HIGH-FAT DIET-INDUCED FERROPTOSIS PROMOTES ENTERIC NEURODEGENERATION: A MECHANISM FOR INTESTINAL MOTILITY DISORDERS

The wiring of the enteric nervous system (ENS) is a dynamic process throughout development and adulthood. In the embryo, enteric neural crest derived cells expand and differentiate into the glia and neurons that form the ENS. In mice, these early neurons are electrically active after 2 days and regulate smooth muscle contraction within 9 days, indicating rapid differentiation into neurons with functional properties. Notably, the birth of new enteric neurons occurs into adulthood and in response to injury or inflammation, supporting the potential of leveraging postnatal neurogenesis for the treatment of enteric neuropathies. Although the ENS is formed rapidly in the embryo, the environmental conditions present in the aged intestine are different in size, structure, cell composition, signalling milieu and extracellular matrix components. Despite advancing knowledge in the mechanisms of endogenous postnatal neurogenesis and the utilization of cultured enteric neural stem cells for regenerative medicine applications, the capabilities of neurons in the postnatal environment to form neuronal circuits and reinnervate the gut have not been resolved. Here, we employ the first pan neuronal-specific Cre driver mouse model (BAF53b-Cre) to understand the potential of post-mitotic enteric neurons to reinnervate the postnatal intestine and elicit functional motor activity. BAF53b expression specifically colocalized with neuronal cell bodies (HuC/D) and fibres (TUBB3), but not enteric glia (Plp1). Cell cultures derived from single cell suspensions of the intestinal muscularis indicated that BAF53b-tdT neurons reform ganglia and interconnected networks within one-week in vitro. Support from mesenchymal cells or glia was required for enteric neurons to project nerve fibres in vitro. BAF53b-Diphtheria Toxin (DT) Receptor mice were generated to perform targeted ablation of colorectal enteric neurons by local delivery of DT. RNA-Seq analysis and wholemount imaging indicated successful and specific neuronal ablation and mice exhibited delayed fecal pellet production. Transplantation of myenteric neurons to the aganglionic environment reinnervated the colon within two weeks, restored contractile activity, and reversed delayed pellet expulsion. This was not dependent on the intestinal region of neuron isolation (colon v. small bowel). Optogenetic experiments using enteric neurons generated from BAF53b- channelrhodopsin mice transplanted to the muscularis indicate that enteric neurons can innervate the muscle with functional activity within 7 days in the postnatal environment. Together, these results demonstrate that enteric neurons are proficient in forming new functional connections in the postnatal environment in a timeframe similar to that of the embryo which highlights the remarkable capabilities of neural network formation and reinnervation in the adult intestine.

Background: Current studies provide compelling evidence that the enteric nervous system (ENS) modulates neuroimmunological processes in the small intestine. Previous work revealed the essential role of enteric glial cells therein. However, the closely associated enteric neurons, which have an essential role in gut homeostasis, including regulation of barrier function and gastrointestinal motility, have not yet been studied in this context. In addition, interleukin-6 (IL-6), a crucial cytokine in the inflamed gut known for its ambivalent effects, was no focus in the context of enteric neuronal function in disease. Therefore, we investigated the role of IL-6 signaling in enteric neurons and its impact on postoperative neuroinflammation and subsequent motility disturbances, e.g. postoperative ileus (POI).
Methods: Neuroinflammation and POI were induced by surgical intestinal manipulation of different transgenic mice. To investigate the transcriptome of enteric neurons during gut inflammation, we generated a neuron-specific RiboTag mouse and performed RNA-Seq analysis. For a closer look at neuronal IL-6 signaling, we used conditional, neuronal IL-6Ra-KO mice (IL-6Ra^neuroKO). To assess differences between the genotypes, we examined gastrointestinal (GI)-motility, immune cell infiltration, and bulk RNA-Seq data in the POI animal model. Moreover, we established primary enteric neuron cultures to evaluate the molecular mechanism of IL-6 signaling in vitro and define the neuronal reaction after IL-6 treatment by qPCR, WB and immunofluorescence microscopy.
Results: IL-6 and its targets are strongly induced during POI on both RNA and protein levels. Enteric neurons show nuclear p-Stat3 activation, and RNA-Seq analysis of neuronal mRNA showed a robust, early induction of IL-6 pathway genes. IL-6Ra^neuroKO mice lacking neuronal IL-6 signaling showed a substantial delay in the recovery of GI motility, elevated neuroinflammation, and increased immune cell infiltration. In addition, the bulk-RNA-Seq analysis revealed significant changes in gene clusters associated with migration, inflammation, and metabolic processes. IL-6 treatment activates Stat3 in primary enteric neurons and induces the expression of IL-6 target genes, inflammatory mediators, and metabolic factors, thereby connecting neuroinflammation with metabolic changes.
Conclusion: IL-6Ra^neuroKO mice showed substantial immune-mediated intestinal damage and delayed recovery in the POI animal model. Both neuroinflammatory induction of p-Stat3 and enrichment of neuronal IL-6 target genes implicate the IL-6 pathway as a central regulator in restoring homeostatic enteric neuronal function in the gut.

Background: Iron plays a vital role in neurogenesis. However, disturbed iron homeostasis can lead to ferroptotic neurodegeneration. We have previously demonstrated that a high-fat diet (HFD) can lead to enteric neuronal degeneration and subsequent delayed gastrointestinal motility. In this study, we investigated the role of iron and ferroptosis in HFD and saturated fatty acid-induced enteric neurodegeneration. We further assessed the clinical relevance of iron overload in isolated networks of human myenteric ganglia and determined the presence of iron overload in myenteric ganglia in obesity.

Methods: Immortal mouse fetal enteric neuronal (IM-FEN) cell line and mouse primary enteric neurons were treated with vehicle, palmitic acid (PA, 0.5 mM), Ferrostatin-1 (Fer-1, 10 μM), or PA together with either Fer-1 or the deferoxamine (DFO). After 24 h, the cells were analyzed by Western blotting and RT-qPCR for ferroptosis markers including ferritin heavy chain 1 (FTH-1) and transferrin receptor 1 (TfR1), and 4-hydroxynonenal (4-HNE). Mitochondrial function was measured using the Seahorse XF Cell Mito Stress Test Kit. In vivo, CF-1 mice were fed a regular diet (RD) or HFD for 4 and 12 weeks. Intestinal motility was measured by dye-transit test. Mice intestines were collected for immunostaining and Western blot analysis. Using freshly isolated networks of human myenteric ganglion cells in culture we assessed changes in iron transporter expression and ferroptosis after exposure to vehicle, PA, and IL1β.

Results: Enteric neurons treated with PA had increased levels of labile iron and FTH-1 and showed evidence of ferroptosis including increased TfR1 expression and 4-HNE levels (P<0.001). PA-induced ferroptosis was prevented by the iron chelator DFO as evidenced by the reduced 4-HNE and labile iron levels. PA-induced loss of enteric neurons was also prevented by the ferroptosis inhibitor Fer-1, (P<0.05). Erastin, an inducer of ferroptosis, led to reduced mitochondrial function (P<0.01). Male and female mice fed HFD for 12 weeks gained more weight and had slower intestinal motility (P<0.001) and reduced number of nitrergic neurons than RD-fed mice. Duodenal myenteric neurons from HFD-fed male and female mice also had higher (P<0.001) FTH-1 levels relative to RD-fed mice indicative of iron overload. Isolated human ganglion networks demonstrated evidence of increased iron transporters, DMT-1, ZIP-14, and TfR1 mRNA (P<0.05) in the presence of PA (or IL1β).

Conclusions: Our findings demonstrate HFD/PA exposure leads to mitochondrial dysfunction and enteric neuronal loss. In vitro PA treatment caused systemic iron excess, lipid peroxidation, and ferroptotic cell death in murine and human enteric neurons. Chronic HFD exposure in vivo leads to iron overload and ferroptosis and can lead to the development of enteric neuropathy and gastrointestinal dysfunction.