Hormonal Signal Amplification Mediates Environmental Conditions during Development and Controls an Irreversible Commitment to Adulthood

Many animals can choose between different developmental fates to maximize fitness. Despite the complexity of environmental cues and life history, different developmental fates are executed in a robust fashion. The nematode Caenorhabditis elegans serves as a powerful model to examine this phenomenon because it can adopt one of two developmental fates (adulthood or diapause) depending on environmental conditions. The steroid hormone dafachronic acid (DA) directs development to adulthood by regulating the transcriptional activity of the nuclear hormone receptor DAF-12. The known role of DA suggests that it may be the molecular mediator of environmental condition effects on the developmental fate decision, although the mechanism is yet unknown. We used a combination of physiological and molecular biology techniques to demonstrate that commitment to reproductive adult development occurs when DA levels, produced in the neuroendocrine XXX cells, exceed a threshold. Furthermore, imaging and cell ablation experiments demonstrate that the XXX cells act as a source of DA, which, upon commitment to adult development, is amplified and propagated in the epidermis in a DAF-12 dependent manner. This positive feedback loop increases DA levels and drives adult programs in the gonad and epidermis, thus conferring the irreversibility of the decision. We show that the positive feedback loop canalizes development by ensuring that sufficient amounts of DA are dispersed throughout the body and serves as a robust fate-locking mechanism to enforce an organism-wide binary decision, despite noisy and complex environmental cues. These mechanisms are not only relevant to C. elegans but may be extended to other hormonal-based decision-making mechanisms in insects and mammals

Dynamic rerouting of the carbohydrate flux is key to counteracting oxidative stress

<p>Abstract</p> <p>Background</p> <p>Eukaryotic cells have evolved various response mechanisms to counteract the deleterious consequences of oxidative stress. Among these processes, metabolic alterations seem to play an important role.</p> <p>Results</p> <p>We recently discovered that yeast cells with reduced activity of the key glycolytic enzyme triosephosphate isomerase exhibit an increased resistance to the thiol-oxidizing reagent diamide. Here we show that this phenotype is conserved in <it>Caenorhabditis elegans </it>and that the underlying mechanism is based on a redirection of the metabolic flux from glycolysis to the pentose phosphate pathway, altering the redox equilibrium of the cytoplasmic NADP(H) pool. Remarkably, another key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is known to be inactivated in response to various oxidant treatments, and we show that this provokes a similar redirection of the metabolic flux.</p> <p>Conclusion</p> <p>The naturally occurring inactivation of GAPDH functions as a metabolic switch for rerouting the carbohydrate flux to counteract oxidative stress. As a consequence, altering the homoeostasis of cytoplasmic metabolites is a fundamental mechanism for balancing the redox state of eukaryotic cells under stress conditions.</p

Mutations in the Key Autophagy Tethering Factor EPG5 Link Neurodevelopmental and Neurodegenerative Disorders Including Early‐Onset Parkinsonism

Objective: Autophagy is a fundamental biological pathway with vital roles in intracellular homeostasis. During autophagy, defective cargoes including mitochondria are targeted to lysosomes for clearance and recycling. Recessive truncating variants in the autophagy gene EPG5 have been associated with Vici syndrome, a severe early‐onset neurodevelopmental disorder with extensive multisystem involvement. Here, we aimed to delineate the extended, age‐dependent EPG5‐related disease spectrum. Methods: We investigated clinical, radiological, and molecular features from the largest cohort of EPG5‐related patients identified to date, complemented by experimental investigation of cellular and animal models of EPG5 defects. Results: Through worldwide collaboration, we identified 211 patients, 97 of them previously unpublished, with recessive EPG5 variants. The phenotypic spectrum ranged from antenatally lethal presentations to milder isolated neurodevelopmental disorders. A novel Epg5 knock‐in mouse model of a recurrent EPG5 missense variant featured motor impairments and defective autophagy in brain areas particularly relevant for the neurological disorders in milder presentations. Novel age‐dependent neurodegenerative manifestations in our cohort included adolescent‐onset parkinsonism and dystonia with cognitive decline, and myoclonus. Radiological features suggested an emerging continuum with brain iron accumulation disorders. Patient fibroblasts showed defects in PINK1‐Parkin‐dependent mitophagic clearance and α‐synuclein overexpression, indicating a cellular basis for the observed neurodegenerative phenotypes. In Caenorhabditis elegans, EPG5 knockdown caused motor impairments, defective mitophagic clearance, and changes in mitochondrial respiration comparable to observations in C. elegans knockdown of parkinsonism‐related genes. Interpretation: Our findings illustrate a lifetime neurological disease continuum associated with pathogenic EPG5 variants, linking neurodevelopmental and neurodegenerative disorders through the common denominator of defective autophagy. ANN NEUROL 2025 ANN NEUROL 202

Mutations in the Key Autophagy Tethering Factor EPG5 Link Neurodevelopmental and Neurodegenerative Disorders Including Early-Onset Parkinsonism

OBJECTIVE: Autophagy is a fundamental biological pathway with vital roles in intracellular homeostasis. During autophagy, defective cargoes including mitochondria are targeted to lysosomes for clearance and recycling. Recessive truncating variants in the autophagy gene EPG5 have been associated with Vici syndrome, a severe early-onset neurodevelopmental disorder with extensive multisystem involvement. Here, we aimed to delineate the extended, age-dependent EPG5-related disease spectrum.METHODS: We investigated clinical, radiological, and molecular features from the largest cohort of EPG5-related patients identified to date, complemented by experimental investigation of cellular and animal models of EPG5 defects.RESULTS: Through worldwide collaboration, we identified 211 patients, 97 of them previously unpublished, with recessive EPG5 variants. The phenotypic spectrum ranged from antenatally lethal presentations to milder isolated neurodevelopmental disorders. A novel Epg5 knock-in mouse model of a recurrent EPG5 missense variant featured motor impairments and defective autophagy in brain areas particularly relevant for the neurological disorders in milder presentations. Novel age-dependent neurodegenerative manifestations in our cohort included adolescent-onset parkinsonism and dystonia with cognitive decline, and myoclonus. Radiological features suggested an emerging continuum with brain iron accumulation disorders. Patient fibroblasts showed defects in PINK1-Parkin-dependent mitophagic clearance and α-synuclein overexpression, indicating a cellular basis for the observed neurodegenerative phenotypes. In Caenorhabditis elegans, EPG5 knockdown caused motor impairments, defective mitophagic clearance, and changes in mitochondrial respiration comparable to observations in C. elegans knockdown of parkinsonism-related genes.INTERPRETATION: Our findings illustrate a lifetime neurological disease continuum associated with pathogenic EPG5 variants, linking neurodevelopmental and neurodegenerative disorders through the common denominator of defective autophagy. ANN NEUROL 2025 ANN NEUROL 2025.</p

<i>daf-9</i> is transcriptionally upregulated in the hypodermis after commitment to reproductive development.

<p>(A,E) Fold change of total <i>daf-9</i> transcripts quantified by qPCR during development of synchronized broods (see Experimental Procedures, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001306#pbio.1001306.s008" target="_blank">Table S2</a>). Bars represent means ± standard deviations of fold change between biological replicates. (A) Development in favorable conditions. (E) Development in unfavorable conditions. (B,F) Fraction of worms expressing hypodermal <i>daf-9</i> during development in (B) favorable and (F) unfavorable conditions. (C,D) Δ7-DA regulates hypodermal <i>daf-9</i> transcription and development. <i>daf-9(dh6)</i> animals carrying the a <i>daf-9</i> promoter construct fused to <i>gfp</i> (<i>pdaf-9::gfp</i>) were grown on 0 to 100 nM DA. (C) Hypodermal GFP intensity. Animals grown in the presence of 0 to 0.5 DA show no hypodermal <i>daf-9</i> expression. Higher DA levels (0.75 to 10 nM DA) result in strong hypodermal expression, whereas higher levels (50 nM) abolish expression. Each bar represents the pixel intensity of a fixed area of the hypodermis of a single animal. (D) Phenotypic distribution of <i>daf-9(dh6)</i> worms expressing hypodermal <i>daf-9</i> when exposed to different concentrations of DA. Hypodermal upregulation is visible only at concentrations of DA that give rise to abnormal adults. (G,I) Hypodermal expression in worms grown in pheromone (red) and shifted to favorable conditions for a specified time window (blue) and shifted back to unfavorable conditions: (G) 6 h with a dauer frequency of 7%±5%, and (I) 1 h with a dauer frequency of 90%±10%. Bars represent the average of three biological replicates ± standard deviation. (H,J) Fold change of total <i>daf-9</i> transcripts quantified by qPCR when worms are shifted from unfavorable to favorable conditions (H), or maintained in unfavorable conditions (J).</p

Hypodermal <i>daf-9</i> expression propagates from anterior to posterior upon commitment to the L3 fate.

<p>(A, top) Fluorescent images of worms at each time point are shown at shift from unfavorable to favorable at 0 (24 hph) h (leftmost image) through 12 hph (rightmost image). Arrowheads mark the XXX cells. (A, bottom) Expression of hypodermal <i>daf-9</i> was quantified along the anterior posterior axis in 4–6 worms in each time point. Each green shaded histogram represents the mean grey value of DAF-9::GFP per worm, normalized to length. Different worms were imaged at each time point (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001306#pbio.1001306.s009" target="_blank">Text S1</a> for details of analysis). (B–D) XXX cells and Δ7-DA are required to initiate hypodermal daf-9 expression and reproductive development. (B) Cells were ablated during L2d and recovered in favorable conditions. (C) L2d ablated worms were let to recover on 1, 5, or 10 nM Δ7-DA. All worms expressing hypodermal <i>daf-9</i> developed into normal adults with no Mig or Cut phenotypes. (D) Worms were grown to L2d, XXX cells were ablated after commitment to L3 at 27 hph. * <i>p</i><1×10⁻⁴, ** <i>p</i><1×10⁻¹⁰.</p

Commitment to dauer or reproductive development.

<p>(A) Developmental molt times of <i>C. elegans</i> N2 strain growing in favorable (blue) or unfavorable (red) conditions. (B–E) Time courses of commitment as a function of environmental conditions (pheromone). Top, representative colored bars indicate shifts to unfavorable conditions (red) or favorable conditions (blue). Bottom, means of dauer frequencies between biological replicates ± standard deviation. Numbers in parentheses indicate total worms per time point. (B) Period of pheromone sensitivity during L1 and L2: worms respond to pheromone between 12 and 18 hph. (C) Point of commitment to dauer: worms commit to dauer 33 hph denoted by the red dashed gridline. (D) Point of commitment from L2d to L3: worms commit to L3 after a 3 h pulse in favorable conditions when shifted at 24 hph. (E) Start time of pulse shifts to favorable conditions during L2d. Pheromone was added to worms 3 h post-shift to favorable conditions. Cultures shifted to favorable conditions at 33 hph show a higher ratio of dauers since worms commit to dauer at 33 hph (red dashed gridline). Control, worms grown without pheromone. Dauer, worms grown in 3% (v/v) pheromone with no shifts.</p

A feedback loop amplifies a DA signal leading to coordinate development.

<p>(A) Environmental conditions overlap with DA time of action. Top, growth in favorable conditions (blue) during the induction period commits worms to adulthood. Committed worms develop into adults even if shifted to unfavorable conditions (red). Commitment to adulthood correlates with the time of action of DA. Middle and bottom: worms grown in unfavorable conditions during the induction period develop into L2d worms which decide between regular development and alternative development during the integration period (between 15 and 33 hph, hashed grey lines). Worms commit to the dauer fate at 33 hph or to adult development if exposed to favorable conditions for 3 h. Development to dauer correlates with no DA production, and development to adulthood correlates with production of DA. (B) Noisy and uncertain environmental information is measured by sensory neurons and reduced in complexity into the four signaling pathways. Information complexity is reduced further to the XXX cells, the primary source of DA. If nascent amounts of DA produced by the XXX cells bypass the dauer DA threshold, worms will develop into reproductive adults, and if DA levels are under the threshold, worms will develop into dauers. Upon commitment to reproductive development, DA originating from the XXX cells will initiate the hypodermal <i>daf-9</i> positive feedback loop, thus increasing the amounts of DA and thus locking the adult decision and producing sufficient amounts of DA for complete adult development. The positive feedback loop canalizes development guaranteeing that sufficient amounts of DA are produced so that abnormal phenotypes are not expressed in adult worms.</p

High amounts of DA are required for normal adult development.

<p>(A) Images of dauer, arrested L3, abnormal development Mig and Cut worms, and normal adults. Yellow hatched area encloses the gonad, v, vulva. (B) Distribution of developmental stages as a function of DA, scored 48 hph. Means of dauer (red), abnormal development (arrested L3, Mig and Cut; yellow) and normal adult (L3, L4, and young adult; blue) phenotype in <i>daf-9(dh6)</i> worms. (C) Distribution of developmental stages in the <i>daf-9(rh50)</i> background. (D) Distribution of stages in the adult fraction of phenotypes. Means of population proportions of stages indicate the relative developmental rate at each concentration of DA scored at 48 hph. Error bars represent means ± standard deviations across three biological replicates, <i>N</i>>500. Mig, gonad migratory defective; Cut, cuticle defective; YA; young adult. * Worms were gravid the next day.</p