Regulation of somatic stem cell proliferation is critical for the maintenance of tissue and organ function throughout the body. Modulators of this process include nutrients and peptides, but the role of an autonomic neural influence on stem cell proliferation has been neglected. This article describes the literature in support of autonomic nervous system (ANS) influence on somatic stem cells, with emphasis on intestinal epithelial stem cells (IESCs) as a representative somatic stem cell. Based on the current available data, models for the direct influence of both branches of the ANS (the sympathetic and parasympathetic nervous systems) on IESCs are outlined. Finally, the prospect of treatments derived from ANS influence on somatic stem cells is explored.

INTRODUCTION Somatic stem cell proliferation is critical for maintaining tissue homeostasis through the regeneration of tissues. Multiple physiological systems modulate somatic stem cell proliferation by altering the rate of renewal, the number of cells produced, or the ratio of differentiated daughter cell types (4, 16, 50, 64, 66). All of these outcomes ultimately lead to functional changes in the tissue. Studies defining modulators of this process have largely concentrated on nutrients and peptides but have traditionally neglected to investigate a role for the autonomic nervous system (ANS). It would seem beneficial for the brain to guide somatic stem cell proliferation via ANS signaling, therefore participating in decisions of tissue regeneration such as the replenishment of cells lost because of normal levels of apoptosis, renewal of tissues after injury, and growth of tissues during development or high nutrient availability. Roles for the ANS in controlling tissue regeneration have been identified after tissue injury in the liver (5, 12, 15, 30) and pancreas (37) and following ANS nerve ablation in the intestine and adipose tissue (11, 31, 33, 40, 57, 58). However, the direct participation of ANS connections with the somatic stem cells in these effects has been overlooked. Investigation of a possible direct autonomic neural influence on somatic stem cell proliferation will allow for a further understanding of the mechanisms regulating stem cells and may reveal novel neurally driven strategies to therapeutically modulate somatic stem cell proliferation to treat tissue abnormalities.

EVIDENCE IN SUPPORT OF A DIRECT AUTONOMIC INFLUENCE ON SOMATIC STEM CELL PROLIFERATION: THE INTESTINAL EPITHELIUM The ANS regulates many functions of the intestinal epithelium, the innermost layer of the intestinal mucosa that lines the lumen, including fluid transport (24) and hormone release (48). A growing body of anatomical and functional evidence suggests that the ANS can also directly modulate proliferation of the intestinal epithelial stem cells (IESCs), which are located within the crypts of the tissue and are responsible for its renewal and growth. IESCs divide constitutively to produce progenitor cells, and these progenitor cells differentiate into the mature, functional daughter cells of the intestinal epithelium (i.e., enterocyte, enteroendocrine, goblet, paneth). Thus, IESCs are critical in maintaining the integrity and function of the tissue and any modulation of their proliferation would certainly alter intestinal homeostasis (6). Surgical or chemical ablation of either branch of the ANS, the sympathetic (SNS) or parasympathetic (PNS), results in a loss of the in vivo source of the autonomic neurotransmitters and alters intestinal epithelial crypt cell proliferation (11, 31, 33, 40, 57, 58), although the direction of the alteration of proliferation is variable based on time point after denervation (17). After SNS or PNS denervation, proliferation has been shown to increase (11, 33, 57) or decrease (11, 31, 33, 40, 58) at different time points, with several studies demonstrating eventual recovery to control levels of proliferation (17, 33, 40). This may be because of other systems capable of modulating proliferation having a greater impact after loss of ANS input. These varied results after chronic ANS denervation may also point to an acute role of the ANS in coordinating bodily functions based on immediate perturbations in homeostasis, instead of a long-term modulatory role. The nerve terminals of the SNS and PNS are found in a position that suggests direct control of IESCs by the ANS is possible. In particular, SNS nerve terminals are densely concentrated at the base of the crypt where the IESCs are located (23, 41), whereas PNS nerves make direct synaptic contact with intestinal epithelial cells (10). The SNS-associated α2A adrenoreceptor and the PNS-associated muscarinic acetylcholine (ACh) receptor subtypes M1 and M3 are also expressed in IESCs and are implicated in controlling intestinal epithelial cell proliferation (18, 25, 44, 51, 59). Moreover, the primary autonomic neurotransmitters norepinephrine (NE) and ACh are ligands of these receptors and are able to modulate intestinal epithelial proliferation in vitro. Application of NE or ACh to intestinal epithelial organoids decreases the expression of a key cell cycle gene, cyclin D1 (18), of which the protein is rate-limiting in proliferation (2, 28, 43, 46, 47, 62). Consistent with these results, an ACh agonist decreases intestinal epithelial organoid proliferation (55). Beyond the primary SNS and PNS neurotransmitters, autonomic cotransmitters are also possible candidates to bind to receptors expressed by IESCs to alter proliferation. The autonomic cotransmitters neuropeptide Y, adenosine triphosphate, and vasoactive peptide modulate proliferation in other cell types, including additive or synergistic effects with the classical autonomic neurotransmitters (20, 21, 29, 32, 42, 56, 60, 63). Together, these studies support a role for the ANS in the direct modulation of IESC proliferation and tissue regeneration through primary and co-neurotransmitters and receptors. Based on the data outlined above, we propose models for the PNS and the SNS to influence IESC proliferation directly (Fig. 1). As IESCs span the length of the small intestine and colon (8), we include both of these gastrointestinal organs in the models. If either model was proven to be correct, it would be the first known instance of the nervous system directly regulating nonneuronal somatic stem cell proliferation. Fig. 1.Proposed models of direct parasympathetic (PNS) or sympathetic (SNS) influence on intestinal epithelial stem cell (IESC) proliferation. Proposed parasympathetic model: preganglionic PNS neurons become activated. Cell bodies of these neurons are located in one of two areas: the dorsal motor nucleus of the vagus (DMV) in the brain stem or the sacral spinal cord. From these areas, preganglionic PNS neurons project to the one of the two ganglionated plexuses of the enteric nervous system, the myenteric plexus, or the submucosal plexus within the small intestine or colon. These neural projections travel via the vagus nerve (supplying the small intestine and proximal colon) or the pelvic splanchnic nerves (supplying the distal colon). Within the myenteric or submucosal plexus ganglia, the preganglionic PNS neurons make synaptic connections with postganglionic PNS neurons, which are subsequently activated. These postganglionic PNS neurons then make synaptic connections with IESCs (slender black cells in figure). Acetylcholine released from the postganglionic PNS nerve terminals binds to muscarinic acetylcholine receptor subtypes M1 and/or M3 located on IESCs. This initiates an intracellular signaling cascade that causes a suppression of cyclin D1 expression and a downstream decrease in proliferation. This model outlines a direct effect of the PNS on IESC proliferation, which is one of many plausible mechanisms through which the PNS could influence this process. Proposed sympathetic model: preganglionic SNS neurons become activated. These neurons have cell bodies located in the thoracic or lumbar spinal cord. These neurons project via splanchnic nerves to synapse onto and subsequently activate postganglionic SNS neurons located in paravertebral sympathetic ganglia. The paravertebral ganglia include the celiac and superior mesenteric ganglia, which supply the small intestine, and the inferior mesenteric ganglion, which supplies the colon. Postganglionic SNS neurons then project to and make synaptic connections with IESCs. Norepinephrine (NE) is released from the SNS nerve terminals and subsequently binds to α2A adrenoreceptors (α 2A -ARs) located on IESCs. Activation of the α 2A -ARs by NE initiates an intracellular signaling cascade that causes a suppression of cyclin D1 expression and a consequential decrease in proliferation. This model outlines a direct effect of the SNS on IESC proliferation, which is one of many plausible mechanisms through which the SNS could influence this process. Download figureDownload PowerPoint



EVIDENCE FOR A ROLE OF THE ANS ON IESC PROLIFERATION INDEPENDENT OF THE ENTERIC NERVOUS SYSTEM The SNS and PNS make synaptic connections with neurons of the enteric nervous system (ENS), the intrinsic neural system of the gastrointestinal tract. The ENS is already known to participate in the control of intestinal epithelial cell proliferation (26, 67, 68) and, thus, can serve as a neural liaison to enact SNS- and PNS-driven functions. Although the ENS is a possible pathway through which SNS and PNS alteration of IESC proliferation might occur, evidence indicates a role for the ANS to alter IESC proliferation independent of ENS participation. As NE is not an enteric neurotransmitter (36), the effect of NE on intestinal epithelial cell proliferation can only occur through SNS signaling, constituting a direct effect. With regard to the PNS, ACh is used by both the PNS and ENS as a neurotransmitter (36), which prohibits the definitive attribution of the effect of ACh on intestinal epithelial cell proliferation to the PNS or the ENS. However, as PNS denervation leaves ENS circuitry intact but still alters intestinal epithelial proliferation (11, 33, 40, 57), this suggests a role for the PNS in controlling IESC proliferation, for which a direct effect cannot be ruled out.

AUTONOMIC INFLUENCE ON SOMATIC STEM CELLS: POTENTIAL APPLICATIONS FOR HUMAN MEDICINE ANS influence on somatic stem cells may be a valuable tool for applications in human medicine. Targeted manipulation of ANS outflow could be used to increase somatic stem cell proliferation and subsequently regenerate a variety of tissue types that are damaged or defective. For example, this strategy could be used to treat short bowel syndrome, which is primarily characterized by a poor absorption of nutrients caused by intestinal resection or tissue damage. Facilitation of IESC proliferation via ANS signaling could increase epithelial tissue size to improve absorptive potential. Another approach could be used to drive somatic stem cell differentiation to purposely alter the ratio of mature cell types, in this case driving the cells toward an absorptive lineage. Alteration of ANS-driven stem cell therapies might also be useful in the treatment of cancer. Many cancers are believed to be of stem cell origin (9), including certain colon cancers, which originate in IESCs and retain similar characteristics to IESCs under neoplastic conditions (7, 14, 38, 52, 54). Autonomic nerves innervate all solid tumors (39), and ANS denervation alters the growth of certain types of tumors in rodent models in vivo (19, 34, 49, 65). Some cancer cells also express autonomic neurotransmitter receptors and subsequently alter their proliferation in response to application of NE, ACh, or related agonists/antagonists in vitro (13, 22, 27, 35, 39, 45, 61), an effect that is also seen in certain types of cancer stem cells (1, 3). Therefore, ANS tone might be modulated to decrease the proliferation of cancer stem cells. In converse to the positive effects of ANS modulation on stem cell proliferation, unintended side effects on tissue regeneration may occur if stimulation or suppression of ANS activity is being used to treat other diseases [e.g., vBloc (53)]. Overall, understanding the effect and mechanism of direct ANS modulation on somatic stem cells may help to add to the current treatments in clinical practice. Perspectives and Significance As technologies improve to be able to identify, isolate, and manipulate somatic stem cells of varying tissues, the role of the ANS in directly controlling proliferation and tissue regeneration may become evident. Characterization of this understudied phenomenon would allow for better understanding of the complex interface between the nervous system and peripheral tissues, possibly allowing for the development of treatments involving neural manipulation of somatic stem cells for clinical use.

GRANTS This work was supported by United States Department of Agriculture National Institute of Food and Agriculture (to M. Dailey) No. ILLU-538–926.

DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS E.A.D. prepared figures; E.A.D. drafted manuscript; M.J.D. edited and revised manuscript; E.A.D. and M.J.D. approved final version of manuscript.