CRH-CRHR1/GABA Signaling Pathways In The Molecular Pathogenesis Of Depression

Depression is one of the most concerned problems of mental diseases, the role of stress-related neuroendocrine system in the pathogenesis of depression is recently attracting more and more attentions. Hyper-activation of the hypothalamic pituitary adrenal (HPA) axis is the characteristic neuroendocrine alteration in depression. As the central driving force of regulating HPA axis activation, the hypothalamic corticotropin releasing hormone (Corticotropin-releasing Hormone, CRH; known as corticotropin-releasing Factor, CRF in Rodents) activate the downstream signal pathway cascades through interacting with its receptor (coticotropin-releasing hormone receptorl, CRHR1), leading to activation of the HPA axis, which plays an crucial role in the pathogenesis of depression. Clinical researches showed that, antidepressants can decrease the expression of CRH in patients and healthy populations as well as inhibit the activity of HPA axis, and suggested that the anti-depressant effects are related to CRHR1.This study had focused on the following two parts, Part I Molecular mechanisms of high susceptibility to stress in the tree shrew model as well as Part II Local circuit of CRHR/GABAR in stress responses, to explore the mechanisms of stress induced molecular pathogeneses in depressive disorders of the brain.In the field on depression research, until recently the progresses on pathological researches, drug discovery and clinical effectiveness basing on rodent models is still very limited and few substantial breakthrough and development has been achieved, which was partly attributed to the large differences of brain functions between rodents and human brains. Tree shrew, a small mammal that is more closely related to primates than rodents and has high vulnerabilities to social and environmental stresses, is a promising animal model of studying stress responses and stress-related disorders. But the mechanisms of its high susceptibility to stress still remain unknown. One of the aims of our research is to explore the molecular mechanisms of the high susceptibility to stress of tree shrew:1. Characterization of the tree shrew CRH gene and their CRH hormones had similar binding affinities to their receptor CRHR1 and similar capabilities to activate downstream signaling pathways.It had been found that mature tree shrew CRH peptide contained a single amino acid mutation compared with that of human in C terminal region, based on our previous gene and protein sequence alignments results. To investigate if the specific mutations between human and tree shrew CRH and CRHR1s had impacts on their physiological pathways of stress responses, we respectively synthesized human CRH(hCRH) and tree shrew CRH(tCRH). followed by comparing their functions in the pituitary cell line, which focused on the activation of downstream signaling pathways. Furthermore, we had over-expressed human CRHR1 (hCRHR1) and tree shrew CRHR1 (tsCRHR1) in cells and further invested the downstream signal activation levels evoked by their corresponding ligands. as well as the time and dose effects between hCRH and tCRH. Simultaneously, we had determined the binding affinities of human and tree shrew CRH to the extracellular domains of their corresponding receptors. Our results had provided informations to preliminarily understand the pathology of depression in tree shrews exposed to stress and to prove the rationality of using tree shrews as animal models in researches of depressive and stress-related disorders.2. GR recruits transcription factors to the active GRE site to form a trans-activation complex to positively regulate CRH gene expressionBased on our initial results of the CRH gene promoter activity analysis in tree shrew and human, we had first confirmed an active GRE loci (GREa) in the CRH promoter region contributes to the higher CRH promoter activity of tree shrew. To explore the biological mechanism of the large amount of CRH synthesis and releasing under stress conditions in tree shrews, we further screened and identified transcription factors that participated in activation of CRH promoter activity in tree shrew at the aGRE site such as SRC-1; Furthermore, by using BiFC dynamic fluorescence imaging we establish an cellular model for real-time observation of CRH and CRH receptor interaction;These findings implicated novel molecular bases of the stress-related diseases in tree shrew and provide foundations to the applications of this animal model in the researches on depression.Numerous studies have demonstrated that in patients of depression there had been aberrations in the structure and functions of the Pre-Frontal Cortex. In addition, there have been close relationships between the pre-frontal cortex and the HPA axis of Hypothalamus. Our hypothesis of the Prefrontal cortex-Hypothalamus circuit suggested that Pyramidal Neurons in the Prefrontal Cortex excite interneurons to negatively regulate the activities of hypothalamic GABAergic neurons across the brain regions.3) CRH signal promote GABAAR membrane insertion. Based on our previouse research findings that acute restraint stress treatment increases both CRH and CRHR1 mRNA expression in Prefrontal Cortex region, we continued to find out that CRHR1 protein levels were also increased by immuno-histochemical process, moreover, the colocalizations of CRHR1 and GABAAR were observed on both prefrontal and PVN interneurons. Binding of CRH to CRHR1 activates its downstream PKA/PKC signal pathway, accordingly GABAAR Beta and Gama subunits could be phosphorylated by PKA/PKC kinases and subsequently promote their membrane stability. In neuronal cell lines, the results of fluorescence internalization Assay of the membrane receptors and membrane protein extraction experiments confirmed that, in cells expressing CRHR1 and GABAAR, short time CRH incubation will block the endocytosis of surface GABAAR that would enter into the intracellular circulation pathway, meanwhile long time CRH incubation will increase the GABAAR insertion into and expression levels on cell membrane.4) Prefrontal Cortex and hypothalamus negative regulation circuit hypothesis. IHC results showed that in the prefrontal regions, increasing of CRH positive neurons were observed in CUMS treated SD rats, as well as the up-regulation of CRHR1 positive immuno-signals on pyramidal neurons and GABAergic inter-neurons, together with the corresponding increased up-regulation and co-localization of both CRHR1 and GABAAR. Meanwhile on the (a) pyramidal neurons in the PVN region and (b) The co-localization of GABAAR and CRHR1 were observed on the interneurons that negatively regulate CRH neuron activity in the PVN region, simultaneously with enhancement of their immunnal staining signals, suggesting that these interneurons were inhibited and consequently reduced the inhibitory functions of GABA, thus the activities of downstream pyramidal and CRH neurons were Amy polumbo FC inhibition of can of GABA neurons were accordingly increased. In the PVN region of hypothalamus, GABAergic neurons that accepted the GABA inhititory projecions were inhibited, decreased the GABA release and reduced their inhibitory regulation activities on the downstream effect cells, resulting in disinhibition of CRH neurons of the hypothalamus PVN region and lead to the hyperactivation of HPA axis.Our research provided molecular physiological basis for understanding the CRH related modulation of neural circuits in the pathogenesis of depressive disorders.