ABSTRACT
Title
Enhanced STAT3 phosphorylation is implicated in the neuroprotection afforded by leptin in rats subjected to permanent middle cerebral artery occlusion
Authors
1,2D. Amantea, 1F. Petrelli, 1,2R. Russo, 1,2G. Bagetta and 3M.T. Corasaniti
1Dept. of Pharmacobiology and 2UCADH Section of Neuropharmacology of Normal and Pathological Neuronal Plasticity, University of Calabria, Rende (CS), Italy
3Dept. of Phamacobiological Sciences, University of Catanzaro “Magna Graecia”, Catanzaro, Italy
1Dept. of Pharmacobiology and 2UCADH Section of Neuropharmacology of Normal and Pathological Neuronal Plasticity, University of Calabria, Rende (CS), Italy
3Dept. of Phamacobiological Sciences, University of Catanzaro “Magna Graecia”, Catanzaro, Italy
Abstract
Leptin is an adipose hormone implicated in the regulation of food intake, energy homeostasis and reproduction. Recent studies suggest that leptin is endowed with neurotrophic and neuroprotective effects [1]. Here we investigated the neuroprotective potential of leptin by using an experimental model of focal brain ischemia induced in rats by permanent occlusion of the middle cerebral artery (MCAo) with an intraluminal filament [2]. Phosphorylated Signal Transducer and Activator of Transcription-3 (p-STAT3) and STAT3 expression in cytosolic and nuclear fractions from brain cortical tissue samples was studied by western blot analysis.
A single, subcutaneous (s.c.) administration of leptin (1 mg/kg, 3 h before MCAo) significantly reduced brain infarct volume measured 24 hours, 3 days or 7 days after permanent MCAo. Neuroprotection was also associated with reduced neurological deficit as assessed up to 7 days following ischemia.
Western blotting and immunofluorescence analysis showed that p-STAT3 immunoreactivity is barely detectable in the cerebral cortex after sham operation, whereas it dramatically (P<0.001) increases in the ipsilateral, ischemic cortex of rats subjected to 3 h of MCAo. Twenty four hours after ischemia, STAT3 phosphorylation further increased in the cerebral cortex ipsilateral to the occluded MCA along with enhanced immunoreactivity also occurring in the contralateral cortex. Interestingly, leptin treatment further increased levels of STAT3 phosphorylation in the ischemic cortex 24 h after MCAo, as compared to vehicle.
Subcellular fractionation studies revealed that the ischemic insult significantly increased levels of STAT3 phosphorylation in both the cytosolic and nuclear fractions of the ipsilateral, ischemic brain cortex at 3 h after MCAo (P<0.05 vs contralateral) and these further increased in the nuclear (P<0.001 vs 3 h) but not in the cytosolic fraction of ischemic cortex at 24 h after occlusion. In comparison to vehicle-treated animals, at 3h after MCAo, leptin further increased STAT3 phosphorylation in the nuclear fraction of the astrocytes populating the penumbra (as confirmed by immunofluorescence). By contrast, after 24 h of ischemia leptin further increased STAT3 phosphorylation in the cytosolic fraction. At this stage pSTAT3 immunoreactivity was mainly detected in neurons and other (presumably microglial) cells of the peri-infarct region of the motor cortex.
Because STAT3 is translocated to the nucleus after phosphorylation-mediated activation, it is conceivable that the early enhancement of nuclear p-STAT3 levels induced by leptin may signal for the induction of neuroprotective genes.
[1] Tang (2008) Biochem Biophys Res Commun 368, 181-185.
[2] Longa et al. (1989) Stroke 20, 84-91.
Financial support by the Ministry of Health (Ricerca Finalizzata 2005, Convenzione n. 105, Titolo del progetto:“Meccanismi di protezione e danno neuronale nella deprivazione energetica”) is gratefully acknowledged. The contribution of Dr Micaela Gliozzi for Western blotting experiments is acknowledged.
A single, subcutaneous (s.c.) administration of leptin (1 mg/kg, 3 h before MCAo) significantly reduced brain infarct volume measured 24 hours, 3 days or 7 days after permanent MCAo. Neuroprotection was also associated with reduced neurological deficit as assessed up to 7 days following ischemia.
Western blotting and immunofluorescence analysis showed that p-STAT3 immunoreactivity is barely detectable in the cerebral cortex after sham operation, whereas it dramatically (P<0.001) increases in the ipsilateral, ischemic cortex of rats subjected to 3 h of MCAo. Twenty four hours after ischemia, STAT3 phosphorylation further increased in the cerebral cortex ipsilateral to the occluded MCA along with enhanced immunoreactivity also occurring in the contralateral cortex. Interestingly, leptin treatment further increased levels of STAT3 phosphorylation in the ischemic cortex 24 h after MCAo, as compared to vehicle.
Subcellular fractionation studies revealed that the ischemic insult significantly increased levels of STAT3 phosphorylation in both the cytosolic and nuclear fractions of the ipsilateral, ischemic brain cortex at 3 h after MCAo (P<0.05 vs contralateral) and these further increased in the nuclear (P<0.001 vs 3 h) but not in the cytosolic fraction of ischemic cortex at 24 h after occlusion. In comparison to vehicle-treated animals, at 3h after MCAo, leptin further increased STAT3 phosphorylation in the nuclear fraction of the astrocytes populating the penumbra (as confirmed by immunofluorescence). By contrast, after 24 h of ischemia leptin further increased STAT3 phosphorylation in the cytosolic fraction. At this stage pSTAT3 immunoreactivity was mainly detected in neurons and other (presumably microglial) cells of the peri-infarct region of the motor cortex.
Because STAT3 is translocated to the nucleus after phosphorylation-mediated activation, it is conceivable that the early enhancement of nuclear p-STAT3 levels induced by leptin may signal for the induction of neuroprotective genes.
[1] Tang (2008) Biochem Biophys Res Commun 368, 181-185.
[2] Longa et al. (1989) Stroke 20, 84-91.
Financial support by the Ministry of Health (Ricerca Finalizzata 2005, Convenzione n. 105, Titolo del progetto:“Meccanismi di protezione e danno neuronale nella deprivazione energetica”) is gratefully acknowledged. The contribution of Dr Micaela Gliozzi for Western blotting experiments is acknowledged.