Nerve Injury-Induced Protein 2 preserves lysosomal membrane integrity to suppress ferroptosis

Reviewer #1 (Public review):

Summary:

This study reports a novel and potentially impactful role for NINJ2 in maintaining lysosomal integrity and regulating cellular susceptibility to ferroptosis. The authors demonstrate that NINJ2 localizes to lysosomes and interacts with LAMP1, a key lysosomal membrane glycoprotein involved in sensing lysosomal stress. Loss of NINJ2 increases lysosomal membrane permeabilization (LMP), resulting in selective leakage of lysosomal contents, including labile iron, into the cytosol. The authors further show that NINJ2 deficiency reduces the expression of ferritin storage proteins, thereby sensitizing cells to ferroptosis induced by RSL3 and erastin. Collectively, the work proposes a mechanistic link between NINJ2-mediated control of LMP, iron homeostasis, and ferroptotic vulnerability, with potential relevance to cancer biology.

Strengths:

This study identifies a novel role for NINJ2 in regulating lysosomal integrity and ferroptosis and establishes a mechanistic link between lysosomal membrane permeabilization, iron homeostasis, and ferroptotic sensitivity, with potential translational relevance in cancer.

Weaknesses:

The results overall support the authors' conclusions and provide a plausible mechanistic framework; however, additional quantification of Western blot data and further discussion of mechanistic questions would strengthen the study.

The findings are likely to have a broad impact by linking lysosomal integrity to ferroptosis and iron homeostasis, both of which are relevant to cancer biology and therapeutic targeting.

Reviewer #2 (Public review):

This manuscript, "Nerve Injury-Induced Protein 2 preserves lysosomal membrane integrity to suppress ferroptosis", identifies a previously unrecognized function of NINJ2 as a regulator of lysosomal membrane integrity and iron homeostasis, thereby suppressing ferroptosis. The authors demonstrate that NINJ2 localizes to lysosomes, interacts with LAMP1, limits lysosomal membrane permeabilization (LMP), stabilizes ferritin, and protects cells from ferroptotic cell death. They further extend these mechanistic findings to human cancer datasets, showing co-overexpression and positive correlation of NINJ2 with ferritin genes in iron-addicted cancers.

Overall, the study is conceptually interesting, technically solid, and integrates cell biology, iron metabolism, and ferroptosis in a coherent framework. The work expands the functional repertoire of the Ninjurin family beyond plasma membrane rupture and inflammation, which will be of interest to researchers in cell death, lysosome biology, and cancer metabolism.

Strengths:

(1) The identification of NINJ2 as a lysosome-associated protein that suppresses ferroptosis represents a meaningful advance beyond its previously described roles in inflammation, pyroptosis, and tumorigenesis.

(2) The work distinguishes NINJ2 functionally from NINJ1, reinforcing the idea that structurally related Ninjurins have divergent membrane-related roles.

(3) The study presents a logically connected pathway:
NINJ2 loss → LMP → labile iron increase → ferritin degradation → ferroptosis sensitization, which is well supported by the data.

(4) The link between LAMP1, ferritin turnover, and ferroptosis is particularly compelling and timely given recent interest in lysosomal contributions to ferroptotic signaling.

(5) The authors use confocal microscopy, proximity ligation assays, biochemical IPs, iron measurements, protein half-life analyses, ferroptosis assays, and TCGA-based analyses, providing convergent evidence for their model.

(6) Use of two distinct cell lines (MCF7 and Molt4) strengthens generalizability.

(7) The integration of cancer expression datasets linking NINJ2 with ferritin expression in hepatocellular and breast carcinomas enhances translational relevance.

(8) Assigning NINJ2 a lysosomal protective function, distinct from NINJ1-mediated plasma membrane rupture, is novel.

(9) Linking NINJ2 to ferroptosis regulation via lysosomal iron handling, rather than canonical GPX4 or system Xc⁻ pathways, is also novel, along with proposing a NINJ2-LAMP1-ferritin axis as a buffering mechanism against iron-driven lipid peroxidation.

(10) These insights are not incremental; they reframe how NINJ2 may function at the intersection of membrane biology, iron metabolism, and regulated cell death.

Areas for improvement:

While the study is strong, several issues should be addressed for mechanistic depth and general relevance.

(1) Although NINJ2 is shown to interact with LAMP1 and LAMP1 knockdown rescues ferritin levels, it remains unclear whether the NINJ2-LAMP1 interaction is required for lysosomal protection. The authors could:
a) Map the NINJ2 domain required for LAMP1 interaction and test whether an interaction-deficient mutant fails to protect against LMP and ferroptosis.
b) Rescue NINJ2 KO cells with wild-type versus mutant NINJ2 to establish causality.

(2) The conclusion that NINJ2 suppresses ferroptosis relies primarily on RSL3 and Erastin sensitivity. A direct assessment of ferroptosis would hence the study, such as:
a) Include ferroptosis rescue experiments using ferrostatin 1 or liproxstatin 1.
b) Assess lipid peroxidation directly (e.g., C11 BODIPY staining) to strengthen the ferroptosis claim.

(3) The manuscript discusses lysosomal ferritin degradation but does not directly examine NCOA4, a central mediator of ferritinophagy. It would be good to:
a) Test whether NCOA4 knockdown rescues ferritin loss and ferroptosis sensitivity in NINJ2 KO cells.
b) This would clarify whether NINJ2 acts upstream of canonical ferritinophagy pathways or via an alternative mechanism.

(4) The study is entirely cell-based, despite references to inflammatory and tumor phenotypes in Ninj2-deficient mice. While not strictly required, even limited in vivo validation (e.g., ferroptosis markers or iron accumulation in existing Ninj2 KO tissues) would substantially strengthen the manuscript.

(5) Finally, most imaging data (e.g., Galectin 3/LAMP1 colocalization, PLA signals) and immunoblot data are presented qualitatively. The authors should provide the qualifications of Western blots and other measurements.

Author response:

Reviewer #1:

We appreciate the reviewer’s insightful suggestions. In the revised manuscript, we will provide quantitative analysis of Western blot data throughout the study to improve data robustness and reproducibility. In addition, we will expand the “Discussion” session to address the following points raised by the reviewer #1: (1) Potential mechanisms underlying the regulation of LAMP1 transcript levels by NINJ2; (2) Whether Ninjurin1 may play a similar role in regulating lysosomal membrane permeabilization (LMP); (3) The potential clinical implications of our findings, particularly in relation to cancer progression and therapeutic targeting.

Reviewer #2:

We thank the reviewer for the insightful and constructive suggestions, which would further deepen the mechanistic understanding of the NINJ2-LAMP1 pathway and its role in ferroptosis regulation. To address the reviewer’s concerns, we will clarify the interpretation of our findings, add quantitative analyses where appropriate, and expand the Discussion to acknowledge these important mechanistic questions and future research directions. Specifically, we will revise the Statistical Analysis section to clearly describe the statistical methods used, including whether corrections for multiple comparisons were applied where appropriate. We will further discuss the potential interaction domain(s) between NINJ2 and LAMP1. We will also discuss the potential role of NCOA4, a central mediator of ferritinophagy, in the NINJ2-FTH1-LAMP1 pathway. Finally, we will include a schematic model summarizing the proposed NINJ2-LAMP1-iron-ferroptosis axis to better illustrate the working model of our study.