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GSK3B has been an interesting drug target in the pharmaceutical industry. Its dysfunctional expression has prognostic significance in the top 3 cause of death associated with non-communicable diseases (cancer, Alzheimer's disease and type 2 diabetes). Previous studies have shown clearly that inhibiting GSK3B has proven therapeutic significance in Alzheimer's disease, but its contribution to various cancers has not been clearly resolved. In this study we report the contribution and prognostic significance of GSK3B to two breast cancer subtypes; ductal carcinoma in-situ (DCIS) and invasive ductal carcinoma (IDC) using the Oncomine platform. We performed high throughput screening using molecular docking. We identified BT-000775, a compound that was subjected to further computational hit optimization protocols. Through computational predictions, BT-000775 is a highly selective GSK3B inhibitor, with superior binding affinity and robust ADME profiles suitable for the patho-physiological presentations.

Glycogen synthase kinase-3 (GSK-3) is a regulator of signaling pathways. KRas is frequently mutated in pancreatic cancers. The growth of certain pancreatic cancers is KRas-dependent and can be suppressed by GSK-3 inhibitors, documenting a link between KRas and GSK-3. To further elucidate the roles of GSK-3β in drug-resistance, we transfected KRas-dependent MIA-PaCa-2 pancreatic cells with wild-type (WT) and kinase-dead (KD) forms of GSK-3β. Transfection of MIA-PaCa-2 cells with WT-GSK-3β increased their resistance to various chemotherapeutic drugs and certain small molecule inhibitors. Transfection of cells with KD-GSK-3β often increased therapeutic sensitivity. An exception was observed with cells transfected with WT-GSK-3β and sensitivity to the BCL2/BCLXL ABT737 inhibitor. WT-GSK-3β reduced glycolytic capacity of the cells but did not affect the basal glycolysis and mitochondrial respiration. KD-GSK-3β decreased both basal glycolysis and glycolytic capacity and reduced mitochondrial respiration in MIA-PaCa-2 cells. As a comparison, the effects of GSK-3 on MCF-7 breast cancer cells, which have mutant PIK3CA, were examined. KD-GSK-3β increased the resistance of MCF-7 cells to chemotherapeutic drugs and certain signal transduction inhibitors. Thus, altering the levels of GSK-3β can have dramatic effects on sensitivity to drugs and signal transduction inhibitors which may be influenced by the background of the tumor.

GSK-3β or tau-kinase I is particularly abundant in the central nervous system (CNS), playing a key role in the pathogenesis of Alzheimer’s disease (AD). Accordingly, transgenic mouse models overexpressing this kinase recapitulate some aspects of this disease, such as tau hyperphosphorylation, neuronal death, and microgliosis. These alterations have been studied in mouse models showing GSK-3β overexpression from birth. In this case, some of these alterations may be due to adaptations that occur during development. Here we explored the potential of the Tet-OFF conditional system in the murine CamKIIα-tTA/GSK-3β model to increase the activity of GSK-3β only during adulthood. To this end, the overexpression of GSK-3β remained OFF during embryonic and postnatal development by administration of doxycycline in drinking water for 6 months, while it was turned ON in adult animals by removal of the treatment for 6 months. In these conditions, the CamKIIα-tTA/GSK-3β mouse is characterized by an increase in phosphorylated tau, cell death, and microgliosis. Furthermore, the increase in GSK-3β expression in the adult animals triggered a cognitive deficit, as determined through the hippocampus-dependent object recognition test (OR). These results demonstrate that the GSK-3β plays a key role in AD and that previously published data with other transgenic models are neither caused by or a consequence of adaptations to high levels of the enzyme during development., Ministry of Economy and Competitiveness (PGC-2018-09177-B-100) and the Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII). Work in the laboratory of FH is funded by grants from the Spanish Ministry of Economy and Competitiveness (Ministerio de Economía, Industria y Competitividad, Gobierno de España, BFU2016-77885-P) and was co-financed from the Comunidad de Madrid through Structural Funds of the European Union [S2017/BMD-3700 (NEUROMETAB-CM)]

Reg-1α belongs to the Reg family of small, secreted proteins expressed in both pancreas and nervous system. Reg-1α is composed of two domains, an insoluble C-type lectin domain and a short soluble N-terminal peptide, which is released from the molecule upon proteolytic N-terminal processing, although the biological significance of this proteolysis remains unclear. We have previously shown that binding of Reg-1α to its receptor Extl3 stimulates axonal outgrowth. Reg-1α and Extl3 genes are expressed in the developing cortex but their expression decreases in adulthood, pointing to a possible function of this signaling system at the early developmental stages. Here, we demonstrate that recombinant Reg-1α increases migration and differentiation of cultured embryonic rat telencephalic progenitors via the activation of GSK-3β activity. In vivo overexpression of Reg-1α by in utero electroporation, has a similar effect, favoring premature differentiation of cortical progenitors. Notably, the N-terminal soluble domain, but not the C-type lectin domain, is largely responsible for Reg-1α effects on cortical neuronal differentiation. We thus conclude that Reg-1α via its proteolytically generated N-terminal domain is required for basic development processes., Institut national de la santé et de la recherche de Médicale

The semi-synthesis of 11 isatin derivatives was achieved through bimolecular nucleophilic substitution and click chemistry. Seven new compounds were obtained. All chemical structures were determined by infrared spectroscopy (IR), nuclear magnetic resonance spectrometry (NMR) and high-resolution mass spectrometry (HRMS) data. These derivatives were evaluated for their anti-GSK-3 activity and all isatin derivatives (N-alkyl and 1,2,3-triazolic) exhibited strong inhibitory activity, with 2b and 4h exhibiting remarkable potency. In addition, docking studies were performed with 2b and 2e models to unravel the molecular mechanism underlying the polar interactions on the GSK-3 ATP-binding site., This study was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil (CAPES), Finance Code 001, by the National Council of Scientific and Technological Development (CNPq) and Foundation of Support to Research and Innovation of Espírito Santo (FAPES PPE-Agro No. 76418880/16). We also would like to acknowledge INCTBioNat (CNPq 465637/2014-0) for additional support and NCQP-UFES, as well as the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020, Programa Operacional Regional do Norte. L. R. R. also acknowledges her sabbatical leave fellowship (SFRH/BSAB/142991/2018) funded by FCT. D. F. is recipient of a doctoral fellowship (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020, Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Summary: Mitochondria are key organelles for brain health. Mitochondrial alterations have been reported in several neurodegenerative disorders, including Alzheimer’s disease (AD), and the comprehension of the underlying mechanisms appears crucial to understand their relationship with the pathology. Using multiple genetic, pharmacological, imaging, and biochemical approaches, we demonstrate that, in different familial AD cell models, mitochondrial ATP synthesis is affected. The defect depends on reduced mitochondrial pyruvate oxidation, due to both lower Ca2+-mediated stimulation of the Krebs cycle and dampened mitochondrial pyruvate uptake. Importantly, this latter event is linked to glycogen-synthase-kinase-3β (GSK-3β) hyper-activation, leading, in turn, to impaired recruitment of hexokinase 1 (HK1) to mitochondria, destabilization of mitochondrial-pyruvate-carrier (MPC) complexes, and decreased MPC2 protein levels. Remarkably, pharmacological GSK-3β inhibition in AD cells rescues MPC2 expression and improves mitochondrial ATP synthesis and respiration. The defective mitochondrial bioenergetics influences glutamate-induced neuronal excitotoxicity, thus representing a possible target for future therapeutic interventions. : Mitochondria are key organelles for brain health. Rossi et al. show that, in different Alzheimer’s disease cell models, lower mitochondrial Ca2+ signal and pyruvate uptake reduce ATP synthesis. GSK-3β hyper-activation contributes to the defect by impairing HK1-mitochondria association, decreasing MPC2 levels and destabilizing MPC complexes. Defective bioenergetics affects neuronal functionality. Keywords: Alzheimer’s disease, presenilin, mitochondrial metabolism, bioenergetics, calcium homeostasis, pyruvate, mitochondrial pyruvate carrier, hexokinase 1, GSK-3b

Growth hormone (GH) overexpression throughout life in transgenic mice is associated with the development of liver tumors at old ages. The preneoplastic pathology observed in the liver of young adult GH-overexpressing mice is similar to that present in humans at high risk of hepatic cancer. To elucidate the molecular pathogenesis underlying the pro-oncogenic liver pathology induced by prolonged exposure to elevated GH levels, the activation and expression of several components of signal transduction pathways that have been implicated in hepatocellular carcinogenesis were evaluated in the liver of young adult GH-transgenic mice. In addition, males and females were analyzed in parallel in order to evaluate sexual dimorphism. Transgenic mice from both sexes exhibited hepatocyte hypertrophy with enlarged nuclear size and exacerbated hepatocellular proliferation, which were higher in males. Dysregulation of several oncogenic pathways was observed in the liver of GH-overexpressing transgenic mice. Many signaling mediators and effectors were upregulated in transgenic mice compared with normal controls, including Akt2, NFκB, GSK3β, β-catenin, cyclin D1, cyclin E, c-myc, c-jun and c-fos. The molecular alterations described did not exhibit sexual dimorphism in transgenic mice except for higher gene expression and nuclear localization of cyclin D1 in males. We conclude that prolonged exposure to GH induces in the liver alterations in signaling pathways involved in cell growth, proliferation and survival that resemble those found in many human tumors. Fil: Miquet, Johanna Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina Fil: Freund, Thomas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina Fil: Martínez, Carolina S.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina Fil: Gonzalez, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina Fil: Díaz, María E.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina Fil: Micucci, Giannina P.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina Fil: Zotta, Elsa. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas; Argentina Fil: Bomparai, Ravneet K.. Southern Illinois University. School Of Medicine; Estados Unidos Fil: Bartke, Andrzej. Southern Illinois University; Estados Unidos Fil: Turyn, Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina Fil: Sotelo, Ana Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina

The dentate gyrus (DG) plays a crucial role in hippocampal-related memory. The most abundant cellular type in the DG, namely granule neurons, are developmentally generated around postnatal day P6 in mice. Moreover, a unique feature of the DG is the occurrence of adult hippocampal neurogenesis, a process that gives rise to newborn granule neurons throughout life. Adult-born and developmentally generated granule neurons share some maturational aspects but differ in others, such as in their positioning within the granule cell layer. Adult hippocampal neurogenesis encompasses a series of plastic changes that modify the function of the hippocampal trisynaptic network. In this regard, it is known that glycogen synthase kinase 3ß (GSK-3ß) regulates both synaptic plasticity and memory. By using a transgenic mouse overexpressing GSK-3ß in hippocampal neurons, we previously demonstrated that the overexpression of this kinase has deleterious effects on the maturation of newborn granule neurons. In the present study, we addressed the effects of GSK-3ß overexpression on the morphology and number of dendritic spines of developmentally generated granule neurons. To this end, we performed intracellular injections of Lucifer Yellow in developmentally generated granule neurons of wild-type and GSK-3ß-overexpressing mice and analyzed the number and morphologies of dendritic spines (namely, stubby, thin and mushroom). GSK-3ß overexpression led to a general reduction in the number of dendritic spines. In addition, it caused a slight reduction in the percentage, head diameter and length of thin spines, whereas the head diameter of mushroom spines was increased., Spanish Ministry of Economy and Competitiveness [SAF-2014-53040-P (JA), SAF 2015-66603-P (JD), and RYC-2015-17189 (MLL-M)], the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, Spain

Glycogen synthase kinase-3b (GSK-3b) is a serine-threonine kinase implicated in multiple processes and signaling pathways. Its dysregulation is associated with different pathological conditions including Alzheimer’s disease (AD). Here we demonstrate how changes in GSK-3b activity and/or levels regulate the production and subsequent secretion of fractalkine, a chemokine involved in the immune response that has been linked to AD and to other different neurological disorders. Treatment of primary cultured neurons with GSK-3b inhibitors such as lithium and AR-A014418 decreased full-length fractalkine in total cell extracts. Opposite effects were observed after neuron transduction with a lentiviral vector overexpressing the kinase. Biotinylation assays showed that those changes mainly affect the plasma membrane-associated form of the protein, an observation that positively correlates with changes in the levels of its soluble form. These effects were confirmed in lithium-treated wild type (wt) mice and in GSK-3b transgenic animals, as well as in brain samples from AD patients, evident as age-dependent (animals) or Braak stage dependent changes (humans) in both the membrane-bound and the soluble forms of the protein. Further immunohistochemical analyses demonstrated how GSK-3b exerts these effects by affecting the trafficking of the chemokine from the Golgi to the plasma membrane, in different and opposite ways when the levels/activity of the kinase are increased or decreased. This work provides for the first time a mechanism linking GSK-3b and fractalkine both in vitro and in vivo, with important implications for neurological disorders and especially for AD, in which levels of this chemokine might be useful as a diagnostic tool., Ministry of Economy and Competitiveness (SAF 2006-02424, BFU-2008-03980, BFU-2010-21507), Comunidad de Madrid (SAL/0202/2006), Fundación M. Botín, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII), and an institutional grant from the Fundación R. Areces

Adult hippocampal neurogenesis (AHN) is a key process for certain types of hippocampal-dependent learning. Alzheimer’s disease (AD) is accompanied by memory deficits related to alterations in AHN. Given that the increased activity of GSK-3β has been related to alterations in the population of hippocampal granule neurons in AD patients, we designed a novel methodology by which to induce selective GSK-3β overexpression exclusively in newborn granule neurons. To this end, we injected an rtTA-IRES-EGFP-expressing retrovirus into the hippocampus of tTO-GSK-3β mice. Using this novel retroviral strategy, we found that GSK-3β caused a cell-autonomous impairment of the morphological and synaptic maturation of newborn neurons. In addition, we examined whether GSK-3β overexpression in newborn neurons limits the effects of physical activity. While physical exercise increased the number of dendritic spines, the percentage of mushroom spines, and the head diameter of the same in tet-OFF cells, these effects were not triggered in tet-ON cells. This observation suggests that GSK-3β blocks the stimulatory actions of exercise. Given that the activity of GSK-3β is increased in the brains of individuals with AD, these data may be relevant for non-pharmacological therapies for AD., Spanish Ministry of Health (SAF-2014-5040-P), and the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII) (J. A ´ vila); the Japan Society for the promotion of Science (post-doctoral fellowship to M. Llorens-Martín); and the Alzheimer’s Association (2015-NIRG-340709

Autophagy delivers cytoplasmic material for lysosomal degradation in eukaryotic cells. Starvation induces high levels of autophagy to promote survival in the lack of nutrients. We compared genome-wide transcriptional profiles of fed and starved control, autophagy-deficient Atg7 and Atg1 null mutant Drosophila larvae to search for novel regulators of autophagy. Genes involved in catabolic processes including autophagy were transcriptionally upregulated in all cases. We also detected repression of genes involved in DNA replication in autophagy mutants compared with control animals. The expression of Rack1 (receptor of activated protein kinase C 1) increased 4.1- to 5.5-fold during nutrient deprivation in all three genotypes. The scaffold protein Rack1 plays a role in a wide range of processes including translation, cell adhesion and migration, cell survival and cancer. Loss of Rack1 led to attenuated autophagic response to starvation, and glycogen stores were decreased 11.8-fold in Rack1 mutant cells. Endogenous Rack1 partially colocalized with GFP-Atg8a and early autophagic structures on the ultrastructural level, suggesting its involvement in autophagosome formation. Endogenous Rack1 also showed a high degree of colocalization with glycogen particles in the larval fat body, and with Shaggy, the Drosophila homolog of glycogen synthase kinase 3B (GSK-3B). Our results, for the first time, demonstrated the fundamental role of Rack1 in autophagy and glycogen synthesis.

Autophagy plays a key role in the maintenance of cellular homeostasis, and autophagy deregulation gives rise to severe disorders. Many of the signaling pathways regulating autophagy under stress conditions are still poorly understood. Using a model of proteasome stress in rat hippocampus, we have characterized the functional crosstalk between the ubiquitin proteasome system and the autophagy-lysosome pathway, identifying also age-related modifications in the crosstalk between both proteolytic systems. Under proteasome inhibition, both autophagy activation and resolution were efficiently induced in young but not in aged rats, leading to restoration of protein homeostasis only in young pyramidal neurons. Importantly, proteasome stress inhibited glycogen synthase kinase-3β in young but activated in aged rats. This age-related difference could be because of a dysfunction in the signaling pathway of the insulin growth factor-1 under stress situations. Present data highlight the potential role of glycogen synthase kinase-3β in the coordination of both proteolytic systems under stress situation, representing a key molecular target to sort out this deleterious effect., This work was supported by grant PI12/00445 (DR) from the Carlos III Health Institute, Spain. EG was supported by a contract from JA.

Umbilical cord blood (UCB) is a well established alternative source of haematopoietic stem/progenitor cells (HSPC) for haematopoietic stem cell transplantation (HSCT). One factor limiting the wider use of UCB for transplantation is the relatively low cell dose from a UCB unit compared to bone marrow (BM) and peripheral blood (PB). To overcome this barrier, many strategies have been tested to expand HSPC numbers ex vivo. The aims of this thesis were to define the phenotypic, functional and transcriptional changes that occur during ex vivo expansion of UCB HSPC and investigate how activation of the Wingless (Wnt) pathway via GSK-3 inhibition can provide a novel approach to improve transplantation outcomes using expanded cells. In vitro and in vivo models of HSPC function were used to evaluate the impact of expansion and GSK-3 inhibition on progenitor function while gene array technology was used to investigate potential molecular mechanisms. We have shown that expansion of UCB HSPC results in a loss of primitive phenotype, an increase in progenitors with reduced function and a shift in transcriptional profile from ‘stem-like quiescent’ to ‘divide and differentiate’. Brief treatment of expansion cultures with a GSK-3 inhibitor resulted in delayed cell cycle progression which translated into improved PB and BM repopulation. Gene expression analysis revealed modulation of Notch and Tie2 pathways, suggesting their potential role in improving stem cell engraftment. In addition, down-regulation of cyclin D1 and upregulation of CDK inhibitor p57 known to be important in maintenance of quiescence was observed. This work is a timely reminder of the medical revolution that E. Donnall Thomas pioneered in the 1950’s, paving the way for successful treatment of those with haematological malignancies and other blood disorders. In addition, the year 2013 marked the 25th anniversary of the first UCB transplant that took place in France in October 1988. Since then over 600,000 UCB units have been banked and more than 30,000 UCB transplants performed worldwide. This thesis contributes to the understanding of HSPC biology and how this can be modulated to improve clinical transplant outcomes in the future.

Dysregulated cell cycling is a universal hallmark of cancer and is often mediated by abnormal activation of cyclin-dependent kinases (CDKs) and their cyclin partners. Overexpression of individual complexes are reported in multiple myeloma (MM), making them attractive therapeutic targets. In this study, we investigate the preclinical activity of a novel small-molecule multi-CDK inhibitor, AT7519, in MM. We show the anti-MM activity of AT7519 displaying potent cytotoxicity and apoptosis; associated with in vivo tumor growth inhibition and prolonged survival. At the molecular level, AT7519 inhibited RNA polymerase II (RNA pol II) phosphorylation, a CDK9, 7 substrate, associated with decreased RNA synthesis confirmed by [(3)H] Uridine incorporation. In addition, AT7519 inhibited glycogen synthase kinase 3beta (GSK-3beta) phosphorylation; conversely pretreatment with a selective GSK-3 inhibitor and shRNA GSK-3beta knockdown restored MM survival, suggesting the involvement of GSK-3beta in AT7519-induced apoptosis. GSK-3beta activation was independent of RNA pol II dephosphorylation confirmed by alpha-amanitin, a specific RNA pol II inihibitor, showing potent inhibition of RNA pol II phosphorylation without corresponding effects on GSK-3beta phosphorylation. These results offer new insights into the crucial, yet controversial role of GSK-3beta in MM and show significant anti-MM activity of AT7519, providing the rationale for its clinical evaluation in MM.

O cérebro é dependente de um fluxo sanguíneo contínuo para suplemento de glicose e oxigênio. A isquemia cerebral resulta na degeneração celular e perda da sua funcionalidade. Culturas organotípicas representam um modelo in vitro que permite estudar o dano cerebral isquêmico. Neste trabalho nós investigamos o efeito neuroprotetor do 17b-estradiol num modelo de isquemia in vitro. Para mimetizar um insulto isquêmico, fatias de cultura organotípica foram expostas à privação de oxigênio e glicose (POG) usando uma câmara anaeróbica desenvolvida em nosso laboratório. A morte celular foi quantificada pela captação de iodeto de propídio. Houve uma diminuição na incorporação de iodeto de propídeo nas fatias submetidas à POG que receberam tratamento agudo e crônico com 17b-estradiol (10 nM) Para examinar um possível mecanismo pelo qual 17b-estradiol previne a morte celular foi avaliado o imunoconteúdo da enzima pGSK-3b por Western Blotting. Esta enzima ativa vias que levam à morte celular e é inibida por fosforilação. O tratamento agudo e crônico com 17b-estradiol aumentou a fosforilação de GSK-3b nas fatias controles e nas submetidas à POG. Esses resultados sugerem que esta é uma possível via pela qual o 17b-estradiol atua como neuroprotetor, visto que a fosforilação de GSK-3b inibe sua ação apoptótica. To examine a possible mechanism by which estradiol prevents cellular death we evaluate the immunocontent of pGSK-3b by western blot analysis. This enzyme has been shown to trigger cellular death. We have found that acute and chronic treatment with 17b-estradiol increased the phosphorylation of GSK-3b in the control and OGD slices. These findings might suggest that this is a possible pathway by which 17b-estradiol acts as neuroprotective agent, once the phosphorylation of GSK-3b inhibits its apoptotic action.