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 4

Ferroptotic injury in response to chronic, but not acute, exposure to 0.5 mM PA is sensitive to the ferroptosis inhibitor Fer-1 in IM-FEN enteric neurons.

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Ferroptotic injury in response to chronic, but not acute, exposure to 0....
(A) Chronic PA exposure abolished Ca2+ responses in IM-FEN in response to EFS. A representative example of a frequency-dependent EFS Ca2+response is shown in IM-FEN; cells were loaded with the Fluo-4 Ca2+ indicator. The Ca2+transients are shown in the panel on the right for the various treatments. PA abolished responses and pretreatment with Fer-1 preserved Ca2+ signaling. TTX eliminated all EFS activity. (B) Identical frequency response Ca2+curves were obtained in mouse IM-FEN neurons and neurons in LMMP mouse preparations in Wnt1:GCaMP Ca2+reporter mice. (C) Pooled data show that chronic exposure (24 hours) to 0.5 mM PA abolished the EFS response in IM-FEN neurons. Fer-1 prevented the chronic effect of PA. EFS responses were blocked by TTX and therefore involved neuronal Nav channels and nerve conduction. (D) Acute PA exposure led to direct concentration-dependent increases in neuronal Ca2+response from 0.01–0.5 mM. Responses were not sensitive to Fer-1. (E) Acute PA exposure (0.01–0.5 mM) did not cause significant neuronal cell death, whereas chronic PA exposure (0.5 mM) caused significant cell death in the IM-FEN population. PI exclusion assay results are shown. (F) Differential effects of acute exposure to different concentrations of PA on neuronal activity. PA (0.1 mM) reduced Ca2+responses. PA (0.5 mM) augmented EFS responses at lower frequencies of stimulation, and the responses were only partially sensitive to TTX. (G) Pooled data for acute PA effects show that PA (0.1 mM) was sufficient to nearly abolish EFS responses, whereas PA (0.5 mM) enhanced responses at low-to-intermediate frequencies, did not block the EFS response, and was associated with a TTX-insensitive component; EFS responses were normally abolished by TTX. (H) Fer-1 had no effect on frequency-dependent responses during acute PA exposure (pooled data). *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way ANOVA for statistical comparisons between curves. ΔF/F0, change in fluorescence intensity relative to baseline fluorescence (F0).