A premature ovarian insufficiency-associated LMD-3 point mutation impairs protein stability and causes autophagic-lysosomal dysfunction in Caenorhabditis elegans

Yile Zhai; Tiantian Wang; Meixi Gong; Yuhan Liang; Wenfei Li; Xin Wang; Zhe Zhang

doi:10.1371/journal.pone.0361215

. 2026 Oct 27;21(10):e0361215. doi: 10.1371/journal.pone.0361215

A premature ovarian insufficiency-associated LMD-3 point mutation impairs protein stability and causes autophagic-lysosomal dysfunction in Caenorhabditis elegans

Yile Zhai ¹, Tiantian Wang ², Meixi Gong ², Yuhan Liang ², Wenfei Li ^2,^*, Xin Wang ^3,^*, Zhe Zhang ^1,^2,^*

Editor: Ahmed Atwa⁴

PMCID: PMC13712518 PMID: 42945204

Abstract

Reproductive longevity decline characterizes both natural aging and premature ovarian insufficiency (POI). While our previous work established C. elegans LMD-3 as a critical regulator of reproductive capacity, the in vivo consequences of specific clinical mutations remain unclear. Here, we modeled the POI-associated human NCOA7 W804C mutation by engineering the orthologous W690C substitution into the C. elegans lmd-3 locus. We demonstrate that this single-nucleotide substitution severely compromises LMD-3 protein stability and reduces transcript levels. This mutational destabilization is associated with profound systemic autophagic-lysosomal dysfunction, which impairs macromolecular degradation and leads to widespread proteotoxic accumulation of misfolded proteins. Coincident with these organismal defects, mutant animals exhibit an early-onset collapse in reproductive capacity driven by elevated germline apoptosis. Crucially, this pathology is consistent with reduced vitamin B12 availability; mecobalamin supplementation can mitigate the lysosomal block, restoring autophagic flux, proteostasis, and fecundity. These findings elucidate how a clinical mutation drives systemic lysosomal failure and reproductive decline, suggesting vitamin B12 supplementation as a promising metabolic intervention worth further translational exploration.

Introduction

Reproductive senescence represents a hallmark of physiological aging, frequently manifesting as the earliest decline in female fecundity across diverse species, from C. elegans to humans [1,2]. In women, ovarian function diminishes prematurely compared to other somatic systems [3,4]. The acceleration of this process underlies premature ovarian insufficiency (POI), an increasingly global health concern [1,5]. A growing body of evidence connects this fertility loss to a breakdown in proteostasis, the integrated network safeguarding protein quality control [6]. Although cellular stress pathways and quality control mechanisms are conserved throughout evolution [7–9], the precise molecular triggers linking these systems to reproductive failure remain largely unmapped.

The nematode C. elegans offers an evolutionary conserved platform to dissect the molecular determinants of reproductive health [10,11]. Because its mid-life reproductive decline and oocyte quality deterioration mirror the human experience, it is highly suitable for investigating reproductive aging [2,12]. This decline is primarily orchestrated by the loss of germline integrity, a state contingent upon robust proteostasis, which is governed by specialized systems such as the lysosomal and autophagic machinery [13–15].

A key component of this protective network is the oxidation resistance 1 (OXR1) protein family, which includes OXR1 and its mammalian ortholog, nuclear receptor coactivator 7 (NCOA7) [16]. These evolutionarily conserved proteins are critical mediators of cellular stress responses [17,18]. Recently, NCOA7 has emerged as a regulator of ovarian longevity, with its orthologs exhibiting conserved protective effects against oxidative damage in zebrafish and C. elegans [19–21]. A defining feature of these proteins is the TLDc domain. This domain physically associates with V-type ATPase, the proton pump governing lysosomal acidification and proteolytic capacity [22,23]. These structural and functional parallels highlight the utility of C. elegans in decoding human reproductive pathology. We recently established that genetic ablation of lmd-3, the sole C. elegans ortholog of the NCOA7/OXR1 family, severely impairs reproductive capacity, yolk protein homeostasis, and lysosomal acidification [24].

However, while the phenotype resulting from the total loss of LMD-3 is well-defined, the biological consequences of clinical missense mutations, such as the POI-associated W804C mutation identified in the human NCOA7 TLDc domain [19], remain to be elucidated in vivo. It is currently unknown whether such site-specific amino acid substitutions function by destabilizing protein structure or by specifically disrupting essential protein-protein interactions.

In this study, we modeled the clinically relevant human mutation by engineering the orthologous W690C substitution into the C. elegans lmd-3 locus to probe its biochemical and pathogenic consequences. Specifically, we sought to determine how this single-amino-acid substitution impacts LMD-3 protein stability, systemic autophagic-lysosomal function, and reproductive capacity. Furthermore, we investigated whether metabolic supplementation could mitigate these mutational defects, thereby providing new insights into the cellular mechanisms underlying early-onset reproductive decline.

Results

Genetic and structural evidence for a shared POI-associated W690C mutation in LMD-3

The C. elegans LMD-3 protein, which serves as the sole OXR1 family member in the nematode, possesses a domain architecture highly conserved with its human orthologs, OXR1 and NCOA7 (Fig 1A). This architecture is defined by three conserved LysM, GRAM, and TLDc domains. Despite the relatively low 26.46% overall amino acid sequence identity between LMD-3 and human NCOA7 [25], the functional domains and critical motifs are remarkably homologous (S1 Fig in S1 File).

Fig 1 — (A) Schematic representation of conserved domains in *C. elegans* LMD-3 and its human orthologs, OXR1 and NCOA7. The schematic diagrams illustrate the conserved LysM, GRAM, and TLDc domains. The total number of amino acids (aa) is indicated for each protein. (B) The conserved tryptophan residue (W804) associated with premature ovarian insufficiency (POI) is highly conserved across species. A multiple sequence alignment of NCOA7 orthologs from five different species demonstrates the conservation of POI-associated W804 residue in human NCOA7. The residue, corresponding to W690 in *C. elegans* LMD-3, is highlighted with a red arrowhead. (C) Comparative structural analysis of the TLDc domain from human NCOA7 and *C. elegans* LMD-3. The TLDc domains are shown in detail to highlight their conserved structure similarity. The intrinsically disordered region (IDR) is colored gray. The conserved POI-associated tryptophan residues (human W804 and *C. elegans* W690) are indicated by black arrows and are colored red in the detailed view. (D) Sequencing confirmation of the *lmd-3*^W690C knock-in allele. Representative DNA sequencing traces confirm the successful creation of the mutation. Red arrows indicate the target base change and the knock-in site. The resulting amino acid substitution (W to C) is highlighted in red, and the synonymous mutation introduced for screening is highlighted in light blue. (E, F) Development defects detection between wild-type and *lmd-3*^W690C animals at D1 and D2 adult stages with bright-field image (E) and quantification (F) of animal body lengths (n ≥ 17 for each group, unpaired t-tests: ns, no significant difference, with effect sizes of Cohen’s d = 0.199 for D1 and Cohen’s d = 0.480 for D2). Scale bar, 50 μm.

This conservation is further underscored by the POI-associated tryptophan residue (W804) located within the TLDc domain of human NCOA7 [19]. Sequence alignment revealed that this residue is strictly conserved across multiple species, corresponding to tryptophan 690 (W690) in C. elegans LMD-3 (Fig 1B and S1 Fig in S1 File) [26]. This remarkable conservation of a clinically relevant residue provides a strong genetic basis for a shared pathogenic mechanism, validating the C. elegans W690C mutant as a suitable model for investigating this specific POI-associated variant.

Following this genetic evidence, we sought to determine if this functional similarity was underpinned by structural conservation. Comparative structural analysis using AlphaFold3 revealed a striking similarity between the predicted C. elegans LMD-3 structure and its human NCOA7 ortholog (Fig 1C). Despite differences in unique α-helices and intrinsic disordered regions (IDRs), the core LysM, GRAM, and TLDc domains are highly conserved. This structural homology is particularly evident in the V-ATPase-interacting TLDc domain, which adopts an almost identical conformation and maintains the conserved W804/W690 site (Fig 1C and S1 Video). These results provide compelling structural insights that the TLDc-V-ATPase interaction is a central, conserved mechanism governing lysosomal activity and proteostasis across species.

Based on structural evaluations, we established an in vivo model of human POI by introducing the orthologous W690C mutation into the C. elegans lmd-3 locus using CRISPR/Cas9 (Fig 1D and S1 Table). Preliminary phenotypic analysis of lmd-3^W690C homozygous mutants revealed no overt developmental defects, as confirmed by body length measurements comparable to those of wild-type animals (Fig 1E and 1F). The absence of significant developmental abnormalities, which is consistent with the localized reproductive pathology observed in human POI, confirms the lmd-3^W690C strain as a highly relevant in vivo model for dissecting the molecular mechanisms of this disease.

The POI-associated W690C mutation impairs fecundity and germline integrity in C. elegans

The deleterious W804C mutation in human NCOA7 has been implicated in POI, primarily by enhancing oxidative stress-induced senescence in granulosa cells [19]. Building on the precise conservation of this tryptophan residue in C. elegans LMD-3 protein and our established findings regarding its role in antioxidant defense [20,27], we sought to determine if the orthologous lmd-3^W690C substitution would recapitulate the reproductive pathology observed in human patients. Our analysis revealed a significant reproductive deficit: the mutant strain exhibited an approximate 15% reduction in total progeny output compared to wild-type animals (Fig 2A, 2B; S2A and S2B Fig in S1 File). Recapitulating the early functional decline characteristic of POI, this reduction was particularly pronounced during the peak reproductive window (24–72 hours of adulthood).

Fig 2 — (A) Progeny production curves show the reproductive output of wild-type (black) and *lmd-3*^W690C (red) hermaphrodites (n ≥ 13 per group, *p < 0.05). (B) Total brood sizes indicate the overall reproductive ability of wild-type and *lmd-3*^W690C mutants. Data are presented as mean ± SEM (n = 10 per group, **p < 0.01, Cohen’s d = 1.414). (C, D) Representative fluorescence images (C) and quantification (D) of DAPI-stained total germline cells in wild-type and *lmd-3*^W690C mutants (n = 14 for each group, ***p < 0.001, Cohen’s d = 1.509). Scale bars, 50 μm. (E) Representative fluorescence images and quantification of DAPI staining for germline stem cells and meiotic cells in wild-type and *lmd-3*^W690C mutants (n = 14 for each group, **p < 0.01, ***p < 0.001, with effect sizes of Cohen’s d = 1.602 for proliferative zone nuclei and Cohen’s d = 0.994 for meiotic prophase nuclei). Scale bars, 20 μm. (F, G) Germline apoptosis in wild-type and *lmd-3*^W690C mutants with SYTO12 staining (F) and a *lim-7p::ced-1::GFP* reporter (G). White arrowheads indicate apoptotic cell corpses. n ≥ 13 per group; unpaired t-tests, ***p < 0.001. D1, Day 1 of adulthood; D2, Day 2 of adulthood, with effect sizes for (F) being Cohen’s d = 2.886 at D1 and Cohen’s d = 1.851 at D2, and for (G) being Cohen’s d = 3.445 at D1 and Cohen’s d = 4.147 at D2. Scale bars, 50 µm.

To dissect the cellular basis of this diminished brood size, we evaluated whether it originated from impaired embryo production or compromised embryonic viability. We quantified both egg-laying and hatching rates, revealing that lmd-3^W690C mutants laid 14.94% fewer eggs than wild-type controls (S2C Fig in S1 File). Conversely, the hatching rate remained robust and statistically indistinguishable between the two strains (S2C Fig in S1 File). This precise delineation suggests that the fecundity defect is driven by impaired oocyte production or ovulation, rather than by post-fertilization embryonic lethality.

Given that standard reproductive aging in C. elegans is typically precipitated by sperm depletion rather than an intrinsic decline in oocyte quality [28,29], cross-fertilization assays with fem-1(hc17) females were performed to test this [30]. These assays confirmed that sperm functionality remains fully intact in the mutants (S2D Fig in S1 File). Furthermore, germline-specific overexpression of the wild-type lmd-3 gene in the mutant background fully rescued progeny counts to wild-type levels (S2E Fig in S1 File). Collectively, these findings indicate that LMD-3 functions cell-autonomously within the germline to sustain early oocyte production.

Having established this germline autonomy, we proceeded to examine the structural integrity of the germline tissue [31]. The C. elegans germline is a dynamic, spatially organized tissue defined by distinct zones of proliferation, meiosis, and differentiation, all of which are critical for sustainable oogenesis (S2A Fig in S1 File). Germ cell pools are maintained by a distal stem cell region regulated by the distal tip cell (DTC), providing a clear spatial framework for visualizing germ cell progression throughout the gonad [32].

Quantitative analysis of DAPI-stained gonads confirmed a significant reduction in the total number of germ cells in Day 1 adult lmd-3^W690C mutants (Fig 2C and 2D). This defect was accompanied by a marked decline in the number of proliferative germline stem cells (Fig 2E), suggesting that the mutation fundamentally compromises the maintenance of the progenitor pool required for ongoing germ cell production and homeostasis [33].

To elucidate the downstream mechanism driving this germ cell loss, we quantified germline apoptosis, a stress-responsive process frequently linked to compromised oocyte quality [34]. We observed a significant elevation in apoptotic cell corpses within the germlines of lmd-3^W690C mutants at Day 1 and Day 2 of adulthood (Fig 2F, 2G; S2F and S2G Fig in S1 File). These findings demonstrate a robust correlation between increased germ cell death and reduced fecundity in this POI model. Our results collectively show that the lmd-3^W690C mutation compromises germline integrity and reproductive capacity, characterized by impaired stem cell maintenance and elevated apoptosis.

The LMD-3 W690C mutation induces widespread and chronic cellular stress

To characterize the physiological implications of the W690C substitution, we first analyzed the expression profile and subcellular localization of LMD-3. An integrated (Is) transcriptional reporter demonstrated a broad tissue distribution of lmd-3, including the nervous system, intestine, and reproductive tract (Fig 3A) [27]. Subcellular fractionation confirmed that N-terminal mCherry-tagged LMD-3, including its TLDc domain, resides primarily within the cytoplasmic and membrane fractions (Fig 3B), consistent with its established lysosomal and V-ATPase-associated functions [22,23]. We also identified a minor nuclear localized fraction, which aligns with the hypothesized transcriptional and antioxidant roles attributed to its human NCOA7 and OXR1 orthologs (Fig 3B) [35,36].

Fig 3 — (A) Expression pattern of *lmd-3* at the L4 larval stage. Representative images show the expression of the *lmd-3p*::GFP transcriptional reporter. Is, integrated transgene. Scale bars, 100 µm. (B) Subcellular localization of full-length LMD-3 and its TLDc domain. The distribution of mCherry-tagged full-length LMD-3 (upper) and mCherry-tagged TLDc domain (lower) was analyzed. The fractions analyzed were cytoplasmic (Cyto), membrane (Memb), soluble nuclear (SolNu), chromatin-bound (ChroB), and cytoskeletal (CytoSkel). (C, D) Transcriptional level of *lmd-3* during different developmental stages with representative fluorescence images (C) and mean fluorescence intensity quantification (D) using the *lmd-3*p::GFP reporters (n = 6 for each group, ***p < 0.001, with effect sizes relative to L4 being Cohen’s d = 0.046 at D1, Cohen’s d = 2.788 at D2, Cohen’s d = 6.934 at D4, and Cohen’s d = 3.827 at D6). Is, integrated transgene. Scale bars, 100 μm. a.u., arbitrary units. (E, F) Representative fluorescence images (E) and mean fluorescence intensity quantification (F) of the UPR^cyto reporter *hsp-16.2*p::GFP in wild type and *lmd-3*^W690C animals at indicated stages (n ≥ 20 for each group, ns, no significant difference with Cohen’s d = 0.368, *p < 0.05 with Cohen’s d = 0.169, ***p < 0.001 with Cohen’s d = 1.117). Scale bars represent 100 μm. a.u., arbitrary units. L4, the fourth larval stage. D1, Day 1 of adulthood. D2, Day 2 of adulthood. (G, H) Representative fluorescence images (G) and mean fluorescence intensity quantification (H) of the UPR^ER reporter *hsp-4*p::GFP in wild-type and *lmd-3*^W690C animals at indicated stages (n = 10 for each group, unpaired t-tests: ns, no significant difference with Cohen’s d = 0.207 at L4, and Cohen’s d = 0.597 at D1; *p < 0.05 with Cohen’s d = 1.259). Scale bars represent 100 μm. a.u., arbitrary units.

Given the established link between oxidative stress and POI [19], and the fact that LMD-3 orthologs exhibit stress-inducible behavior in yeast and humans we investigated whether lmd-3 expression serves as a protective mechanism during aging. We observed a progressive, age-dependent increase in lmd-3 transcription (Fig 3C and 3D).

To evaluate whether the W690C mutation impairs global stress resilience, we monitored systemic cytoplasmic and endoplasmic reticulum (ER) stress from the late larval to early adult stages. The cytosolic unfolded protein response (UPR^cyto), tracked via the hsp-16.2p::GFP reporter [37], showed a gradual yet distinct induction in the mutants (Fig 3E, 3F and S2 Table in S1 File), signifying elevated systemic oxidative stress. Concurrently, the ER unfolded protein response (UPR^ER), visualized by the hsp-4p::GFP reporter [38], exhibited a progressive activation profile (Fig 3G, 3H and S2 Table in S1 File).

These findings indicate that the POI-associated W690C mutation triggers widespread, chronic cellular stress, evidenced by the activation of both cytosolic and ER stress pathways. Notably, at the onset of adulthood (Day 1), a time point marked by severe lysosomal dysfunction and reproductive decline, ER stress remains relatively stable, while cytosolic stress is only modestly elevated.

The W690C-associated lysosomal dysfunction correlates with proteostasis collapse

Building on the established interaction between the TLDc domain and V-ATPases [22,23], and given the central role of lysosomes in aging, we investigated the impact of the W690C mutation on general lysosomal function. Transcriptional profiling of whole-animal lysates revealed a significant downregulation of a broad spectrum of lysosome-related genes in the mutant (Fig 4A). This signature included key components required for lysosomal acidification (V-ATPases), structural integrity (LMP-2, NCR-1), and proteolysis (cathepsin proteases ASP-3, ASP-4).

Fig 4 — (A) Transcriptional analysis of lysosome-related genes in wild type and *lmd-3*^W690C animals at the D1 adult stage (n = 4 independent biological replicates per group, each performed in technical triplicates, unpaired t-tests: ***p < 0.001; genes were analyzed as predefined candidate targets; the complete set of Cohen’s d for each gene is provided in S1 Data). (B, C) Representative fluorescence images (B) and mean fluorescence intensity quantification (C) of NUC-1::pHTomato expression in the hypodermis driven by a heat-shock (hs) promoter (n ≥ 20 per group, ***p < 0.001, Cohen’s d = 1.477). a.u., arbitrary units. Scale bar, 10 μm. (D, E) Representative confocal fluorescence images (D) and quantification (E) of intestines co-stained with Lysosome-Specific Green (LSG DND-189) and Lysosome-Targeting Red (LTR DND-99) in wild type and *lmd-3*^W690C animals at the D1 adult stage. White arrowheads indicate vesicular lysosomes stained by both dyes. Relative intensity of LSG/LTR were quantified (n ≥ 13 per group, ***p < 0.001, Cohen’s d = 1.615). Scale bars, 10 μm. (F, G) Representative fluorescence images (F) and mean fluorescence intensity quantification (G) of secreted protein accumulation (*myo-3*p::ssGFP) in the coelomocytes of wild type and *lmd-3*^W690C animals at the D1 and D2 stages (n ≥ 20 per group, unpaired t-tests: **p < 0.01, ***p < 0.001, with effect sizes of Cohen’s d = 1.047 at D1 and Cohen’s d = 0.764 at D2). Scale bars, 50 μm. a.u., arbitrary units. (H) Confocal fluorescence images and quantification of NMY-2::GFP accumulation in wild type and *lmd-3*^W690C animals at D1 stage (mean ± SEM, n = 3 biological replicates, n ≥ 12 per condition per replicate, unpaired t-tests: ***p < 0.001, Cohen’s d = 14.471). (I) Fluorescence images and quantification of Q40::YFP accumulation in the body wall muscles in wild type and *lmd-3*^W690C animals at the L4 stage (n ≥ 16 per group, unpaired t-tests: ***p < 0.001, Cohen’s d = 3.539). Scale bars, 50 μm.

To determine whether these transcriptional defects result in a functional loss of lysosomal activity, we assessed lysosomal acidification, which is essential for efficient protein degradation [39]. Analysis using a heat-shock-inducible pH-sensitive reporter, NUC-1::pHTomato [40], revealed a significantly higher fluorescence intensity in the mutant hypodermis, signifying a less acidic, more neutralized intralysosomal pH (Fig 4B, 4C; S3A and S3B Fig in S1 File). This finding was further validated by co-staining with the acidotropic dye LysoSensor Green (LSG) and the lysosome-specific dye LysoTracker Red (LTR) [41]. The reduced LSG/LTR fluorescence ratio in the mutant definitively confirmed that the transcriptional alterations translate into a severe impairment of lysosomal acidity in C. elegans (Fig 4D, 4E; S3C and S3D Fig in S1 File).

This systemic acidification defect coincided with a failure in degradative capacity, which we assessed by monitoring the clearance of secreted ssGFP [42]. The reporter exhibited a significant accumulation of fluorescence in the coelomocytes of the mutant (Fig 4F and 4G), demonstrating a block in lysosomal degradation. This defect was associated with a broader collapse in proteostasis, characterized by extensive aggregation of the age-related protein NMY-2::GFP in oocytes [43] (Fig 4H) and the polyglutamine reporter Q40::YFP in body wall muscles [44] (Fig 4I). The vital role of LMD-3 in maintaining protein homeostasis was further underscored by the accumulation of vitellogenin—a primary egg yolk protein—in the mutant’s intestine, pseudocoelom, and germline (S3E Fig in S1 File), confirming that LMD-3 deficiency disrupts the processing of crucial reproductive proteins.

Building upon the broad genetic and biochemical framework established in our recent work [24], genetic compromise V-ATPase activity using the vha-12(ok821) loss-of-function allele not only recapitulated the reproductive defects observed in the W690C mutant but also exacerbated the decline (S3F Fig in S1 File). Consistent with the established synergistic network between LMD-3 and V-ATPases [24], these findings support a working model in which LMD-3 functionally interacts with V-ATPase-dependent pathways to maintenance lysosomal homeostasis [6,19,45].

Vitamin B12 supplementation mitigates LMD-3(W690C)-associated autophagic-lysosomal defects

Building on our findings of systemic lysosomal dysfunction and proteostasis collapse in the lmd-3^W690C mutant, which exhibits phenotypes reminiscent of the defective autophagy and senescence linked to NCOA7 dysfunction in human POI [19], we next investigated the integrity of the autophagic pathway. We first utilized a transcriptional reporter for tts-1, a stress-inducible long non-coding RNA (lncRNA) that represses global translation and serves as a direct transcriptional target of HLH-30/TFEB, the master regulator of autophagy [46]. In contrast to wild-type animals, lmd-3W690C mutants exhibited a significant global reduction in tts-1p::GFP fluorescence (Fig 5A). This was corroborated by the systemic downregulation of essential autophagy genes, including hlh-30 [47], and genes required for autophagosome formation, cargo recognition, fusion, and degradation (Fig 5B). Given that HLH-30 activity relies on nuclear translocation, we examined the subcellular localization of HLH-30::GFP; we found that stress-induced nuclear entry of HLH-30 was significantly impaired in lmd-3^W690C mutants (S4A Fig in S1 File), thereby compromising HLH-30-dependent transcriptional programs. Together, these findings indicate that the W690C mutation severely compromises both the biogenesis and function of the autophagic system.

Fig 5 — (A) Representative fluorescence images and mean fluorescence intensity quantification of the autophagy reporter *tts-1*p::GFP in wild type and *lmd-3*^W690C animals at the indicated stages (n ≥ 14 per group, ***p < 0.001, Cohen’s d = 1.417). a.u., arbitrary units. Scale bars represent 100 μm. (B) Transcriptional analysis of key autophagy genes was performed in wild type and *lmd-3*^W690C animals, and gene expression was quantified by RT-qPCR (n = 4 independent biological replicates per group, each performed in technical triplicates, unpaired t-tests: analyzed as predefined candidate targets; **p < 0.01, ***p < 0.001, ns, no significant difference; Cohen’s d values for each gene is provided in S1 Data). (C) Representative fluorescence images and quantification of SQST-1::GFP accumulation in wild type and *lmd-3*^W690C animals at D1 adult stage (n ≥ 14 per group, ***p < 0.001, Cohen’s d = 4.017). White arrowheads denote fluorescent protein aggregates. Scale bars, 50 μm. (D, E) Representative confocal images (D) and quantification (E) of the mCherry::GFP::LGG-1 reporter in wild type and *lmd-3*^W690C animals at D1 and D2 adult stages. The ratio of autophagosomes (yellow puncta, yellow arrow) to total puncta and autolysosomes (red puncta, red arrow) to total puncta was quantified (n ≥ 11 per group, ***p < 0.001; Cohen’s d values are provided in S1 Data). Scale bar, 10 μm. (F) Representative Western blot and corresponding quantification of the ratio of lipidated LGG-1-II to unlipidated LGG-1-I protein in wild-type and *lmd-3*^W690C mutant animals (n = 3 biological replicates, ***p < 0.001, Cohen’s d = 24.773). a.u., arbitrary units. IB, immunoblotting.

To confirm that these transcriptional defects correlated with functional impairment, we assessed the accumulation of SQST-1, the C. elegans ortholog of p62. Efficient clearance of SQST-1 is indicative of a healthy autophagic flux, and we observed a massive accumulation of SQST-1::GFP aggregates in the adult W690C mutants (Fig 5C), providing strong evidence that the autophagic degradation pathway is severely disrupted. Autophagic flux was assessed using the tandem fluorescent reporter mCherry::GFP::LGG-1 [48]. Compared to wild-type animals, W690C mutants exhibited a dramatic increase in autophagosomes (yellow puncta) and a concomitant decrease in mature autolysosomes (red puncta) (Fig 5D, 5E; S4B and S4C Fig in S1 File). These findings indicate that autophagic flux is impaired at the maturation and degradation stage. Furthermore, Western blot analysis of GFP::LGG-1 in whole-animal lysates revealed a simultaneous defect in autophagosome biogenesis [49], evidenced by a significant reduction in the ratio of membrane-associated LGG-1-II (lipidated) to cytosolic LGG-1-I (unlipidated) (Fig 5F). These findings confirm that the W690C mutation induces a comprehensive, multi-stage failure of the autophagic-lysosomal pathway.

Given that lmd-3^W690C dysfunction impairs lysosomal activity and reproductive health, we hypothesized that enhancing cellular resilience could restore cellular and reproductive homeostasis. Since lysosomes are critical for the uptake and processing of dietary vitamin B12, the lysosomal failure in lmd-3 mutants likely induces conditions consistent with reduced vitamin B12 availability [50]. We therefore assessed the capacity of mecobalamin (meCbl), an active form of vitamin B12, to mitigate these defects. Strikingly, treatment with 150 nM meCbl fully restored the autophagosome-to-autolysosome ratio to wild-type levels, indicating rescued autophagic flux (Fig 6A and 6B).

Consistent with this recovery, meCbl supplementation significantly reduced NMY-2::GFP aggregates in the germline (Fig 6C and 6D) and enhanced ssGFP clearance in coelomocytes (S5A, S5B Fig in S1 File). Crucially, meCbl treatment restored the diminished brood size of lmd-3^W690C mutants to wild-type levels (Fig 6E). Finally, using a dietary B12 sensor strain carrying an acdh‑1p::GFP reporter [51], a marker for the B12-independent propionate shunt pathway [52], we observed significant activation of acdh-1 transcription in lmd-3^W690C animals (Fig 6F and 6G), strongly indicating that the W690C mutation is consistent with impaired B12-dependent metabolism.

The W690C mutation impairs LMD-3 expression and structural integrity

Having established the link between the W690C mutation and profound systemic lysosomal and reproductive dysfunction, we sought to delineate the biochemical consequences of this specific point mutation—a key distinction from previous complete loss-of-function models. We first investigated whether the mutation affected lmd-3 transcript level. Transcriptional analysis across three distinct regions of the lmd-3 gene (Fig 7A and S3 Table) revealed a significant and consistent reduction in overall lmd-3 transcript levels in the mutant compared to wild-type animals (Fig 7B), indicating that the mutation results in reduced steady-state transcript levels.

Fig 7 — (A) The schematic diagram shows the genomic structure of *lmd-3*, including the positions of its conserved LysM, GRAM, and TLDc domains. The locations of the qRT-PCR primers (P1, P2, P3) designed to quantify *lmd-3* mRNA are also shown. (B) qRT-PCR measurements for endogenous *lmd-3* mRNA levels in wild type (wt) and *lmd-3*^W690C animals using primer sets P1, P2, and P3 (n = 4 independent biological replicates per group, each performed in technical triplicates, unpaired t-tests: ***p < 0.001, with effect sizes of Cohen’s d = 5.548 for P1, Cohen’s d = 13.762 for P2, and Cohen’s d = 6.039 for P3). (C, D) Structural analysis of the LMD-3^W690C protein. Predicted three-dimensional structures of wild-type (C) and W690C (D) proteins are presented to illustrate the effects of the W690C mutation on protein folding. Key domains are color-coded: LysM (cornflower blue), GRAM (light salmon), and TLDc (pale violet red). A magnified view shows the trimer-like region, where the mutation leads to altered inter-subunit distances in the p.W690C protein (49.97 Å, 53.43 Å, 36.33 Å) compared to the wild-type (47.58 Å, 52.70 Å, 39.58 Å). (E, F) The W690C mutation reduces LMD-3 protein stability. Representative Western blot images (E) and semi-quantitative analysis (F) show the turnover of 1xFLAG-tagged LMD-3 in HEK-293T cells treated with cycloheximide (CHX) for 0, 6, and 12 h (n = 3 biological replicates, *p < 0.05, ***p < 0.001, with effect sizes for 0 vs 12 h being Cohen’s d = 3.740 for wild type and Cohen’s d = 13.122 for *lmd-3*^W690C). a.u., arbitrary units.

Given the established link between human POI-associated NCOA7 mutations and protein stability [19], we reasoned that the W690C mutation might also destabilize the LMD-3 protein. Based on three-dimensional structural modeling of C. elegans wild-type and W690C-mutated LMD-3 [53], LMD-3 is predicted to form a conserved trimer-like assembly comprising LysM, GRAM, and TLDc domains (Fig 7C). While the overall core conformation remained largely preserved, the W690C mutation significantly altered the inter-subunit distances within the predicted trimer interface (Fig 7D), suggesting a disruption of structural integrity. Complementary analysis using six machine learning-based algorithms corroborated that the W690C mutation compromises overall protein stability (S4 Table in S1 File) [54–58], consistent with findings in human orthologs [19].

Evaluation of LMD-3 protein turnover in a heterologous expression system revealed that while wild-type LMD-3 protein remained relatively stable, the mutant protein exhibited a significant decrease in steady-state levels, showing marked degradation following 12 hours of CHX treatment (Fig 7E, 7F and S6A Fig in S1 File). This provides supportive biochemical evidence that the W690C missense mutation fundamentally compromises intrinsic LMD-3 protein stability, mechanistically linking the structural defect to rapid degradation.

Finally, we confirmed that RNAi knockdown of lmd-3 in wild-type animals phenocopied all observed defects, including reduced brood size, elevated ER stress, and impaired lysosomal degradation, indicating that the W690C mutation acts primarily through a loss of LMD-3 function rather than gain-of-function or dominant-negative effects (S6B–S6E Fig in S1 File). Collectively, these findings establish that the POI-associated W690C mutation severely impairs both steady-state lmd-3 transcript levels and protein stability.

Discussion

Reproductive longevity is a fundamental pillar of healthy aging, yet the molecular basis of premature ovarian insufficiency (POI) remains largely elusive. Recent translational and in vivo evaluations emphasize that preserving ovarian follicular reserve and mitigating cellular stress are critical for combating POI-associated reproductive decline [59]. Our previous work established LMD-3 as a critical regulator of reproductive capacity in C. elegans [24]. Building upon this foundation, the current study leverages a novel POI-associated W690C knock-in model. This approach allows us to dissect the pathophysiological mechanisms of fertility decline at single-amino-acid resolution. We demonstrate that this pathogenic lesion compromises reproductive health not merely through a simple loss-of-function, but by triggering a profound collapse in cellular proteostasis and lysosomal integrity, which can be partially rescued by vitamin B12 supplementation.

A core finding of our work is the mechanistic distinction between the W690C mutation and complete lmd-3 loss. Our data reveal that W690C compromises the intrinsic stability of the LMD-3 protein (based on heterologous data) and concurrently reduces its mRNA levels (Fig 7B). This observation contrasts with the human NCOA7(W804C) variant, where transcript levels remain stable [19]. The exact mechanism underlying this mRNA reduction in C. elegans requires further investigation, and may involve transcript-level destabilization or surveillance pathways. Nevertheless, such species-specific variations in mRNA regulation are widely documented in comparative biology [60]. Furthermore, at the functional level, the existence of the mammalian ortholog OXR1 may provide a functional buffer that masks the physiological impact of NCOA7 destabilization. By contrast, the non-redundant nature of the C. elegans lmd-3 locus renders the organism uniquely sensitive to the protein-level destabilization caused by W690C, making it an ideal system to map the core pathogenic consequences of this specific mutation.

The structural and functional destabilization of LMD-3(W690C) precipitates systemic lysosomal dysfunction, characterized by defective acidification and impaired degradative capacity. These results align with the evolving concept of the lysosome as a central signaling hub whose failure is intrinsically linked to aging and proteotoxicity [61–63]. We provide evidence that this organelle failure creates a detrimental environment for non-dividing cells like oocytes [6,64], leading to increased germline apoptosis and depletion of the progenitor pool. By linking impaired proteostasis, a hallmark of systemic aging [65], to early-onset ovarian dysfunction, our model bridges the gap between general cellular quality control and specific reproductive health.

Beyond mapping the pathogenic cascade, our study introduces a pioneering therapeutic perspective. Through experimental demonstration, we show that the lmd-3^W690C mutation causes primary lysosomal and autophagic dysfunction, which correlates with metabolic alterations consistent with reduced vitamin B12 availability and impaired propionate metabolism [66]. Given that methylmalonyl-CoA mutase requires cobalamin to prevent the accumulation of toxic propionate metabolites [67], we propose a working mechanistic model wherein impaired B12-dependent metabolism acts as a significant compounding factor alongside primary proteostatic collapse, rather than a strict linear sequence where lysosomal failure exclusively causes B12 deficiency which subsequently drives all downstream defects.

Within this conceptual framework, supplementation with mecobalamin (meCbl) effectively bypassed the lysosomal-metabolic block, restoring autophagic flux, clearing protein aggregates, and rescuing reproductive output. Parallel studies on metabolic and tissue-protective interventions similarly highlight the efficacy of targeted restorative strategies in rescuing compromised physiological and endocrine functions under stress conditions [68]. This finding underscores the potential for simple nutritional interventions to mitigate the cellular consequences of specific genetic lesions in C. elegans reproductive tissues, though its direct clinical applicability to human POI requires rigorous evaluation in mammalian models and clinical settings.

Despite its utility, our model has several limitations when extrapolating to human POI. First, the continuously proliferating syncytial nematode germline contrasts fundamentally with the finite human primordial follicle pool [1,32,33]. Second, mammalian ovarian aging is driven by oocyte exhaustion, whereas nematode reproductive decline involves broader systemic aging factors [28,29]. Third, our systemic assays lack the resolution to model cell-type-specific microenvironments, such as granulosa-oocyte interactions [5,19]. Finally, we rely on the acdh-1 biosensor to infer impaired B12-dependent metabolism [51,52]; future direct biochemical quantification is required to confirm these metabolic shifts. Nevertheless, our model captures conserved core mechanisms of lysosomal collapse, offering metabolic insights that warrant mammalian validation.

Looking forward, the conservation of the TLDc domain and its V-ATPase-interacting interface suggests that the LMD-3/NCOA7 pathway is a promising target for broad genetic screening in idiopathic POI. Furthermore, our findings open new questions regarding whether the natural decline of wild-type LMD-3/NCOA7 expression contributes to age-related oocyte quality decline [19]. The high-throughput nature of our C. elegans model facilitates the screening of compounds that modulate TFEB-mediated lysosomal activity or autophagic flux [48,69,70], potentially expanding the therapeutic landscape for reproductive longevity.

In summary, this study proposes a working mechanistic model linking a clinical POI-associated mutation to intrinsic protein instability, systemic lysosomal failure, and functionally associated alterations in B12-dependent metabolism. By distinguishing our experimentally demonstrated phenotypes from this broader working framework, we validate our C. elegans model as a platform for mechanistic discovery. Furthermore, we highlight vitamin B12 as a valuable conceptual framework for understanding and potentially managing early-onset reproductive decline, pending future translational studies.

Materials and methods

C. elegans strains and maintenance

All C. elegans strains (S1 Table), including the wild-type N2 Bristol isolate, were reared at 20 °C on standard nematode growth medium (NGM) plates. Bacterial lawns were established by seeding plates with Escherichia coli OP50. To ensure synchronized development for experiments, gravid adults were treated with a standard bleaching solution to harvest eggs.

CRISPR-Cas9 mediated mutagenesis

The lmd-3^W690C missense mutation was engineered by SunyBiotech using CRISPR-Cas9 technology. Sanger sequencing was employed to verify the specific point mutation (S3 Table). To minimize potential off-target effects and ensure genetic purity, the resulting mutant line was backcrossed three times against the wild-type N2 strain prior to all downstream phenotypic characterizations.

Plasmid construction

To enable biochemical assessment in mammalian systems, we developed 1xFLAG-tagged LMD-3 expression constructs. PCR was used to amplify the full-length lmd-3 cDNA sequences from both wild-type and lmd-3^W690C mutant backgrounds. These products were subsequently cloned into the pcDNA3.1 vector. Primer sequences are listed (S3 Table).

Cross-fertilization assay

To assess sperm functionality, cross-fertilization assays were conducted utilizing the spermless fem-1(hc17) mutant strain. Synchronized L4 fem-1(hc17) hermaphrodites were co-cultured with either wild-type or lmd-3^W690C males at a 1:4 ratio on 35 mm NGM plates at the restrictive temperature of 25 °C. Following a 20-hour mating period, the hermaphrodites were individually transferred to fresh plates at the same temperature, and the total number of cross-progeny was subsequently quantified.

Transgene generation and germline rescue

For germline-specific rescue experiments, the pie-1p::mCherry::lmd-3 expression plasmid was introduced into the lmd-3^W690C mutant background via standard microinjection techniques. The injection mixture comprised the rescue construct, 200 ng/μL of sheared salmon sperm DNA to mitigate transgene silencing, and 5 ng/μL of a myo-2p::Tomato Red co-injection marker. Transgenic F1 progeny, identified by red fluorescence in the pharynx, were subsequently isolated for brood size evaluations.

Quantitative real-time PCR (qRT-PCR)

For transcript quantification, 200 age-synchronized Day 1 adults were collected in M9 buffer, subjected to lysis using Trizol Reagent (Invitrogen, 15596026CN), and flash-frozen in liquid nitrogen. Total RNA was isolated through chloroform/isopropanol extraction. Subsequently, 1.2 μg of RNA was reverse-transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) (Vazyme, R312-01). qRT-PCR was conducted on an ABI QuantStudio 1 system (Thermo Fisher Scientific) utilizing ChamQ SYBR Color qPCR Master Mix (Vazyme, Q411-02). Data were normalized to the act-1 endogenous control.

Reproductive capacity analysis

In accordance with established protocols [71], a minimum of 10 synchronized L4 hermaphrodites per strain were individually isolated on fresh NGM plates. Worms were transferred to new plates every 12 hours until the cessation of egg-laying. The total progeny output per individual was determined by counting the offspring collected from each transfer. Experiments were executed in three biological replicates.

Measurement of body length

Worms at specified developmental stages were anesthetized in levamisole (Sangon Biotech, China) and immobilized on agarose pads. Bright-field images were captured via a Nexcope 910 microscopy system. Body length (head-to-tail distance) was calculated using Image View software, with a minimum of 22 animals analyzed per experimental cohort.

Microscopy and imaging analysis

For all fluorescence and bright-field imaging, animals were anesthetized with levamisole and mounted on agarose pads. Depending on the resolution requirements, images were acquired using either a Nexcope 910 fluorescence microscope or a Zeiss LSM 980 confocal microscope. Quantitative image metrics, such as fluorescence intensity and puncta characteristics, were assessed using ImageJ (1.54J, NIH). For fluorescence intensity, the mean signal (total intensity per region of interest) was recorded. Growth-related variation was mitigated by normalizing intensity to the animal’s body area. All quantification was conducted by researchers blinded to the experimental genotype to prevent observer bias.

Germline staining and quantification

To assess germline integrity, synchronized Day 1 adults were fixed in ice-cold methanol for 5 minutes, followed by three washes in M9 buffer. Nuclei were visualized by incubating samples in DAPI (Macklin, D807022) for 30 minutes in the dark, followed by additional washing steps. Confocal microscopy was used to examine the gonads. The total germ cell population and proliferative stem cell counts were determined quantitatively using ImageJ (1.54J, NIH).

SYTO12 staining for germline apoptotic cells

Synchronized larvae were reared to Day 1 and Day 2 of adulthood. Animals were incubated in SYTO 12 dye (Thermo Fisher Scientific, S7575) for 3 hours in the dark. Post-incubation, worms underwent three washes via centrifugation (4,000 rpm, 1 minute) and were allowed a 1-hour recovery period on NGM plates to clear residual dye from the gut. Anesthetized animals were examined via green fluorescence microscopy to score apoptotic corpses.

Dual lysosomal staining and quantification

As previously described [72], synchronized Day 1 adults were co-incubated with 10 mM LysoSensor Green DND-189 and LysoTracker Red DND-99 (Yeasen) for 1 hour at 20 °C in the dark. A 1-hour recovery phase on OP50-seeded NGM plates followed. Lysosomal acidity was assessed by calculating the fluorescence ratio of LysoSensor Green to LysoTracker Red via ImageJ (1.54J, NIH); a reduced ratio indicates diminished lysosomal acidity.

Cell culture and Transient transfection

HEK-293T cells were cultured in DMEM (C119955-00BT, Grand Island Biological Company) containing 10% fetal bovine serum (Excell Media) at 37 °C in a 5% CO² atmosphere. Cells were transfected using polyethyleneimine (PEI) (Sigma-Aldrich, 408727) at a DNA-to-PEI ratio of 1:4.5, with 1 µg of total DNA used per 10^6 cells.

Cycloheximide pulse chase assay

Protein turnover was evaluated via a Cycloheximide (CHX) chase assay [73]. Thirty-six hours post-transfection, cells were treated with 50 µg/mL CHX to inhibit new protein synthesis. At specified intervals, cells were harvested to quantify protein levels via Western blot analysis.

RNA interference (RNAi)

RNAi experiments were conducted utilizing an RNAi-competent E. coli OP50 strain (iOP50) transformed with either the L4440 empty vector (control) or an lmd-3 RNAi construct. iOP50 cultures were grown in LB broth containing 100 µg/mL ampicillin, then seeded onto NGM plates supplemented with 100 µg/mL ampicillin and 1 mM IPTG to initiate dsRNA production. Plates were incubated overnight at 25 °C. Synchronized L4 larvae were transferred to these plates and maintained at 20 °C until they reached the young adult stage for downstream analysis.

Autophagy flux and aggregation analysis

Autophagic flux was monitored using the tandem reporter mCherry::GFP::LGG-1. In anterior intestinal cells, autophagosomes (AP) were identified as GFP/mCherry-positive (yellow) puncta, while autolysosomes (AL) were identified as mCherry-only (red) puncta (due to GFP quenching). SQST-1::GFP aggregates in head neurons were similarly quantified. To ensure stringency, quantification was performed manually by two independent, blinded investigators, and results were averaged. A minimum of 11 worms per condition were analyzed across three biological replicates.

Mecobalamin supplementation

A 150 µM stock of meCbl (Macklin, M812748) was dissolved in H₂O (also used for control plates). For treatment, the stock was diluted into the NGM agar medium to a final concentration of 0.15 µM prior to plate pouring, ensuring continuous exposure throughout the lifecycle.

Quantification of myo-3p::ssGFP intensity

Day 1 adult C. elegans expressing the secreted myo-3p::ssGFP reporter were imaged. Coelomocytes were manually identified as regions of interest (ROI) using ImageJ (1.54J, NIH). Mean fluorescence intensity was calculated for each ROI, and values from all coelomocytes in an individual animal were averaged. Background fluorescence from non-coelomocyte regions was subtracted. At least 18 animals were evaluated per group.

Quantification of NMY-2::GFP aggregates in germline oocytes

Day 1 adults expressing NMY-2::GFP were selected for evaluation. Using a Zeiss LSM 980 confocal microscope, we determined the presence or absence of aggregates in the two most proximal oocytes of the gonad. Scoring was performed by a blinded investigator. The aggregate proportion was calculated from three independent biological replicates, with at least 10 animals analyzed per replicate.

Quantification of NUC-1::pHTomato intensity

Lysosomal acidification was assessed using the pH-sensitive reporter PhsNUC-1::pHTomato [40,43]. Expression was induced by a 30-minute heat shock at 33 °C, followed by a 24-hour recovery at 20 °C. Mean pHTomato fluorescence intensity per unit area in the hypodermis was quantified using ImageJ (1.54J, NIH) in at least 20 animals per strain.

Western blotting

Day 1 adults (n = 200) were lysed in 1 × sample buffer and boiled at 95 °C for 10 minutes. Protein extracts were separated via SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST and probed with primary antibodies: anti-GFP (1:1000, abclonal, AE011), anti-α-tubulin (1:5000, Sigma-Aldrich, T5168), anti-flag DDDDK-tag (1:10000, MBL, M185), and anti-GAPDH (1:500, Absin, abs830030ss). Signals were detected using ECL (Tanon 5200). Due to stripping constraints, parallel gels were used for target proteins and loading controls. Raw, uncropped blot data are included (S2 Data).

Protein sequence and structure prediction

Human NCOA7 (NP_001186548.1) and C. elegans LMD-3 (NP_505173.2) sequences were retrieved from NCBI. 3D modeling was performed using AlphaFold3 [53]. Predicted structures were analyzed via ChimeraX 1.10.1 (University of California, San Francisco).

Inter-domain distance measurement

To quantify the structural impact of the mutation on the predicted LMD-3 trimer-like region, the distances between the conserved LysM, GRAM, and TLDc domains were measured. The centroid of each respective domain was defined as a vertex of a triangle. The distances between these centroids were then calculated using the “Distance” function within the “Annotation” module of ChimeraX 1.10.1.

Protein stability prediction

To assess the effect of the point mutation on protein stability, we employed six distinct machine learning-based prediction tools [54–58]. The LMD-3 protein sequence and 3D structural model were the same as those used for the structural analysis above. For each web-based tool, the W690C substitution was specified to calculate the change in free energy (ΔΔG). The definition of a “Destabilizing” prediction varied among the different websites, corresponding to specific ΔΔG thresholds.

Statistical analysis

All data were statistically analyzed using GraphPad Prism 9 (La Jolla, CA, USA). Data are presented as the mean, with error bars representing the standard error of the mean (SEM). Student’s t-tests were utilized for comparisons between two distinct groups, while one-way ANOVA tests were performed for multiple group comparisons. To evaluate the magnitude of differences between groups, effect sizes were calculated using Cohen’s d where appropriate using SPSS 27 (Chicago, USA). Statistical significance was established at p < 0.05 (indicated by *), p < 0.01 (indicated by **), and p < 0.001(indicated by ***); ns, no significant difference. For targeted transcript analyses (such as lysosomal and autophagy-related genes in Figs 4A and 5B), the evaluated genes were treated as predefined candidate targets and assessed using targeted individual hypothesis tests without multiple testing corrections.

Supporting information

S1 File. Supplementary Figures and Tables.

This file contains S1 to S6 Figs, S2 Table, and S4 Table.

(PDF)

pone.0361215.s001.pdf^{(1.2MB, pdf)}

S1 Video. Structural conservation of the TLDc domains.

This movie presents a 360-degree rotation of the aligned TLDc domains from human NCOA7 (pink) and C. elegans LMD-3 (light blue). The movie highlights the remarkable structural conservation, with key conserved tryptophan residues (human W804 and C. elegans W690) highlighted by black arrows and colored bright green and yellow, respectively.

(MP4)

Download video file^{(4.4MB, mp4)}

S1 Table. C. elegans strains used in this study.

(XLSX)

pone.0361215.s003.xlsx^{(12KB, xlsx)}

S3 Table. Primers and oligos used in this study.

(XLSX)

pone.0361215.s004.xlsx^{(16.8KB, xlsx)}

S1 Data. Comprehensive record of experimental raw data.

(XLSX)

pone.0361215.s005.xlsx^{(140.2KB, xlsx)}

S2 Data. Uncropped scans of western blot membranes.

(DOCX)

pone.0361215.s006.docx^{(18.2MB, docx)}

Acknowledgments

We thank the Caenorhabditis Genetics Center (CGC), Dr. Xiaochen Wang (Southern University of Science and Technology), Dr. Dengke Ma (UCSF), and Dr. Chenggang Zou (Yunnan University) for strains. Dr. Yingying Qin, Dr. Ting Dong, and Dr. Xue Jiao for their valuable suggestions (Shandong University).

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This research was supported by a grant (no. 2022KF006) from the State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, and the Natural Science Foundation of Shandong Province (Grant No. ZR2026MS0275 to Z.Z.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0361215.r001

Decision Letter 0

Ahmed Atwa

29 Jul 2026

Dear Dr. Zhang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Dear Authors,

Thank you for submitting your manuscript entitled “A premature ovarian insufficiency-associated LMD-3 point mutation impairs protein stability and causes autophagic-lysosomal dysfunction in Caenorhabditis elegans ” to PLOS ONE.

The reviewers found the study interesting and biologically relevant, and they acknowledged the value of establishing a C. elegans model carrying a POI-associated LMD-3/NCOA7 point mutation. The manuscript provides potentially important insights into the relationship between LMD-3 protein stability, lysosomal/autophagic dysfunction, proteostasis, germline apoptosis, and reproductive decline.

However, both reviewers identified several issues that must be addressed before the manuscript can be considered further. In particular, the mechanistic and translational conclusions require moderation and/or stronger support. The claims linking LMD-3 mutation, V-ATPase dysfunction, autophagy impairment, functional vitamin B12 deficiency, and reproductive decline should be presented more cautiously unless additional direct or genetic evidence is provided. The reviewers also requested clarification of the reduced LMD-3 transcript level, stronger support for the proposed vitamin B12-related mechanism, improved distinction between Results and Discussion, and greater emphasis on the limitations of extrapolating findings from C. elegans to human POI.

Therefore, I invite you to submit a substantially revised manuscript. Please provide a detailed point-by-point response to all reviewer comments and revise the manuscript accordingly. In particular, please ensure that:

The manuscript should moderate its translational claims: the current C. elegans data support a disease-relevant model, but they do not yet establish vitamin B12/mecobalamin as a directly translatable therapeutic intervention for human POI.
The proposed LMD-3–V-ATPase–lysosome–autophagy–reproductive decline pathway is interesting, but the causal hierarchy remains incompletely demonstrated; the authors should either add genetic/functional validation or clearly frame this as a proposed mechanistic model.
The reported reduction in both LMD-3 protein stability and transcript expression requires clarification, as the mechanism underlying decreased mRNA levels is not addressed and may affect interpretation of the mutation as primarily protein-destabilizing.
The vitamin B12 deficiency claim should be softened unless supported by direct biochemical measurements of B12 status or related metabolic markers; current evidence appears functional and indirect.
The Results section should be revised to remove methodological explanations and mechanistic interpretation, reserving such interpretation for the Discussion, with clearer separation between observed data and proposed biological mechanisms.

==============================

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Kind regards,

Ahmed Atwa, Ph.D

Academic Editor

PLOS One

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: N/A

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Recommendation: Major Revision

Major Comments

1. Although the study successfully establishes a C. elegans model carrying the POI-associated mutation, the translational relevance to human disease remains insufficiently supported. The manuscript would benefit from additional experimental evidence or a more cautious interpretation of the findings regarding human POI.

2. The authors report that the W690C mutation reduces both LMD-3 protein stability and transcript levels. However, the mechanism underlying the reduction in mRNA expression is not investigated. Additional experiments or a more detailed discussion addressing this observation would strengthen the mechanistic conclusions.

3. The conclusion that lysosomal dysfunction induces a functional vitamin B12 deficiency is based primarily on indirect evidence. Direct biochemical measurements of vitamin B12 levels or related metabolic markers would provide stronger support for this important claim.

4. The proposed mechanistic pathway linking LMD-3, V-ATPase dysfunction, autophagy impairment, and reproductive decline would be strengthened by additional genetic interaction (epistasis) experiments or complementary functional analyses.

5. Several conclusions, particularly those suggesting that vitamin B12 represents a "translatable therapeutic intervention," appear stronger than the presented data support. Since all experiments were performed in C. elegans, these statements should be moderated.

Minor Comments

1. The quality of several fluorescence microscopy images should be improved to facilitate data interpretation.

2. Some quantitative analyses, particularly RT-qPCR experiments with only three biological replicates, would benefit from additional replication to improve statistical robustness.

3. The Discussion should further emphasize the limitations of the C. elegans model when extrapolating findings to human ovarian biology.

4. Several sentences in the Introduction and Discussion are overly long and could be revised for improved readability.

5. Reporting effect sizes in addition to p-values would improve the statistical presentation of the results.

Overall Evaluation

This manuscript addresses an interesting and biologically relevant question using a comprehensive experimental approach. The data are generally of good quality, and the findings provide valuable insights into the role of LMD-3 in lysosomal function and reproductive aging. However, several mechanistic claims require stronger experimental support, and some conclusions should be presented more cautiously. Therefore, I recommend Major Revision before the manuscript can be considered for publication.

Reviewer #2: This manuscript addresses a relevant topic—the impact of LMD-3 point mutation on premature ovarian insufficiency in C. elegans.

The introduction is well structured and allows a comprehensive overview of the recent advances related to the topic. However, authors described the findings of the current study at the end of the introduction section. These data are not relevant to the introduction, they should be removed “Our data show that this point mutation significantly reduces early reproductive capacity…..a potential therapeutic intervention”.

Authors should clearly formulate the aim of the study at the end of the introduction section.

Some methodological points are stated in the results section (example: Employing two complementary approaches—the nucleic acid dye SYTO12 and a lim-7p::ced-1::GFP reporter [35,36], we observed….These data are to remove as they are related to the methods section.

The sesults section should not contain data interpretation. Authors are invited to describe experiments results without anay interpretation. “This temporal dissociation suggests that the global UPR is likely a concurrent or secondary response rather than the primary early trigger of reproductive failure. Instead, our data sugges that early reproductive senescence is more likely driven by a breakdown in localized germline protein quality control, specifically through impaired lysosomal degradative capacity [6,19,41].”

“These data indicate that the W690C mutation imposes a substantial burden on lysosomal biogenesis and system-wide function. “

The discussion section is well written and appropriatly explains the interest of the current study in establishing a comprehensive mechanistic framework for the link between LMD-3 mutation and premature ovarian insufficiency in C. elegans.

**********

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Reviewer #1: Yes: Ali Salehi

Reviewer #2: No

**********

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PLoS One. 2026 Oct 27;21(10):e0361215. doi: 10.1371/journal.pone.0361215.r002

Author response to Decision Letter 1

13 Aug 2026

Response to Reviews:

We are grateful to the reviewers for their constructive and insightful feedback on our manuscript. We are encouraged that they recognized the value of our C. elegans POI model. In response to their suggestions, we have increased RT-qPCR biological replicates, optimized image quality, and included effect size measurements. More importantly, we have restructured the Results and Discussion sections, systematically moderated our translational and vitamin B12-related claims, framed the proposed pathway as a working mechanistic model, and resolved all technical formatting and raw data requirements.

A detailed, point-by-point response to all comments, along with the marked-up and clean manuscript files, has been fully uploaded as an attachment ('Response to Reviewers'). For quick reference, a summary of our point-by-point responses is also provided below:

Reviewer #1: Recommendation: Major Revision

Major Comments:

Regarding the translational relevance to human disease, we have systematically softened our claims on vitamin B12 as a direct clinical intervention, framing it instead as a conceptual metabolic lead, and expanded the Discussion to explicitly outline the biological and physiological boundaries of extrapolating C. elegans data to human ovarian biology.

Regarding the reduced lmd-3 mRNA expression, we increased our RT-qPCR biological replicates from n=3 to n=4 (now in updated Fig 7B), added a discussion on theoretical transcript surveillance mechanisms, and addressed species-specific variations.

Regarding the indirect evidence of vitamin B12 deficiency, we softened the language in the Results to "strongly indicating" and explicitly acknowledged the reliance on the acdh-1 biosensor as a limitation in the Discussion.

Regarding the genetic interaction pathway, we have contextualized the proposed cascade as a working mechanistic model rather than proven linear epistasis, keeping the focus on variant-specific biochemical collapse and metabolic rescue.

Regarding the moderation of therapeutic claims, we have thoroughly tempered our conclusions in the Abstract and Discussion to avoid overstating clinical efficacy.

Minor Comments:

We have re-evaluated and optimized the resolution, contrast, and clarity of key fluorescence images (Fig 4H-I, Fig 5C, S3F Fig).

We have increased RT-qPCR biological replicates from n=3 to n=4 across all transcriptional analyses (Fig 4A, 5B, 7B).

We have expanded the Discussion to thoroughly emphasize the limitations of the C. elegans model.

We have systematically shortened and clarified overly long sentences in the Introduction and Discussion.

We have calculated and reported effect sizes (Cohen's d) for quantitative comparisons in the figure legends and S1 Data.

Reviewer #2:

1 & 2. We have removed the summary of findings from the Introduction and replaced it with a clear formulation of the study's aims.

3. We have removed methodological statements from the Results section.

4. We have removed data interpretations and speculations from the Results section to ensure purely objective data reporting.

5. We sincerely thank the reviewer for the encouraging assessment of our Discussion section.

Attachment

Submitted filename: 4. Response to Reviews_PLoS one_RV1.pdf

pone.0361215.s008.pdf^{(245.8KB, pdf)}

PLoS One. doi: 10.1371/journal.pone.0361215.r003

Decision Letter 1

Ahmed Atwa

5 Oct 2026

Dear Dr. Zhang,

Thank you for submitting your manuscript to PLOS One. After careful consideration, we feel that it has merit but does not fully meet PLOS One’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

The main points that should be addressed are as follows:

Please soften all statements referring to vitamin B12 “deficiency” and instead frame the findings as being “consistent with reduced vitamin B12 availability” or “consistent with impaired B12-dependent metabolism.”
Please avoid presenting AlphaFold3-based structural modeling as experimental evidence.
Please clarify that the cycloheximide-chase experiment performed in HEK293T cells supports an effect of the mutation on protein stability, but does not directly demonstrate altered endogenous LMD-3 stability in C. elegans.
Please revise the interpretation of the qRT-PCR data to indicate “reduced steady-state transcript abundance,” rather than implying transcriptional repression unless direct evidence is available.
Please clarify whether correction for multiple comparisons was performed. Alternatively, if the analyzed genes were predefined candidate targets, this should be explicitly stated in the Methods and/or figure legends.
Please ensure that the Discussion is appropriately contextualized within the relevant recent literature on lysosomal dysfunction, autophagy, vitamin B12-related metabolism, and reproductive decline. Where directly relevant, the authors may consider incorporating additional recent studies, including 10.33594/000000744 and 10.1186/s43088-025-00604-2. These references should be discussed in relation to the manuscript’s mechanistic interpretation, rather than added as isolated citations.

Overall, the appropriate decision is Minor Revision, with editorial assessment after revision. The manuscript appears close to acceptance, but the remaining mechanistic and interpretative statements should be refined before final acceptance.

==============================

Please submit your revised manuscript by Nov 04 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS One offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Ahmed Atwa, Ph.D

Academic Editor

PLOS One

Journal Requirements:

1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

**********

Reviewer #1: The authors have made substantial and constructive revisions in response to the previous review. The increased number of biological replicates, inclusion of effect-size measurements, improvement of figure quality, provision of raw Western blot data, and expanded discussion of the limitations of the C. elegans model have strengthened the manuscript considerably. I also appreciate the authors’ efforts to moderate several of the translational and therapeutic statements.

Nevertheless, a few points should be addressed before publication:

1. **Vitamin B12 interpretation:** The acdh-1p::GFP reporter provides evidence consistent with altered vitamin B12 availability, but it does not directly demonstrate systemic vitamin B12 deficiency. Although this limitation is now acknowledged in the Discussion, some statements in the Results and Conclusion remain stronger than the available evidence. Please consistently frame the B12 phenotype as being *consistent with reduced vitamin B12 availability or impaired B12-dependent metabolism*, rather than as definitively demonstrating systemic B12 deficiency.

2. **Causal relationship between lysosomal dysfunction and B12 deficiency:** The mecobalamin rescue experiments are interesting and support a functional relationship between B12 metabolism and the observed phenotypes. However, they do not establish a definitive linear pathway in which lysosomal dysfunction causes B12 deficiency, which then causes autophagic and reproductive defects. Please distinguish clearly between experimentally demonstrated findings and the proposed mechanistic model.

3. **Structural modeling:** The AlphaFold3-based structural analyses and predicted trimer-like assembly are useful for generating mechanistic hypotheses. However, computational predictions should not be presented as experimental evidence of oligomerization, structural disruption, or a direct mechanism of protein degradation. Please moderate the relevant statements accordingly.

4. **Protein stability experiment:** The cycloheximide-chase experiment was performed in HEK293T cells using exogenously expressed FLAG-tagged LMD-3. This is supportive evidence for an intrinsic effect of the W690C substitution on protein stability, but it does not directly demonstrate altered endogenous LMD-3 stability in C. elegans. Please maintain this distinction throughout the manuscript.

5. **Interpretation of qRT-PCR:** Reduced steady-state lmd-3 mRNA abundance does not by itself demonstrate transcriptional repression. Statements such as “the mutation suppresses gene expression” should therefore be replaced with more precise wording indicating reduced steady-state transcript abundance, unless additional transcriptional evidence is available.

6. **Autophagy interpretation:** The tandem mCherry::GFP::LGG-1 reporter and LGG-1 measurements support impaired autophagic maturation and/or degradation. However, the precise molecular step at which the defect occurs is not completely established. Please avoid overly definitive statements regarding a block specifically at autophagosome–lysosome fusion or degradation.

7. **Genetic interaction with vha-12:** The vha-12 genetic experiment supports a functional relationship between LMD-3 and lysosomal V-ATPase-dependent function, but it does not establish direct molecular regulation or definitive linear epistasis. The interpretation should remain consistent with the authors’ revised description of this relationship as a working mechanistic model.

8. **Statistical analysis:** For the multiple lysosomal and autophagy-related genes analyzed individually, please clarify whether correction for multiple comparisons was performed. If these genes were predefined candidate targets and individual hypothesis tests were intentionally used, this should be explicitly stated in the Methods and/or figure legends.

Subject to appropriate clarification and moderation of these points, I consider the revised manuscript substantially improved. The remaining issues are primarily related to the precision of mechanistic interpretation rather than fundamental experimental deficiencies.

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PLoS One. 2026 Oct 27;21(10):e0361215. doi: 10.1371/journal.pone.0361215.r004

Author response to Decision Letter 2

6 Oct 2026

Responses to Editorial Requests:

We sincerely thank the Academic Editor and the reviewer for their careful reading of our manuscript and for their valuable feedback. We have meticulously addressed each of the points raised in the editorial report, as detailed below:

1. Please soften all statements referring to vitamin B12 “deficiency” and instead frame the findings as being “consistent with reduced vitamin B12 availability” or “consistent with impaired B12-dependent metabolism.

We have thoroughly screened the manuscript to remove definitive or absolute references to systemic vitamin B12 "deficiency". All relevant statements across the Abstract, Results, and Discussion have been appropriately moderated and reframed as being "consistent with reduced vitamin B12 availability" or "consistent with impaired B12-dependent metabolism."

2. Please avoid presenting AlphaFold3-based structural modeling as experimental evidence.

We have carefully revised our text to ensure that the AlphaFold3-based structural predictions are strictly presented as computational models and hypothesis-generating analyses rather than direct experimental evidence of protein oligomerization or structural disruption.

3. Please clarify that the cycloheximide-chase experiment performed in HEK293T cells supports an effect of the mutation on protein stability, but does not directly demonstrate altered endogenous LMD-3 stability in C. elegans.

We have clarified throughout the text (including the Results and Discussion) that the cycloheximide (CHX)-chase assay was performed in heterologous HEK293T cells using exogenously expressed FLAG-tagged LMD-3, providing supportive evidence for intrinsic protein stability effects rather than direct measurement of endogenous LMD-3 stability in C. elegans.

4. Please revise the interpretation of the qRT-PCR data to indicate “reduced steady-state transcript abundance,” rather than implying transcriptional repression unless direct evidence is available.

We have replaced absolute terms implying transcriptional repression (such as "suppresses gene expression") with more precise wording indicating "reduced steady-state transcript abundance" across the manuscript.

5. Please clarify whether correction for multiple comparisons was performed. Alternatively, if the analyzed genes were predefined candidate targets, this should be explicitly stated in the Methods and/or figure legends.

We have explicitly clarified in the Materials and Methods and figure legends that the analyzed lysosomal and autophagy-related genes were predefined candidate targets evaluated using targeted individual hypothesis tests.

6. Please ensure that the Discussion is appropriately contextualized within the relevant recent literature on lysosomal dysfunction, autophagy, vitamin B12-related metabolism, and reproductive decline. Where directly relevant, the authors may consider incorporating additional recent studies, including 10.33594/000000744 and 10.1186/s43088-025-00604-2. These references should be discussed in relation to the manuscript’s mechanistic interpretation, rather than added as isolated citations.

We have significantly enriched the Discussion section by appropriately contextualizing our findings within the broader literature on lysosomal dysfunction, autophagy, and reproductive decline. Specifically, we have incorporated and discussed the suggested recent studies (DOI: 10.1186/s43088-025-00604-2 and DOI: 10.33594/000000744, as new References [59] and [68] respectively) in direct relation to our mechanistic interpretations. In addition, all other minor interpretative caveats raised by Reviewer #1, including distinguishing the mechanistic model from linear epistasis for vha-12, moderating explicit steps in autophagic flux, and separating experimentally demonstrated findings from proposed models, have been fully integrated and refined throughout the revised manuscript.

Response to Reviewer #1:

We sincerely thank the Reviewer for the constructive feedback and positive evaluation of our previous revisions. We have fully addressed the remaining points raised, focusing on refining mechanistic phrasing, moderating absolute claims, and enhancing interpretive precision. Our point-by-point responses are detailed below:

The authors have made substantial and constructive revisions in response to the previous review. The increased number of biological replicates, inclusion of effect-size measurements, improvement of figure quality, provision of raw Western blot data, and expanded discussion of the limitations of the C. elegans model have strengthened the manuscript considerably. I also appreciate the authors’ efforts to moderate several of the translational and therapeutic statements. Nevertheless, a few points should be addressed before publication:

1. Vitamin B12 interpretation: The acdh-1p::GFP reporter provides evidence consistent with altered vitamin B12 availability, but it does not directly demonstrate systemic vitamin B12 deficiency. Although this limitation is now acknowledged in the Discussion, some statements in the Results and Conclusion remain stronger than the available evidence. Please consistently frame the B12 phenotype as being “consistent with reduced vitamin B12 availability or impaired B12-dependent metabolism”, rather than as definitively demonstrating systemic B12 deficiency.

We completely agree with the reviewer’s insightful critique. While the robust induction of the acdh-1p::GFP biosensor indicates an impairment in B12-dependent metabolism, we acknowledge that it does not directly prove absolute systemic deficiency.

To ensure consistency and scientific rigor across the entire manuscript, we have conducted a thorough, systematic screening of the Results, Discussion, and Conclusion sections. All definitive or absolute references to systemic vitamin B12 "deficiency" have been removed or moderated and reframed as being "consistent with reduced vitamin B12 availability" or "consistent with impaired B12-dependent metabolism." We believe these modifications accurately represent our experimental findings while strictly adhering to the limits of our in vivo biosensor data.

2. Causal relationship between lysosomal dysfunction and B12 deficiency: The mecobalamin rescue experiments are interesting and support a functional relationship between B12 metabolism and the observed phenotypes. However, they do not establish a definitive linear pathway in which lysosomal dysfunction causes B12 deficiency, which then causes autophagic and reproductive defects. Please distinguish clearly between experimentally demonstrated findings and the proposed mechanistic model.

We appreciate this insightful distinction. We agree that the mecobalamin rescue experiments demonstrate a clear functional link rather than a rigid linear pathway. To address this, we have revised the Discussion to clearly separate our solid experimental findings, such as lysosomal failure, B12 biosensor activation, and meCbl-mediated rescue, from our working mechanistic model. We now frame altered B12 metabolism as a significant contributing factor operating alongside primary proteostatic collapse, rather than as a strict, single-track linear chain of events.

3. Structural modeling: The AlphaFold3-based structural analyses and predicted trimer-like assembly are useful for generating mechanistic hypotheses. However, computational predictions should not be presented as experimental evidence of oligomerization, structural disruption, or a direct mechanism of protein degradation. Please moderate the relevant statements accordingly.

We completely agree with the reviewer’s prudent distinction. While computational modeling provides valuable structural insights for hypothesis generation, it cannot replace direct experimental proof.

To ensure absolute scientific rigor, we have carefully reviewed and refined all relevant text across the manuscript. We have explicitly ensured that the AlphaFold3-based structural analyses, predicted trimer assemblies, and interface distance measurements are strictly framed as computational predictions and hypothesis-generating models rather than direct experimental evidence of oligomerization, structural disruption, or protein degradation mechanisms. We believe these modifications appropriately calibrate the interpretation of our structural modeling data.

4. Protein stability experiment: The cycloheximide-chase experiment was performed in HEK293T cells using exogenously expressed FLAG-tagged LMD-3. This is supportive evidence for an intrinsic effect of the W690C substitution on protein stability, but it does not directly demonstrate altered endogenous LMD-3 stability in C. elegans. Please maintain this distinction throughout the manuscript.

We completely agree with this important distinction. While the cycloheximide (CHX)-chase assay in heterologous HEK293T cells provides valuable supportive evidence regarding the intrinsic impact of the W690C mutation on protein stability, we acknowledge that it does not directly measure endogenous LMD-3 protein turnover in C. elegans.

To ensure absolute scientific rigor and precision, we have carefully reviewed the text across the manuscript, particularly in the Results and Discussion sections, to maintain this exact distinction. We have ensured that our description strictly frames these heterologous results as supportive biochemical evidence for intrinsic protein stability effects, rather than overstating them as direct in vivo measurements of endogenous protein degradation within nematodes.

5. Interpretation of qRT-PCR: Reduced steady-state lmd-3 mRNA abundance does not by itself demonstrate transcriptional repression. Statements such as “the mutation suppresses gene expression” should therefore be replaced with more precise wording indicating reduced steady-state transcript abundance, unless additional transcriptional evidence is available.

We completely agree with the reviewer’s perceptive distinction. qRT-PCR measures steady-state mRNA abundance, which reflects a net balance between transcription and degradation rates rather than strictly proving direct transcriptional repression.

To ensure absolute scientific rigor and precision, we have systematically screened the entire manuscript. All terms implying direct transcriptional repression or suppression, such as "suppresses gene expression" or "transcriptional downregulation", have been replaced with precise phrasing indicating "reduced steady-state transcript abundance" or "decreased steady-state mRNA levels".

6. Autophagy interpretation: The tandem mCherry::GFP::LGG-1 reporter and LGG-1 measurements support impaired autophagic maturation and/or degradation. However, the precise molecular step at which the defect occurs is not completely established. Please avoid overly definitive statements regarding a block specifically at autophagosome–lysosome fusion or degradation.

We completely agree with the reviewer’s suggestion. While our tandem mCherry::GFP::LGG-1 reporter data and LGG-1 measurements clearly show impaired autophagic maturation and reduced degradation, we acknowledge that confirming the exact molecular step between autophagosome-lysosome fusion and subsequent degradation requires further study.

To ensure scientific rigor, we have carefully reviewed the manuscript and softened all overly definitive statements regarding the precise locus of the autophagic block. We now describe the defect more broadly as impaired autophagic maturation and degradation or compromised late-stage autophagic flux, rather than overstating a specific block at the fusion or degradation stage.

7. Genetic interaction with vha-12: The vha-12 genetic experiment supports a functional relationship between LMD-3 and lysosomal V-ATPase-dependent function, but it does not establish direct molecular regulation or definitive linear epistasis. The interpretation should remain consistent with the authors’ revised description of this relationship as a working mechanistic model.

We completely agree with the reviewer’s suggestion. While our vha-12 genetic data demonstrate a clear functional relationship between LMD-3 and V-ATPase, we acknowledge that these experiments do not establish direct molecular regulation or definitive linear epistasis.

To ensure scientific rigor, we have carefully reviewed the manuscript to ensure that this relationship is consistently framed as a working mechanistic model rather than overstating direct molecular regulation or strict epistasis.

8. Statistical analysis: For the multiple lysosomal and autophagy-related genes analyzed individually, please clarify whether correction for multiple comparisons was performed. If these genes were predefined candidate targets and individual hypothesis tests were intentionally used, this should be explicitly stated in the Methods and/or figure legends. Subject to appropriate clarification and moderation of these points, I consider the revised manuscript substantially improved. The remaining issues are primarily related to the precision of mechanistic interpretation rather than fundamental experimental deficiencies.

We thank the reviewer for this constructive comment. These genes were predefined candidate targets, so we intentionally used individual hypothesis tests without correction for multiple comparisons.

We have added this statement to the Statistical analysis section of the Methods:

"For targeted transcript analyses (such as lysosomal and autophagy-related genes in Figs 4A and 5B), the evaluated genes were treated as predefined candidate targets and assessed using targeted individual hypothesis tests without multiple testing corrections."

We appreciate the reviewer's positive feedback.

Attachment

Submitted filename: 4. Response to Reviewers_RV2.pdf

pone.0361215.s009.pdf^{(186.7KB, pdf)}

PLoS One. doi: 10.1371/journal.pone.0361215.r005

Decision Letter 2

Ahmed Atwa

8 Oct 2026

A premature ovarian insufficiency-associated LMD-3 point mutation impairs protein stability and causes autophagic-lysosomal dysfunction in Caenorhabditis elegans

PONE-D-26-28276R2

Dear Dr. Zhang,

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Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0361215.r006

Acceptance letter

Ahmed Atwa

PONE-D-26-28276R2

PLOS One

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File. Supplementary Figures and Tables.

This file contains S1 to S6 Figs, S2 Table, and S4 Table.

(PDF)

pone.0361215.s001.pdf^{(1.2MB, pdf)}

S1 Video. Structural conservation of the TLDc domains.

(MP4)

Download video file^{(4.4MB, mp4)}

S1 Table. C. elegans strains used in this study.

(XLSX)

pone.0361215.s003.xlsx^{(12KB, xlsx)}

S3 Table. Primers and oligos used in this study.

(XLSX)

pone.0361215.s004.xlsx^{(16.8KB, xlsx)}

S1 Data. Comprehensive record of experimental raw data.

(XLSX)

pone.0361215.s005.xlsx^{(140.2KB, xlsx)}

S2 Data. Uncropped scans of western blot membranes.

(DOCX)

pone.0361215.s006.docx^{(18.2MB, docx)}

Attachment

Submitted filename: 4. Response to Reviews_PLoS one_RV1.pdf

pone.0361215.s008.pdf^{(245.8KB, pdf)}

Attachment

Submitted filename: 4. Response to Reviewers_RV2.pdf

pone.0361215.s009.pdf^{(186.7KB, pdf)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

A premature ovarian insufficiency-associated LMD-3 point mutation impairs protein stability and causes autophagic-lysosomal dysfunction in Caenorhabditis elegans

Yile Zhai

Tiantian Wang

Meixi Gong

Yuhan Liang

Wenfei Li

Xin Wang

Zhe Zhang

Roles

Abstract

Introduction

Results

Genetic and structural evidence for a shared POI-associated W690C mutation in LMD-3

Fig 1. Characterization of the POI-associated W690C mutation in the conserved TLDc domain of C. elegans LMD-3.

The POI-associated W690C mutation impairs fecundity and germline integrity in C. elegans

Fig 2. The POI-associated W690C mutation in C. elegans LMD-3 is associated with impaired fecundity and germline integrity.

The LMD-3 W690C mutation induces widespread and chronic cellular stress

Fig 3. LMD-3 expression pattern and the impact of the W690C mutation on stress responses.

The W690C-associated lysosomal dysfunction correlates with proteostasis collapse

Fig 4. The W690C mutation correlates with proteostasis collapse and lysosomal dysfunction in C. elegans.

Vitamin B12 supplementation mitigates LMD-3(W690C)-associated autophagic-lysosomal defects

Fig 5. lmd-3W690C mutation is associated with impaired autophagy and autophagic flux in C. elegans.

Fig 6. meCbl supplementation restores proteostasis and mitigates autophagic-lysosomal defects in lmd-3W690C mutants.

The W690C mutation impairs LMD-3 expression and structural integrity

Fig 7. The W690C mutation compromises LMD-3 protein structure and stability.

Discussion

Materials and methods

C. elegans strains and maintenance

CRISPR-Cas9 mediated mutagenesis

Plasmid construction

Cross-fertilization assay

Transgene generation and germline rescue

Quantitative real-time PCR (qRT-PCR)

Reproductive capacity analysis

Measurement of body length

Microscopy and imaging analysis

Germline staining and quantification

SYTO12 staining for germline apoptotic cells

Dual lysosomal staining and quantification

Cell culture and Transient transfection

Cycloheximide pulse chase assay

RNA interference (RNAi)

Autophagy flux and aggregation analysis

Mecobalamin supplementation

Quantification of myo-3p::ssGFP intensity

Quantification of NMY-2::GFP aggregates in germline oocytes

Quantification of NUC-1::pHTomato intensity

Western blotting

Protein sequence and structure prediction

Inter-domain distance measurement

Protein stability prediction

Statistical analysis

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Ahmed Atwa

Roles

Author response to Decision Letter 1

Decision Letter 1

Ahmed Atwa

Roles

Author response to Decision Letter 2

Decision Letter 2

Ahmed Atwa

Roles

Acceptance letter

Ahmed Atwa

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Fig 5. lmd-3^W690C mutation is associated with impaired autophagy and autophagic flux in C. elegans.

Fig 6. meCbl supplementation restores proteostasis and mitigates autophagic-lysosomal defects in lmd-3^W690C mutants.