The role of endogenous neural stem cells (eNSCs) in metabolic syndrome and aging

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 8

Wormholes and islands

This thesis explores the topic of spacetime wormholes and islands. Wormholes can be thought of as smooth bridges between two far away, disconnected points in spacetime. A wormhole that allows information to travel through it is called traversable. For a wormhole to be traversable it needs to be supported by negative energy, which is not easily produced. In recent developments, a non-local coupling connecting two sides of a double sided black hole was constructed by Gao, Jafferis and Wall, which allows holographic wormholes to become traversable. In this thesis, we investigate various features of this geometry, such as the amount of information that can be sent through. We also construct a new wormhole geometry in higher dimensions. Additionally, we study islands, a much newer concept in physics. Recently, the Page curve of Hawking radiation was recovered within semi-classical gravity, using the island formula. Essentially, what was realized is that in order to correctly calculate the fine grained entropy of the radiation we need to include in the computation a disconnected region that usually lies inside the black hole, called the island. The island formula naturally has been applied mostly to black holes, where we have a clear notion of the information paradox. Moreover, there have been found some conditions that are necessary in order to have an island. In this thesis, we apply these conditions to open and closed sliced FRW cosmologies and investigate in which cases it is possible to have an island

Egnatia Motorway: Analysing the impacts on the economic and social sustainability of urban areas in Greece

Mobility is one of the most important key factors that determine economic development and growth in most modern countries all over the world. Therefore, investments in infrastructure can have multiple effects when it comes to economic and social growth. What is more, economic development is directly associated with quality of life, as people tend to satisfy their advancing need for mobility with the aid of transport infrastructures. Transport infrastructure is internationally considered as an instrument of regional policy, on a national level, but their exact contribution to even regional disparities is not yet determined in the literature. Although a high quality transport system is not sufficient by itself for the development of regions, it assists in bypassing obstacles and creates the conditions for their development. Moreover, it does not create significant long-term regional changes. Egnatia Motorway is a recently constructed infrastructure project in the northern Greece and it is a part of the Trans - European Transport Network E-90. This major infrastructure project enhances the connectivity of the cities that are located across the northern part of the country while they are becoming more accessible to other greater urban agglomerations. This is achieved by connecting the reference area with the central part of the country. The aim of this paper is to identify and evaluate the impacts of the project with regards to sustainability. Sustainability is a multidimensional term, when combined with economic prosperity and social cohesion. Besides, increased accessibility is positively associated with regional development, but this is not the only factor that determines that. In particular, it will focus on whether the role of the local economies, of the different scale urban areas Egnatia crosses, has been strengthened or weakened when raised on intra-region level. For that purpose, the paper focuses on a case study of three different types of urban centres; Thessaloniki, a metropolis city, the medium scale city of Kavala and the smaller, rural city of Grevena. Although it seems to be obvious that there is a relationship between economy, urban growth and society, this research will be a tool which will help understanding how these concepts are related

Neural and synaptic plasticity in the chick brain after passive avoidance learning

The avian hippocampus (Hp) is believed to be homologous to mammalian hippocampus both from a developmental and an anatomical view-point. In one-day old domestic chicks (Gallus domesticus), studies have demonstrated that it may play a key role in the acquisition of one trial passive avoidance learning (PAL) where the aversive experience is exposure to a bitter tasting substance, methyl anthranilate. In the present study, following PAL the numerical density of asymmetric axospinous synapses decreased by 36% in the dorsal Hp of the right hemisphere of MeA trained compared to control birds, 6 hours after training. In contrast, 24h post PAL there is a 33% decrease in numerical density of asymmetric axodendritic synapses in the MeA trained group in relation to the same area of control chicks. Cell proliferation studies using the thymidine analogue, bromodeoxyuridine, (BrdU) demonstrated a 47% reduction in cell proliferation in the dorsal Hp of the MeA trained group 24h later in comparison with controls, which disappears after 9 days. In nucleus taeniae of amygdala and the arcopallium dorsale and intermediale, there are no differences between birds 24h or 9 days post BrdU injection but in olfactory bulb of MeA trained chicks cell labelling increases by 95% and 71.4% respectively, compared to control and water-trained birds, 24 h after PAL. The increase is more dramatic 9 days post PAL, when the MeA-trained group shows a 259% and 314% increase respectively in relation to control and water-trained animals. Following PAL apoptotic studies in the Hp 24h, 5 and 9 days post BrdU injection demonstrated that there were no differences in cell death between the different groups. Radioimmunoassay measurements of cortisol in chick forebrain tissue demonstrated longer term increase in levels of steroid in the chick Hp compared to arcopallium and striatum mediale 20 minutes after training, indicating that PAL is a stressful experience which may explain synaptic density and cell proliferation reduction observed after PAL

Aegean Identities and Interactions in the Late Bronze Age Mediterranean

Interaction in Mediterranean protohistory is generally considered via the core-periphery model, with greater influence being ascribed to the complex polities of the eastern Mediterranean than to those of the Aegean and central Mediterranean. This is despite archaeological evidence attesting that they actively participated in material and cultural exchanges. In this paper, we focus on Minoan Crete and Mycenaean Greece, reflecting on their interaction spheres and on the meaning of ‘central’ and ‘peripheral’ places. We consider two case studies: Thera and the Aeolian Islands. These islands functioned as maritime hubs in both inter-regional and regional networks. We propose a model of ‘cycles of integration’, as a more accurate and less static representation of interaction

Astrocytic Ephrin-B1 Regulates Synapse Remodeling Following Traumatic Brain Injury.

Traumatic brain injury (TBI) can result in tissue alterations distant from the site of the initial injury, which can trigger pathological changes within hippocampal circuits and are thought to contribute to long-term cognitive and neuropsychological impairments. However, our understanding of secondary injury mechanisms is limited. Astrocytes play an important role in brain repair after injury and astrocyte-mediated mechanisms that are implicated in synapse development are likely important in injury-induced synapse remodeling. Our studies suggest a new role of ephrin-B1, which is known to regulate synapse development in neurons, in astrocyte-mediated synapse remodeling following TBI. Indeed, we observed a transient upregulation of ephrin-B1 immunoreactivity in hippocampal astrocytes following moderate controlled cortical impact model of TBI. The upregulation of ephrin-B1 levels in hippocampal astrocytes coincided with a decline in the number of vGlut1-positive glutamatergic input to CA1 neurons at 3 days post injury even in the absence of hippocampal neuron loss. In contrast, tamoxifen-induced ablation of ephrin-B1 from adult astrocytes in ephrin-B1(loxP/y)ERT2-Cre(GFAP) mice accelerated the recovery of vGlut1-positive glutamatergic input to CA1 neurons after TBI. Finally, our studies suggest that astrocytic ephrin-B1 may play an active role in injury-induced synapse remodeling through the activation of STAT3-mediated signaling in astrocytes. TBI-induced upregulation of STAT3 phosphorylation within the hippocampus was suppressed by astrocyte-specific ablation of ephrin-B1 in vivo, whereas the activation of ephrin-B1 in astrocytes triggered an increase in STAT3 phosphorylation in vitro. Thus, regulation of ephrin-B1 signaling in astrocytes may provide new therapeutic opportunities to aid functional recovery after TBI

Investigation of infertility using endometrial organoids.

Infertility is a common problem in modern societies with significant socio-psychological implications for women. Therapeutic interventions are often needed which, depending on the cause, can either be medical treatment, surgical procedures or assisted reproductive technology (ART). However, the treatment of infertility is not always successful due to our limited understanding of the preparation of the lining of the uterus, the endometrium, for pregnancy. The endometrium is of central importance for successful reproduction as it is the site of placental implantation providing the interface between the mother and her baby. Due to the dynamic, structural and functional changes the endometrium undergoes throughout the menstrual cycle, it is challenging to study. A major advancement is the establishment of 3D organoid models of the human endometrium to study this dynamic tissue in health and disease. In this review, we describe the changes that the human endometrium undergoes through the different phases of the menstrual cycle in preparation for pregnancy. We discuss defects in the processes of endometrial repair, decidualization and acquisition of receptivity that are associated with infertility. Organoids could be utilized to investigate the underlying cellular and molecular mechanisms occurring in non-pregnant endometrium and early pregnancy. These studies may lead to therapeutic applications that could transform the treatment of reproductive failure

Wormholes from averaging over states

An important question about black holes is to what extent a typical pure state differs from the ensemble average. We show that this question can be answered within semiclassical gravity. We focus on the quantum deviation, which measures the fluctuations in the expectation value of an operator in an ensemble of pure states. For a large class of ensembles and observables, these fluctuations are calculated by a correlation function in the eternal black hole background, which can be reliably calculated within semi-classical gravity. This implements the idea of [arXiv:2002.02971] that wormholes can arise from averages over states rather than theories. As an application, we calculate the size of the long-time correlation function〈A(t)A(0).</p