Obligatory Activation of SRC and JNK by GDNF for Survival and Axonal Outgrowth of Postnatal Intestinal Neurons.

The neurotrophin GDNF acts through its co-receptor RET to direct embryonic development of the intestinal nervous system. Since this continues in the post-natal intestine, co-cultures of rat enteric neurons and intestinal smooth muscle cells were used to examine how receptor activation mediates neuronal survival or axonal extension. GDNF-mediated activation of SRC was essential for neuronal survival and axon outgrowth and activated the major downstream signaling pathways. Selective inhibition of individual pathways had little effect on survival but JNK activation was required for axonal maintenance, extension or regeneration. This was localized to axonal endings and retrograde transport was needed for central JUN activation and subsequent axon extension. Collectively, GDNF signaling supports neuronal survival via SRC activation with multiple downstream events, with JNK signaling mediating structural plasticity. These pathways may limit neuron death and drive subsequent regeneration during challenges in vivo such as intestinal inflammation, where supportive strategies could preserve intestinal function.

GDNF requires HIF-1α and RET activation for suppression of programmed cell death of enteric neurons by metabolic challenge.

Intestinal inflammation challenges both function and structure of the enteric nervous system (ENS). In the animal model of TNBS-induced colitis, an influx of immune cells causes early neuron death in the neuromuscular layers, followed by axonal outgrowth from surviving neurons associated with upregulation of the neurotrophin GDNF (glial cell line-derived neurotrophic factor). Inflammation could involve ischemia and metabolic inhibition leading to neuronal damage, which might be countered by a protective action of GDNF. This was examined in a primary co-culture model of rat myenteric neurons and smooth muscle, where metabolic challenge was caused by dinitrophenol (DNP), O-methyl glucose (OMG) or hypoxia. These caused the specific loss of 50% of neurons by 24 h that was blocked by GDNF both in vitro and in whole mounts. Neuroprotection was lost with RET inhibition by vandetanib or GSK3179106, which also caused neuron loss in untreated controls. Thus, both basal and upregulated GDNF levels signal via RET for neuronal survival. This includes a key role for upregulation of HIF-1α, which was detected in neurons in colitis, since the inhibitor chetomin blocked rescue by GDNF or ischemic pre-conditioning in vitro. In DNP-treated co-cultures, neuron death was not inhibited by zVAD, necrosulfonamide or GSK872, and cleaved caspase-3 or - 8 were undetectable. However, combinations of inhibitors or the RIP1kinase inhibitor Nec-1 prevented neuronal death, evidence for RIPK1-dependent necroptosis. Therefore, inflammation challenges enteric neurons via ischemia, while GDNF is neuroprotective, activating RET and HIF-1α to limit programmed cell death. This may support novel strategies to address recurrent inflammation in IBD.

Analgesia and mouse strain influence neuromuscular plasticity in inflamed intestine.

BACKGROUND: Mouse models of inflammatory bowel disease (IBD) identify an impact on the enteric nervous system (ENS) but do not distinguish between Crohn's disease and ulcerative colitis phenotypes. In these models, analgesia is required, but its influence on different strains and disease outcomes is unknown. Therefore, changes to the ENS and intestinal smooth muscle were studied in trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) induced colitis to identify the effects of analgesia, and compared between two mouse strains. METHODS: Colitis was induced in CD1 or BALB/c mice receiving analgesia with either buprenorphine or tramadol. Euthanasia was on Day 8 (DSS) or Day 4 (TNBS). Outcomes were Disease Activity Index and cytokine assay, and quantitative histology and immunocytochemistry were used to evaluate effects of inflammation on neurons and smooth muscle. KEY RESULTS: In BALB/c mice, both models of colitis caused >2-fold increase in smooth muscle cell number. DSS caused axon proliferation without neuron loss while TNBS caused significant neuron loss and axonal damage. Buprenorphine (but not tramadol) was generally anti-inflammatory in both strains, but correlated with lethal outcomes to TNBS in BALB/c mice. CONCLUSIONS AND INFERENCES: Smooth muscle growth is common to both models of colitis. In contrast, ENS damage in TNBS is correlated with the severe response of a Crohn's disease-like phenotype, while DSS correlates with a milder, ulcerative colitis-like outcome in the deeper tissues. Analgesia with tramadol over buprenorphine is supported for mouse studies of IBD.