Orexin-A protects against oxygen-glucose deprivation/reoxygenation-induced cell damage by inhibiting endoplasmic reticulum stress-mediated apoptosis via the Gi and PI3K signaling pathways

The neuropeptide orexin-A (OXA) has a neuroprotective effect, acting as an anti-apoptotic factor in response to multiple stimuli. Apoptosis induced by endoplasmic reticulum stress (ERS) underlies oxygen-glucose deprivation and reoxygenation (OGD/R)-induced cell damage, an in vitro model of ischemia/reperfusion injury. However, that OXA inhibits ERS-induced apoptosis in the OGD/R model has not been reported. In the present study, we investigated the neuroprotective effect of OXA (0.1 μM) on OGD/R-induced damage in the human neuroblastoma cell line SH-SY5Y. After OXA treatment following 4 h oxygen-glucose deprivation (OGD) and then 4 h reoxygenation (R), cell morphology, viability, and apoptosis were analyzed by histology, Cell Counting Kit-8 assay, and flow cytometry, respectively. Western blotting was used to measure expression levels of ERS- and apoptosis-related proteins. To determine signaling pathways involved in OXA-mediated neuroprotection, the Gi pathway inhibitor pertussis toxin (PTX; 100 ng/mL) and PI3K inhibitor LY294002 (LY; 10 μM) were added. In addition, in order to prove the specificity of these characteristics, the OXA antagonist Suvorexant (DORA; Ki of 0.55 nM and 0.35 nM for OX1R and OX2R) was used for intervention. Our results showed that OGD/R induced cell damage, manifested as morphological changes and a significant decrease in viability. Furthermore, Western blotting detected an increase in ERS-related proteins GRP78, p-IRE1α, p-JNK, and Cleaved caspase-12, as well as apoptosis-related proteins Cleaved caspase-3 and Bax, and a decrease in the anti-apoptosis factor Bcl-2. OXA intervention alleviated the degree of cellular damage, and protein expression was also reversed. In addition, the protective effect of OXA was reduced by adding PTX and LY. Meanwhile, after the use of DORA, changes in the expression of related proteins were detected, and it was found that the protective effect of OXA was weakened. Collectively, our results indicate that OXA has a neuroprotective effect on OGD/R-induced cell damage by inhibiting ERS-induced apoptosis through the combined action of Gi and PI3K signaling pathways. These findings help to clarify the mechanism underlying the neuroprotective action of OXA, which should aid the development of further candidate drugs, and provide a new therapeutic direction for the treatment of ischemic stroke

Transmembrane peptide 4 and 5 of APJ are essential for its heterodimerization with OX1R

Increasing evidence indicates some G protein-coupled receptors function as a heterodimer, which provide a novel target for therapeutics investigation. However, study on the receptor-receptor interaction interface, a potent target on interfering dimer formation, are still limited. Here, using bioluminescence resonance energy transfer (BRET) combined with co-immunoprecipitation (Co-IP), we found a new constitutive GPCR heterodimer, apelin receptor (APJ)-orexin receptor type 1 (OX1R). Both APJ and OX1R co-internalized when constantly subjected to cognate agonist (apelin-13 or orexin-A) specific to either protomer. Combined with BRET and immunostaining, the in vitro synthesized transmembrane peptides (TMs) interfering experiments suggests that TM4 and 5 of APJ act as the interaction interface of the APJ-OX1R heterodimer, and co-internalization could be disrupted by these peptides as well. Our study not only provide new evidence on GPCR heterodimerization, but address a novel heterodimerization interface, which can be severed as a potential pharmacological target

The orexin/receptor system : molecular mechanism and therapeutic potential for neurological diseases

Orexins, also known as hypocretins, are two neuropeptides secreted from orexin-containing neurons, mainly in the lateral hypothalamus (LH). Orexins orchestrate their effects by binding and activating two G-protein-coupled receptors (GPCRs), orexin receptor type 1 (OX1R) and type 2 (OX2R). Orexin/receptor pathways play vital regulatory roles in many physiological processes, especially feeding behavior, sleep-wake rhythm, reward and addiction and energy balance. Furthermore several reports showed that orexin/receptor pathways are involved in pathological processes of neurological diseases such as narcolepsy, depression, ischemic stroke, drug addiction and Alzheimer's disease (AD). This review article summarizes the expression patterns, physiological functions and potential molecular mechanisms of the orexin/receptor system in neurological diseases, providing an overall framework for considering these pathways from the standpoints of basic research and clinical treatment of neurological diseases

Ghrelin through GHSR1a and OX1R heterodimers reveals a Gαs–cAMP-cAMP response element binding protein signaling pathway in vitro

Growth hormone secretagogue receptor 1α (GHSR1a) and Orexin 1 receptor (OX1R) are involved in various important physiological processes, and have many similar characteristics in function and distribution in peripheral tissues and the central nervous system. We explored the possibility of heterodimerization between GHSR1a and OX1R and revealed a signal transduction pathway mechanism. In this study, bioluminescence and fluorescence resonance energy transfer and co-immunoprecipitation (Co-IP) analyses were performed to demonstrate the formation of functional GHSR1a/OX1R heterodimers. This showed that a peptide corresponding to the 5-transmembrane domain of OX1R impaired heterodimer construction. We found that ghrelin stimulated GHSR1a/OX1R heterodimer cells to increase the activation of Gαs protein, compared to the cells that express GHSR1a. Stimulation of GHSR1a/OX1R heterodimers with orexin-A did not alter GPCR interactions with Gα protein subunits. GHSR1a/OX1R heterodimers induced Gαs and downstream signaling pathway activity, including increase of cAMP-response element luciferase reporter activity and cAMP levels. In addition, ghrelin induced a higher proliferation rate in SH-SY5Y cells than in controls. This suggests that ghrelin GHSR1a/OX1R heterodimers promotes an upregulation of a Gαs-cAMP-cAMP-responsive element signaling pathway in vitro and an increase in neuroblastoma cell proliferation

Heterodimerization of mouse orexin type 2 receptor variants and the effects on signal transduction

Orexin-A and Orexin-B play important roles in many physiological processes in which Orexins orchestrate diverse downstream effects via two G-protein coupled receptors: Orexin1R and Orexin2R. Two alternative C-terminus splice variants of the mouse Orexin receptors mOX2αR and mOX2βR have recently been identified. This study explored the possibility of heterodimerization between mOX2αR and mOX2βR, and investigated novel signal transduction characteristics after stimulation. The dimerization of mOX2αR and mOX2βR was confirmed by BRET and co-immunoprecipitation assays. Meanwhile, in HEK293 cells, co-expression of mOX2αR and mOX2βR resulted in a strengthened increase in activation of ERK1/2, with maximal activation at 5 min and 100 nM. Furthermore, heterodimerization also elicits stronger intracellular Ca2 + elevation after Orexin(s) stimulation, followed by a slower decline in intracellular Ca2 + to a steady endpoint. Protein Kinase C Inhibitor significantly inhibited these downstream effects. In addition, the cAMP response element reporter activities were significantly reduced, whereas the serum response element luciferase and the T-lymphocyte activation of nuclear factor-responsive element reporter activity were significantly up-regulated after Orexin(s) stimulation. Besides, Orexin-A/-B induced a significantly higher rate of HEK293 cell proliferation in cells co-expressing mOX2αR/mOX2βR compared to the control group. Taken together, we provide conclusive evidence that mOX2αR can form a functional heterodimer with mOX2βR and this leads to increased PKC and decreased protein kinase A activity by ERK signal pathway leading to a significant increase in cell proliferation. The nature of this signaling pathway has significant implications for the role of Orexin in the regulation of physiological processes including the homeostasis of feeding