Key words STAT3 - Hes3 - stem cells
Introduction
The existence of neural stem cells (NSCs) in the adult mammalian brain was inferred in 1962 by Joseph Altman who demonstrated the incorporation of tritiated thymidine in adult neurons [1 ]. Later, Kaplan, Gould, and colleagues provided supporting evidence [2 ]
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[6 ]. Goldman and Nottebohm reported similar phenomena in the adult songbird brain, demonstrating massive seasonal neuronal turnover as male birds develop new songs [7 ]. Hockfield and McKay identified nestin as a biomarker of fetal NSCs [8 ], while Cattaneo and McKay developed cell culture techniques that allowed the maintenance of these cells in vitro [9 ]. Subsequently, Reynolds and Weiss were able to isolate and culture NSCs from the adult mammalian brain, which exhibited proliferation capability, as well as the potential of differentiation into neurons and glia after treatment with appropriate growth factors in vitro [10 ]. These and other studies led to efforts aimed at promoting brain repair by either transplanting newly formed neurons into the brain, therefore replacing lost cells, or by stimulating endogenous NSCs (eNSCs) to facilitate their neuroprotective role following brain injury or against degenerative disease [11 ]
[12 ].
Cell replacement strategies have recently received an enormous boost by reprogramming technologies that allow patient-specific cell sources to be converted into immature cells (somatic cells into embryonic stem cell-like cells), which can then be directed to differentiate towards specific cell fates and then be grafted into the patient [13 ]. However, the immense cytoarchitectural complexity of the brain hinders their successful implementation. Graft-derived neurons need to be precisely placed and they oftentimes should connect brain areas that are centimeters away from each other. In parallel to these efforts, scientists have been exploring the possibility to pharmacologically target eNSCs to increase their number and putative neuroprotective functions. These cells are already naturally incorporated into the tissue, within specific microenvironments (the neurovascular niche) and by understanding the signals that these cells respond to, it is possible to increase their number several fold through direct local injection of a number of soluble factors. Such treatments have proven highly consequential, leading to long-term behavioral improvement in models of ischemic stroke and Parkinson’s disease [14 ]
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[18 ].
To take full advantage of eNSCs, many aspects of their biology need to be further understood, including the molecular mechanisms that regulate their number and fate specification, to allow the direction of their fate to appropriate cell types for replacement, neuroprotection, or immunomodulation. Here we focus on a recently identified signaling pathway that regulates the number of NSCs in vitro and in vivo [19 ]. A major input into the pathway is insulin, providing a new molecular mechanism via which the pancreas may regulate aspects of brain function. New evidence points towards the operation of this pathway in the hypothalamus and adrenal gland [20 ], suggesting that it has a broad role in the neuroendocrine system, deserving further study.
The STAT3-Ser/Hes3 Signaling Axis in NSCs
The STAT3-Ser/Hes3 Signaling Axis in NSCs
NSCs apparently interpret signaling pathways in their own distinct ways. For example, whereas many cell types in the body utilize the JAK/STAT pathway for growth and survival, this leads to the differentiation of NSCs [21 ]
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[24 ]. Specifically, tyrosine phosphorylation of STAT3 is a key component of the survival pathway of most cell types, but NSCs avoid this phosphorylation in order to prevent differentiation into glia. Instead, NSCs utilize the serine phosphorylation of STAT3 for their survival, which is largely redundant for many other cell types [16 ]. Several inputs into STAT3-serine phosphorylation have been identified, including a noncanonical branch of Notch signaling (activated by Notch receptor ligands such as Delta4 and Jagged1), angiopoietin 2 (a ligand of the Tie2 receptor), basic fibroblast growth factor (bFGF), and insulin [16 ]
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[25 ]
[26 ]. These treatments lead to the transcriptional induction of Hes3, a transcription factor/passive repressor belonging to the Hes/Hey family of basic helix-loop-helix genes [27 ]
[28 ]. This pathway is opposed by JAK and p38 MAP kinases and, therefore, inhibitors of these kinases promote its activation and NSC survival. Hes3 itself mediates many of these functions, as adult cultured NSCs show a reduced response to Delta4 and insulin stimulation, and to JAK inhibition [17 ]. Hes3 RNA interference also opposes the growth of cultured cancer stem cells from human glioblastoma multiforme biopsies [29 ].
Pharmacological activation of this pathway has powerful consequences as it directly promotes the survival of cultured NSCs from many sources. In vivo, the same treatments increase the number of putative eNSCs and induce long-lasting neuroprotection in models of ischemic stroke and Parkinson’s disease [16 ]. One of these treatments is the direct injection of insulin into the lateral ventricle of adult rats, suggesting that circulating insulin may also regulate eNSC number and function, and rendering the endocrine pancreas a key regulator of the eNSC population in both established neurogenic zones, such as the subventricular zone (SVZ) and the subgranular zone (SGZ) of the dentate gyrus of the hippocampus, but also in the brain parenchyma [17 ]. The disease-modifying functions of this pathway may involve, in part, cell replacement but more likely, the increased release of neuroprotective factors [30 ]. Hes3, in particular, may also be involved in the reprogramming of induced pluripotency cells and adult somatic cells to the NSC fate, suggesting not only roles in promoting NSC growth but also specification [31 ]
[32 ].
STAT3-Ser/Hes3 Signaling Axis in the Pancreatic Islet
STAT3-Ser/Hes3 Signaling Axis in the Pancreatic Islet
It appears likely, then, that the endocrine pancreas can regulate aspects of brain functions, such as neuroprotection and regeneration, via the release of insulin and its actions on endogenous NSCs. Molecular mechanisms that regulate insulin production/release and pancreatic islet beta cell properties may thus be directly relevant to brain function [33 ]. Remarkably, the same signaling pathway also regulates key functions of pancreatic islets, as well as their resistance to damage. These observations suggest that the STAT3-Ser/Hes3 Signaling Axis in the pancreas regulates the same signaling pathway in eNSCs and with it, the function of both of these cell types.
Specifically, Hes3 is expressed in pancreatic islets and Hes3 null mice exhibit more pronounced damage to the pancreatic islet beta cell population when treated with the toxin streptozotocin (STZ), relative to wild type controls [34 ], resulting in a greater loss of beta cells and an earlier diabetic phenotype, as assessed by blood glucose levels. Following STZ-induced damage, Hes3 promoter activity (in this study the activity of only one of the 2 promoters, Hes3a, was assessed) is elevated, suggesting an involvement in pancreatic islet regeneration. In fact, Hes3 null mice exhibit reduced islet regeneration following partial STZ-induced damage [Masjkur et al., in press]. In the exocrine pancreas, Hes3 null mice fail to upregulate neurogenin3, an indicator of regenerative responses in these STZ models [35 ].
Mouse insulinoma cell lines cultured under defined conditions also express Hes3 and Hes3 RNA interference regulates gene expression, reduces cell growth, and opposes glucose-stimulated insulin release. In the absence of an insult to pancreatic islet cells (STZ), no phenotype has yet been reported [36 ]. For example, homozygous null mice for Hes3 are healthy, breed well, have normal blood glucose levels and glucose tolerance test (GTT) scores, and normal gross pancreatic islet morphology, suggesting that this pathway has relevance in conditions of damage and regeneration. These observations also showcase that the lack of obvious phenotypes in genetic models does not preclude the manifestation of deficiencies in paradigms of damage, stress, or other insults. These signaling concepts may be of relevance to diabetes-mediated encephalopathy which, at the early stages exhibits damage in the neurovascular niche in the brain through oxidative and vascular damage and at later stages exhibits impaired neurogenic potential [37 ].
STAT3-Ser/Hes3 Signaling Axis in the Adrenal Cortex and Medulla
STAT3-Ser/Hes3 Signaling Axis in the Adrenal Cortex and Medulla
We recently reported Hes3 expression in the bovine adrenal medulla and in cultures of chromaffin progenitor cells from this tissue [38 ]. Using a reporter mouse strain where the Hes3 gene has been replaced by the lacZ gene under the control of the Hes3a promoter (one of the 2 known promoters of Hes3) [39 ], we also showed the expression of Hes3 in the mouse medulla, corroborating the bovine data ([Fig. 1 ]). In addition, we reported Hes3a promoter activity in the adrenal cortex. In the cortex, Hes3 expression was most prominent in the zona glomerulosa, where sonic hedgehog (Shh)-expressing progenitor cells reside [40 ]. Because in NSCs, Hes3 is a positive regulator of Shh expression [16 ], this observation raises the question of whether Hes3 is expressed in adrenocortical progenitors and whether it functions to induce Shh expression. Copper chelation by a cuprizone-rich diet induces massive upregulation of Hes3a promoter activity further suggesting that different insult paradigms should be investigated to unveil roles of the STAT3-Ser/Hes3 Signaling Axis ([Fig. 1 ]).
Fig. 1 Hes3 is expressed in different organs of the endocrine and neuroendocrine systems. Hes3 is expressed in pancreatic islet cells where it regulates insulin release (in vitro data) and regenerative responses (in vivo). Insulin (direct injections into the lateral ventricle) regulates the number of endogenous neural progenitor cells in the adult brain. Hes3-expressing cells in the adult brain are found in a number of areas, including the SVZ, cerebral cortex, hippocampus, and hypothalamus (green arrows and red box). In the hypothalamus, Hes3+cells are found in the median eminence where a proportion co-localize with vimentin-expressing β2 tanycytes [In this image, Hes3+cells are identified by immunolabeling for beta-galactosidase in a mouse line where the Hes3 gene has been replaced by beta-galactosidase paced under the control of one of the 2 Hes3 gene promoters (Hes3a)]. Hes3 expression in these cells raises the question of possible roles of Hes3 in the neuroendocrine system, for example, through the regulation of adrenal function via the HPA Axis. The adrenal gland also contains Hes3-expressing cells. In the adult mouse adrenal medulla it co-localizes with a subpopulation of nestin positive cells (nestin-GFP). The magnification in the right side of the panel shows a perinuclear localization of Hes3 in nestin-GFP positive cells (yellow arrow), whereas cells negative for nestin-GFP displayed a nuclear localization of Hes3 (blue arrow; Scale 50 μm). Hes3+cells in the adrenal medulla exhibit a scattered distribution and may represent adrenomedullary chromaffin progenitors, as suggested by in vitro studies showing the expression of Hes3 in isolated cultured progenitors. In the adrenal cortex, Hes3+cells are located in the general area where progenitor cells are also thought to reside. Copper chelation by a cuprizone-containing diet induces massive activation of Hes3a promoter activity in the adrenal. (The “X-Gal (Hes3a)” image is reproduced from Masjkur et al., J Biol Chem, 2014). (Color figure available online only).
STAT3-Ser/Hes3 Signaling Axis in Tanycytes of the Hypothalamus?
STAT3-Ser/Hes3 Signaling Axis in Tanycytes of the Hypothalamus?
The STAT3-Ser/Hes3 Signaling Axis regulates pancreatic beta cells as well as neural and neuroendocrine stem cells and progenitor cells in the adult brain and adrenal gland. Given this broad range of cells that utilize the pathway, it is reasonable to speculate that hypothalamic tanycytes may also be subject to regulation by this pathway. Tanycytes are involved in the transport of chemicals from the cerebrospinal fluid to the brain and which participate in the metabolic regulation of the organism [41 ]. In addition, they have putative progenitor cell properties [42 ]
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[44 ]; why a cell with such defined function may also have progenitor properties is a subject of great interest with potential ramifications to the fields of neurodegenerative disease and metabolic control.
Indeed, tanycytes of the median eminence express Hes3 as demonstrated by immunolabeling for beta-galactosidase that is expressed under the control of one of the 2 Hes3 promoters (the one that induces the transcription of the Hes3a product) ([Fig. 1 ]). It remains to be determined how this pathway may affect the stem/progenitor cell properties of tanycytes and, as a consequence, metabolic regulatory functions of the hypothalamus.
The adult hypothalamus is derived from the ventral diencephalon, which contains 2 distinct proliferative zones; the lateral and the midline zone, which form the peripheral and the core region of the hypothalamus, respectively [45 ]
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[47 ], and it is a key regulator of many physiological functions and homeostatic mechanisms including growth, feeding/energy expenditure and reproduction. Impaired hypothalamic maturation is responsible for a variety of behavioral and somatic diseases like obesity, infertility, and mood disorders [48 ]. The hypothalamus consists of a variety of nuclei that form a highly complex neuronal network controlling functions. To regulate body weight, the hypothalamus must interpret and integrate incoming signals such as levels of glucose, free fatty acids and amino acids but also hormones like leptin, ghrelin, and insulin from the periphery (stomach, pancreas, and adipose tissue) [49 ]. It then needs to synthesize and secrete hormones that will affect other endocrine glands such as the pituitary and, subsequently, the adrenal, affecting a wide range of functions, including stress adaptation, mood/emotions, digestion, the immune system, sexual function, and energy storage/expenditure [48 ].
In the more traditionally established neurogenic zones of the adult brain, the SVZ and SGZ, it is becoming increasingly appreciated that immature, highly plastic cell populations are able to play important roles in brain function by providing new cells and, possibly, signals to the tissue [50 ]
[51 ]. Similar concepts appear to hold true in the hypothalamus as well. Many of the key nuclei controlling energy balance located in close proximity to the 3rd ventricle contain a population of ependymal cells. This population can be further divided into 2 subgroups: (i) the ciliated ependymal cells with very short processes lining the dorsal part of the ventricle; (ii) the tanycytes, which lack cilia but have microvilli, distinct apical specializations, as well as long processes projecting into the brain parenchyma, lining the ventral part of the ventricle [52 ]
[53 ]. The tanycytes are further divided into 4 distinct subgroups; the α1 and α2 tanycytes, which are located in the dorsal part of the ventricle and are close to the ventromedial hypothalamic nuclei and the β1 and β2 tanycytes, which are located more ventrally and in close proximity to the arcuate nucleus and the median eminence. β2 tanycytes have direct access to circulating plasma due to the unique position of the median eminence outside the blood-brain barrier; with their processes in close proximity to neuronal populations controlling energy homeostasis (e. g., AgRP and POMC neurons in the arcuate nucleus), they may play a particularly important role in energy homeostasis and body weight balance [41 ]
[52 ]. The discovery of tanycytes can be traced to decades ago when Rita Levi Montalcini together with her colleague Stanley Cohen discovered a specific cell population with stem cell like characteristics that were highly responsive to nerve growth factor (NGF) [54 ]. A striking morphological characteristic of tanycytes is their long processes reaching several hundred microns in length making them capable of contacting the hypothalamic nuclei and the neurons within them that play key roles in the control of energy homeostasis.
The question of how the stem cell/progenitor properties of tanycytes come into play in hypothalamic function is still under intense investigation. Tanycytes may be non-terminally specified immature cells tasked with the ability for metabolic sensing (a complex biochemical function) and, at the same time, the capacity to contribute to tissue remodeling by generating new neurons. In this way, tanycytes may be able to dynamically contribute to tissue plasticity, in response to dietary and other changes [41 ]
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[56 ]. Over the last decade, a variety of studies support this view and demonstrate that tanycytes express a number of markers in common with neural stem cells/progenitor cells including nestin, vimenting, doublecortin (for early neurons), and Sox2 [43 ]
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Central administration of ciliary neurotrophic factor (CNTF) results in weight loss and induces neurogenesis in the arcuate nucleus and the ventromedial hypothalamus, suggesting a relation between the 2 processes [64 ]. High fat diet-induced inflammation results in increased apoptosis and neuronal depletion in the hypothalamus [65 ]. The neuronal deficit may involve both increased apoptosis of existing neurons, as well as the depletion of neurogenic tanycytes, through an IKKβ/NF-κβ-mediated mechanism in β2 tanycytes [44 ]
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[67 ]. The median eminence (containing β2 tanycytes) appears to be a particularly neurogenic zone that responds to high fat diet by increasing neurogenesis. Such plasticity may be of consequence to metabolic regulation as blocking neurogenesis by focused irradiation of the hypothalamus attenuated the body weight increases that are normally induced by high fat diet [43 ].
The lineage relation among different tanycyte subpopulations is not clear, although α tanycytes seem to be more robust at self-renewing than β tanycytes, as suggested by lineage tracing [68 ] and neurosphere formation experiments [69 ]. Whereas there is still uncertainty as to whether β2 tanycytes in the median eminence represent bona fide stem cells or neural progenitors with limited self-renewal ability, several lines of evidence support their immature state and neurogenic potential and demonstrate expression of biomarkers shared with neural stem cells, including nestin and Rax [43 ]. The preferential regulation of different tanycyte subpopulations by different stimuli (including cytokine treatment, diets, etc.) correlates with different neuronal differentiation patterns and body weight outcomes, suggesting that perhaps, α tanycytes associate with body weight loss or maintenance, while β tanycytes associate with body weight gain [41 ].
It is important to understand the molecular mechanisms that regulate both chemosensing and the response of tanycytes to various stimuli. Tanycytes possess the molecular machinery that pancreatic beta cells use for their glucosensing. As the STAT3-Ser/Hes3 Signaling Axis is now implicated in glucosensing in mouse insulinoma cell lines that are able to release insulin in response to glucose stimulation, and as tanycytes also express Hes3, it will be of great interest to assess whether Hes3 in tanycytes is utilized for their chemosensing properties. It is possible, therefore, that this signaling pathway is used for both cell growth and glucose sensing in both pancreatic beta cells and tanycytes. The median eminence is innervated by neurosecretory cells, positioning β2 tanycytes in an ideal location for metabolic sensing. Cerebrospinal fluid insulin and choroid plexus-derived angiopoietin 2 may play a role in the maintenance of Hes3 expression and the immature state of β2 tanycytes, as both factors are powerful activators of the STAT3-Ser/Hes3 Signaling Axis [16 ]
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[25 ].
Conclusions
It is becoming increasingly appreciated that stem/progenitor cell populations in the adult brain play important roles in the function of the nervous system. Immature cells can sense environmental cues and determine whether to quiesce, proliferate, or differentiate, contributing to the plasticity of the tissue they reside in. Elucidating the molecular mechanisms that these cells utilize will help us understand how local and endocrine signals regulate these cells, how to manipulate them, and it will also help explain how they are affected in disease. The STAT3-Ser/Hes3 Signaling Axis regulates several cell types with stem/progenitor properties not only in the brain and adrenal, but also pancreatic islet beta cells that exhibit highly plastic properties and powerful regenerative potential. Several brain areas may be involved in responses to peripheral insulin and the control of behaviors that contribute to obesity [70 ], including the prefrontal cortex, where Hes3+ cells also reside [71 ]. Here we focus on the most well studied of these areas, the hypothalamus. We present data that Hes3 is also expressed in tanycytes in the median eminence of the hypothalamus and propose that this pathway may be a new metabolic regulator and that it may be utilized by different brain regions and organs that are part of the endocrine and neuroendocrine systems.
