It is established that growth factors support neuronal survival through the phosphoinositide 3-kinase (PI3K)/Akt pathway but little is known about factors that inhibit Akt signaling in neurons. Given ...that the sst2 type somatostatin receptor exerts pro-apoptotic effects in tumor cells by inhibiting PI3K/Akt, we examined whether neuronal sst2 has similar effects. In primary cortical cultures heterozygously expressing a sst2 knockout/lacZ knockin allele, beta-galactosidase staining revealed expression of the sst2 gene in the vast majority of the cultured neurons. Somatostatin was identified in a subpopulation of neurons by immunocytochemistry. Immunoblots showed a strong reduction of Akt phosphorylation at S473 in wild type cultures undergoing stimulation with the sst2 agonist BIM-23244. While the sst2 agonist did not cause neuronal death under control conditions, it promoted hypoxic/ischemic neuronal death in cortical cultures subjected to oxygen/glucose deprivation. Treatment of wild type cultures with the sst2 antagonist BIM-23627 and homozygous deletion of the sst2 gene were protective in this paradigm, suggesting that endogenous somatostatin signals through sst2 during hypoxia/ischemia. In fact, examination of sst2 phosphorylation and sst2 internalization provided evidence for sst2 activation in neurons subjected to oxygen/glucose deprivation. Thus, somatostatin acts as a sensor of hypoxia/ischemia, inhibits Akt activity through sst2 and aggravates hypoxic/ischemic neuronal death. sst2-selective antagonists are proposed as neuroprotectants in stroke.
•sst2 somatostatin receptor and somatostatin are expressed in cultured cortical neurons.•Neuronal sst2 is activated by endogenous somatostatin during oxygen/glucose deprivation.•sst2 activation reduces Akt phosphorylation in cortical neuronal cultures.•sst2 signaling aggravates hypoxic/ischemic neuronal death.•Use of sst2 antagonists in stroke might maintain Akt signaling and protect from neuronal death.
Abstract Morphine modulates neuronal and immune cell functions via μ-opioid receptors. In primary and Jurkat T cells, and Raji B cells μ-opioid receptor transcripts were detected only after ...stimulation of the cells with IL-4 or TNF-α. Moreover, the amount of the induced μ-opioid receptor mRNA in the immune cells was 15 to 200 times less than those in primary cortical and SH SY5Y neuronal cells. Nevertheless, μ-opioid receptor mRNA in immune cells is processed to functional receptors, as demonstrated by morphine-mediated phosphorylation of mitogen activated protein kinase, morphine-mediated up-regulation of IL-4 mRNA and coupling to adenylyl cyclase in Jurkat cells.
To test the hypothesis of an involvement of tachykinins in destabilization and hyperexcitation of neuronal circuits, gliosis, and neuroinflammation during cerebral ischemia, we investigated ...cell-specific expressional changes of the genes encoding substance P (SP), neurokinin B (NKB), and the tachykinin/neurokinin receptors (NK1, NK2, and NK3) after middle cerebral artery occlusion (MCAO) in the rat. Our analysis by quantitative in situ hybridization, immunohistochemistry, and confocal microscopy was concentrated on cerebrocortical areas that survive primary infarction but undergo secondary damage. Here, SP-encoding preprotachykinin-A and NK1 mRNA levels and SP-like immunoreactivity were transiently increased in GABAergic interneurons at 2 d after MCAO. Coincidently, MCAO caused a marked expression of SP and NK1 in a subpopulation of glutamatergic pyramidal cells, and in some neurons SP and NK1 mRNAs were coinduced. Elevated levels of the NKB-encoding preprotachykinin-B mRNA and of NKB-like immunoreactivity at 2 and 7 d after MCAO were confined to GABAergic interneurons. In parallel, the expression of NK3 was markedly downregulated in pyramidal neurons. MCAO caused transient NK1 expression in activated cerebrovenular endothelium within and adjacent to the infarct. NK1 expression was absent from activated astroglia or microglia. The differential ischemia-induced plasticity of the tachykinin system in distinct inhibitory and excitatory cerebrocortical circuits suggests that it may be involved in the balance of endogenous neuroprotection and neurotoxicity by enhancing GABAergic inhibitory circuits or by facilitating glutamate-mediated hyperexcitability. The transient induction of NK1 in cerebrovenular endothelium may contribute to ischemia-induced edema and leukocyte diapedesis. Brain tachykinin receptors are proposed as potential drug targets in stroke.
Pituitary adenylate cyclase activating peptide (PACAP) and the chemokine stromal cell-derived factor (SDF-1) have been implicated in neuroprotection, neurogenesis, and regeneration. Focal ischemia is ...associated with rapid upregulation of PACAP in perifocal neurons and delayed induction of SDF-1 in hypoxic/ischemic tissues, the latter process being involved in the recruitment of stem cells and inflammatory cells. Here, we studied mRNA patterns of PACAP, SDF-1 and the cognate receptors PAC1 and CXCR4 by in situ hybridization in the rat hippocampus after transient global ischemia, a rat model for programmed death of CA1 pyramidal neurons. Cell death in CA1 was not associated with local induction of PACAP and SDF-1 expression or recruitment of CXCR4-expressing infiltrates. However, there was a transient, almost complete loss of SDF-1 expression in microvessels in all hippocampal regions. Granule cells transiently showed a decrease of SDF-1 and an increase of PACAP expression. While PAC1 mRNA was moderately decreased throughout the hippocampus, CXCR4 expression was selectively increased in the subgranular layer. We propose that altered PACAP and SDF-1 gene expression in granule cells plays a role in regulated neurogenesis after global ischemia. The finding that programmed neuronal death after global ischemia was not associated with SDF-1 upregulation or recruitment of CXCR4-expressing cells is in sharp contrast to SDF-1/CXCR4-mediated infiltration of infarct tissue after focal ischemia. Hence, the different modes of neuronal death after focal and global ischemia are associated with distinct SDF-1 and PACAP gene regulation patterns and distinct reorganization mechanisms.
The induction of growth factor synthesis in brain tissue by β
2-adrenoceptor agonists, such as clenbuterol, is a promising approach to protect brain tissue from ischemic damage. Clenbuterol (0.01–0.5 ...mg/kg) reduced the cortical infarct volume in Long–Evans rats as measured 7 days after permanent occlusion of the middle cerebral artery. Dosages of clenbuterol higher than 1 mg/kg showed no cerebroprotective effect due to a decrease in blood pressure and an increase in plasma glucose level. The increase in the mRNA level of nerve growth factor (NGF), basic fibroblast growth factor (basic FGF), and transforming growth factor-β
1 (TGF-β
1) mRNA in cortical and hippocampal tissue occurred earlier after middle cerebral artery occlusion and was more pronounced in animals treated with clenbuterol than in controls. In addition, glial fibrillary acidic protein (GFAP) mRNA expression was enhanced in astrocytes 6 h after ischemia in clenbuterol-treated animals. The results suggest that growth factor synthesis is enhanced in activated astrocytes and that this could be the mechanism of clenbuterol-induced cerebroprotection after ischemia.
The olfactory epithelium (OE) possesses unique lifelong neuroregenerative capacities and undergoes constitutive neurogenesis throughout mammalian lifespan. Two populations of stem cells, frequently ...dividing globose basal cells (GBCs) and quiescent horizontal basal cells (HBCs), readily replace olfactory neurons throughout lifetime. Although lineage commitment and neuronal differentiation of stem cells has already been described in terms of transcription factor expression, little is known about external factors balancing between differentiation and self‐renewal. We show here that expression of the CXC‐motif chemokine receptor 4 (CXCR4) distinguishes both types of stem cells. Extensive colocalization analysis revealed exclusive expression of CXCR4 in proliferating GBCs and their neuronal progenies. Moreover, only neuronal lineage cells were derived from CXCR4‐CreER‐tdTomato reporter mice in the OE. Furthermore, Cre‐tdTomato mice specific for HBCs (Nestin+ and Cytokeratin14+) did not reduce CXCR4 expression when bred to mice bearing floxed CXCR4 alleles, and did not show labeling of the neuronal cells. CXCR4 and its ligand CXCL12 were markedly upregulated upon induction of GBC proliferation during injury‐induced regeneration. in vivo overexpression of CXCL12 did downregulate CXCR4 levels, which results in reduced GBC maintenance and neuronal differentiation. We proved that these effects were caused by CXCR4 downregulation rather than over‐activation by showing that the phenotypes of CXCL12‐overexpressing mice were highly similar to the phenotypes of CXCR4 knockout mice. Our results demonstrate functional CXCR4 signaling in GBCs regulates cell cycle exit and neural differentiation. We propose that CXCR4/CXCL12 signaling is an essential regulator of olfactory neurogenesis and provide new insights into the dynamics of neurogenesis in the OE.
CXCL12 is secreted by cells of the lamina propria and activates the chemokine receptor CXCR4 in globose basal cells of the olfactory epithelium. Receptor activation promotes division of these stem cells, and thereby regulates the size of the stem cell pool. Absence of CXCR4 signaling leads to exit from the cell cycle and increased neuronal differentiation.
Somatostatin receptor 2 (SSTR2) mediates neuromodulatory signals of somatostatin and cortistatin in the cerebral cortex. Recently, SSTR2 has been shown to enhance conserved death ligand- and ...mitochondria-mediated apoptotic pathways in non-neuronal cells. Whether somatostatin receptors are activated in cerebrocortical neurons and contribute to neurodegeneration after experimental focal ischemia was unknown until now. Here we examined internalization of SSTR2 in a rat model of middle cerebral artery occlusion (MCAO) by confocal microscopy. At 3 and 6 hr after MCAO, SSTR2 was internalized excessively in cerebrocortical neurons adjacent to the infarct, which was prevented by intracerebroventricular application of the SSTR2-selective antagonist BIM-23627. SSTR2 internalization was associated with a transient depletion of somatostatin from axonal terminals and increased expression of SSTR2 mRNA. The initial loss of somatostatin was followed by an increase in somatostatin mRNA levels, whereas cortistatin mRNA expression was decreased. In SSTR2-deficient mice with lacZ under the control of the SSTR2 promoter, MCAO-induced upregulation of SSTR2 gene expression was less pronounced than in wild types. SSTR2-deficient mice exhibited a 40% reduction of infarct size after permanent distal MCAO and a 63% reduction after transient proximal MCAO. In summary, we provide direct evidence for activation of SSTR2 by an endogenous ligand after focal ischemia. Activation of functional SSTR2 receptors contributes to increased SSTR2 gene expression and postischemic neurodegeneration.
The interaction of mu-opioid receptor (MOPr) with the neuronal membrane glycoprotein M6a is known to facilitate MOPr endocytosis in human embryonic kidney 293 (HEK293) cells. To further study the ...role of M6a in the post-endocytotic sorting of MOPr, we investigated the agonist-induced co-internalization of MOPr and M6a and protein targeting after internalization in HEK293 cells that co-expressed HA-tagged MOPr and Myc-tagged M6a. We found that M6a, MOPr, and Rab 11, a marker for recycling endosomes, co-localized in endocytotic vesicles, indicating that MOPr and M6a are primarily targeted to recycling endosomes after endocytosis. Furthermore, co-expression of M6a augmented the post-endocytotic sorting of delta-opioid receptors into the recycling pathway, indicating that M6a might have a more general role in opioid receptor post-endocytotic sorting. The enhanced post-endocytotic sorting of MOPr into the recycling pathway was accompanied by a decrease in agonist-induced receptor down-regulation of M6a in co-expressing cells. We tested the physiological relevance of these findings in primary cultures of cortical neurons and found that co-expression of M6a markedly increased the translocation of MOPrs from the plasma membrane to intracellular vesicles at steady state and significantly enhanced both constitutive and agonist-induced receptor endocytosis. In conclusion, our results strongly indicate that M6a modulates MOPr endocytosis and post-endocytotic sorting and has an important role in receptor regulation.
Phosphorylation of G protein-coupled receptors (GPCRs) is a key event for cell signaling and regulation of receptor function. Previously, using tandem mass spectrometry, we identified two ...phosphorylation sites at the distal C-terminal tail of the chemokine receptor CXCR4, but were unable to determine which specific residues were phosphorylated. Here, we demonstrate that serines (Ser) 346 and/or 347 (Ser-346/7) of CXCR4 are phosphorylated upon stimulation with the agonist CXCL12 as well as a CXCR4 pepducin, ATI-2341. ATI-2341, a G
heterotrimer-biased CXCR4 agonist, induced more robust phosphorylation of Ser-346/7 compared with CXCL12. Knockdown of G protein-coupled receptor kinase (GRK) 2, GRK3, or GRK6 reduced CXCL12-induced phosphorylation of Ser-346/7 with GRK3 knockdown having the strongest effect, while inhibition of the conventional protein kinase C (PKC) isoforms, particularly PKC
, reduced phosphorylation of Ser-346/7 induced by either CXCL12 or ATI-2341. The loss of GRK3- or PKC-mediated phosphorylation of Ser-346/7 impaired the recruitment of
-arrestin to CXCR4. We also found that a pseudo-substrate peptide inhibitor for PKC
effectively inhibited CXCR4 phosphorylation and signaling, most likely by functioning as a nonspecific CXCR4 antagonist. Together, these studies demonstrate the role Ser-346/7 plays in arrestin recruitment and initiation of receptor desensitization and provide insight into the dysregulation of CXCR4 observed in patients with various forms of WHIM syndrome.
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