorylation mechanisms. This is compatible with the rapid hyperpolarizing effects observed in electrophysiological recordings from AH neurons in which the specific inhibition of Src with PP2 blocked the effects of IL-1β (Fig. 1). The putative second messenger role for ceramide in mediating the rapid effects of IL-1β at IL-1RI in AH neurons is further strengthened by the increased Src phosphorylation induced by C2-ceramide, and by the hyperpolarizing effects of C2-ceramide on AH neurons in culture. In support of both these effects, C2-ceramide also mimicked the effects of IL-1β in terms of time dependence (Fig. 6). In addition, a membrane-impermeable analog of ceramide, dihydroceramide, failed to reproduce the effects of the cell-permeable C2-ceramide, suggesting that ceramide is activating Src and hyperpolarizing neurons through intracellular mechanisms. In vivo, ceramide contributes to the rapid induction of fever in rats within minutes, while it is PGE2 that sustains the fever response over hours (Dinarello 2004; Sanchez-Alavez et al. 2006).

IL-1β increased the phosphorylation of CAPK/KSR in a spiroepoxide-sensitive and MyD88-dependent fashion (Fig. 5), confirming that ceramide acts as a second messenger of fast IL-1β actions. The use of CAPK/KSR phosphorylation as a measure of ceramide formation is indirect and it would have been preferable to obtain direct measurements of rapid elevation of ceramide levels in AH neurons upon IL-1 exposure. However, direct enzyme activity (N-SMase) measurements had a high background and a low degree of stimulation by IL-1, probably because only a fraction of neurons will have this effect. We can, however, follow these effects when studying individual neurons electrophysiologically, or when following phosphorylation of CAPK/KSR and Src. The fact that C2-ceramide mimics, and spiroepoxide inhibits pSrc phosphorylation, and that dihydroceramide fails to mimic IL-1 effects, nevertheless provides strong evidence for the IL-1-mediated activation of N-SMase and for ceramide production in AH/POA neurons.

It is possible that stimulation of the IL-1β/ceramide pathway can affect Tyr and/or Ser/Thr phosphorylation of ion channels through the activation of Src kinase and the activation of MAPK/ERK by its recruitment to the KSR/Raf/MEK complex (Conway et al. 2000), thus leading to functional modification of the electrophysiological properties of AH neurons.

In an attempt to define the rapid effects of IL-1β in neurons, we have characterized one pathway by which IL-1β produces its effects in the CNS. Increasing evidence is showing that the IL-1 system is involved in mediating rapid neuronal changes in the brain, including epileptic seizures and febrile seizures (reviewed in Allan et al. 2005). Recent studies have shown that IL-1 is a potent regulator of febrile seizures and potentially contributes to long-lasting hyperexcitability and excitotoxicity associated with hippocampal epilepsy (Dube et al. 2005). Furthermore, blockade of the IL-1 system with the use of an IL-1 receptor antagonist (IL-1Ra) has been shown to reduce epileptic seizures in rodents (Vezzani et al. 2000). It is important to determine which pathways are activated following IL-1 stimulation in neurons, and to what degree these pathways contribute to either rapid non-transcriptional changes or transcription-dependent long-term changes in the brain. The relative contribution of the pathways described in this report to the IL-1β/ceramide-mediated changes in membrane properties in hypothalamic neurons requires further investigation. These IL-1β/ceramide-activated pathways, individually or jointly, may underlie the fast non-transcription-dependent electrophysiological effects of IL-1β observed in AH neurons in vivo.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This study was supported by National Institutes of Health Grant R01 NS043501 (TB). This article is manuscript no. 17813-MIND from The Scripps Research Institute.

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  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Fig. S1 Sphingosine-1-phosphate (S-1P) has no effect of the rapid electrophysiologcal properties of POA neuons.

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JNC3951+Supplementary+figure+with+legend.doc773KSupporting info item
JNC3951+supplementary+figure.tif1105KSupporting info item

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