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 3

PA drives mitochondrial ferroptosis via ROS accumulation, mitochondrial disruption, and Mfrn2 upregulation.

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PA drives mitochondrial ferroptosis via ROS accumulation, mitochondrial ...
IM-FEN and primary enteric neurons were treated with vehicle, PA, 0.5 mM, Fer-1, 10 μM, or PA+Fer-1 for 24 hours to examine mitochondrial oxidative stress and ferroptosis-related mitochondrial changes. (A) Heatmap of mitochondrial dysfunction and ferroptosis-related markers in enteric neurons treated with vehicle or PA. (B) Heatmap of mitochondrial complex I–associated regulators of ferroptosis in the same conditions. (C) MitoFerroGreen (green) and MitoTracker Red (red) staining in IM-FEN cells showing increased mitochondria-associated labile Fe²+ after PA treatment, which is partially reduced by co-treatment with Fer-1; histogram shows fold change in MitoFerroGreen fluorescence colocalized with MitoTracker relative to vehicle. (D) Immunofluorescence staining of primary enteric neurons for TUBB3 (cyan) and MitoBrilliant 646 (magenta) with magnified insets showing PA-induced mitochondrial disruption, which was rescued by Fer-1. (E) Immunofluorescence staining of primary enteric neurons for TUBB3 (green) and Mfrn2 (brown) showing increased Mfrn2 expression with PA, which was reversed by Fer-1. Histograms in D and E represent the fold change in fluorescence intensity relative to vehicle. Scale bars: 50 μm. Data represent 3 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA with Tukey’s multiple-comparison test (C–E).