To ensure success in reproduction, organisms dedicate substantial resources to supporting the germline. In testes, somatic gonadal cells form a barrier that isolates germ cells from circulating nutrients, raising the que...To ensure success in reproduction, organisms dedicate substantial resources to supporting the germline. In testes, somatic gonadal cells form a barrier that isolates germ cells from circulating nutrients, raising the question of how germ cell metabolism is sustained and how somatic cells ensure that sufficient resources are directed to the germline. Here, we use lineage-specific genetic manipulations and metabolite reporters to show that Drosophila somatic gonadal cells break down circulating sugars to produce and shuttle lactate to germ cells in vivo, thus sustaining their survival. Further, we uncover that somatic cells ensure the allocation of carbohydrate metabolites specifically to germ cell support and that increasing autonomous consumption of carbohydrates in somatic cells increases germ cell death. Thus, germ cell survival depends on functional metabolic compartmentalisation within gonadal somatic support cells.
Davies EL, Calderon Nuñez A, Ward AL
… +11 more, Sowar H, Rivers E, Balci A, Mair D, Moorhouse E, Towers J, Wickenhagen A, Turnbull ML, Palmarini M, Wilson SJ, Fletcher AJ
Interferons (IFN) are cytokines that regulate the expression of hundreds of genes during viral infections to generate a broadly antiviral environment in the stimulated cell. Antiviral breadth is provided by the concurren...Interferons (IFN) are cytokines that regulate the expression of hundreds of genes during viral infections to generate a broadly antiviral environment in the stimulated cell. Antiviral breadth is provided by the concurrent expression of many individual IFN-stimulated genes (ISG), each encoding a protein with often exquisite antiviral specificity. Here, we identify mechanistic plasticity at a single genetic locus as a novel mechanism to diversify the antiviral profile of human cells. Through alternative splicing, the OAS2 gene encodes two antiviral molecules with distinct target specificities. The shorter OAS2 p69 isoform restricts seasonal human coronavirus OC43 (HCoV-OC43), whereas the longer p71 isoform restricts picornavirus Cardiovirus A (EMCV). The restriction profile is determined by the variable length OAS2 C-terminal tails. Notably, these antiviral activities differ in their dependence on RNase L, suggesting that alternative splicing separates canonical restriction and virus sensing functions across two distinct OAS2 polypeptides. Together, these findings show how alternative splicing expands antiviral diversity at the human OAS2 locus.
The origin of insect wings marked a pivotal evolutionary innovation that enabled their extraordinary ecological success, yet the metabolic mechanisms sustaining this transition remain elusive. Here, we identify apolipopr...The origin of insect wings marked a pivotal evolutionary innovation that enabled their extraordinary ecological success, yet the metabolic mechanisms sustaining this transition remain elusive. Here, we identify apolipoprotein D2 (ApoD2)-a neofunctionalized paralog of apolipoprotein D-as a key metabolic regulator that spatially coordinates lipid allocation during lepidopteran wing development. Comparative phylogenomics across 791 metazoan genomes revealed that ApoD2 emerged as a lepidopteran duplicate exhibiting sustained, wing-enriched expression across developmental stages. Using Bombyx mori as a model, we show that ApoD2 is indispensable for wing morphogenesis, coupling lipid compartmentalization to local energetic demands. Loss of ApoD2 disrupts mitochondrial bioenergetics and fatty acid oxidation, leading to depletion of wing muscle cells. Lipidomic profiling further revealed that ApoD2 deficiency causes systemic lipid misallocation-characterized by hemolymph fatty acid accumulation and depletion of diglycerides and morphogenic lipids in wings, triggering AMPK-dependent autophagy. Mechanistically, duplicated ApoD2 integrates systemic lipid transport with organ-specific energy deployment, linking metabolic rewiring to morphological innovation. Together, these findings reveal how the neofunctionalization of a metabolic regulator resolved evolutionary trade-offs between energy efficiency and structural complexity, illuminating a general principle by which metabolic innovation drives the evolution of complex traits in insects.
The ability of cells to sense and respond to metabolic signals is fundamental to life, yet the molecular mechanisms underlying metabolite surveillance remain incompletely understood. Here, we elucidate the structural bas...The ability of cells to sense and respond to metabolic signals is fundamental to life, yet the molecular mechanisms underlying metabolite surveillance remain incompletely understood. Here, we elucidate the structural basis of metabolite recognition by OXGR1, a G Protein-Coupled Receptor (GPCR) that senses key intermediates in the tricarboxylic acid (TCA) cycle. Using cryo-electron microscopy, we determined cryo-EM structures of OXGR1 bound to α-ketoglutarate (AKG), itaconate (ITA), and structurally related metabolites succinate (SUC) and maleate (MA). These structures reveal a positively charged binding pocket and an extensive hydrogen-bond network that mediate selective recognition of dicarboxylic acids. In addition, we identify a distinct arrangement of hydrophobic residues that modulates ligand potency and selectivity. Mutational analysis and molecular dynamics simulations further demonstrate that noncanonical micro-switch motifs, including FRY and NLxxY, are essential for ligand recognition and receptor activation. Comparative structural and evolutionary analyses indicate that these mechanisms are conserved across species, underscoring the critical role of OXGR1 in maintaining metabolic homeostasis. Together, our findings define a mechanistic framework for metabolite sensing by OXGR1 and provide a framework for therapeutic modulation of metabolic and inflammatory diseases.
Type VI secretion systems (T6SSs) are nanomachineries used by bacteria to inject toxic effectors into neighbouring cells during interbacterial competition or infection. Distinct paralogues of the structural proteins VgrG...Type VI secretion systems (T6SSs) are nanomachineries used by bacteria to inject toxic effectors into neighbouring cells during interbacterial competition or infection. Distinct paralogues of the structural proteins VgrG and PAAR and specific accessory proteins allow the same T6SS to deliver a wide range of effectors. We describe a new set of accessory proteins required for assembly of T6SSs containing the VgrG1-Paar1 spike and delivery of a novel VgrG1-associated membrane-targeting effector in Serratia marcescens. TvpAB, TvpB, TvpC and Paar1 form a pre-complex essential for T6SS assembly, with VgrG1 replacing TvpC in the final accessory complex. Phylogenetic analysis and structural modelling reveal that Tvp proteins are widely conserved, but Tvp-containing complexes vary in organisation and complexity. We define three classes of Tvp complex, all sharing a DUF2169-containing TvpA protein which stabilises a DUF4150/PAAR-containing protein. Class I, which includes only TvpA, functions without a pre-complex, whereas Classes II and III involve additional Tvp components and two-step assembly. Our findings highlight how modular effector recruitment strategies underlie the versatility of the T6SS and suggest how alternative Tvp systems are tailored to promote assembly of secretion-competent, effector-loaded VgrG-PAAR spikes.
Microglia are critical regulators of neuroinflammation and neurodegeneration. Haploinsufficiency of C9orf72, the most frequently mutated gene in amyotrophic lateral sclerosis and frontotemporal dementia, has been linked...Microglia are critical regulators of neuroinflammation and neurodegeneration. Haploinsufficiency of C9orf72, the most frequently mutated gene in amyotrophic lateral sclerosis and frontotemporal dementia, has been linked to autophagy-lysosomal pathway defects, but the role of C9orf72 in microglia remains unclear. Here, we identify the C9orf72/SMCR8 complex as a key regulator of microglial homeostasis through promoting lysosomal membrane repair. Loss of C9orf72 and SMCR8 in mice causes age‑dependent neuroinflammation and microgliosis, with microglia adopting a disease-associated state. In aged brain and spinal cord tissue, microglia display lysosomal damage marked by galectin‑3 accumulation. Using a lysosomotropic agent to induce lysosomal damage in microglia, we find that C9orf72/SMCR8-deficient cells accumulate damaged lysosomes and show defective recruitment of phosphorylated RAB8A and the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery to damaged lysosomes. Notably, mutant microglia accumulate GTP‑bound RAB8A, which becomes hyperphosphorylated and mislocalized to RAB7-positive, LAMP1-negative vesicles. The GTPase-activating activity of the C9orf72/SMCR8 complex is essential for lysosomal repair. Our findings reveal that the C9orf72/SMCR8 complex coordinates RAB8A-ESCRT-mediated lysosomal repair to safeguard microglial homeostasis and limit neuroinflammation.
Coronaviruses establish infection by reorganizing the host endoplasmic reticulum (ER) to form double-membrane vesicles (DMVs), which function as viral replication platforms. However, the role of other cellular organelles...Coronaviruses establish infection by reorganizing the host endoplasmic reticulum (ER) to form double-membrane vesicles (DMVs), which function as viral replication platforms. However, the role of other cellular organelles in this process remains incompletely understood. Here, we uncover a self-reinforcing cycle between viral replication organelles and mitochondrial damage that sustains coronavirus replication. We show that DMV formation disrupts ER-mitochondria contact sites (ERMCs), causing mitochondrial damage. This injury initiates a feed-forward mechanism wherein mitochondria release the matrix enzyme ECHS1 into the cytosol. Cytosolic ECHS1 then binds and stabilizes the DMV inducer protein NSP3 by blocking its K963 ubiquitination via the host E3 ligase RBBP6, thereby promoting further DMV formation. Disrupting this cycle, either through enhanced ER-mitochondria tethering or targeted interference with ECHS1-NSP3 binding, effectively suppresses viral replication. Our findings reveal that coronaviruses exploit an inter-organellar feedback loop linking mitochondrial damage to DMV formation, identifying new potential therapeutic targets for inhibition of coronaviral replication.
Ahmed T, Zhang H, Mushraf GM
… +22 more, Pan Y, Zhang Z, Sun Z, Yin M, Xu X, Wu X, Gao Y, Li Y, Li P, Ding L, Zhang Q, Lin R, Ullah K, Huang Y, Mirza B, Huang Y, Sammad A, Kong X, Qin D, Esteban MA, Wang L, Qin B
The electron transport chain (ETC) is essential for NAD regeneration and proliferation. While many cell types tolerate ETC inhibition when pyruvate or aspartate is supplied, pluripotent stem cells (PSCs) enter a reversib...The electron transport chain (ETC) is essential for NAD regeneration and proliferation. While many cell types tolerate ETC inhibition when pyruvate or aspartate is supplied, pluripotent stem cells (PSCs) enter a reversible paused state even at abundant pyruvate levels. Here, we show that ETC inhibition triggers severe NADH reductive stress in mouse embryonic stem cells (mESCs), driven mainly by threonine dehydrogenase (TDH). TDH-derived NADH establishes a metabolic environment that disfavors cells with compromised mitochondrial function, maintains inhibition of pyruvate dehydrogenase (PDH), and is associated with increased genomic and epigenetic stability at the cellular population level. ETC inhibition similarly induces pausing in early mouse embryos and in human pluripotent stem cells (hPSCs). In hPSCs, combined inhibition of the one-carbon metabolism enzymes serine hydroxymethyltransferase (SHMT1/2) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) effectively reduced reductive stress and rescued the paused phenotype. Together, these findings support a model in which limited mitochondrial redox adaptability represents a conserved metabolic feature of pluripotent stem cells and in which NADH reductive stress is associated with genomic and epigenetic stability.
In many viruses, intrinsically disordered proteins (IDPs) drive the formation of replicative organelles via liquid-liquid phase separation (LLPS). In species A rotaviruses, the disordered protein NSP5 forms condensates w...In many viruses, intrinsically disordered proteins (IDPs) drive the formation of replicative organelles via liquid-liquid phase separation (LLPS). In species A rotaviruses, the disordered protein NSP5 forms condensates with NSP2, but its high sequence diversity raises questions about whether this mechanism is conserved across strains. Using a machine learning approach, we show that NSP5 variants differ significantly in LLPS propensity. We engineered an NSP5 variant with features derived from strains with low-LLPS propensity (low-LLPS). Despite lacking the ability to phase separate in vitro unless phosphorylated, this variant nevertheless supported condensate formation and viral replication in cells. We found that low-LLPS variants require phosphorylation to nucleate phase separation, whereas high-LLPS variants do not, suggesting distinct nucleation mechanisms between viral strains. Hydrogen-deuterium exchange mass spectrometry revealed a phosphorylation-driven allosteric switch that alters NSP2 interactions depending on the NSP5 variant. These findings suggest that phosphorylation plays a context-dependent role in condensate formation, tuning NSP5-NSP2 interactions in a strain-specific manner and highlighting the mechanistic diversity underpinning replicative organelle formation among viral strains.
Endoplasmic reticulum autophagy (ER-phagy) is a selective autophagy pathway in which receptor proteins target ER membranes and proteins for degradation, yet its role in Alzheimer's disease (AD) remains unclear. Here, we...Endoplasmic reticulum autophagy (ER-phagy) is a selective autophagy pathway in which receptor proteins target ER membranes and proteins for degradation, yet its role in Alzheimer's disease (AD) remains unclear. Here, we identify FAM134B/RETREG1 as a specific ER-phagy receptor mediating amyloid precursor protein (APP) degradation. FAM134B directly interacts with ER-localized wild-type and familial mutant APP via their C-terminal domains and recruits LC3 through its LC3-interacting region (LIR) to promote APP delivery to phagophores for lysosomal degradation. In AD, epigenetic silencing at the FAM134B promoter suppresses its transcription by limiting TFEB/TFE3 binding despite their nuclear enrichment. This transcriptional suppression impairs ER-phagy, leading to APP accumulation and exacerbated AD pathology. AAV-mediated hippocampal expression of wild-type, but not LIR-mutant, FAM134B in 5XFAD mice restores ER-phagy, enhances APP clearance, reduces Aβ deposition, preserves synaptic and myelin integrity, and improves cognitive performance. These findings establish FAM134B downregulation as an upstream pathogenic event in AD, suggesting ER-phagy enhancement as a promising strategy to suppress Aβ generation at its source.
Schmieder SS, Cordara G, Kersten F
… +10 more, Steiner K, Samim CH, Plaza DF, Ali-Ahmad A, Boeggild A, Karlsen JL, Sokolowska BO, Boesen T, Krengel U, Künzler M
Fungal defense against predators largely relies on protein toxins, many of which are lectins. We previously showed that the production of the nematotoxin CCTX2 is upregulated in the Agaricomycete Coprinopsis cinerea upon...Fungal defense against predators largely relies on protein toxins, many of which are lectins. We previously showed that the production of the nematotoxin CCTX2 is upregulated in the Agaricomycete Coprinopsis cinerea upon predation by nematodes. Here, we classify CCTX2 as the founding member of a previously unknown family of fungal chimerolectins. Cryo-EM analysis to 3.2 Å resolution reveals five domains, with the four N-terminal β-trefoil fold (BTF) domains cradling a C-terminal domain, which exhibits an unusual α + β protein fold with some similarity to killer protein 4. Mutational analysis suggests that both terminal domains are functionally required for nematotoxicity. The first two BTF domains enable CCTX2 binding to glycosphingolipids with LacNAc or LacdiNAc glycoepitopes on nematode intestinal epithelial cells, whereas the biochemical function of the C-terminal domain remains unknown. Experiments in the model nematode Caenorhabditis elegans demonstrate that CCTX2 exploits the endocytic and retrograde trafficking machinery of cells in the intestinal epithelium to exert its toxicity and access the yet-to-be-identified intracellular target of the non-lectin domain. Our findings thus show that the molecular architecture and mode of action of CCTX2 is reminiscent of bacterial and plant AB toxins.
Endothelial tissues are essential mechanosensors in the vasculature, and defects in their response to mechanical cues such as blood flow can lead to endothelial dysfunction and cardiovascular diseases like atherosclerosi...Endothelial tissues are essential mechanosensors in the vasculature, and defects in their response to mechanical cues such as blood flow can lead to endothelial dysfunction and cardiovascular diseases like atherosclerosis. Here, we explore how mechanoresponses tune endothelial tissue physiology and function. By bulk RNA sequencing in endothelial cells experiencing varying flow profiles, we identify a set of novel mechanosensitive genes associated with the cytoskeleton and adhesion structures. We focus on a cytoskeletal protein, Four-and-a-half LIM protein 2 (FHL2), which is consistently enriched in endothelial tissues experiencing atherosclerosis-prone disturbed flow, both in vitro and in vivo. We demonstrate that increased FHL2 expression is necessary and sufficient to induce hallmarks of atherosclerosis-like endothelial phenotypes, including aberrant cell morphology, discontinuous cell junctions, hypercontractility, and increased tissue permeability. Strikingly, this atherosclerosis-like phenotype requires the force-sensitive binding of FHL2 to the actin cytoskeleton. Mechanistically, we show that FHL2 controls endothelial tissue phenotypes by promoting RhoGTPase-dependent actomyosin contractility via release of the microtubule-bound RhoGTPase effector, GEF-H1. These findings reveal a positive mechanochemical feedback wherein FHL2 force-sensitivity tunes multi-scale mechanoresponses and endothelial tissue physiology.
The roles of translational control in the immune system are incompletely understood. Using a CRISPR/Cas9-mediated functional screen of RNA helicases in an in vitro system of plasma cell differentiation, we identify in th...The roles of translational control in the immune system are incompletely understood. Using a CRISPR/Cas9-mediated functional screen of RNA helicases in an in vitro system of plasma cell differentiation, we identify in this study DHX29 as a critical regulator of this process. Mice with B-cell-specific deletion of Dhx29 exhibit severely impaired germinal center B-cell formation, plasma cell differentiation, and antibody production. Mechanistically, DHX29 promotes translation of Tcf3 and Tle3 mRNAs via binding to their 5' untranslated regions (UTRs). In the absence of DHX29, B cells exhibit normal proliferation but fail to undergo class switch to IgG1 and differentiation into plasma cells, resulting in impaired antibody production. Ectopic expression of TCF3 and TLE3 largely restores plasma cell differentiation of Dhx29-deficient B cells. Our study provides insights into the functional importance of translational control in the immune system by unraveling critical roles of the RNA helicase DHX29 in the translation of key transcription factors controlling germinal center response and plasma cell differentiation.
Immunotherapy has reshaped melanoma treatment, yet the majority of patients fail to respond or develop resistance. Nerve growth factor receptor (NGFR/p75NTR/CD271) has been linked to melanoma aggressiveness and therapeut...Immunotherapy has reshaped melanoma treatment, yet the majority of patients fail to respond or develop resistance. Nerve growth factor receptor (NGFR/p75NTR/CD271) has been linked to melanoma aggressiveness and therapeutic resistance, but the underlying mechanisms remain unclear. Here, we demonstrate that pharmacologic inhibition of NGFR with THX-B significantly reduces distant metastasis and restores sensitivity in immunotherapy-resistant tumors. THX-B treatment also enhances intratumoral CD8⁺ T-cell infiltration. Further, we show that immune-resistant melanomas acquire an invasive, ameboid phenotype characterized by elevated NGFR, PD-L1, and activation of non-muscle myosin light chain 2 (NM II; MLC2). Mechanistically, NGFR regulates RhoA/ROCK-dependent MLC2 phosphorylation and proteasomal stabilization. Analysis of melanoma patient samples reveals that high NGFR expression correlates with reduced progression-free survival following immunotherapy. Moreover, NGFR and phosphorylated MLC2 are consistently enriched at the invasive fronts of primary tumors, metastases, and in immunotherapy-resistant cell lines from melanoma patients. Altogether, these findings identify the NGFR-MLC2 axis as a mediator of invasive immune resistance and support NGFR inhibition as a strategy to enhance immunotherapy efficacy.
Mitochondrial targeting of the PINK1 kinase results, under normal conditions, in membrane-potential-driven inner membrane penetration and cleavage by the resident protease PARL before retro-translocation and proteasomal...Mitochondrial targeting of the PINK1 kinase results, under normal conditions, in membrane-potential-driven inner membrane penetration and cleavage by the resident protease PARL before retro-translocation and proteasomal degradation. In compromised mitochondria, with reduced membrane potential, inner membrane incorporation is not achieved, which leads to surface activation of the full-length protein, Parkin recruitment and mitophagy. Here, we identify a third pathway in which PINK1 is imported into the mitochondrial matrix. Structural modelling predicts that PINK1's transmembrane domain (TMD) is conformationally plastic, forming either an α-helix or α/β-hybrid at the interface between Tim17 of the TIM23-complex for engagement of either ROMO1 or PARL. These mutually exclusive assemblies define distinct protein-import channels with differing biological roles. PINK1's α-helical TMD adopts a pose suggestive of translocation through the ROMO1/Tim17-channel, while the α/β-hybrid engages PARL and is cleaved. We propose that TMD structural plasticity determines whether PINK1 is imported into the matrix or cleaved and retro-translocated. The results expand the role of PINK1 beyond that of a damage sensor and imply a role in healthy mitochondrial function with potential relevance to Parkinson's disease.
Loss of Ca efflux capacity contributes to the pathogenesis and progression of Alzheimer's disease (AD) by promoting mitochondrial Ca (Ca) overload. Here, we utilized loss-of-function genetic mouse models to causally eval...Loss of Ca efflux capacity contributes to the pathogenesis and progression of Alzheimer's disease (AD) by promoting mitochondrial Ca (Ca) overload. Here, we utilized loss-of-function genetic mouse models to causally evaluate the role of Ca uptake by conditionally deleting the mitochondrial calcium uniporter channel (mtCU) in a robust mouse model of AD. Loss of neuronal Ca uptake reduced Aβ and tau-pathology, synaptic dysfunction, and cognitive decline in 3xTg-AD mice. Knockdown of Mcu in an in vitro model of AD significantly reduced matrix Ca content, redox imbalance, and mitochondrial dysfunction. The preservation of mitochondrial function rescued the AD-dependent decline in autophagic capacity and protected neurons against amyloidosis and cell death. This was corroborated by in vivo data showing improved mitochondrial structure and apposition in AD mice with loss of neuronal Mcu. These results suggest that inhibition of neuronal Ca uptake represents a powerful therapeutic target to impede AD progression.
Yamauchi YY, Sheu XD, Tarfder R
… +10 more, Kumamoto T, Hatakeyama J, Sato H, Rouillard P, Bilgic M, Deguchi S, Nakamura T, Kishi Y, Emoto K, Suzuki IK
Mammals share a laminar cerebral cortex, with excitatory neuron subtypes organized in distinct layers. Although this framework is conserved, subtype balance varies markedly between species due to largely unknown mechanis...Mammals share a laminar cerebral cortex, with excitatory neuron subtypes organized in distinct layers. Although this framework is conserved, subtype balance varies markedly between species due to largely unknown mechanisms. Here, we show that species-specific neuronal composition arises from non-uniform scaling of the temporal dynamics of neurogenesis. Comparative histology of eight mammalian species reveals a significant, rat-specific expansion of the deep layer in the somatosensory cortex. This feature of the rat cortex results from a specific extension of the early neurogenetic phase of deep-layer neuron production before transitioning to the upper layer, as confirmed by neuronal birthdating and single-cell transcriptomics. The duration of deep-layer neuron production is regulated by a genetic program controlling neural progenitor cell aging, including canonical Wnt signaling. Comparative single-cell transcriptomics revealed that cortical progenitor cells in rats exhibit significantly elevated Wnt ligand expression. Therefore, while sequential cortical neurogenesis is conserved, its progression is non-uniformly scaled between species. Precise heterochronic fine-tuning allows evolutionary refinement of cellular configuration without drastic remodeling of the conserved corticogenesis program.
Programmed degradation of maternal proteins is essential for the oocyte-to-embryo transition (OET). While pharmacological inhibition studies have established the importance of proteasomes in ovarian reserve maintenance,...Programmed degradation of maternal proteins is essential for the oocyte-to-embryo transition (OET). While pharmacological inhibition studies have established the importance of proteasomes in ovarian reserve maintenance, oocyte maturation and fertilization, the physiological impact of intrinsic proteasome insufficiency and underlying molecular mechanisms remain poorly understood. In mice, endolysosomal vesicular assemblies (ELVAs), specialized membraneless compartments composed of proteasomes, endolysosomes and autophagosomes, facilitate protein degradation during oocyte maturation and early embryogenesis. In this study, we generated mice with oocyte-specific deletion of the proteasomal core subunit Psma7, to investigate the physiological function of the 20S proteasome and its roles in ELVAs-mediated protein degradation. PSMA7-deficiency destabilized 20S proteasomes and disrupted translocation of ELVAs, leading to pronounced accumulation of ubiquitinated proteins in oocytes and zygotes. Consequently, maternal Psma7 deletion resulted in female infertility, manifested by impaired oocyte maturation and developmental arrest at one- to two-cell stage. Furthermore, we observed reduced proteasome abundance and dysfunction of ELVAs in aged oocytes, providing a mechanistic explanation for the decline in developmental competence associated with oocyte aging. Taken together, our findings elucidate the critical function of proteasome-regulated proteostasis within ELVAs in maintaining oocyte quality during OET and reproductive aging.
Skin aging, the most visible and accessible manifestation of organismal aging, reflects systemic physiological decline, compromising barrier integrity, immune defense, and regenerative capacity-functions essential for ov...Skin aging, the most visible and accessible manifestation of organismal aging, reflects systemic physiological decline, compromising barrier integrity, immune defense, and regenerative capacity-functions essential for overall tissue homeostasis and longevity. Understanding why and how the skin ages offers crucial insights into tissue homeostasis and systemic aging. Here, we dissect the multi-layered mechanisms of skin aging across the epidermis, dermis, and appendages, highlighting how intrinsic cellular senescence, disrupted inter-compartmental communication, and dysregulation of the skin microbiome and hormonal signaling collectively undermine epithelial structure and function. We also summarize advances in quantitative evaluation of skin aging, from molecular signatures to morphological, microbial, and phenotypic indices, enabling objective assessment of biological age and intervention efficacy. Finally, we highlight rejuvenation strategies, encompassing rewiring of gene expression programs, metabolic modulation, microenvironmental remodeling, microbiome modulation, and hormone regulation, offering a framework for precision interventions and next-generation regenerative therapies.