Leptin Is Induced in the Ischemic Cerebral Cortex and Exerts Neuroprotection Through NF-κB/c-Rel–Dependent Transcription
Background and Purpose— Leptin is an adipose hormone endowed with angiopoietic, neurotrophic, and neuroprotective properties. We tested the hypothesis that leptin might act as an endogenous mediator of recovery after ischemic stroke and investigated whether nuclear transcription factors κB activation is involved in leptin-mediated neuroprotection.
Methods— The antiapoptotic effects of leptin were evaluated in cultured mouse cortical neurons from wild-type or NF-κB/c-Rel−/− mice exposed to oxygen–glucose deprivation. Wild-type, c-Rel−/− and leptin-deficient ob/ob mice were subjected to permanent middle cerebral artery occlusion. Leptin production was measured in brains from wild-type mice with quantitative reverse transcriptase–polymerase chain reaction and immunostaining. Mice received a leptin bolus (20 μg/g) intraperitoneally at the onset of ischemia.
Results— Leptin treatment activated the nuclear translocation of nuclear transcription factors κB dimers containing the c-Rel subunit, induced the expression of the antiapoptotic c-Rel target gene Bcl-xL in both control and oxygen–glucose deprivation conditions, and counteracted the oxygen–glucose deprivation-mediated apoptotic death of cultured cortical neurons. Leptin-mediated Bcl-xL induction and neuroprotection against oxygen–glucose deprivation were hampered in cortical neurons from c-Rel−/− mice. Leptin mRNA was induced and the protein was detectable in microglia/macrophage cells from the ischemic penumbra of wild-type mice subjected to permanent middle cerebral artery occlusion. Ob/ob mice were more susceptible than wild-type mice to the permanent middle cerebral artery occlusion injury. Leptin injection significantly reduced the permanent middle cerebral artery occlusion-mediated cortical damage in wild-type and ob/ob mice, but not in c-Rel−/− mice.
Conclusions— Leptin acts as an endogenous mediator of neuroprotection during cerebral ischemia. Exogenous leptin administration protects against ischemic neuronal injury in vitro and in vivo in a c-Rel-dependent manner.
Leptin, the product of the ob gene, is a peptide hormone primarily secreted by adipocytes, which plays multiple roles in the brain.1 Besides modulating hypothalamic function to inhibit food intake and increase energy expenditure,1 leptin displays neurotrophic effects.2 In particular, we showed that leptin increases the axonal growth of mouse cortical neurons through the phosphatidylinositol 3-kinase/Akt, MEK/extracellular signal regulated kinase 1/2, and protein kinase C pathways, which converge in the inactivation of glycogen synthase kinase-3β (GSK3β).2 All these signals can mediate protection against cerebral ischemia.3,4 Indeed, leptin has been found to exert neuroprotection in a mouse model of transient focal cerebral ischemia.5
The nuclear transcription factors κB (NF-κB) are key regulators of neuron death or survival. NF-κB signaling relies on the nuclear translocation of homo- or heterodimers composed of different members of the NF-κB family (RelA [p65], RelB, c-Rel, p50, and p52) to specifically regulate gene transcription.6 Although the activation of NF-κB p50/p65 dimers contributes to neurodegeneration in brain ischemia,7,8 c-Rel–containing dimers promote neuron survival, mainly due to enhanced expression of the c-Rel target gene Bcl-xL,9 an antiapoptotic factor that protects against cerebral ischemia.10 Activation of c-Rel is dependent on phosphatidylinositol 3-kinase, MEK, and protein kinase C in brain tissues.11 An inverse association between GSK3β activity and NF-κB signaling has also been described,12 but knowledge of specific NF-κB subunit(s) affected by GSK3β activity is lacking.
Cerebral ischemia induces adaptation responses aimed at facilitating oxygen and glucose delivery and neural plasticity. According to its proangiogenic13 and neurotrophic2 properties, leptin might take part in the adaptive processes to ischemic brain injury. Hypoxia induces the transcription of the leptin gene in cells that do not express the hormone in normal conditions.14 Leptin is strongly upregulated in several tissues when rodents are exposed in vivo to hypoxia,15 but its expression in the ischemic brain, which might elicit signals facilitating recovery, has not been evaluated yet.
We provide evidence that leptin exposure of mouse cortical neurons activates an antiapoptotic program involving c-Rel–mediated Bcl-xL induction that counteracts oxygen–glucose deprivation (OGD) damage. We also show that after permanent middle cerebral artery occlusion (pMCAO), leptin expression is induced in the peri-infarcted microglia/macrophage cells. Permanent MCAO causes higher damage in leptin-deficient ob/ob mice than in wild-type mice. When exogenously administered, leptin reduces infarct size in wild-type and ob/ob mice but not in c-Rel−/− mice.
Pregnant C57BL/6J mice and male 8-week-old Lepob/Lepob (ob/ob) mice in the C57BL/6J strain, along with wild-type C57BL/6J mice, were purchased from Charles River (Caleo, Cosmo, Italy). C-Rel−/− mice16 were backcrossed for 7 generations on a C57BL/6J background. Procedures involving animals were approved by the Institutional Animal Care Committee in compliance with the Italian guidelines for animal care (DL 116/92) and the European Communities Council Directive (86/609/EEC).
Neuronal Cultures and Treatments
Fifteen-day embryonic mice were taken with cesarean section from anesthetized pregnant C57BL/6J or cRel−/− dams. Primary cortical neurons were purified and cultured 11 to 13 days in Neurobasal medium containing 2% B27 supplement (Invitrogen Corporation).2 Cells were treated with murine recombinant leptin (National Hormone and Peptide Program, NHPP-NIDDK, Dr A.F. Parlow), LY294002 (Cell Signaling Technology), PD98059 (Calbiochem), GF109203X (Calbiochem), or SB216763 (Tocris) at concentrations and times indicated in the figure legends.
Primary cortical neurons were transferred into glucose-free balanced salt solution (116 mmol/L NaCl, 5.4 mmol/L KCl, 0.8 mmol/L MgSO4, 1.1 mmol/L NaH2PO4, 26.2 mmol/L NaHCO3, 1.8 mmol/L CaCl2) previously saturated with 95% N2/5% CO2 and incubated in an anaerobic chamber at 37°C for 3 hours. Oxygen concentration was <0.4% throughout the OGD period as assessed by an oxygen analyzer (Servomex 580A). Control neurons were transferred in balanced salt solution containing 5.5 mmol/L glucose (saturated with 95% O2/5% CO2) and incubated at 37°C for 3 hours under normoxic conditions. Neurons were allowed to recover in culture medium in normoxia for 24 hours. Unless otherwise specified, leptin was added 15 minutes before OGD and maintained during OGD and recovery. The release of lactate dehydrogenase was measured with the CytoTox 96 Assay (Promega). Duplicate lactate dehydrogenase measurements were done on OGD experiments run in triplicate from at least 3 different cultures. Alternatively, neurons on coverslips were fixed and neuronal apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated DNA nick end labeling (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling [TUNEL] kit from Roche). After counterstaining with the DNA-binding dye Hoechst 33258 (Invitrogen), coverslips were mounted and visualized using an Olympus IX51 microscope. The percentage of apoptotic nuclei (number of TUNEL-positive nuclei against the total number of nuclei identified by Hoecsht staining) was blindly counted from 10 randomly selected fields. Greater than 95% of TUNEL-positive nuclei were condensed or fragmented. Mean values were calculated from 3 separate experiments.
Protein extracts were quantified and processed for Western blot analysis with enhanced chemiluminescence (ECL plus kit; Amersham Biosciences) as previously described.2 Primary antibodies were anti-Bcl-xL (1:1000 dilution; Cell Signaling Technology) or anti-Bax (sc-493, 1:1000; Santa Cruz Biotechnology). Quantification was performed by densitometric scanning of exposed films using Gel-Pro Analyzer software (Media Cybernetics). The ratio between Bcl-xL and Bax band intensities was calculated from at least 3 immunoblots with samples from separate cell preparations. Mean values were referred to control values taken as 1.0.
DNA-Binding Activity of Nuclear Factor κB
Detection of NF-κB activity was performed with the BD Mercury TransFactor Kit (Clontech BD Biosciences) as described.9 Aliquots of nuclear lysates (5 μg) were transferred to 96-well plates containing high-density immobilized κB oligonucleotides. The active forms of p50, p65, or c-Rel NF-κB subunits bound to the target DNA were detected using specific antibodies followed by an horseradish peroxidase-conjugated secondary antibody and revealed according to manufacturer’s instructions. Data are expressed as the difference of absorbance in the presence or absence of nuclear extracts.
Immunoprecipitation of Nuclear Factor κB
Coimmunoprecipitation studies were carried out in 30 μg of nuclear extracts from cortical neurons as described.9 Antibodies used for immunoprecipitation were anti-p50 (sc-1190), anti–c-Rel (sc-71-G), or normal goat IgG (Santa Cruz Biotechnology). Immunoprecipitated proteins were electrophoresed and detected by Western blotting as reported previously using the following primary antibodies: anti-p50 (1:500; Abcam); anti-p65 (1:200; sc-372), anti-RelB (1:500; sc-226) anti-p52 (1:50; sc-848), or anti–c-Rel (1:200; sc-71), all from Santa Cruz Biotechnology. To confirm equal amounts of immunoprecipitated proteins, 30 μg of nuclear extracts were electrophoresed and detected by Western blotting with anti-β-actin primary antibody (1:1000; Sigma-Aldrich).
Permanent Focal Cerebral Ischemia
Permanent distal MCAO was induced as previously described.17 Mice were anesthetized (1.5% isoflurane in air) and rectal temperature was maintained between 36.5°C and 37.5°C. Rectal temperature, mean arterial blood pressure, pH, PaO2, and Paco2 did not differ among the experimental groups before, during, and 1 hour after ischemia. Mortality was below 3% without differences among groups. Laser Doppler (PF2B; Perimed) analysis carried out for 30 minutes after ischemia revealed that drug treatment did not affect basal cerebral blood flow and drop on artery cauterization. Mice were euthanized 24 hours after pMCAO unless otherwise specified and brains snap-frozen in nitrogen vapor. For infarct determination, toluidine blue-stained coronal sections were imaged using the Image-Pro Plus 3.0 analysis software (Media Cybernetics). Infarct measurement was conducted as described.17 Mouse recombinant leptin (NHPP-NIDDK) was dissolved in 100 μL of phosphate-buffered saline and injected intraperitoneally at a dose of 20 μg/g at the onset of ischemia. Control animals received an equal amount of saline.
Quantitative Reverse Transcriptase–Polymerase Chain Reaction
For the analysis of mRNA levels,18 1 μg of total RNA isolated using the RNeasy kit (Qiagen) was reverse-transcribed using iScript cDNA Synthesis Kit (Bio-Rad Laboratories). Triplicate polymerase chain reactions were carried out on an iCycler iQ Real Time PCR Detection System (Bio-Rad Laboratories). The cycle number at which the leptin transcript was detectable was compared with that of acidic ribosomal phosphoprotein P0 (Arbp/36B4). Relative gene expression was calculated as described.18 Primers sequences were designed using Beacon Designer 2.6 software (Premier Biosoft International).
Immunohistochemistry and Immunofluorescence
Peroxidase immunohistochemistry was performed on 40-μm-thick free-floating brain coronal sections as described.2 Leptin was detected with a primary antiserum against mouse recombinant leptin (1:400; NHPP-NIDDK). Negative controls (not shown) were obtained by (1) using preimmune serum instead of primary antiserum; and (2) preadsorbing the primary antibody with antigen excess. Immunostained sections were observed under a motorized Leica DM6000 microscope (Leica Microsystems). Sections for double fluorescence were incubated overnight at 4°C with a mixture of rabbit antileptin (1:200) and rat anti-CD11b (1:500; Serotec) antisera followed by a cocktail of fluorescein isothiocyanate donkey antirabbit and TRITC donkey antirat antibodies (1:100; Jackson ImmunoResearch) for 1 hour at room temperature. Sections were mounted in Vectashield (Vector). Fluorescence was detected with a Leica TCS SL spectral confocal microscopy. Fluorescein isothiocyanate and TRITC were excited with the 488- and 543-nm lines, respectively, and imaged separately.
The results were analyzed by unpaired 2-tailed t test or one-way analysis of variance followed by a Bonferroni post hoc test for multiple comparison. Data are presented as means±SE.
Leptin Counteracts Oxygen–Glucose Deprivation–Induced Neuronal Apoptosis Through Signals Converging on Glycogen Synthase Kinase-3β Inhibition
Leptin dose-dependently reduced OGD-induced neuronal death, as assessed by lactate dehydrogenase release with significant inhibition observed at 0.1 to 500 ng/mL and maximal inhibition at 100 ng/mL (Figure 1A). A similar pattern of U-shaped dose–response to leptin has been reported in other experimental settings.19 At 100 ng/mL, even delayed application of leptin provided neuroprotection (Figure 1B). Leptin significantly reduced the rate of OGD-induced neuronal apoptosis measured by means of TUNEL/Hoechst 33258 nuclear staining (Figure 1C).
Coexposure of neurons to the phosphatidylinositol 3-kinase inhibitor LY294002 (10 to 50 μmol/L),20 the MEK inhibitor PD98059 (10 to 50 μmol/L),21 or the protein kinase C inhibitor GF109203X (1 to 5 μmol/L)22 impaired the protective effect of leptin against OGD damage (Figure 1D). The pharmacological inhibitors per se did not significantly modify lactate dehydrogenase release in control nor in OGD conditions (Figure 1D). Leptin-mediated neuroprotection was mimicked by treatment with 10 μmol/L SB216763 (Figure 1E), a selective inhibitor of GSK3β.23 Coexposure of cortical neurons to leptin and SB216763 further provided a small, but not significant, enhancement of neuroprotection (Figure 1E).
Leptin Induces a Prosurvival Profile of Nuclear Transcription Factors κB Activation
Leptin activated NF-κB in cortical neurons, as shown by the nuclear translocation of NF-κB factors p50, p65, and c-Rel, in a time-dependent manner (Figure 2A). Nuclear translocation of p50, p65, and c-Rel induced by leptin treatment (60 minutes) was prevented by coexposure to the pharmacological inhibitors LY294002, PD98059, or GF109203X (Figure 2B). Exposure to SB216763 to inhibit GSK3β induced the nuclear translocation of p50, p65, and c-Rel NF-κB subunit with a time course superimposable to that observed after leptin treatment (Figure 2C).
By means of coimmunoprecipitation studies, we investigated the effects of leptin on the activation of various NF-κB complexes. We analyzed the p50/p65 complex playing a major role in neuronal death7–9 and the c-Rel-containing complexes mediating neuroprotection.9 Leptin treatment left unchanged the nuclear content of the p50/p65 dimer and increased the nuclear amount of the c-Rel-containing dimers cRel/p50 and cRel/p65 (Figure 2D). Furthermore, leptin treatment activated the cRel/RelB and cRel/p52 complexes (Figure 2D).
Nuclear Factor κB Subunit c-Rel Is Necessary to Leptin-Mediated Bcl-xL Induction and Neuroprotection
We reported that silencing neuronal c-Rel abolishes the induction of the c-Rel target gene Bcl-xL and its prosurvival effects.9 Bcl-xL confers protection toward cerebral ischemia10 by counteracting the proapoptotic effects of Bax. According to its ability to induce c-Rel nuclear translocation, leptin strongly increased the expression of Bcl-xL in cortical neurons; furthermore, it slightly reduced the expression of Bax, thus significantly increasing the Bcl-xL/Bax ratio (Figure 3A). Leptin failed to induce Bcl-xL expression and to affect the Bcl-xL/Bax ratio in cortical neurons obtained from c-Rel−/− mice (Figure 3A).
To assess the relevance of c-Rel in leptin-mediated neuroprotection, we performed OGD in cultures from c-Rel−/− mice. OGD increased lactate dehydrogenase release from c-Rel−/− cortical neurons at levels similar to those observed after OGD in wild-type neurons (mean fold increase, 2.19±0.22 in wild-type neurons; 2.04±0.11 in c-Rel−/− neurons; Figure 3B). However, the leptin-mediated neuroprotection against OGD, which was observed in wild-type neurons, was completely lost in cortical neurons from c-Rel−/− mice (Figure 3B). Likewise, exposure to 100 ng/mL leptin reversed the decrease in Bcl-xL/Bax ratio produced by OGD insult in wild-type neurons (Figure 3C) but not in neurons from c-Rel−/− mice (Figure 3C).
Leptin mRNA and Protein Are Upregulated After Ischemic Stroke
Because leptin has the potential to be synthesized by resident brain cells under pathological conditions,24 we analyzed leptin production in brain tissue from middle cerebral artery-occluded normal mice. Leptin mRNA levels were increased in the perilesional cerebral tissue from the ischemic hemisphere when compared with the contralateral, nonischemic hemisphere within 6 hours (data not shown) and remained higher up to 24 hours after pMCAO (Figure 4A). The production of leptin was further investigated by means of immunohistochemistry. No leptin immunoreactivity was detectable in cortical tissue contralateral to the lesion (Figure 4B). However, a marked leptin expression was found in cells from the peri-infarcted cortical brain tissue 24 (Figure 4C) and 48 hours (Figure 4D) after pMCAO. All the leptin-immunoreactive cells were also positive to CD11b, a marker of microglia/macrophage cells,25 as assessed by confocal microscopy (Figure 4E).
Leptin Administration Counteracts Cerebral Infarct Damage in a c-Rel–Dependent Manner
The neuroprotective effect of leptin was then assessed in the pMCAO model. Intraperitoneal injection of leptin (20 μg/g) at the onset of pMCAO significantly reduced the cortical infarct size in wild-type mice (Figure 5). Ob/ob mice, that cannot synthesize leptin in response to the ischemic challenge, sustained significantly greater infarcts relative to wild-type mice (Figure 5). Leptin administration significantly counteracted the ischemic brain damage in the ob/ob mice (Figure 5). As previously described,8 the infarct size in c-Rel−/− mice did not differ from that of wild-type mice. However, leptin injection failed to modify the pMCAO injury in c-Rel−/− mice (Figure 5).
In the present study, we demonstrate that the neuroprotective effect of leptin against an in vitro ischemic insult is dependent on the phosphatidylinositol 3-kinase, MEK, and protein kinase C signaling pathways that are known to mediate GSK3β inactivation in mouse cortical neurons2 and is mimicked by the selective GSK3β inhibitor SB216763. The lack of additive effects of leptin and SB216763 suggests that the 2 molecules share most of the protective mechanisms.
We also show that leptin modulates the activity of NF-κB transcription factors in a phosphatidylinositol 3-kinase-, MEK- and protein kinase C-dependent manner and that the leptin’s pattern of NF-κB activation is mirrored by SB216763. Leptin treatment does not affect the subcellular localization of the p50/p65 dimer but enhances the nuclear translocation of p65 and p50 when complexed to the c-Rel subunit. This pattern of NF-κB activation is typical of neuroprotective molecules9,26 and is associated with the induction of the c-Rel-responsive gene Bcl-xL.9 Accordingly, we show that leptin induces Bcl-xL and modifies the Bcl-xL/Bax ratio toward an antiapoptotic state. We also observed that leptin induces the nuclear translocation of the c-Rel/RelB and c-Rel/p52 complexes, which could possibly contribute to neuroprotection. Worth noting, molecular activation of c-Rel-containing NF-κB dimers was responsible for the leptin-mediated modulation of the Bcl-xL/Bax ratio and neuroprotection, because both effects were completely suppressed in c-Rel−/− neurons.
We further applied an in vivo model of focal cerebral ischemia, which produces cortical damage, and observed the de novo production of leptin and its localization in microglia and/or macrophage cells of the penumbra. Ob/ob mice lacking endogenous leptin have a worse outcome in terms of infarct size when undergoing pMCAO, as already described in models of ischemia and reperfusion.27 The observation that db/db mice lacking the functional leptin receptor are also more prone to cerebral ischemic damage independently of their glycemic levels28 suggests that leptin signaling is required to exert the protective effect.
Slow-rate administration of a very low leptin dose (5 μg/d) 2 days before transient MCAO, to reconstitute basal levels normally detected in wild-type mice, did not reduce the damage but tended to enhance inflammatory responses in ob/ob mice.27 On the contrary, we found that a single 20 μg/g intraperitoneal leptin injection at the onset of pMCAO is able to reverse the increased ischemic damage in these mice. This discrepancy might be attributable to the different dosing regimen, suggesting the need of a bolus injection to reach therapeutic leptin levels in the brain, in line with the results by Zhang and coworkers in normal mice subjected to transient MCAO.5 Noticeably, leptin efficiently reduced infarct size also in wild-type mice undergoing permanent ischemic injury. As we and other authors19 showed that optimal leptin effects in vitro are at intermediate doses, the therapeutic range of leptin in vivo remains to be assessed. Finally, although our study does not exclude a contribution of systemic effects to the in vivo benefits of leptin, diverse points are in favor of its direct central action: (1) leptin crosses the blood–brain barrier both in normal and in ob/ob mice29; and (2) peripheral leptin administration activates neuroprotective signals in mouse cerebral cortex2 and modifies the morphology of cerebral cortex30 as well as the activity of cortical circuits regulating behavior31 in genetically leptin-deficient human subjects.
In line with previous reports,8 we found that germline deletion of c-Rel per se has no effect on the infarct size in the pMCAO model. However, because c-Rel−/− mice were insensitive to the effects of leptin on pMCAO, the present study demonstrates that c-Rel is necessary to the neuroprotective effect of leptin in vivo. Leptin is efficacious and well tolerated in the treatment of rare human conditions.32 We suggest that leptin administration, by targeting NF-κB/c-Rel–mediated transcription, may have the potential as a safe therapeutic approach to enhance endogenous repair mechanisms in focal ischemic stroke.
Sources of Funding
This work was supported by funds from the Italian Ministry of Research (grant 2005068257 to E.N., M.P., and A.G. and grant 2005067481 to M.O.C.) and from the Italian Ministry of Health (E.N. and M.O.C.).
M.P. and E.N. contributed equally to this work.
- Received June 12, 2008.
- Accepted June 19, 2008.
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