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 5

PA suppresses p-Nfe2l2 in enteric neurons, while AAV-mediated Nfe2l2 overexpression restores redox signaling in vivo.

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PA suppresses p-Nfe2l2 in enteric neurons, while AAV-mediated Nfe2l2 ove...
To assess the effect of palmitate on Nfe2l2 signaling and the therapeutic potential of AAV-mediated Nfe2l2 overexpression, we performed in vitro and in vivo analyses in enteric neurons and colon tissues. (A) Immunofluorescence staining of primary enteric neurons for TUBB3 (green) and p-Nfe2l2 (orange) showing reduced p-Nfe2l2 expression with PA treatment (0.5 mM, 24 hours), rescued by Fer-1 (10 μM). Data represent 3 independent experiments. (B) Experimental timeline for in vivo study: male and female mice were fed a CD or a WD for 12 weeks, received retro-orbital AAV-EGFP or AAV-Nfe2l2 at week 2, and were phenotyped at week 11 before tissue collection. (C) Immunofluorescence staining of colon sections from CD-fed male and female mice treated with AAV-EGFP or AAV-Nfe2l2, costained for TUBB3 (cyan) and t-Nfe2l2 (red). Merged images show enhanced neuronal Nfe2l2 expression in AAV-Nfe2l2-treated mice. Histogram shows the fold change in t-Nfe2l2 fluorescence intensity within TUBB3+ neurons. (D and E) GI motility measured by bead expulsion time in male and female. n = 4 mice for AAV-EGFP groups; n = 3 mice for AAV-Nfe2l2 groups. Scale bars: 50 μm. Quantification histograms represent the fold change relative to vehicle or AAV-EGFP group as appropriate. *P < 0.05 and ***P < 0.001, by 1-way ANOVA with Tukey’s multiple-comparison test (A), unpaired, 2-tailed t test (C), and 2-way ANOVA with Tukey’s multiple-comparison test (D and E).