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Cancer cells can switch between signaling pathways to regulate growth under different conditions. In the tumor microenvironment, this likely helps them evade therapies that target specific pathways. We must identify all possible states and utilize them in drug screening programs. One such state is characterized by expression of the transcription factor Hairy and Enhancer of Split 3 (HES3) and sensitivity to HES3 knockdown, and it can be modeled in vitro. Here, we cultured 3 primary human brain cancer cell lines under 3 different culture conditions that maintain low, medium, and high HES3 expression and characterized gene regulation and mechanical phenotype in these states. We assessed gene expression regulation following HES3 knockdown in the HES3-high conditions. We then employed a commonly used human brain tumor cell line to screen Food and Drug Administration (FDA)-approved compounds that specifically target the HES3-high state. We report that cells from multiple patients behave similarly when placed under distinct culture conditions. We identified 37 FDA-approved compounds that specifically kill cancer cells in the high-HES3-expression conditions. Our work reveals a novel signaling state in cancer, biomarkers, a strategy to identify treatments against it, and a set of putative drugs for potential repurposing.-Poser, S. W., Otto, O., Arps-Forker, C., Ge, Y., Herbig, M., Andree, C., Gruetzmann, K., Adasme, M. F., Stodolak, S., Nikolakopoulou, P., Park, D. M., Mcintyre, A., Lesche, M., Dahl, A., Lennig, P., Bornstein, S. R., Schroeck, E., Klink, B., Leker, R. R., Bickle, M., Chrousos, G. P., Schroeder, M., Cannistraci, C. V., Guck, J., Androutsellis-Theotokis, A. Controlling distinct signaling states in cultured cancer cells provides a new platform for drug discovery.

Cancer stem cells (CSCs) are responsible for drug resistance, tumor recurrence, and metastasis in several cancer types, making their eradication a primary objective in cancer therapy. Glioblastoma Multiforme (GBM) tumors are usually composed of a highly infiltrating CSC subpopulation, which has Nestin as a putative marker. Since the majority of these infiltrating cells are able to elude conventional therapies, we have developed gold nanorods (AuNRs) functionalized with an engineered peptide capable of specific recognition and selective eradication of Nestin positive infiltrating GBM-CSCs. These AuNRs generate heat when irradiated by a near-infrared laser, and cause localized cell damage. Nanoparticle internalization assays performed with GBM-CSCs or Nestin negative cells cultured as two-dimensional (2D) monolayers or embedded in three-dimensional (3D) biodegradable-hydrogels of tunable mechanical properties, revealed that the AuNRs were mainly internalized by GBM-CSCs, and not by Nestin negative cells. The AuNRs were taken up via energy-dependent and caveolae-mediated endocytic mechanisms, and were localized inside endosomes. Photothermal treatments resulted in the selective elimination of GBM-CSCs through cell apoptosis, while Nestin negative cells remained viable. Results also indicated that GBM-CSCs embedded in hydrogels were more resistant to AuNR photothermal treatments than when cultured as 2D monolayers. In summary, the combination of our engineered AuNRs with our tunable hydrogel system has shown the potential to provide an in vitro platform for the evaluation and screening of AuNR-based cancer therapeutics, leading to a substantial advancement in the application of AuNRs for targeted GBM-CSC therapy. Statement of Significance There is an urgent need for reliable and efficient therapies for the treatment of Glioblastoma Multiforme (GBM), which is currently an untreatable brain tumor form with a very poor patient survival rate. GBM tumors are mostly comprised of cancer stem cells (CSCs), which are responsible for tumor reoccurrence and therapy resistance. We have developed gold nanorods functionalized with an engineered peptide capable of selective recognition and eradication of GBM-CSCs via heat generation by nanorods upon NIR irradiation. An in vitro evaluation of nanorod therapeutic activities was performed in 3D synthetic-biodegradable hydrogel models with distinct biomechanical cues, and compared to 2D cultures. Results indicated that cells cultured in 3D were more resistant to photothermolysis than in 2D systems.

Cancer cells can switch between signaling pathways to regulate growth under different conditions. In the tumor microenvironment, this likely helps them evade therapies that target specific pathways. We must identify all possible states and utilize them in drug screening programs. One such state is characterized by expression of the transcription factor Hairy and Enhancer of Split 3 (HES3) and sensitivity to HES3 knockdown, and it can be modeled in vitro. Here, we cultured 3 primary human brain cancer cell lines under 3 different culture conditions that maintain low, medium, and high HES3 expression and characterized gene regulation and mechanical phenotype in these states. We assessed gene expression regulation following HES3 knockdown in the HES3-high conditions. We then employed a commonly used human brain tumor cell line to screen Food and Drug Administration (FDA)-approved compounds that specifically target the HES3-high state. We report that cells from multiple patients behave similarly when placed under distinct culture conditions. We identified 37 FDA-approved compounds that specifically kill cancer cells in the high-HES3–expression conditions. Our work reveals a novel signaling state in cancer, biomarkers, a strategy to identify treatments against it, and a set of putative drugs for potential repurposing.—Poser, S. W., Otto, O., Arps-Forker, C., Ge, Y., Herbig, M., Andree, C., Gruetzmann, K., Adasme, M. F., Stodolak, S., Nikolakopoulou, P., Park, D. M., Mcintyre, A., Lesche, M., Dahl, A., Lennig, P., Bornstein, S. R., Schroeck, E., Klink, B., Leker, R. R., Bickle, M., Chrousos, G. P., Schroeder, M., Cannistraci, C. V., Guck, J., Androutsellis-Theotokis, A. Controlling distinct signaling states in cultured cancer cells provides a new platform for drug discovery. FASEB J. 33, 9235–9249 (2019). www.fasebj.org. © FASEB

Diabetes mellitus is a group of disorders characterized by prolonged high levels of circulating blood glucose. Type 1 diabetes is caused by decreased insulin production in the pancreas whereas type 2 diabetes may develop due to obesity and lack of exercise; it begins with insulin resistance whereby cells fail to respond properly to insulin and it may also progress to decreased insulin levels. The brain is an important target for insulin, and there is great interest in understanding how diabetes affects the brain. In addition to the direct effects of insulin on the brain, diabetes may also impact the brain through modulation of the inflammatory system. Here we investigate how perturbation of circulating insulin levels affects the expression of Hes3, a transcription factor expressed in neural stem and progenitor cells that is involved in tissue regeneration. Our data show that streptozotocin-induced β-cell damage, high fat diet, as well as metformin, a common type 2 diabetes medication, regulate Hes3 levels in the brain. This work suggests that Hes3 is a valuable biomarker helping to monitor the state of endogenous neural stem and progenitor cells in the context of diabetes mellitus. © 2018, The Author(s).

Encapsulation of primary bovine adrenocortical cells in alginate is an efficacious model of a bioartificial adrenal cortex. Such a bioartificial adrenal cortex can be used for the restoration of lost adrenal function in vivo as well as for in vitro modeling of the adrenal microenvironment and for investigation of cell-cell interactions in the adrenals. The aim of this work was the optimization of a bioartificial adrenal cortex, that is the generation of a highly productive, self-regenerating, long-term functioning and immune tolerant bioartificial organ. To achieve this, it is necessary that adrenocortical stem and progenitor cells are present in the bioartificial gland, as these undifferentiated cells play important roles in the function of the mature gland. Here, we verified the presence of adrenocortical progenitors in cultures of bovine adrenocortical cells, studied the dynamics of their appearance and growth and determined the optimal time point for cell encapsulation. These procedures increased the functional life span and reduced the immunogenicity of the bioartificial adrenal cortex. This model allows the use of the luteinizing hormone-releasing hormone (LHRH) agonist triptorelin, the neuropeptide bombesin, and retinoic acid to alter cell number and the release of cortisol over long periods of time.

Interest is great in the new molecular concepts that explain, at the level of signal transduction, the process of reprogramming. Usually, transcription factors with developmental importance are used, but these approaches give limited information on the signaling networks involved, which could reveal new therapeutic opportunities. Recent findings involving reprogramming by genetic means and soluble factors with well-studied downstream signaling mechanisms, including signal transducer and activator of transcription 3 (STAT3) and hairy and enhancer of split 3 (Hes3), shed new light into the molecular mechanisms that might be involved. We examine the appropriateness of common culture systems and their ability to reveal unusual (noncanonical) signal transduction pathways that actually operate in vivo. We then discuss such novel pathways and their importance in various plastic cell types, culminating in their emerging roles in reprogramming mechanisms. We also discuss a number of reprogramming paradigms (mouse induced pluripotent stem cells, direct conversion to neural stem cells, and in vivo conversion of acinar cells to β-like cells). Specifically for acinar-to-β-cell reprogramming paradigms, we discuss the common view of the underlying mechanism (involving the Janus kinase-STAT pathway that leads to STAT3-tyrosine phosphorylation) and present alternative interpretations that implicate STAT3-serine phosphorylation alone or serine and tyrosine phosphorylation occurring in sequential order. The implications for drug design and therapy are important given that different phosphorylation sites on STAT3 intercept different signaling pathways. We introduce a new molecular perspective in the field of reprogramming with broad implications in basic, biotechnological, and translational research. Significance Reprogramming is a powerful approach to change cell identity, with implications in both basic and applied biology. Most efforts involve the forced expression of key transcription factors, but recently, success has been reported with manipulating signal transduction pathways that might intercept them. It is important to start connecting the function of the classic reprogramming genes to signaling pathways that also mediate reprogramming, unifying the sciences of signal transduction, stem cell biology, and epigenetics. Neural stem cell studies have revealed the operation of noncanonical signaling pathways that are now appreciated to also operate during reprogramming, offering new mechanistic explanations.

The adrenal is a highly plastic organ with the ability to adjust to physiological needs by adapting hormone production but also by generating and regenerating both adrenocortical and adrenomedullary tissue. It is now apparent that many adult tissues maintain stem and progenitor cells that contribute to their maintenance and adaptation. Research from the last years has proven the existence of stem and progenitor cells also in the adult adrenal medulla throughout life. These cells maintain some neural crest properties and have the potential to differentiate to the endocrine and neural lineages. In this article, we discuss the evidence for the existence of adrenomedullary multi potent progenitor cells, their isolation and characterization, their differentiation potential as well as their clinical potential in transplantation therapies but also in pathophysiology.

The neural crest-derived adrenal medulla is closely related to the sympathetic nervous system; however, unlike neural tissue, it is characterized by high plasticity which suggests the involvement of stem cells. Here, we show that a defined pool of glia-like nestin–expressing progenitor cells in the adult adrenal medulla contributes to this plasticity. These glia-like cells have features of adrenomedullary sustentacular cells, are multipotent, and are able to differentiate into chromaffin cells and neurons. The adrenal is central to the body's response to stress making its proper adaptation critical to maintaining homeostasis. Our results from stress experiments in vivo show the activation and differentiation of these progenitors into new chromaffin cells. In summary, we demonstrate the involvement of a new glia-like multipotent stem cell population in adrenal tissue adaptation. Our data also suggest the contribution of stem and progenitor cells in the adaptation of neuroendocrine tissue function in general. Stem Cells 2015;33:2037–2051

Omic science is rapidly growing and one of the most employed techniques to explore differential patterns in omic datasets is principal component analysis (PCA). However, a method to enlighten the network of omic features that mostly contribute to the sample separation obtained by PCA is missing. An alternative is to build correlation networks between univariately-selected significant omic features, but this neglects the multivariate unsupervised feature compression responsible for the PCA sample segregation. Biologists and medical researchers often prefer effective methods that offer an immediate interpretation to complicated algorithms that in principle promise an improvement but in practice are difficult to be applied and interpreted. Here we present PC-corr: a simple algorithm that associates to any PCA segregation a discriminative network of features. Such network can be inspected in search of functional modules useful in the definition of combinatorial and multiscale biomarkers from multifaceted omic data in systems and precision biomedicine. We offer proofs of PC-corr efficacy on lipidomic, metagenomic, developmental genomic, population genetic, cancer promoteromic and cancer stem-cell mechanomic data. Finally, PC-corr is a general functional network inference approach that can be easily adopted for big data exploration in computer science and analysis of complex systems in physics. C.V.C. thanks Jennifer Labus from the UCLA Oppenheimer Center for Neurobiology of Stress and Resilience for the precious discussions and valuable suggestions on the unsupervised pattern analysis. We thank Gregorio Alanis Lobato from the Institute of Molecular Biology Mainz for the useful suggestions on the population genetics dataset. We thank Miguel Angel del Pozo from the Spanish National Center for Cardiovascular Research (CNIC) for kind support. We thank Piero Carnici, Alistair Forrest, Timothy Ravasi, the RIKEN Omics Science Center (OSC) in Yokohama and the FANTOM consortium for their kind support. Work in the CVC laboratory was supported by the Klaus Tschira Stiftung (KTS) gGmbH, Germany (Grant number: 00.285.2016). S.C. PhD fellowship is supported by Lipotype GmbH. V.J.J. PhD scholarship has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 641639. We acknowledge support by the German Research Foundation and the Open Access Publication Funds of the TU Dresden. Sí

The adrenal gland is a highly plastic organ with the capacity to adapt the body homeostasis to different physiological needs. The existence of stem-like cells in the adrenal cortex has been revealed in many studies. Recently, we identified and characterized in mice a pool of glia-like multipotent Nestin-expressing progenitor cells, which contributes to the plasticity of the adrenal medulla. In addition, we found that these Nestin progenitors are actively involved in the stress response by giving rise to chromaffin cells. Interestingly, we also observed a Nestin-GFP-positive cell population located under the adrenal capsule and scattered through the cortex. In this article, we discuss the possibility of a common progenitor giving rise to subpopulations of cells both in the adrenal cortex and medulla, the isolation and characterization of this progenitor as well as its clinical potential in transplantation therapies and in pathophysiology.

// Akio Soeda 1 , Justin Lathia 2 , Brian J. Williams 3 , Qiulian Wu 2 , Joseph Gallagher 2 , Andreas Androutsellis-Theotokis 4 , Amber J. Giles 5 , Chunzhang Yang 5 , Zhengping Zhuang 5 , Mark R. Gilbert 5 , Jeremy N. Rich 2 and Deric M. Park 5 1 Department of Neurosurgery, Gifu University, Gifu, Japan 2 Department of Stem Cell Biology and Regenerative Medicine, Cleveland Clinic Foundation, Cleveland, OH, USA 3 Department of Neurosurgery, University of Louisville, Louisville, KY, USA 4 Division of Stem Cell Biology, Technische Universitat Dresden, Dresden, Germany 5 Neuro-Oncology Branch, National Cancer Institute, NIH, Bethesda, MD, USA Correspondence to: Deric M. Park, email: deric.park@nih.gov Keywords: glioma, cancer stem cell, p38 MAPK, EGFR, quiescence Received: July 10, 2016 Accepted: March 19, 2017 Published: March 31, 2017 ABSTRACT EGFR pathway is upregulated in malignant gliomas, and its downstream signaling is important for self-renewal of glioma cancer stem-like cells (GSC). p38 mitogen-activated protein kinase (MAPK) signaling, a stress-activated signaling cascade with suppressive and permissive effects on tumorigenesis, can promote internalization and ubiquitin ligase mediated degradation of EGFR. In this study, we investigated the role of p38 MAPK signaling on the self-renewal of GSCs with the hypothesis that inhibition may lead to enhanced self-renewal capacity by retention of EGFR. Inhibition of p38 MAPK pathway led to increase in EGFR expression but surprisingly, reduced proliferation. Additional functional evaluation revealed that p38 inhibition was associated with decrease in cell death and maintenance of undifferentiated state. Further probing the effect of p38 inhibition demonstrated attenuation of EGFR downstream signaling activity in spite of prolonged surface expression of the receptor. In vitro observations were confirmed in xenograft in vivo experiments. These data suggest that p38 MAPK control of EGFR signaling activity may alter GSC cell cycle state by regulating quiescence and passage into transit amplifying state.

Loss of insulin-producing pancreatic islet β-cells is a hallmark of type 1 diabetes. Several experimental paradigms demonstrate that these cells can, in principle, be regenerated from multiple endogenous sources using signaling pathways that are also used during pancreas development. A thorough understanding of these pathways will provide improved opportunities for therapeutic intervention. It is now appreciated that signaling pathways should not be seen as “on” or “off” but that the degree of activity may result in wildly different cellular outcomes. In addition to the degree of operation of a signaling pathway, noncanonical branches also play important roles. Thus, a pathway, once considered as “off” or “low” may actually be highly operational but may be using noncanonical branches. Such branches are only now revealing themselves as new tools to assay them are being generated. A formidable source of noncanonical signal transduction concepts is neural stem cells because these cells appear to have acquired unusual signaling interpretations to allow them to maintain their unique dual properties (self-renewal and multipotency). We discuss how such findings from the neural field can provide a blueprint for the identification of new molecular mechanisms regulating pancreatic biology, with a focus on Notch, Hes/Hey, and hedgehog pathways.

The endocrine system involves communication among different tissues in distinct organs, including the pancreas and components of the Hypothalamic-Pituitary-Adrenal Axis. The molecular mechanisms underlying these complex interactions are a subject of intense study as they may hold clues for the progression and treatment of a variety of metabolic and degenerative diseases. A plethora of signaling pathways, activated by hormones and other endocrine factors have been implicated in this communication. Recent advances in the stem cell field introduce a new level of complexity: adult progenitor cells appear to utilize distinct signaling pathways than the more mature cells in the tissue they co-reside. It is therefore important to elucidate the signal transduction requirements of adult progenitor cells in addition to those of mature cells. Recent evidence suggests that a common non-canonical signaling pathway regulates adult progenitors in several different tissues, rendering it as a potentially valuable starting point to explore their biology. The STAT3-Ser/Hes3 Signaling Axis was first identified as a major regulator of neural stem cells and, subsequently, cancer stem cells. In the endocrine/neuroendocrine system, this pathway operates on several levels, regulating other types of plastic cells: (a) it regulates pancreatic islet cell function and insulin release; (b) insulin in turn activates the pathway in broadly distributed neural progenitors and possibly also hypothalamic tanycytes, cells with important roles in the control of the adrenal gland; (c) adrenal progenitors themselves operate this pathway. The STAT3-Ser/Hes3 Signaling Axis therefore deserves additional research in the context of endocrinology.

The endocrine system involves communication among different tissues in distinct organs, including the pancreas and components of the Hypothalamic-Pituitary-Adrenal Axis. The molecular mechanisms underlying these complex interactions are a subject of intense study as they may hold clues for the progression and treatment of a variety of metabolic and degenerative diseases. A plethora of signaling pathways, activated by hormones and other endocrine factors have been implicated in this communication. Recent advances in the stem cell field introduce a new level of complexity: adult progenitor cells appear to utilize distinct signaling pathways than the more mature cells in the tissue they co-reside. It is therefore important to elucidate the signal transduction requirements of adult progenitor cells in addition to those of mature cells. Recent evidence suggests that a common non-canonical signaling pathway regulates adult progenitors in several different tissues, rendering it as a potentially valuable starting point to explore their biology. The STAT3-Ser/Hes3 Signaling Axis was first identified as a major regulator of neural stem cells and, subsequently, cancer stem cells. In the endocrine/neuroendocrine system, this pathway operates on several levels, regulating other types of plastic cells: (a) it regulates pancreatic islet cell function and insulin release; (b) insulin in turn activates the pathway in broadly distributed neural progenitors and possibly also hypothalamic tanycytes, cells with important roles in the control of the adrenal gland; (c) adrenal progenitors themselves operate this pathway. The STAT3-Ser/Hes3 Signaling Axis therefore deserves additional research in the context of endocrinology.

Loss of insulin-producing pancreatic islet beta-cells is a hallmark of type 1 diabetes. Several experimental paradigms demonstrate that these cells can, in principle, be regenerated from multiple endogenous sources using signaling pathways that are also used during pancreas development. A thorough understanding of these pathways will provide improved opportunities for therapeutic intervention. It is now appreciated that signaling pathways should not be seen as “on” or “off” but that the degree of activity may result in wildly different cellular outcomes. In addition to the degree of operation of a signaling pathway, noncanonical branches also play important roles. Thus, a pathway, once considered as “off” or “low” may actually be highly operational but may be using noncanonical branches. Such branches are only now revealing themselves as new tools to assay them are being generated. A formidable source of noncanonical signal transduction concepts is neural stem cells because these cells appear to have acquired unusual signaling interpretations to allow them to maintain their unique dual properties (self-renewal and multipotency). We discuss how such findings from the neural field can provide a blueprint for the identification of new molecular mechanisms regulating pancreatic biology, with a focus on Notch, Hes/Hey, and hedgehog pathways.

The article depicts in two infographics a summary of what has been presented in the Tools of the Trade column over the past 10 years. The first figure categorizes the major points of each column into information, opinion, and prescriptive guidelines. The second figure associates with each broad theme specific indicative tools.

Hes3 is a component of the STAT3-Ser/Hes3 Signaling Axis controlling the growth and survival of neural stem cells and other plastic cells. Pharmacological activation of this pathway promotes neuronal rescue and behavioral recovery in models of ischemic stroke and Parkinson's disease. Here we provide initial observations implicating Hes3 in the cuprizone model of demyelination and remyelination. We focus on the subpial motor cortex of mice because we detected high Hes3 expression. This area is of interest as it is impacted both in human demyelinating diseases and in the cuprizone model. We report that Hes3 expression is reduced at peak demyelination and is partially restored within 1 week after cuprizone withdrawal. This raises the possibility of Hes3 involvement in demyelination/remyelination that may warrant additional research. Supporting a possible role of Hes3 in the maintenance of oligodendrocyte markers, a Hes3 null mouse strain shows lower levels of myelin basic protein in undamaged adult mice, compared to wild-type controls. We also present a novel method for culturing the established oligodendrocyte progenitor cell line oli-neu in a manner that maintains Hes3 expression as well as its self-renewal and differentiation potential, offering an experimental tool to study Hes3. Based upon this approach, we identify a Janus kinase inhibitor and dbcAMP as powerful inducers of Hes3 gene expression. We provide a new biomarker and cell culture method that may be of interest in demyelination/remyelination research.

Introduction Aromatic (ar-) turmerone is a major bioactive compound of the herb Curcuma longa. It has been suggested that ar-turmerone inhibits microglia activation, a property that may be useful in treating neurodegenerative disease. Furthermore, the effects of ar-turmerone on neural stem cells (NSCs) remain to be investigated. Methods We exposed primary fetal rat NSCs to various concentrations of ar-turmerone. Thereafter, cell proliferation and differentiation potential were assessed. In vivo, naïve rats were treated with a single intracerebroventricular (i.c.v.) injection of ar-turmerone. Proliferative activity of endogenous NSCs was assessed in vivo, by using noninvasive positron emission tomography (PET) imaging and the tracer [18F]-fluoro-L-thymidine ([18F]FLT), as well as ex vivo. Results In vitro, ar-turmerone increased dose-dependently the number of cultured NSCs, because of an increase in NSC proliferation (P

Correction to: Molecular Psychiatry; advance online publication, 26 January 2016; doi: 10.1038/mp.2015.230 Following publication of the above article, the authors noticed that the first author’s name was presented incorrectly. The author’s name should have appeared as MF Rubin de Celis. The publisher regrets the error.

Introduction The adult brain exhibits low regenerative ability. Stem cell-based transplantation approaches have been largely unsuccessful, due to the difficulty to recapitulate the complex cytoarchitecture of the central nervous system (CNS). eNSCs are a new therapeutic option as pharmacological activation and increase of their number in vivo is accompanied by powerful neuroprotection in various disease models. Hes3 is expressed in both proliferating and quiescent NSCs, which makes it a useful biomarker for NSC identification. Direct injections of insulin in the adult brain increase the number of eNSCs and promote rescue of injured neurons via a novel molecular mechanism, the STAT3-Ser/Hes3 Signaling Axis. This molecular pathway with the STAT3-Ser phosphorylation at its core regulates Hes3 and together they form a merging point for several signals including insulin receptor activation. Main aim and Hypothesis Beyond the brain, STAT3-Ser/Hes3 signaling regulates various plastic cell populations in other organs of the endocrine/neuroendocrine system. In the pancreas, Hes3 is expressed in islets cells and regulates their growth, regeneration, and insulin release. Hes3 is also expressed in mouse hypothalamic tanycytes, which are diet responsive cells and play a very crucial role for the communication between the brain and the endocrine system. Also, Hes3 is expressed in the adrenal gland (both in the cortex and medulla); cultured adrenal progenitors express Hes3 and various treatments that induce Hes3 expression promote their growth. Therefore, STAT3-Ser/Hes3 Signaling may be involved in tissue problems that result from metabolic dysfunction. Metabolic syndrome often results in diabetes (Type I, Type II) and insulin resistance, suggesting that eNSCs may be affected by the condition. There is evidence that obesity induces inflammatory reactions in the hypothalamus, leading to NSC loss. However, it is not clear if damage to NSCs is also directly linked to insulin signaling disruption. Results Our results show that various parameters affect Hes3 levels in the brain. Aging decreased Hes3 mRNA expression. Type I diabetes increased Hes3 expression. Type II diabetes decreased Hes3 expression. Thus, we conclude that eNSCs are modulated by diabetes in an age-dependent manner. We also investigated whether common medication for metabolic related dysfunction also affects Hes3 expression in the adult brain. Indeed, our results show that metformin decreases Hes3 expression in the mouse hypothalamus. To address whether metformin has a direct effect on NSCs we treated primary mouse fNSCs with metformin. Metformin decreases cell number, proliferation and affects cell morphology, giving a more differentiated appearance (large, flat cell body with wider projections). Hes3 expression increases significantly at 72 hours of treatment. The metformin result opens the question if the increase in the Hes3 expression is a direct effect of the signal transduction pathways activated by metformin or due to a stress reaction. To address this we treated NSCs with exendin-4, another diabetes drug that we previously showed to both elevate Hes3 expression and cell number using a mouse insulinoma cell line (MIN6). Exendin-4 increases fNSC cell number but it did not affect the morphology. Similar to metformin proliferation was not affected. Hes3 expression increased significantly at 72 hours of treatment as well. This result indicates the distinctive action of the drugs on the STAT3-Ser/Hes3 signaling pathway. Specifically it dissociates Hes3 levels from other cellular parameters. Importantly it shows that two common diabetes medications have very different effects on NSCs. Because Hes3 is strongly regulated by metabolic parameters and medication we addressed potential roles of Hes3 using an established Hes3 null mouse line. Hes3 null mice exhibit no obvious phenotypes under normal conditions. However, we previously showed that when stressed by chemical induced damage, they exhibit low regenerative potential in the pancreas and brain. To identify additional phenotypes, we performed a phenotypic analysis of the Hes3 null mouse line under normal diet and HFD conditions (which induced type II diabetes). We found mild phenotypes that relate to the nervous system, the immune system and metabolism. At the molecular level, Hes3 deletion affects the expression of other genes within the Hes superfamily in the adult mouse brain. However, we did not observe these molecular differences in the HFD condition, suggesting an interplay between metabolic parameters (possibly, circulating insulin) and the regulation of Hes/Hey genes in the brain. Our data suggest a broad range of roles for Hes3, particularly under abnormal conditions. Conclusions Our work establishes that multiple parameters of metabolic state as well as diabetes medication affect Hes3 expression in the brain. Metabolic syndrome is a risk factor for many neurological disorders such as Alzheimer’s disease, Parkinson’s disease and Multiple Sclerosis. It is important to understand at the molecular and cellular level how metabolic dysfunction affects the brain. Here, we introduced a new cellular biomarker and signaling component that is greatly regulated in metabolic dysfunction.:1 Introduction 18 1.1 The ''plastic brain'': Neural Stem Cells, progenitors and precursors 19 1.2 Functional adult neurogenesis 19 1.3 NSCs in conventional and nonconventional regions of the adult brain 20 1.4 Neurodegenerative diseases, cell replacement and endogenous NSCs 21 1.5 The STAT3-Ser/Hes3 signaling axis in NSCs 24 1.6 Beyond the brain: The STAT3-Ser/Hes3 signaling axis operates in plastic cells 27 1.6.1 STAT3-Ser/Hes3 Signaling Axis in the pancreatic islet 27 1.6.2 STAT3-Ser/Hes3 Signaling Axis in the adrenal cortex and medulla 28 1.6.3 STAT3-Ser/Hes3 Signaling Axis in tanycytes of the hypothalamus? 28 1.6.4 STAT3-Ser/Hes3 Signaling: A new molecular component of the neuroendocrine system? 29 1.7 Metabolic syndrome and neurological disease 31 1.7.1 Metabolic dysfunction and Alzheimer's disease 31 1.7.2 Metabolic dysfunction and Parkinson's disease 31 1.7.3 Metabolic dysfunction and Multiple Sclerosis 32 1.7.4 Metabolism and neurodegenerative disease: Are they connected? 32 1.8 Main Aim – Hypothesis 33 2 Materials and Methods 34 2.1 Animal experiments 34 2.1.1 Animal use authorization 34 2.1.2 Genotyping 34 2.1.3 In vivo models 36 2.1.4 In vivo metabolic Analyses 36 2.1.5 Nociception 37 2.1.6 Histology 38 2.1.7 PCR and Real-Time quantitative PCR (qPCR) 39 2.1.8 Western Blot 41 2.2 Mouse phenotyping 42 2.3 Neural stem cell cultures 43 2.3.1 Preparation – Coatings 43 2.3.2 Cell Isolation and Cell Culture 43 2.3.3 Pharmacological Manipulation (Metformin – Exendin-4) 43 2.4 Heat maps 44 2.5 Statistical analyses 44 3 Results 45 3.1 Hes3 is expressed in the mouse brain 46 3.2 Aging and diabetes models alter Hes3 in the brain 48 3.2.1 Hes3 expression decreases with age 48 3.2.2 Pancreatic islet damage by streptozotocin increases Hes3 expression in the brain 48 3.2.3 High Fat Diet reduces Hes3 expression in the brain 49 3.3 Common diabetes medication affect neural stem cells (NSCs) in the brain 53 3.3.1 Metformin decreases Hes3 expression in the brain 53 3.3.2 Metformin opposes growth but increases Hes3 expression in cultured NSCs 54 3.3.3 Exendin-4 promotes growth and increases Hes3 expression in cultured NSCs 54 3.3.4 Metformin and Exendin-4 affect the STAT3-Ser/Hes3 signaling axis 59 3.4 Hes3 null mice exhibit a quasi-normal phenotype 60 3.4.1 Phenotypic Analysis - Normal Diet (ND) 60 3.4.2 Metabolism Relevant Phenotypes – HFD challenge 63 3.4.3 Phenotypic Analysis – Molecular 67 4 DISCUSSION 70 4.1 Diabetes affects the brain 71 4.2 STAT3-Ser/Hes3: a putative mediator 71 4.3 Hes3 is a special member of the Hes/Hey gene family 72 4.4 Patterns of Hes3 expression may be specific to cell type and microenvironment 72 4.5 Metabolic dysfunction and diabetes medication affect brain Hes3 73 4.5.1 Age regulates Hes3 73 4.5.2 Diabetes models regulate Hes3 expression in the brain 74 4.5.3 Metformin regulates Hes3 expression in the brain 74 4.6 Hes3 phenotyping provides clues to Hes3 functions 76 4.7 Hes3 and metabolic dysfunction: Are they connected? 77 5 Conclusions and Future Remarks 79 References 81