| Activation of the hypothalamic-pituitary-adrenal (HPA) axis is recognizedas a defining endocrine feature of the stress response. Corticotropin releasinghormone (CRH) plays a central regulatory role in the hypothalamic-pituitary-adrenal stress response system. Recently, many studies reported that as animportant neuromitter, CRH played an important role on emotional reactions,learning and memory, and damage and growth of nervous system.The hippocampus is an important part of limbic system and is crucial forlearning and memory in mammals. Stress affects neuronal function andviability to a different extent, and severe, prolonged stress is even thought toinduce neuronal death, possibly involved in apoptosis. Because numerousglucocorticoid receptors locate at the pyramidal cells of hippocampus, someresearchers thought that stress affected hippocampal neuron function throughthe glucocorticoid receptors. However, lots of researches don't support thisviewpoint. For example, chronic stresses still can affect hippocampal neuronfunction when the adrenal glands are ectomied. Recent findings support CRHand its receptor could participate in the stress-induced hippocampaldysfunction, but the underlying mechanisms are not fully understood.Cell apoptosis is an essential event in normal life and development, as wellas in the pathophysiological processes that lead to disease. It has become clearthat each of the main cellular organelles can participate in cell death signalingpathways, including mitochondrial-and death-receptor-mediated apoptoticpathways, and recent advances have highlighted the importance of theendoplasmic reticulum (ER) in cell death processes. The endoplasmicreticulum (ER) is an important subcellular organelle that is responsible for theproper folding and sorting of proteins. It is susceptible to various stresses that provoke the accumulation of unfolded proteins in the ER lumen. ER stressresponse, also known as the unfolded protein response (UPR), is triggered byaccumulation of unfolded protein in the ER lumen. UPR signaling can protectcells from such ER stress by expanding the amount of ER in the cell,enhancing the degradation of misfolded proteins, and reducing the synthesis ofnew proteins. If homeostasis cannot be reestablished, however, UPR signalingeventually induces cell death by apoptosis. ER stress is considered an early orinitial response of cells under stress or damage and linked to neuronal death invarious neurodegenerative diseases, such as Alzheimer's disease, Parkinson'sdisease and amyotrophic lateral sclerosis. In these processes, ER stress causescell damage and apoptosis but inhibiting ER stress response pathways mayprovide nervous system protection.Based on these findings, we proposed that CRH might play an essential rolein stress-induced hippocampal dysfunction through endoplasmic reticulumstress. In this study, we used the rat restraint stress model and primary culturedhippocampal neurons to investigate the effects of CRH on hippocampalneuron in vivo and in vitro. Furthermore, we also examined the roles ofendoplasmic reticulum stress in CRH-induced neuron apoptosis. It is alsoworth mentioning that, dissimilar to other stress responses, the mediators ofER stress are specific and well defined. Therefore, our study will provideimportant views in deeply understanding the roles of CRH in stress andplausible strategies for diagnostics and therapy in stress induced nervoussystem defects.Part1: The endoplasmic reticulum stress mediates hippocampus cellapoptosis of restraint stress rats.Objective: Through rat restraint stress model, to explore the effects of CRHand its receptor on hippocampus cell apoptosis in vivo. Moreover, themechanism of endoplasmic reticulum stress is also examined.Methods:1CP154526(the CRH receptor1antagonist), salubrinal (the endoplasmicreticulum stress inhibitor), or normal saline was injected into the lateral ventricle30min before restraint stress, using a stereotaxic instrument.2The plasma CRH and corticosterone levels were tested by ELISA.3The protein expressions of GRP78and CHOP, the endoplasmic reticulumstress markers, were determined by immunohistochemistry.4The protein levels of GRP78, CHOP and caspase12were examined bywestern blotting.5Apoptotic cells of different groups in vivo were determined by AnnexinV/PI apoptosis detection kit according to manufacturer's protocol.Results:1Compared with control group, the body weight were significantlydecreased in restraint stress rats. However, compared with restraint stressgroup or normal saline administration before restraint stress, CP154526orsalubrinal administration before restraint stress did not affect the body weight.2The plasma CRH and corticosterone levels tested by ELISA were bothsignificantly increased in restraint stress rats, compared with control group.However, compared with restraint stress group or normal saline administrationbefore restraint stress, CP154526administration by intracerebroventricularinjection didn't attenuate these increases.3Immunohistochemistry staining indicated that GRP78and CHOP, thebiomarkers of ER stress, positive cells were significantly increased in the rathippocampus of restraint stress model, compared with control group.Salubrinal administration by intracerebroventricular injection attenuatedGRP78and CHOP-positive expression.4Similarly, the protein levels of GRP78and CHOP detected by westernblotting were also elevated in restraint stress group, and the increases of theseproteins were effectively inhibited by salubrinal. Furthermore, caspase12wascleaved in restraint stress group, and the cleavage of caspase12was attenuatedby salubrinal administration.5Apoptosis was determined by flow cytometry. This assay was dividedapoptotic cells into two stages: early apoptotic (Annexin V+/PI) and lateapoptotic (Annexin V+/PI+) cells, the two parts of cells represented the total cells of apoptosis. Compared with control group, apoptosis rate wassignificantly increased in restraint stress group. CP154526or salubrinaladministration both effectively reduced the apoptotic rate, although it was notcompletely abolished.Conclusion: In restraint stress rats, CRH can exert hippocampus defects byactivating CRHR1. Moreover, the endoplasmic reticulum stress response isactivated and plays an important role in cell apoptosis.Part2: The endoplasmic reticulum stress mediates CRH-inducedprimary cultured rat hippocampal neuron apoptosis.Objective: Through primary cultured hippocampal neurons, to explore theeffects of CRH on hippocampal neuron apoptosis in vitro. Furthermore, themechanism of endoplasmic reticulum stress is also examined.Methods:1The primary cultured hippocampal neurons were cultured for7d inneurobasal medium with2%B27supplement as described previously.Cultures were maintained at37℃in a humidified atmosphere of95%air and5%CO2. Then, the neurons were identified by immunocytochemistry orimmunofluorescence with antibody against microtubule associated protein-2(MAP2), which is marker for neurons.2Neuron survival rates were tested after different concentration of CRH(10-9M-10-5M) or different times (1,4,8,16,24,48h) treatment by MTTassay.3In order to examine the involvement of CRH receptor in CRH-inducedapoptosis, CP154526, CRHR1antagonist, was co-treated with10-8M CRH.Apoptosis was determined by TUNEL method. Hoechst, the nuclear dye, wasused to identify the cells in the field. Labeled neurons were analyzed with afluorescent microscope.4The effects of CRH on protein levels of ER stress biomarkers, GRP78,CHOP and caspase12, were investigated in hippocampal neurons by westernblotting. Furthermore, the mRNA levels of GRP78and CHOP were examinedby real time RT-PCR. 5To investigate CRH-induced apoptosis and its relevance to ER stress,Annexin V and PI staining was used to measure the hippocampal neuronapoptotic rate.Results:1The primary cultured hippocampal neurons were matured when culturedfor7days, and cell body showed chubbiness and dendrites connected eachother. The cultured neurons were identified with antibody against MAP2, andmore than90%neurons were used in the following experiments.2CRH decreased cell viability in a concentration-and time-dependentmanner. Compared with untreated controls, CRH (10-8M-10-5M) graduallydecreased cell viability. Therefore, the lower cytotoxic concentration,10-8M,was selected to investigate the time-dependent manner of CRH and itsrelevance to apoptosis. For16,24,48h, CRH produced a significant15.3%,17.8%, and24.6%decrease in neuronal viability as compared with untreatedcontrols. Therefore, the shortest action time of CRH was16h, which wasselected to examine cell apoptosis.3The result of TUNEL test demonstrated that the number ofTUNEL-positive cells was significantly increased in neurons treated withCRH, compared with untreated controls, and CP154526markedly abolishedthe increase in apoptosis. These data suggested that CRHR1participated in theCRH-induced apoptosis.4CRH significantly increased the protein and mRNA levels of GRP78andCHOP, compared to the untreated control, and the increases by CRH wereeffectively inhibited by salubrinal. As expected, the characterized ER stressinducer, tunicamycin robustly increased the protein and mRNA levels ofGRP78and CHOP.Caspase-12, which is identified as the first ER-associated member of thecaspase family, is activated by ER stress. This novel caspase is regarded as arepresentative molecule implicated in the cell death-executing mechanismsrelevant to ER stress. In cultured neurons, the cleavage of caspase-12occurredafter CRH addition, and decreased when co-treated salubrinal. Tunicamycin, also significantly increased the cleavage of caspase-12.5To investigate CRH-induced apoptosis and its relevance to ER stress,Annexin V and PI staining was used to measure the hippocampal neuronapoptotic rate. Compared with untreated control, the apoptotic rate wassignificantly increased when CRH treatment and decreased when co-treatedwith salubrinal. Tunicamyxin also induced observable apoptosis.Conclusion: In primary cultured hippocampal neurons, CRH can decreasecell viability in a concentration-and time-dependent manner. CRHR1participated in the CRH-induced apoptosis. Furthermore, there is a strongassociation between endoplasmic reticulum stress and apoptotic cell death inprimary cultured hippocampal neurons exposed to CRH.Part3: The IRE1/ASK1/JNK signaling pathway of endoplasmicreticulum stress is involved in CRH-induced hippocampal neuronapoptosis.Objective: The above two part studies have showed that CRH, throughactivating CRHR1, induced the ER stress response both in restraint stress ratsand in primary cultured hippocampal neurons. However, the mechanism ofendoplasmic reticulum stress-induced apoptosis has still been complicated. Inthis part, the IRE1/ASK1/JNK signaling pathway of endoplasmic reticulumstress is examined to elucidate the plausible strategies for diagnostics andtherapy in stress induced nervous system defects.Methods:1Salubrinal (ER stress inhibitor), thioredoxin (TR, ASK1inhibitor) orSP600125(SP, JNK inhibitor) was added to CRH to co-culture thehippocampal neurons.2The total or phosphorylated protein levels of IRE1, ASK1and JNK wereexamined by western blotting.3Annexin V and PI staining was used to measure the hippocampal neuronapoptotic rate.Results:1In order to determine the signaling pathway of CRH-induced ER stress, the IRE1/ASK1/JNK cascade of ER stress was investigated in hippocampalneurons by western blotting. CRH stimulated phosphorylation, i.e., activationof IRE1, ASK1and JNK, and the phosphorylation of these proteins by CRHwere effectively inhibited by salubrinal. Tunicamycin also enhanced activationof these proteins. However, the expressions of total proteins did not vary.2Compared with untreated control, the percentage of cells including earlyand late apoptotic cells was increased significantly when CRH treatment.Co-treated with thioredoxin (TR, ASK1inhibitor) or SP600125(SP, JNKinhibitor), the apoptotic rates were decreased, although they were notcompletely abolished.Conclusion: The IRE1/ASK1/JNK signaling pathway of endoplasmicreticulum stress is involved in CRH-induced hippocampal neuron apoptosis.In summary, our studies provide important views in deeply understandingthe roles of ER stress and apoptosis in CRH-induced neuron damage. It willoffer a preliminary rationale for studies assessing the effects of CRH onstress-mediated physiological dysfunction. It is also worth mentioning that,dissimilar to other stress responses, the mediators of ER stress are specific andwell defined; therefore, they could be useful targets for therapy. Inhibitor ofthe IRE1-TRAF2interaction by small chemicals or by antagonisticIRE1-interacting adaptor proteins will be a plausible strategy forstress-induced nervous system defects. |
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