Western diet induces iron-dependent enteric neurodegeneration via ferroptosis
Arun Balasubramaniam, Dmitrii Pavlov, Yunpeng Du, Jeremy Reeves, Alan Harzman, Yunshan Liu, Francesca Cingolani, Xinxu Yuan, Jay M. Patel, Simon Musyoka Mwangi, Peijian He, C. Michael Hart, Wenhui Hu, Fievos L. Christofi, Shanthi Srinivasan
Arun Balasubramaniam, Dmitrii Pavlov, Yunpeng Du, Jeremy Reeves, Alan Harzman, Yunshan Liu, Francesca Cingolani, Xinxu Yuan, Jay M. Patel, Simon Musyoka Mwangi, Peijian He, C. Michael Hart, Wenhui Hu, Fievos L. Christofi, Shanthi Srinivasan
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Research Article Gastroenterology Neuroscience

Western diet induces iron-dependent enteric neurodegeneration via ferroptosis

Abstract

The Western diets (WD), high in saturated fats such as palmitic acid (PA), promotes enteric neurodegeneration and motility disorders. Using murine models, in vitro systems, and human myenteric ganglia, we investigated whether a WD and PA drive iron-dependent ferroptotic injury in the enteric nervous system (ENS). Mice were fed a control diet (CD) or a WD for 12 weeks, with or without systemic AAV9-MaCPNS2 delivery of Nfe2l2 to enteric neurons. Colonic motility was assessed by a bead expulsion assay. We assessed ferroptosis using convergent readouts including iron dysregulation (transferrin receptor 1 [TfR1], ferritin heavy chain 1 [FTH1], labile and mitochondrial iron [Fe2+]), lipid peroxidation (C11-BODIPY and 4-hydroxynonenal [4-HNE]), glutathione peroxidase 4 (GPX4) suppression, and pharmacologic inhibition by ferrostatin 1 (Fer-1) in primary enteric neurons, murine myenteric plexuses, and human networks of myenteric ganglia (nhMPG). WD-fed mice exhibited delayed colonic transit, increased TfR1 and FTH1, and vulnerability of nNOS neurons; these changes were reversed by nuclear factor erythroid 2–related factor 2; (Nfe2l2, also known as Nrf2) overexpression. RNA-seq of PA-treated immortalized murine fetal enteric neurons (IM-FENs) revealed disrupted neurotransmitter signaling, reduced mitochondrial and antioxidant programs, and increased iron import and lipid peroxidation signatures. PA increased labile Fe2+, mitochondrial ROS, membrane depolarization, Ca2+ dysregulation, 4-HNE, and mitoferrin 2 (Mfrn2), whereas Fer-1 preserved mitochondrial integrity, viability, and ENS function. In human nhMPG, PA induced enteric neuronal iron loading and ferroptosis, supporting the translational relevance to diet-associated enteric neuropathy.

Authors

Arun Balasubramaniam, Dmitrii Pavlov, Yunpeng Du, Jeremy Reeves, Alan Harzman, Yunshan Liu, Francesca Cingolani, Xinxu Yuan, Jay M. Patel, Simon Musyoka Mwangi, Peijian He, C. Michael Hart, Wenhui Hu, Fievos L. Christofi, Shanthi Srinivasan

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Figure 1

PA induces ferroptosis-associated iron accumulation in enteric neuronal cells.

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PA induces ferroptosis-associated iron accumulation in enteric neuronal ...
IM-FEN and primary enteric neurons were treated with vehicle (Veh), PA (0.5 mM), Fer-1 (10 μM), or PA+Fer-1 for 24 hours to assess ferroptosis-related iron dysregulation. (A) Heatmap of RNA-seq data showing differential expression of ferroptosis- and iron-regulatory genes in enteric neuron cell lines treated with vehicle or PA. (B) qRT-PCR analysis of TfR1, DMT1, GPX4, IL6, and SLC40A1 mRNA levels in enteric neuron cell lines treated with vehicle or PA, normalized to Hprt1. (C and D) Western blot analysis of FTH1 protein levels in IM-FEN cells treated with vehicle, PA, Fer-1, or PA+Fer-1; β-actin served as a loading control. (E) Immunofluorescence staining for TUBB3 (green) and FTH1 (red) in primary enteric neurons treated with vehicle, PA, or PA+Fer-1. (F) Immunofluorescence staining for TUBB3 (green) and TfR1 (red) in primary enteric neurons treated as in E, showing that PA-induced TfR1 upregulation was reversed by Fer-1. (G) FeRhoNox-1 staining for labile Fe²+ (red) with DAPI (blue) in primary enteric neurons treated as in E, showing that PA-induced iron accumulation was blocked by Fer-1. Histograms represent the fold change in signal intensity relative to vehicle. Scale bars: 50 μm. Data represents 3 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 2-tailed t test (B and C) and 1-way ANOVA with Tukey’s multiple-comparison test (D–G).