PREreview of Disruption of Iron Metabolism Resulting from Dmt1/Slc11A2 Deficiency Compromises Notch Protein Degradation and Transcriptional Activation

Summary In this manuscript, “Disruption of Iron Metabolism Resulting from Dmt1/Slc11A2 Deficiency Compromises Notch Protein Degradation and Transcriptional Activation,” the authors present a thoughtful and well-executed study exploring how Dmt1 loss disrupts iron handling and influences Notch signaling in mouse embryonic fibroblasts (MEFs). The combination of biochemical assays, fluorescent reporters, and gene expression analyses provides a solid foundation for investigating this important biological intersection.

Overall, the manuscript makes an important contribution by highlighting a mechanistic link between intracellular iron dynamics and Notch pathway regulation. The data support the authors’ central conclusions, and the study’s systematic approach is a clear strength. Below, I offer several suggestions aimed at further clarifying the mechanistic model and enhancing data presentation so readers can fully appreciate the significance of the findings.

Major Points

1. Clarifying iron availability and strengthening mechanistic interpretation

The observation that Dmt1 KO MEFs exhibit increased labile iron alongside reduced ferritin is intriguing and highlights a potentially nuanced shift in iron distribution. Expanding this discussion could help readers more fully understand how these measurements inform the proposed model of reduced iron availability.

To further reinforce the mechanistic link between iron handling and Notch signaling, the authors might consider iron-modulation experiments such as:

• TfR1 overexpression to enhance transferrin-dependent iron import

• TfR1 knockout as a complementary approach to test iron-dependence

• PCBP1/PCBP2 overexpression to facilitate intracellular iron trafficking

• Chemical iron supplementation (e.g., ferric ammonium citrate)

Each would offer additional support for the central conclusion and help distinguish DMT1-specific effects from broader disruptions in iron availability.

2. Considering alternative interpretations of lysosomal and endosomal phenotypes

The manuscript attributes several observations to lysosomal dysfunction, and the data are compelling. One additional perspective that may enrich the discussion is the possibility that these effects arise from impaired iron export from endosomes or ferritinophagic lysosomes. For example:

• Tf-containing endosomes lacking DMT1 may accumulate iron and undergo oxidative stress.

• Cells experiencing relative iron deficiency might increase ferritinophagy, leading to iron-loaded lysosomes if DMT1-mediated export is impaired.

Offering this expanded interpretation could help readers appreciate how DMT1 influences multiple iron-trafficking pathways and how these might converge on Notch processing.

3. Opportunities to further probe the iron–Notch connection

The authors present a compelling model in which altered iron availability impacts iron-dependent enzymes regulating Notch target transcription. As an additional way to explore this relationship, testing whether DFO treatment rescues or exacerbates the Dmt1 KO phenotypes could help distinguish between iron maldistribution and iron excess within specific compartments.

The manuscript may also benefit from briefly discussing whether Notch1 trafficking intersects with TfR1-containing endosomes, or whether NICD handling might interface with autophagy pathways such as ferritinophagy. These ideas are not necessary to test experimentally but could enrich the conceptual model.

Minor Points

1. Figure 1C — The distinct rescue patterns of FTH versus FTL by different Dmt1 isoforms are interesting. Clarifying relative protein levels of the isoforms would help readers understand whether this difference reflects biological function or expression efficiency.

2. Figure 2C — The staining pattern appears more widespread than solely lysosomal; a brief note in the figure or text might help clarify this.

3. Flow cytometry data — Including representative plots consistently (as done in Figure 2B) would improve transparency and aid interpretation.

4. Figure 3D — The differences in p97–EGFP localization between WT and KO cells are notable. A brief comment on whether these reflect biological differences or imaging variability would be helpful, and additional representative images could further clarify this point.

5. Figure 5 caption — A minor spacing correction between “Hes1” and “Hes5” would improve readability.

6. Introduction — It might be helpful to note that IRPs regulate mRNA translation or stability depending on IRE location, providing additional context for the Dmt1 3′-IRE.

7. Discussion phrasing — “Iron maldistribution” may better capture the phenotype than “iron accumulation,” given decreased ferritin levels.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.