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
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:33485 |
| Date | 11 March 2019 |
| Creators | Nikolakopoulou, Polyxeni |
| Contributors | Androutsellis-Theotokis, Andreas, Morawietz, Henning, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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