Autophagy

Autophagy is a fundamental intracellular degradation pathway with vital physiological functions. Although it is well known that autophagy is activated during starvation, the extent of basal autophagy remains unclear owing to challenges in measuring autophagic flux in vivo. In this study, we developed autophagy reporter (GFP-LC3-RFP) mice and quantified basal autophagic flux across tissues by comparing normal and autophagy-deficient conditions. Comparative analyses revealed uniformly low basal autophagic flux during embryogenesis, but significant tissue-specific variation in adult mice. In contrast to previous assumptions that basal autophagy in the brain is low, the brain, along with the liver and kidney, exhibited higher basal autophagic flux than the heart, skeletal muscle, and intestine. These data serve as foundational information on basal autophagic flux in mammals and provide a plausible explanation for the severe neurological phenotypes linked to autophagy gene mutations in mice and humans.

Autophagy is a critical cellular process, yet its genomic definition remains inconsistent across digital repositories. This lack of standardisation hinders reproducibility in high-throughput studies and clinical research. Here, we present Au_Sus, a high-confidence human autophagy census established through a frequency-based majority consensus of seven primary databases and literature sources. After rigorous manual curation and nomenclature standardisation, we defined a tiered framework: Maxim_Au (2,581 genes), Au_Sus (the 201-gene core consensus), and Minim_Au (77 universal genes). Functional enrichment and protein-protein interaction analysis confirm that Au_Sus captures a highly integrated and purified autophagic machinery, with significant associations to neurodegeneration and oncology. Furthermore, an analysis of nearly 100 published cancer gene signatures revealed profound functional dilution, with 60% of signature genes absent from our consensus. These findings suggest that many of these models incorporate peripheral stress markers rather than core autophagic effectors. Hence, Au_Sus (freely accessible at ausis.uniovi.es) provides a reliable, ready-to-use benchmark to standardise the study of autophagy in health and disease.

In plant cytokinesis, the partitioning membrane is made by homotypic fusion of secretory vesicles, progressing in a centre-to-periphery direction. In Arabidopsis, this process is mediated by a cytokinesis-specific fusion machinery involving Qa-SNARE KNOLLE which is made during G2/M phase and degraded at the end of cytokinesis. Here we analyse how the turnover of KNOLLE protein is regulated. KNOLLE is ubiquitinated, which is best detected after combined treatment with inhibitors of endocytosis and de-ubiquitination. Site-directed mutagenesis of three clustered lysine residues prevented ubiquitination and internalisation, resulting in stable accumulation of KNOLLE at the plasma membrane in all cells of the seedling root. This is in stark contrast to the transient accumulation of wild-type KNOLLE in dividing cells only. Partial-substitution mutant lines revealed redundancy of lysine residues in both KNOLLE ubiquitination and turnover. KNOLLE ubiquitination resulted in K63-linked ubiquitin chains known to be involved in endocytosis whereas K48-linked chains were not detected. To explore the spatio-temporal conditions, we analysed KNOLLE ubiquitination in cis-SNARE and trans-SNARE complexes during membrane traffic and cell-plate formation. Our findings suggest that KNOLLE protein turnover is caused by a ubiquitination process that depends on successful membrane fusion generating the cell plate.

Coxiella burnetii is a Gram-negative, obligate intracellular pathogen and the causative agent of the zoonotic disease Q fever. Resident alveolar macrophages are the first target cells, but C. burnetii spreads to other cell types. While we have information about C. burnetii uptake and the establishment of the replication-competent phagolysosomal-like C. burnetii-containing vacuole (CCV), it is not well studied how C. burnetii exits its host cell. Here, we show that an infection with C. burnetii also triggers the activation of TFEB, a master regulator of autophagy and lysosomal development. The activation occurs in a time-dependent manner and depends on the size of the CCV. Importantly, TFEB activation during C. burnetii infection depend on MCOLN1, which channels Ca2+ across the lysosomal membrane into the cytosol. Knock-down of MCOLN1 resulted in reduced TFEB activation and smaller CCVs, while MCOLN1 activation boosted bacterial egress. Indeed, peripheral CCVs are positive for LAMP1/2 and release bacteria, without inducing host cell death. Importantly, LAMP1/2 and C. burnetii were stainable in non-permeabilized cells at sites of bacterial release, demonstrating fusion of the lysosome with the plasma membrane. Importantly, while replication of C. burnetii is not inhibited in cells lacking LAMP1/2, egress is impaired. Taken together, our data indicates that with increasing CCV size, TFEB is activated by the release of Ca2+ from lysosomes via the MCOLN1 channel, which in turn enables further CCV development and damage of the CCV membrane. This triggers lysosomal exocytosis and egress of C. burnetii without cell death induction.

Alzheimers disease (AD) is a neurodegenerative disorder affecting millions of patients globally. Despite significant efforts from researchers in recent decades, there are still many unanswered questions about AD pathogenesis. AD patient brains manifest changes in extracellular vesicles (EVs) secreted from diseased neurons, and the effect of this phenomenon remains poorly understood. EVs contain a variety of biomolecules and play a critical role in cell-to-cell communication in all eukaryotic organisms. Here, we report a thorough characterization of small EVs purified from cultures of human cerebrocortical organoids. These organoids are differentiated from human patient-derived stem cells that bear a familial AD mutation in the presenilin 1 (PSEN1) gene, or from an isogenic wildtype (WT) control. The organoid conditioned media was aspirated from cultures and processed for EV enrichment using a non-invasive technique that requires no cellular disruption. EVs purified from AD organoid conditioned media have a wider size distribution and show differential expression of tetraspanins CD63, CD9, and CD81 when compared to WT organoid-derived EVs. AD organoid-derived EVs can have single, double, and even triple membranes and display luminal fibrillar material. A deep proteomic profiling of the EVs reveals several statistically significant differences, including evidence for modifications in secretory autophagy. EV isolates from both WT and AD organoids show strong binding to amyloid detecting dyes, both in bulk fluorescence and fluorescence microscopy assays. After a 1-week co-culture of AD organoids with WT organoids, there is evidence of endosomal membrane transfer between the isogenic cultures with an increase in amyloid-{beta} peptides in the WT organoids. These observations support the notion that non-cell-autonomous spread of amyloid-containing EVs in human AD brains can be modeled in a cerebral organoid system.

Circadian regulation of proteostasis, a key determinant of muscle health, remains poorly understood. Here, we identified DNAJB6, an Hsp40 (DnaJ) co-chaperone, as a substrate of the circadian E3 ligase FBXL21. FBXL21 mediated the ubiquitination-dependent proteasomal degradation of both DNAJB6 and its client proteins including Desmin; causative mutations of DNAJB6 in myopathies, however, rendered resistance to FBXL21-directed degradation. Fbxl21 KO C2C12 cells displayed aberrant accumulation of Desmin, and showed aggravated cytoplasmic accumulation of TDP-43, another DNAJB6 client protein, in heat shock response. Under timed exercise as a physiological stressor, WT mice displayed robust diurnal rhythms in the levels of stress granule markers (G3BP1 and FUS) and TDP-43 as a function of exercise timing. In contrast, the Fbxl21 hypomorph Psttm mutant mice showed elevated expression of these proteins without exercise, which was exacerbated under exercise-induced stress conditions; importantly, these abnormalities were rescued by skeletal muscle-specific FBXL21 expression. Our study elucidates a novel diurnal regulatory mechanism of skeletal muscle proteostasis via FBXL21 as a chaperone-linked E3 ligase, highlighting the FBXL21-DNAJB6 axis as a potential therapeutic target for myopathies.

Plasma Membrane Calcium ATPase (PMCA) is essential for maintaining intracellular calcium homeostasis. Previously, we used constitutive PMCA downregulation in Drosophila melanogaster dopaminergic neurons as a model to increase intracellular calcium and mimic early neuronal alterations associated with Parkinsons disease. Here, we examined the mechanisms underlying the effects mediated by the conditional, adult-specific downregulation of PMCA in dopaminergic neurons in Drosophila melanogaster, both in vivo and in primary neuronal cultures. Adult-specific conditional silencing of PMCA in dopaminergic neurons reduced lifespan but to a lesser extent than the constitutive model and impaired locomotor performance. At the cellular level, PMCA-downregulated dopaminergic neurons exhibited elevated basal calcium, indicating disrupted calcium regulation. This was associated with a progressive increase in presynaptic vesicles and extracellular dopamine levels, suggesting enhanced neurotransmitter release. Notably, the synaptic active zone structure was preserved, indicating primarily functional rather than structural alterations. In primary neuronal cultures, PMCA downregulation reduced dopaminergic neuron survival and induced transient increases in neurite branching. Together, these findings show that PMCA downregulation leads to calcium dysregulation and presynaptic dysfunction without overt neurodegeneration in vivo, while promoting premature neuronal death in culture, indicating increased vulnerability and supporting a pre-degenerative state in which synaptic alterations precede neuronal loss.

The tight regulation of iron homeostasis is of great importance for cellular health. An increase in intracellular iron levels results in the formation of free radicals, which damages macromolecules and membranes, eventually resulting in cell death by Ferroptosis. Recently, we showed that patients with mutations in VPS41 display a severe neurodegenerative phenotype with iron deposition in the brain. VPS41 is well known as subunit of the HOPS complex required for fusion of late endosomes and autophagosomes with lysosomes. However, VPS41 has also been identified as inhibitor of Ferroptosis and regulator of redox homeostasis. How VPS41 exerts these functions and if these are dependent on the HOPS complex is unknown. Here we show that depletion of VPS41 results in increased intracellular iron levels, ROS formation and mitochondrial fission. Our findings indicate an important role for VPS41 in the regulation of iron homeostasis and mitochondrial fission and suggest Ferroptosis as a possible cause for neurodegeneration in VPS41 patients.

Expression of the chloroplast AAA+ chaperone CLPD gene increases during senescence and drought, but its functional role in chloroplast proteostasis is poorly understood. This study provides a comprehensive analysis of Arabidopsis CLPD protein accumulation across development from early seedlings to senescence, and compares results to its homologs CLPC1,2, as well as CLPB3 and cpHSP90. The developmental consequences of complete loss of CLPD expression (clpd-1), as well as overexpression of functional CLPD or CLPD impaired in ATP hydrolysis (CLPD-TRAP), were determined in Arabidopsis. clpd-1 has accelerated seedling development while functional CLPD overexpression lines, but not CLPD-TRAP, have delayed development. To determine if CLPD is a bona fide CLP chaperone associating with the CLPPRT protease and to identify in vivo candidate substrates, we employed the CLPD-TRAP line during the vegetative and flowering (senescent) growth stages. Affinity purification of CLPD-TRAP followed by mass spectrometry showed high enrichment of the CLP protease complex, thus providing direct support for the role of CLPD in substrate delivery to the CLP protease. CLPC1,2 were also highly enriched in the CLPD-TRAP interactome, suggesting hetero-oligomerization and cooperation between the three chaperones is likely. Nine chloroplast candidate substrates were identified in the CLPD-interactomes, including: FHY2 involved in riboflavin synthesis, THI1 and THIC involved in thiamin metabolism, and four proteins of unknown function. Several of these have been previously identified as potential CLPC1 substrates; however, others appear to be specific to CLPD. CLPD acts in substrate selection within a heteromeric CLPC-CLPD hexamer, likely to make unique contributions through its divergent N-terminus.

Batten disease, also known as neuronal ceroid lipofuscinoses, is one of the most common causes of childhood dementia. It is characterized by the accumulation of lipofuscin in lysosomes, leading to loss of brain cell function, onset of dementia-like symptoms, vision loss and seizures and has extremely limited treatment options. Here, we performed computational drug repurposing analysis to identify existing compounds that may target Batten disease risk genes. A total of 81 candidate compounds were identified, 6 of which were selected based on clinical tractability for downstream testing in Batten disease (CLN3) iPSC-derived models. After confirming disease phenotype and drug candidate safety, CLN3 brain cell cultures treated with and without drug candidates underwent bulk RNA-seq to identify drug responses. One of the candidate drugs N-acetylglucosamine (GlcNAc) significantly upregulated Batten disease risk gene CLN5 expression and several other lysosomal markers within CLN3 brain cells, and modulated several pathways implicated in lysosomal storage disorders. Importantly, GlcNAc significantly reduced lipofuscin burden in both CLN3 iPSC-derived neurons and astrocytes, supporting its investigation as an additional therapy for Batten disease.

The bone marrow (BM) vascular network plays crucial roles in driving bone development and supporting hematopoiesis, yet the mechanisms governing its specialized architecture, particularly sinusoidal morphogenesis, remain inadequately characterized. We show in this study that TIE2 (Tek) was highly expressed by BM sinusoidal endothelial cells (SEC) and the endothelial Tek excision led to BM sinusoidal capillarization. Particularly, the BM sinusoids displayed thinner vessel diameter with the aberrant mural cell coverage in the Tek mutants. Mechanistically, TIE2 insufficiency led to a dramatic decrease of VEGFR3 in BM-SECs while its expression in hepatic sinusoids was not obviously altered. The RNA-seq analysis showed that GO terms enriched for the downregulated genes were related to the biological processes including sinusoidal development while pathways related to arterial ECs and angiogenesis were upregulated in the bone marrow of Tek mutants. The alteration of sinusoidal VEGFR3 expression occurred within 48 h after the induced endothelial deletion of Tek. Consistently, the defective BM sinusoidal formation was validated with the induced Tek deletion in VEGFR3+ SECs. The insufficiency of TIE2 ligand ANGPT1 also led to reduced sinusoidal VEGFR3, accompanied by similar BM sinusoidal defects. Furthermore, disruption of sinusoidal morphogenesis was observed in mutant mice with the endothelial excision of Nr2f2 (COUP-TFII), displaying a decreased expression of BM sinusoidal TIE2 and VEGFR3. These findings suggest that ANGPT1/TIE2 and COUP-TFII form a reciprocal regulatory loop to coordinate BM sinusoidal specification via regulating VEGFR3.

Inflammation and oxidative stress (OS) are key to Parkinsons disease (PD). We performed a cross-dataset integrative transcriptomic analysis to identify OS- and inflammation-related hub genes persistently dysregulated in PD and to evaluate their response to nutrigenomic interventions using publicly available datasets. Four GEO datasets (GSE7621, GSE20141, GSE20146, GSE49036) were analysed to identify differentially expressed genes (DEGs), which were intersected with GeneCards OS-inflammation gene sets. Functional enrichment analyses, including gene ontology (GO), pathway over-representation analysis (ORA), and protein-protein interaction (PPI) analysis, were used to identify key pathways and hub genes. Gene-food bioactive compound (FBC) association was explored by integrating PD signatures with nutrigenomic profiles from NutriGenomeDB. We identified 183 DEGs in PD, enriched in synaptic, dopaminergic, OS, and inflammatory pathways. Intersection analysis yielded 26 OS-inflammation-related genes and 10 central regulators, including TH, DDC, SNCA, LRRK2, HSPB1, and HSPA1B. revealed opposing transcriptional patterns, with several FBCs suppressing stress-related genes and upregulating dopaminergic markers such as TH, GCH1, and DDC. Overall, this integrative analysis highlights OS-inflammation gene networks in PD and identifies candidate diet-gene interactions that warrant further experimental validation

Chaperonins complex into double-ringed octamers to aid peptide folding. Recent evidence implicates dysfunctional chaperonin subunits in cancer and neurodegenerative diseases because their deregulation exacerbates cellular injury. Nevertheless, a gap of knowledge exists regarding the expression and localization of chaperonin subunits in relation to amyloidogenic processes in Alzheimers disease (AD). Here, we show that reduced levels of chaperonin-containing TCP-1 subunits 2 (CCT2) and 3 (CCT3) stratify AD, with the subcellular distribution of their residua being mutually exclusive with both {beta}-amyloid and hyperphosphorylated tau in neurons. We find CCT3 localized to a subset of glial fibrillary acidic protein-positive astrocytes in AD. Increased oxidative stress in vitro upregulated CCT3 expression in astrocyte-like U251 cells. Conversely, CCT3, but not CCT2, loss-of-function in neuron-like SH-SY5Y cells increased intracellular {beta}-amyloid load. These data suggest that CCT2/CCT3 are faithful disease-state indicators and implicate CCT3 in oxidative stress-dependent cellular damage pathways.

To better illustrate the genetic etiology of Parkinson's disease (PD), we integrated xQTL weights derived from bulk RNA-seq (n=931), single-nucleus RNA-seq (n=415), and bulk proteomics (n=716) data of dorsolateral prefrontal cortex (DLPFC) with the largest available GWAS summary data of PD. Through integrative Omnibus TWAS and PWAS analyses, we detected risk genes whose genetic effects are mediated through bulk or cell-type-aware gene expression, or bulk protein abundances in DLPFC. We detected 39 significant risk genes by bulk TWAS, 66 by cell-type-aware TWAS across six brain cell types, and 17 by bulk PWAS. Importantly, 57.9% bulk and 62.5% cell-type-aware independent TWAS risk genes are replicated by bulk PWAS. Protein-protein interaction analyses reveal strong connectivity of our detected risk genes with known PD risk genes such as MAPT, SNCA, and LRRC37A. Our detected TWAS and PWAS risk genes are shown enriched in apoptosis signaling and T-cell activation pathways.

WD40 domains share a widespread {beta}-propeller fold, and often act as versatile scaffold proteins. Despite their central role in organizing dynamic cellular complexes, the molecular and structural mechanisms of many WD40 proteins remain poorly understood. Among them, DCAF7, an ubiquitously expressed and essential gene in human, also encodes a highly conserved WD40 protein in eukaryotic organisms. It is known to interact with multiple and functionnally diverse partners to coordinates cellular activity of several protein kinases as well as transcriptional regulators, thereby modulating key cellular processes such as cell growth, differentiation, and transcriptional regulation. However, the precise mode of action of DCAF7 is unknown and its important divergence in sequence from better characterize WD40 prevent information transfer by similarity. Structural interactomic can reveal how protein-protein interactions (PPIs) occur within an organism and are essential for understanding biological functions and developing new therapeutic strategies. Using SLiMAn2, AlphaFold2/3 and PSSMsearch, we identified a conserved -helical short linear motif (SLiM) in several well known DCAF7 partners that binds to the top surface of its {beta}-propeller. This motif was subsequently used to generate a regular expression, to identify potential new direct binders across the DCAF7 meta-interactome and the human proteome. Domain-domain interactions were also predicted for some other partners. Finally, modeling of oligomeric complexes with such new hits reveals the structural basis of DCAF7 scaffolding, with links to neurodevelopmental disorders such as autism.

BackgroundCLN3 disease, also known as juvenile neuronal ceroid lipofuscinosis, is a rare and neurodegenerative disorder characterized by the accumulation of lipopigments in the cells, progressive cognitive decline, seizures, and vision loss. Biomarker discovery in CLN3 disease is essential for enabling early and accurate diagnosis, which is critical given its neurodegenerative course. Biomarkers provide objective measures to track disease progression, stratify patients, and serve as surrogate endpoints in clinical trials, thereby accelerating therapeutic development. They also offer valuable insights into underlying disease mechanisms and treatment response, ultimately advancing individualized medicine and improving clinical outcomes. MethodsWe developed various machine learning models to predict potential protein biomarkers in CLN3 disease using proteomics data and laboratory tests collected from participants in a prospective, observational cohort. To prioritize and evaluate these candidates, we conducted protein-protein interaction (PPI) network analysis and pathway enrichment, ranking proteins based on their topological importance. The top 20 proteins were selected as candidate biomarkers and corroborated using a publicly available CLN3 transcriptomic dataset. Receiver operating characteristic (ROC) curve analysis was performed to assess the discriminative power of each candidate, with AUROC values calculated to quantify their classification performance. ResultsOur computational approach identified six promising biomarker candidates: OSM, IL6R, LMNB1, HIF1A, NPM1, and CSF1. Among them, OSM and HIF1A showed marked differential expression in CLN3 patients, particularly those with slow disease progression. LMNB1 expression was elevated in patients with faster disease progression, suggesting its utility as a prognostic biomarker. These findings highlight the robustness of our biomarker selection, indicating that these six genes may serve as effective diagnostic markers for CLN3 disease. ConclusionsOur findings demonstrate the utility of data-driven approaches for biomarker discovery in CLN3 and offer new insights into the molecular mechanisms of the disease, with broader implications for improving diagnosis and prognosis in other rare diseases.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of poor prognosis, for which age is the strongest risk factor. Despite significant progress in the discovery of ALS-associated mutations, no model explains how such a diversity of mutations converges in a common pathology. In addition, most ALS cases are sporadic and lack known genetic drivers. We recently reported that arginine-rich peptides arising from the C9ORF72 mutation trigger a widespread accumulation of orphan ribosomal proteins (oRP). Here, we show that oRP accumulation is also observed upon expression of other RNA-related ALS mutations, such as hnRNPA2D290V and TDP-43A315T, as well as upon exposure to the ALS-related neurotoxin {beta}-N-methylamino-L-alanine (BMAA). Furthermore, the transcriptional signature of patients with sporadic ALS resembles that of Diamond-Blackfan anemia (DBA), a known ribosomopathy. Supporting the usefulness of our in vitro data, a transcriptional signature defined from these models provides diagnostic and prognostic value in ALS patients. We propose that the accumulation of oRPs due to dysfunctional ribosome biogenesis is a molecular hallmark of ALS that can contribute to the progressive loss of motor neurons in the disease.

Dormancy is a reversible cellular state characterised by suspended proliferation and increased stress resilience, enabling long-term viability under adverse conditions. Although dormant cells are critical for the life cycle of diverse organisms, from microbes to humans, they are understudied compared to proliferating cells. We present a comparative investigation of dormant cells in two divergent species: spores of fission yeast and diapause embryos of turquoise killifish. A genome-wide screen for genes affecting the lifespan and heat-shock resilience of spores uncovered a trade-off between longevity and heat resistance, and considerable differences in the genetic basis for lifespan between spores and chronologically aging yeast cells. RNA-seq and mass-spectrometry analyses revealed substantial transcriptomic and proteomic changes in spores and diapause embryos, with ribosomal proteins induced as transcripts but repressed as proteins. Transcriptomic regulation of biological processes, but less so of specific genes, is broadly conserved across yeast spores, killifish diapause, and human dormant cancer cells, including the induction of autophagy- and translation-related processes and the repression of cell cycle-related processes. Spores and diapause embryos modulate their transcriptomes and proteomes in response to heat stress and prolonged time. These RNA and protein expression changes are uncoupled and differ from aging-related expression signatures in yeast cells and adult fish. Cells derived from older or stressed spores retain phenotypic differences for several cell divisions, reflected in altered expression signatures, lifespan and stress resilience. Similarly, diapause duration and heat exposure are associated with long-term expression signatures in post-diapause embryos before hatching. This study highlights core biological processes and principles that are remarkably conserved in distinct types of dormant cells.

Lipopolysaccharides (LPSs) are pathogen-associated molecular patterns (PAMPs) of Gram-negative pathogenic bacteria recognized by plants, triggering typical pattern-triggered immunity (PTI) responses. However, a LPS sensing receptor for the recognition of plants remains largely undefined. A plant receptor for lipopolysaccharide (LPS) has not yet been identified. Here, we identify a plant protein, OsML1, with homologies to animal MD-2, which is capable of binding LPS. Furthermore, it may act as a molecular chaperone to assist CK1 in perceiving LPS signals. Our results show that OsML1 functions as an LPS-binding protein recognizing LPS and participates in downstream rice immune response activation. Structural modeling and sequence analysis revealed that OsML1 contains both a typical ML domain and a conserved three-dimensional {beta}-barrel structure as mammalian MD-2 proteins. Microscale thermophoresis assays confirmed that OsML1 binds LPS with high affinity. Functional analyses further demonstrated that OsML1 knockout plants show reduced resistance to the rice bacterial blight pathogen, as well as attenuated ROS bursts upon LPS treatments, whereas overexpression plants show enhanced immune responses. Metabolomic profiling indicated significant metabolic changes in OsML1 knockout plants, particularly in immune-related pathways involving lipids, amino acids, and antimicrobial compounds. OsML1 is consequently a structurally conserved and functional LPS-binding protein linking lipid metabolism, LPS perception, immune activation, and metabolic regulation. Phylogenetic and structural analyses revealed that OsML1 likely arose from a duplication of OsML2, forming an independently functional subgroup within the PITP family. Our study identifies OsML1 as a LPS recognition factor involved in LPS sensing and downstream ROS bursts activation, callose deposition, and broad-spectrum gene expression of resistance. These findings expand our knowledge of bacterial LPS perception and immune regulation in plants, offering novel targets and strategies for disease-resistant breeding.

The adult skeletal muscle regenerates robustly upon injury, but this regenerative capacity rapidly declines with age. In this study, we identify the lanthionine synthetase C-Like (LanCL) proteins, mammalian homologs of the bacterial peptide cyclase LanC, as positive regulators of muscle regeneration in middle-aged mice. In a barium chloride-induced injury model, we found the protein levels of LanCL1 and LanCL2 to increase during an early phase of regeneration in middle-aged (12-month-old) but not young adult (4-month-old) mice. Utilizing a mouse line lacking all three LanCL proteins (LanCL triple KO or LTKO), we examined a potential role of LanCL in injury-induced muscle regeneration. Consistent with an age-dependent function of LanCL, we observed a delayed regeneration of the tibialis anterior (TA) muscle after injury, as reflected by reduced sizes of regenerating myofibers in middle-aged (but not young) LTKO compared to age-matched WT mice. Although the pool size of quiescent satellite cells (Pax7+) was comparable between 12-month-old LTKO and WT muscles without injury, the number of Pax7+ cells was significantly higher in regenerating LTKO muscles at day 5 after injury, accompanied by drastically decreased numbers of MyoD+ and MyoG+ cells, as well as increased numbers of proliferating cells. In addition, we detected elevated expression of pro-inflammatory cytokines in regenerating LTKO muscles, while the number of macrophages was similar comparing LTKO and WT muscles. Taken together, our observations suggest that in aging muscles LanCLs are important for proper timing of inflammation resolution and regeneration upon injury. New & NoteworthyPhysiological roles of the mammalian homologs of bacterial LanC, LanCLs, are poorly understood. Our work uncovers a function of LanCLs in post-injury regeneration of aging skeletal muscles. Middle-aged LanCL triple KO mice displayed a delay in satellite cell differentiation and regenerative myofiber formation, as well as persistent inflammatory cytokine expression, suggesting that LanCLs may have an age-dependent role in modulating inflammation in the injured muscles to facilitate regeneration.