Endocrinology and the brain: Corticotropin-Releasing Hormone signaling

Corticotropin-releasing hormone (CRH) is a key player of basal and stress activated responses in the hypothalamic-pituitary-adrenal axis (HPA) and in extrahypothalamic circuits, where it functions as a neuromodulator to orchestrate humoral and behavioral adaptive responses to stress. This review describes molecular components and cellular mechanisms involved in CRH signaling downstream of its G protein-coupled receptors (GPCRs) CRHR1 and CRHR2, and summarizes recent findings that challenge the classical view of GPCR signaling, and impact on our understanding of CRHRs function. Special emphasis is placed on recent studies of CRH signaling that revealed new mechanistic aspects of cAMP generation and ERK1/2 activation in physiologically relevant contexts of the neurohormone action. In addition, we present an overview of the pathophysiological role of the CRH system, which highlights the need for a precise definition of CRHRs signaling at molecular level to identify novel targets for pharmacological intervention in neuroendocrine tissues and specific brain areas involved in CRH-related disorders.Fil: Inda, María Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Armando, Natalia Giannina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Dos Santos Claro, Paula Ayelen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Silberstein Cuña, Susana Iris. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentin

The Impact of Glucose Variation on Human Astrocytes

Diabetes is a metabolic disorder dysregulating glucose homeostasis. The role of astrocytes in central glucose sensing is poorly understood. But it is recognised they take part in whole-body energy homeostasis, specifically as glucose sensors necessary for the counterregulatory response (CRR) to hypoglycaemia. Iatrogenic hypoglycaemia is the limiting factor to glycaemic control in people with type 1 or type 2 diabetes. Severe hypoglycaemia occurs approximately once per year, whereas, the incidence of minor hypoglycaemia is much greater. Hypoglycaemia impairs awareness of future hypoglycaemia and blunts the CRR, eventually causing hypoglycaemia-associated autonomic failure. The mechanisms of this process are poorly understood. This thesis utilised isolated human astrocytes exposed to acute or recurrent low glucose (RLG) in vitro to mimic glucose variation in diabetes. Cellular responses were characterised of three key astrocyte functions. Firstly, is astrocyte metabolism altered by acute and RLG treatment? Secondly, do isolated human astrocytes become activated by low glucose treatment, and is this affected by RLG? Thirdly, are astrocytic inflammatory pathways altered by acute or RLG? The key findings from this thesis shows for the first time that astrocytic mitochondrial oxidation is increased following RLG, with a concurrent increase in fatty acid dependency but decreased coupling efficiency; glycolytic function is also enhanced. Together, this indicates that astrocytes successfully adapt to low glucose to sustain intracellular nucleotide ratios. Contrary to previous work, these human astrocytes do not respond to low glucose by Ca2+-dependent activation. However, the astrocytes do increase inflammatory cytokine release following acute and RLG. Lastly, for the first time an RNA-sequencing approach has been used to identify low glucose-induced differential gene expression. Together these findings support the argument that astrocytes are sensitive to low glucose and may be important in glucose sensation and the CRR

Regulation of Extracellular Signal-Regulated Kinase During Long-term Potentiation in Area CA1 of the Rat Hippocampus IN VIVO

The extracellular signal-regulated kinase (ERK) cascade can transduce cell-surface signals to the nucleus in post-synaptic neurons during hippocampus-dependent learning and hippocampus-dependent synaptic plasticity, yet, whether the cascade can convey information about stimulus frequency or synaptic modification direction to the nucleus during plasticity events has not been addressed. The objective of the current study was to investigate whether ERK regulation differs as a function of stimulus frequency and in accordance with synaptic modification direction by comparing ERK regulation during LTP in area CA1 of the hippocampus in vivo to previous findings for ERK regulation during LTD in area CA1 in vivo (Thiels et al., 2002). The ultimate goal was to determine whether ERK functions as a general or as a specific plasticity kinase during synaptic plasticity events in the hippocampus. Using a combination of in vivo electrophysiology, pharmacology and Western blot analysis, I demonstrate that: (1) LTP induced by high-frequency stimulation applied to commissural fiber inputs to area CA1 pyramidal cells in the adult hippocampus in vivo is accompanied by a rapid yet transient increase in ERK2 activation; (2) blockade of NMDA receptors by MK-801 blocks both LTP induction and the associated increase in ERK2 activation; (3) HFS delivered in the presence of the ERK kinase inhibitor SL327 fails to produce a persistent potentiation; (5) phosphorylation of the transcriptional regulator cAMP response element-binding protein (CREB) is increased after HFS; and (6) inhibition of ERK2 activation by SL327 blocks this observed increase in pCREB. The similarity of the current findings with previous findings for ERK2 activation and regulation during LTD in area CA1 in vivo, suggests that the ERK cascade conveys a general as opposed to a specific plasticity signal during these two forms of synaptic plasticity in area CA1 in vivo. Differences in the coupling of ERK2 activation to CREB phosphorylation between LTP and LTD (Thiels et al., 2002), suggest that other signaling cascades are most likely operative in determining the direction of synaptic modification during bidirectional synaptic plasticity in the hippocampus

Systemic adiponectin malfunction as a risk factor for cardiovascular disease.

Adiponectin (Ad) is an abundant protein hormone regulatory of numerous metabolic processes. The 30 kDa protein originates from adipose tissue, with full-length and globular domain circulatory forms. A collagenous domain within Ad leads to spontaneous self-assemblage into various oligomeric isoforms, including trimers, hexamers, and high-molecular-weight multimers. Two membrane-spanning receptors for Ad have been identified, with differing concentration distribution in various body tissues. The major intracellular pathway activated by Ad includes phosphorylation of AMP-activated protein kinase, which is responsible for many of Ad\u27s metabolic regulatory, anti-inflammatory, vascular protective, and anti-ischemic properties. Additionally, several AMP-activated protein kinase-independent mechanisms responsible for Ad\u27s anti-inflammatory and anti-ischemic (resulting in cardioprotective) effects have also been discovered. Since its 1995 discovery, Ad has garnered considerable attention for its role in diabetic and cardiovascular pathology. Clinical observations have demonstrated the association of hypoadiponectinemia in patients with obesity, cardiovascular disease, and insulin resistance. In this review, we elaborate currently known information about Ad malfunction and deficiency pertaining to cardiovascular disease risk (including atherosclerosis, endothelial dysfunction, and cardiac injury), as well as review evidence supporting Ad resistance as a novel risk factor for cardiovascular injury, providing insight about the future of Ad research and the protein\u27s potential therapeutic benefits

Molecular Mechanisms Underlying Ca2+/Calmodulin-Dependent Protein Kinase Kinase Signal Transduction

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is the activating kinase for multiple downstream kinases, including CaM-kinase I (CaMKI), CaM-kinase IV (CaMKIV), protein kinase B (PKB/Akt), and 5'AMP-kinase (AMPK), through the phosphorylation of their activation-loop Thr residues in response to increasing the intracellular Ca2+ concentration, as CaMKK itself is a Ca2+/CaM-dependent enzyme. The CaMKK-mediated kinase cascade plays important roles in a number of Ca2+-dependent pathways, such as neuronal morphogenesis and plasticity, transcriptional activation, autophagy, and metabolic regulation, as well as in pathophysiological pathways, including cancer progression, metabolic syndrome, and mental disorders. This review focuses on the molecular mechanism underlying CaMKK-mediated signal transduction in normal and pathophysiological conditions. We summarize the current knowledge of the structural, functional, and physiological properties of the regulatory kinase, CaMKK, and the development and application of its pharmacological inhibitors

EVT-701 : inhibition du complexe I de la chaine respiratoire comme stratégie thérapeutique en cancer

Cibler le métabolisme énergétique a récemment été identifié comme une approche thérapeutique prometteuse pour lutter contre le cancer. Les cellules cancéreuses ont des caractéristiques métaboliques particulières qui les rendent différentes des cellules "normales". Le métabolisme oxydatif mitochondrial (OxPhos) a été proposé comme une vulnérabilité métabolique et un des mécanismes de résistance thérapeutique impliquée dans la rechute pour plusieurs types de tumeurs. Le fonctionnement de la chaine respiratoire mitochondriale a pour finalité la production de l'ATP mitochondrial, source d'énergie pour les cellules. Elle a également pour but de soutenir la prolifération cellulaire grâce au maintien des niveaux des intermédiaires métaboliques essentiels pour la biosynthèse nucléotidique. Différents types de cancer du sous-type OxPhos montrent une surexpression des gènes du métabolisme mitochondrial. Ces cancers OxPhos sont ainsi plus vulnérables au ciblage thérapeutique de la respiration mitochondriale. De plus, les cellules cancéreuses peuvent augmenter leur métabolisme oxydatif en réponse à la chimiothérapie, comme démontré pour les leucémies aigües myéloïdes (LAM). Ceci ouvre une opportunité unique pour lutter contre la chimiorésistance et les rechutes. Il existe ainsi un besoin évident de développer des agents thérapeutiques sûrs et sélectifs pour cibler la dépendance à l'OxPhos des cellules cancéreuses. Ces travaux doctoraux présentent l'EVT-701, un nouvel inhibiteur du complexe I de la chaine respiratoire (ETC I) et ses effets anti-cancéreux et notamment anti-leucémiques. Ce composé est sélectif, toléré et démontre une activité significative dans plusieurs modèles de tumeurs solides. Nous avons aussi évalué l'effet de l'EVT-701 dans un contexte de chimiorésistance et caractérisé son effet immuno-modulateur dans un modèle murin de LAM. Ainsi, nous avons mis en évidence un nouveau rôle de l'ETC I dans la régulation de l'expression des immune checkpoints molécules PD-L1 et CD39 dans des cellules de LAM. Enfin, ces travaux confirment le rôle de l'ETC I dans le cancer, renforcent l'intérêt thérapeutique de moduler son activité et montrent son rôle dans l'immuno-modulation, jusqu'alors méconnu pour les LAM.Targeting energetic metabolism has emerged in the recent years as an attractive and promising anti-cancer therapy. Cancer cells show metabolic features that distinguish them from normal cells, going beyond the initially described Warburg effect. Reliance on oxidative phosphorylation (OxPhos) has been identified as a vulnerability of many cancer types. Functional electron transport chain (ETC) is essential for mitochondrial ATP production, as well as for supporting cell proliferation by maintaining appropriate levels of metabolic intermediates essential for nucleotide biosynthesis. Cells from different cancer types overexpress OxPhos and mitochondria genes, displaying enhanced vulnerability to the inhibition of mitochondrial respiration. Furthermore, some molecular alterations such as LKB1 deficiency also render cancer cells susceptible to ETC inhibition. Interestingly, chemotherapy has also been reported to increase OxPhos metabolism in many cancer types including acute myeloid leukemia (AML), paving the way for a novel therapeutic opportunity to eradicate chemoresistance and prevent patient relapse. Overall, this enhanced OxPhos dependence strengths the need for new selective and safe therapeutic agents. This thesis introduces a novel ETC I inhibitor, EVT-701, that is selective and safer than other drug candidates in this target class. It shows anti-cancer activity in several solid cancer tumor models as single agent. Furthermore, we assessed the anti-leukemic effects of EVT-701 in an AML model of resistance to chemotherapy and we discovered a new regulatory role of ETC I on immune checkpoints PD-L1 and CD39 in leukemic cells. In summary, my PhD work highlights the role of ETC I in cancer, the interest to target it, as well as it identifies an immunomodulatory role for ETC I in AML

Adenylyl cyclase 5/6 underlie PIP3 dependent regulation

A wide variety of signaling substances such as hormones, neurotransmitters, odorants and chemokines control intracellular signaling by regulating the production of the second messenger cAMP. By activating Epac, PKA and cyclic nucleotide-gated ion channels, the production of cAMP alters a wide range of biological processes including cell division and metabolism. A number of GPCRs controls intracellular cAMP levels via stimulatory or inhibitory G proteins via adenylyl cyclases. The function of the broadly expressed AC5 and AC6 is enhanced by stimulatory (Gαs) or attenuated by inhibitory (Gαi) G proteins. Mechanistically both inhibition and stimulation is mediated via a direct protein-protein interaction. In addition to this direct regulation, several previous studies reported a cAMP rebound stimulation after withdrawal of Gi stimulation in cardiac myocytes for which the mechanism is debated (Hartzell, 1988; Wang & Lipsius, 1995). A similar cAMP rebound response was observed previously in our lab after termination of α2A-AR adrenergic receptor activation in HEK293T cells (Markus et al., 2013). The present study was aimed at investigating mechanisms underlying Gi-induced cAMP rebound effects. Many genetically encoded biosensors have been developed based on fluorescence resonance energy transfer (FRET) to visualize the spatiotemporal dynamics of various intracellular signals including second messengers. FRET-based cAMP biosensor (Epac1-camps) as well as heterologous overexpression system was used to investigate the mechanisms underlying Gi-mediated cAMP rebound stimulation in cardiac myocytes and also in heterologous expression system. When studying the mechanism of the long-known phenomenon of cAMP rebound stimulation after withdrawal of Gi stimulation in cardiac myocytes, we observed a PTX-sensitive/Gi-mediated/ adenylyl cyclase (type 5/6)/ cAMP-dependent pathway for this cAMP rebound stimulation. In addition, we observed that inhibition of Gβγ by gallein led to an attenuation of the AC5- mediated cAMP rebound response, although, overexpression of AC4 did not produce additional cAMP stimulation. This implies that different Gβγ-mediated signaling pathways may exist. Interestingly, we observed that PI3K inhibitor attenuates AC5/6-dependent cAMP rebound effects. This indicated that Gi-mediated cAMP rebound response was mediated via the PI3K-dependent pathway. Indeed, overexpression of PIP3-specific phosphatase PTEN confirmed that PIP3 itself either directly or indirectly mediated Gi-dependent cAMP rebound responses. Additionally, inhibition of PIP2-specific phosphatase SHIP and downstream events of PIP3-dependent regulation of Akt further confirm the influence of PIP3 on cAMP rebound levels. Indeed, surpassing Gi-mediated PI3K activation through PDGF-receptor stimulation strengthens this pathway. In addition, we confirmed that inhibition of PI3K also prevented cAMP rebound response after withdrawal of ACh in atrial myocytes. We suppose that the novel PIP3 dependent regulation of AC5/6 might represent a missing mechanism that explains physiological phenomena such as post vagal tachycardia