Endan Li

Ghrelin attenuates kainic acid-induced neuronal cell death in the mouse hippocampus

Ghrelin is an endogenous ligand for GH secretagogue receptor type 1a (GHSR1a), and is produced and released mainly from the stomach. It has been recently demonstrated that ghrelin can function as a neuroprotective factor by inhibiting apoptotic pathways. Kainic acid (KA), an excitatory amino acid l-glutamate analog, causes neuronal death in the hippocampus; previous studies suggest that activated microglia and astrocytes actively participate in the pathogenesis of KA-induced hippocampal neurodegeneration. However, it is unclear whether ghrelin has neuroprotective effect in KA-induced hippocampal neurodegeneration. I.p. injection of KA produced typical neuronal cell death in the CA1 and CA3 pyramidal layers of the hippocampus, and the systemic administration of ghrelin significantly attenuated KA-induced neuronal cell death in these regions through the activation of GHSR1a. Ghrelin prevents KA-induced activation of microglia and astrocytes, and the expression of proinflammatory mediators tumor necrosis factor alpha, interleukin-1beta, and cyclooxygenase-2. The inhibitory effect of ghrelin on the activation of microglia and astrocytes appears to be associated with the inhibition of matrix metalloproteinase-3 expression in damaged hippocampal neurons. Our data suggest that ghrelin has a therapeutic potential for suppressing KA-induced pathogenesis in the brain.

Multiple signaling pathways mediate ghrelin-induced proliferation of hippocampal neural stem cells

Ghrelin, an endogenous ligand for the GH secretagogue receptor (GHS-R) receptor 1a (GHS-R1a), has been implicated in several physiologic processes involving the hippocampus. The aim of this study was to investigate the molecular mechanisms of ghrelin-stimulated neurogenesis using cultured adult rat hippocampal neural stem cells (NSCs). The expression of GHS-R1a was detected in hippocampal NSCs, as assessed by western blot analysis and immunocytochemistry. Ghrelin treatment increased the proliferation of cultured hippocampal NSCs assessed by BrdU incorporation. The exposure of cells to the receptor-specific antagonist d-Lys-3-GHRP-6 abolished the proliferative effect of ghrelin. By contrast, ghrelin showed no significant effect on cell differentiation. The expression of GHS-R1a was significantly increased by ghrelin treatment. The analysis of signaling pathways showed that ghrelin caused rapid activation of ERK1/2 and Akt, which were blocked by the GHS-R1a antagonist. In addition, ghrelin stimulated the phosphorylation of Akt downstream effectors, such as glycogen synthase kinase (GSK)-3β, mammalian target of rapamycin (mTOR), and p70(S6K). The activation of STAT3 was also caused by ghrelin treatment. Furthermore, pretreatment of cells with specific inhibitors of MEK/ERK1/2, phosphatidylinositol-3-kinase (PI3K)/Akt, mTOR, and Jak2/STAT3 attenuated ghrelin-induced cell proliferation. Taken together, our results support a role for ghrelin in adult hippocampal neurogenesis and suggest the involvement of the ERK1/2, PI3K/Akt, and STAT3 signaling pathways in the mediation of the actions of ghrelin on neurogenesis. Our data also suggest that PI3K/Akt-mediated inactivation of GSK-3β and activation of mTOR/p70(S6K) contribute to the proliferative effect of ghrelin.

Ghrelin Protects Spinal Cord Motoneurons Against Chronic Glutamate Excitotoxicity by Inhibiting Microglial Activation

Glutamate excitotoxicity is emerging as a contributor to degeneration of spinal cord motoneurons in amyotrophic lateral sclerosis (ALS). Recently, we have reported that ghrelin protects motoneurons against chronic glutamate excitotoxicity through the activation of extracellular signal-regulated kinase 1/2 and phosphatidylinositol-3-kinase/Akt/glycogen synthase kinase-3β pathways. Previous studies suggest that activated microglia actively participate in the pathogenesis of ALS motoneuron degeneration. However, it is still unknown whether ghrelin exerts its protective effect on motoneurons via inhibition of microglial activation. In this study, we investigate organotypic spinal cord cultures (OSCCs) exposed to threohydroxyaspartate (THA), as a model of excitotoxic motoneuron degeneration, to determine if ghrelin prevents microglial activation. Exposure of OSCCs to THA for 3 weeks produced typical motoneuron death, and treatment of ghrelin significantly attenuated THA-induced motoneuron loss, as previously reported. Ghrelin prevented THA-induced microglial activation in the spinal cord and the expression of pro-inflammatory cytokines tumor necrosis factor-α and interleukin-1β. Our data indicate that ghrelin may act as a survival factor for motoneurons by functioning as a microglia-deactivating factor and suggest that ghrelin may have therapeutic potential for the treatment of ALS and other neurodegenerative disorders where inflammatory responses play a critical role.

Ghrelin protects spinal cord motoneurons against chronic glutamate-induced excitotoxicity via ERK1/2 and phosphatidylinositol-3-kinase/Akt/glycogen synthase kinase-3β pathways.

Excitotoxic degeneration of spinal cord motoneurons has been proposed as a pathogenic mechanism in amyotrophic lateral sclerosis (ALS). Recently, we have reported that ghrelin, an endogenous ligand for growth hormone secretagogue receptor (GHS-R) 1a, functions as a neuroprotective factor in various animal models of neurodegenerative diseases. In this study, the potential neuroprotective effects of ghrelin against chronic glutamate-induced cell death were studied by exposing organotypic spinal cord cultures (OSCC) to threohydroxyaspartate (THA), as a model of excitotoxic motoneuron degeneration. Ghrelin receptor was expressed on spinal cord motoneurons. Exposure of OSCC to THA for 3 weeks resulted in a significant loss of motoneurons. However, THA-induced loss of motoneurons was significantly reduced by treatment of ghrelin. Exposure of OSCC to the receptor-specific antagonist D-Lys-3-GHRP-6 abolished the protective effect of ghrelin against THA. Treatment of spinal cord cultures with ghrelin caused rapid phosphorylation of extracellular signal-regulated kinase 1/2, Akt, and glycogen synthase kinase-3β (GSK-3β). The effect of ghrelin on motoneuron survival was blocked by the MEK inhibitor PD98059 and the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002. Taken together, these findings indicate that ghrelin has neuroprotective effects against chronic glutamate toxicity by activating the MAPK and PI3K/Akt signaling pathways and suggest that administration of ghrelin may have the potential therapeutic value for the prevention of motoneuron degeneration in human ALS. Our data also suggest that PI3K/Akt-mediated inactivation of GSK-3β in motoneurons contributes to the protective effect of ghrelin.

Insulin-Like Growth Factor-1 Inhibits 6-Hydroxydopamine-Mediated Endoplasmic Reticulum Stress-Induced Apoptosis via Regulation of Heme Oxygenase-1 and Nrf2 Expression in PC12 Cells

ABSTRACT Endoplasmic reticulum (ER) stress and oxidative stress appear to play a critical role in the progression of Parkinsons disease (PD). Insulin-like growth factor (IGF)-1, a 70-amino acid polypeptide trophic factor, acts as a potent neurotrophic, neurogenic, and neuroprotective/anti-apoptotic factor. In this study, we investigated the protective mechanisms of IGF-1 in rat pheochromocytoma PC12 cells exposed to the PD-related neurotoxin 6-hydroxydopamine (6-OHDA). The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) coordinates expression of genes required for free radical scavenging, detoxification of xenobiotics, and maintenance of redox potential. Exposure of cells to 6-OHDA resulted in an increase in ER-stress-induced apoptotic cell death, which was significantly reduced by treatment of cells with IGF-1. IGF-1 treatment significantly increased BiP and C/EBP homologous protein expression in 6-OHDA-treated cultures. IGF-1 protected cells from 6-OHDA-induced insult by inhibiting intracellular reactive oxygen species generation. Compared with vehicle-treated controls, the expression of Nrf2 and heme oxygenase-1 (HO-1) was increased in 6-OHDA-treated cells. IGF-1 significantly up-regulated HO-1 in cells exposed to 6-OHDA. These results suggest that IGF-1 augment cellular anti-oxidant defense mechanism, at least in part, through the up-regulation of HO-1 expression.

Ghrelin stimulates proliferation, migration and differentiation of neural progenitors from the subventricular zone in the adult mice.

Ghrelin has been shown to regulate neurogenesis in the hippocampus. The aim of this study was to investigate the possible influence of ghrelin on cell proliferation and neuroblast formation in the subventricular zone (SVZ) and rostral migratory system (RMS) and generation of interneurons in the olfactory bulb (OB). We found that ghrelin receptors were expressed in the SVZ-RMS-OB system. Ghrelin knockout (GKO) mice have fewer proliferating neural progenitor cells and neuroblasts in the SVZ, while ghrelin administration attenuated these changes. We also found that not only the number of BrdU-labeled cells but also the fraction of migratory neuroblasts in the RMS was decreased in the GKO mice compared with controls. Treatment of GKO mice with ghrelin restored these numbers to the wild-type control values. Far fewer BrdU/NeuN double-labeled cells were found in the OB of GKO mice than in wild-type mice 4 weeks after labeling, which were increased by ghrelin replacement. GKO mice showed less numbers of BrdU/calbindin, BrdU/calretinin and BrdU/tyrosine hydroxylase double-labeled cells in the periglomerular layer of the OB. However, these numbers were increased to wild-type values after ghrelin administration. Finally, in the GH-deficient spontaneous dwarf rats, ghrelin increased the number of progenitor cells and neuroblasts in the SVZ, without significant effect on the differentiation in the OB. These findings suggest that ghrelin is involved in the regulation of proliferation of progenitor cells in the SVZ, the number of migratory neuroblasts in the SVZ, and the differentiation of interneurons in the OB.