Retinoblastoma protein prevents enteric nervous system defects and intestinal pseudo-obstruction

Abstract

The retinoblastoma 1 (RB1) tumor suppressor is a critical regulator of cell cycle progression and development. To investigate the role of RB1 in neural crest-derived melanocytes, we bred mice with a floxed Rb1 allele with mice expressing Cre from the tyrosinase (Tyr) promoter. TyrCre + ;Rb1 fl/fl mice exhibited no melanocyte defects but died unexpectedly early with intestinal obstruction, striking defects in the enteric nervous system (ENS), and abnormal intestinal motility. Cre-induced DNA recombination occurred in all enteric glia and most small bowel myenteric neurons, yet phenotypic effects of Rb1 loss were cell-type specific. Enteric glia were twice as abundant in mutant mice compared with those in control animals, while myenteric neuron number was normal. Most myenteric neurons also appeared normal in size, but NO-producing myenteric neurons developed very large nuclei as a result of DNA replication without cell division (i.e., endoreplication). Parallel studies in vitro found that exogenous NO and Rb1 shRNA increased ENS precursor DNA replication and nuclear size. The large, irregularly shaped nuclei in NO-producing neurons were remarkably similar to those in progeria, an early-onset aging disorder that has been linked to RB1 dysfunction. These findings reveal a role for RB1 in the ENS. IntroductionChronic intestinal pseudo-obstruction (CIPO) is the clinical indication for 9 percent of small bowel transplants (1), yet the etiology for these disorders remains poorly understood. CIPO is diagnosed when bowel motility defects cause functional, but not mechanical, obstruction, leading to abdominal distension, pain, malnutrition, and, in severe cases, dependence on parenteral nutrition or intestinal transplantation for survival. It is likely that diverse genetic, infectious, autoimmune, metabolic, and toxic insults all contribute to CIPO etiology.Intestinal motility and many other aspects of bowel function are controlled by an interconnected intrinsic network of neurons and glia called the enteric nervous system (ENS) (2-4). The ENS forms from neural crest-derived cells that migrate through fetal bowel, proliferate extensively, and then exit the cell cycle and differentiate into many different neuronal subtypes (5-7). Defects in specific types of enteric neurons may cause life-threatening disease, and, like in the central nervous system, neurons in the ENS must be present in the proper ratios and with correct patterning for the bowel to work well. Signals that control ENS precursor proliferation and cell cycle exit are incompletely understood. Furthermore, the ENS can be damaged in many ways, often with preferential effects on NO-producing enteric neurons that inhibit bowel contraction (8). There are also regional differences in the susceptibility of enteric neurons to damage that are not well understood (9).We initially intended to develop a model of melanoma by deleting the tumor suppressor retinoblastoma 1 (Rb1) in melanocytes,

Emerging roles for enteric glia in gastrointestinal disorders

“…Vagal nerve activation of enteric glia has been linked to enhanced barrier function, and recently nicotinic cholinergic receptor signaling pathways have been shown to downregulate NF-κB in enteric glia, providing a potential intracellular gain weight slowly and suffer from intestinal pseudo-obstruction and severe colonic motility defects. Surprisingly, the most striking alteration in the ENS of these mice is a doubling of the enteric glial cell populations in the myenteric plexus of the ileum and colon, coupled with an enlargement of the size of some NOS-expressing neurons in the distal ileum (41). Other populations of enteric neurons are similar to those in control mice.…”

“…This is due in part to a limited understanding of the factors that regulate the maintenance of enteric glia cell numbers in the GI tract. However, one factor that has recently been described is the retinoblastoma 1 tumor suppressor gene (Rb1) (41). Rb1 knockout mice These studies raise the question of which neurotransmitters enteric glia respond to.…”

Genetic variants of IL‐11 associated with risk of Hirschsprung disease

“…Furthermore, there are other clues suggesting the potential association of IL‐11 with the ENS and HSCR. Firstly, the loss of the retinoblastoma 1 tumor suppressor, which is an effective target of IL‐11, causes intestinal obstruction, severe defects in the ENS, and abnormal intestinal motility . Secondly, guanine nucleotide synthesis, which is also related to IL‐11, may play an important role in the ENS development .…”

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies

“…Few animal models of CIPO exist (Clarke et al, 2007; Fu et al, 2013; Puig et al, 2009). A transgenic mouse with enteric neuropathy, delayed gastrointestinal transit, and selective loss of nNOS-expressing neurons has been described (Wangler et al, 2014) as well as a model of disordered ENS network formation and intestinal dysmotility secondary to mutations in planar cell polarity genes (Sasselli et al, 2013).…”