Autophagy

Heat shock protein family B member 8 (HSPB8) is a chaperone involved in the chaperone-assisted selective autophagy (CASA) complex. HSPB8 in conjunction with cochaperone BAG3, promotes autophagy-mediated removal of misfolded proteins associated with various neurodegenerative diseases. Mutations in HSPB8, previously associated with Charcot Marie Tooth disease type 2L, have recently been linked to an autosomal dominant rimmed vacuolar myopathy (MFM13), and is considered a multisystem proteinopathy. Patients have distal and proximal limb girdle myopathy with muscle biopsy showing fatty replacement, endomysial fibrosis, and rimmed vacuoles leading to muscle atrophy and early demise. We have demonstrated reduced expression of HSPB8, altered autophagy and TDP-43 accumulation in patient fibroblasts. Using CRISPR technology, we generated a knock-in Hspb8 mouse model of the c.515dupC hot spot frameshift variant to study disease pathology. Overexpressed murine Hspb8 frameshift mutant (c.515dupC, fs) displays insolubility and aggregation propensity in Murine Neuroblastoma X Spinal Cord 34 (NSC-34) cells. Mutant Hspb8 mice developed late-onset muscle weakness beginning at 15 months. Muscle biochemical analyses revealed reduced HSPB8 levels, increased TDP-43, and altered autophagy markers, partially recapitulating the human phenotype. Fiber type analysis, neuromuscular junction integrity, and motor neurons show mild myopathy without neurodegeneration. Given the lack of available treatments, we evaluated trehalose, a natural disaccharide that induces HSPB8 and enhances autophagy. Administration of 2% trehalose in drinking water improves motor performance, restores HSPB8 expression, and ameliorates autophagic and TDP-43 pathology in mutant mice. These findings support the value of our preclinical models for translational studies, and autophagy enhancement as a potential therapeutic strategy for HSPB8-related myopathy.

Cellular senescence combines stable proliferative arrest with extensive changes in secretory, metabolic and organelle programs. Procyanidin C1 (PCC1) has dose-dependent senomorphic and senolytic activity, but the structure of its transcriptome-wide response is not well defined. We reanalyzed published RNA-sequencing counts from bleomycin-induced senescent PSC27 human stromal cells treated with 50 {micro}M PCC1. The primary contrast contained three independent biological replicates per condition. PCC1 altered 8,420 genes at adjusted P<0.05 and absolute log2 fold change[&ge;]1. Ribosomal genes showed the clearest coordinated response, followed by lysosomal genes. Analysis across the full ontologies also identified lysosomal-membrane, proton-transport, late-endosome, lipid-localization, cholesterol and fatty-acid programs. Prespecified analyses revealed two trajectories relative to senescence. Lipid-transport, lipid-binding and plasma-membrane programs decreased during senescence and increased after PCC1, whereas lysosomal-membrane, mitochondrial-membrane, cholesterol and ion-transport programs increased in both contrasts. Senescence-associated outputs were more selective. A prespecified SASP-effector score decreased, but a broad Reactome SASP set did not pass false-discovery correction. AP-1-family expression shifted, and an HSP90/HSF1/proteostasis panel increased. Together, these data support a model in which PCC1 induces coordinated transcriptomic remodeling in senescent stromal cells, encompassing membrane-lipid remodeling, changes in cellular infrastructure, and selective modulation of senescence-associated outputs.

Autophagy is a degradative pathway that is critical in neurons to maintain their homeostasis and to direct nervous system development. Neurons are large, highly polarized cells with distinct compartments that perform discrete functions. How an individual neuron can differentially mobilize autophagy in the axon, dendrite, and cell body is unknown. Here we interrogate the role of autophagy in the neurodevelopment of a single neuron in Caenorhabditis elegans to identify how the spatial compartmentalization of neuronal autophagy ultimately restricts neurite outgrowth in multiple neuronal compartments. However, while canonical autophagy restricts dendrite outgrowth, noncanonical forms of autophagy appear to restrain neurite outgrowth in the axon and soma. Through mutant analysis of the autophagy pathway, we identify that WIPI2-independent autophagy modulates ectopic neurite formation in the soma and that ATG9-independent autophagy regulates axon arborization. Further, we find that unrelated lipid scramblases can compensate for the loss of ATG9 in axon arborization. Our data indicate that neurons marshal both canonical and non-canonical autophagy to spatially control development of separate compartments.

Lipid droplets (LDs) are unique organelles, surrounded by a phospholipid monolayer. They are present in most eukaryotic cells including the unicellular model organism S. cerevisiae. LDs store neutral lipids which serve as precursors for amphipathic membrane lipids and as an energy reserve. Loss of LDs in S. cerevisiae results in multiple cellular defects impairing lipid homeostasis and the biogenesis and function of other organelles. Here, we find that the expression levels of many proteins in isolated mitochondrial fractions are altered in cells that cannot synthesize neutral lipids and therefore lack LDs. In addition, among several downregulated proteins, we identified the previously uncharacterized Ylr001c (which we name Vlf1 for Vacuolar Lipophagy Factor 1). We show that Vlf1 is glycosylated and, in contrast to some previous reports, is actually localized to the vacuole. Furthermore, we demonstrate that changes in Vlf1 expression alter growth sensitivity to rapamycin, and detected a physical interaction of Vlf1 with Atg15, a lipase involved in autophagy. Additionally, we observe higher levels of autophagy/lipophagy in the absence of Vlf1 and a reduction upon overexpression of the protein. Taken together, the effects on lipohagy by Vlf1 makes it, according to our knowledge, the first vacuolar lipophagy regulator identified in S. cerevisiae.

Intracellular parasites cause over 226 million disability-adjusted life years (DALY) globally each year. Current treatment methods fall short of desirable impact due to adverse effects and growing resistance, shifting focus to parasitic effectors for future therapeutic development. Unfortunately, the divergent nature of parasitic proteins has impeded in silico discovery of - and functional inference for - parasitic effectors. Here, we present SecretTarget, a pipeline designed to identify host-interacting effector candidates through secondary localization features. By truncating signal peptides from predicted extracellular proteins ({Delta}SP), we unmask potential underlying localization features that dictate subcellular trafficking within the host. Applying our pipeline to a set of Toxoplasma gondii secreted effectors with known localizations and interactions, we propose a novel host ER-parasite interaction critical for parasite survival, recapitulate published localizations, and provide meaningful biological insights aligning with recent host-parasite interaction discoveries.

The SARS-CoV-2 envelope (E) protein is a virulence factor that remodels host endomembranes, but mechanisms remain incompletely understood. We recently demonstrated that E protein interacts with and inhibits the sarco/endoplasmic reticulum Ca2-ATPase (SERCA), disrupting ER calcium homeostasis. Here, we investigated how this perturbation affects autophagy-associated membrane organization. E protein expression induced lipidated LC3 accumulation and enlarged p62-positive structures, consistent with dysregulated autophagic turnover. Although E protein partially colocalized with LC3 and p62, enlarged p62-positive structures were also observed in cells retaining the reticular ER distribution of E protein, indicating that their formation does not require association with E protein or ER reorganization. E protein also increased the association of p62-positive structures with lysosomes without altering lysosome abundance. Pharmacological SERCA activation attenuated E protein-induced remodeling of autophagy-associated structures, demonstrating that SERCA inhibition contributes to these alterations. Together, our findings establish SERCA-dependent ER calcium homeostasis as a host pathway linking E protein expression to remodeling of autophagy-associated membrane compartments, providing a mechanistic framework for how the SARS-CoV-2 E protein promotes ER membrane remodeling associated with coronavirus replication.

Aquaporins at the plant plasmalemma are divided into two highly conserved subclasses, PLASMA MEMBRANE INTINSIC PROTEINs 1 (PIP1) and PIP2. Arabidopsis thaliana encodes five PIP1 and eight PIP2 isoforms. Individual loss-of-function mutants had been employed for functional analyses. Here, we observe that the pip2;1 pip2;2 pip2;4 pip2;6 pip2;7 quintuple mutant defective of major PIP2 isoforms concomitantly leads to a strongly reduced PIP1 protein level. Lower order mutants pip2;1 pip2;2 and pip2;1 pip2;2 pip2;7 still harbor only 60% and 20% residual PIP1, respectively. This repression is established post-translationally, since neither PIP1s steady-state transcripts nor polysome-associated PIP1 mRNAs are suppressed by pip2;1 pip2;2 pip2;7. Thus, the two major pathways operating in eukaryotes for removal of aberrant proteins, ubiquitin proteasome system (UPS)-dependent ER-associated degradation (ERAD) and autophagy/vacuole-linked degradation, were assessed. Introgression of atg7 blocking autophagy-mediated degradation does not affect the PIP1 protein level of pip2;1 pip2;2 pip2;7. In contrast, introgression of ERAD loss-of-function mutations hrd1A hrd1B and dln1 into pip2;1 pip2;2 pip2;7 partially stabilizes its PIP1 protein level. PIP1 accumulates intracellularly upon pharmacological inhibition of proteasomal degradation by MG132. Nevertheless, the lack of a full PIP1 recovery by these means suggests the flexible operation of parallel ERAD components or unknown pathways. In conclusion, the essential dependence of PIP1 expression on PIP2 isoforms intrinsically interconnects the two PIP subclades at the protein level and will thereby affect their mutual functions. Significance statementPlasma membrane intrinsic proteins constituting the most homogenous plant aquaporin family are nonetheless split into two highly conserved subfamilies, PIP1 and PIP2. The loss of major Arabidopsis PIP2 isoforms does not lead to compensation by PIP1 members, but rather to PIP1s concomitant, post-translational repression. This dependence of PIP1 isoforms inevitably ties the two PIP subfamilies and their function.

BackgroundTuberous Sclerosis Complex (TSC) is a genetic disorder caused by variants in TSC1 or TSC2, leading to mTORC1 hyperactivation and autophagy suppression. Although TSC tumorigenesis typically follows a "two-hit" model, the role of TSC2 haploinsufficiency in autophagy regulation remains unclear. We evaluated autophagy markers in haploinsufficient and gene-edited TSC2 primary cells and investigated the role of metformin in modulating autophagy levels. MethodsPrimary fibroblast cultures were obtained from one healthy individual and three from patients carrying heterozygous germline TSC2 variants: the pathogenic variants c.1008T>G and c.4375C>T.A variant of uncertain significance (VUS) c.724A>T. CRISPR/Cas9-RNP editing was used to model loss of heterozygosity (LOH) in cell pools carrying each variant. Cultures were treated with rapamycin, HBSS, metformin, bafilomycin A1, or vehicle controls, and autophagy was assessed by autolysosomes formation by flow cytometry (acridine orange) and autophagosomes immunofluorescence (LC3 and p-S6K). ResultsIn wild-type cells, only HBSS increased autophagy-positive (acridine orange-positive) cells versus control (15.6% vs. 7.5%; p=0.003). In heterozygous pathogenic cells, rapamycin and metformin increased autophagic cells: c.1008T>G (16.2%, p=0.006; 17.6%, p=0.002) and c.4375C>T (12.5%, p=0.003; 13.3%, p=0.001), versus DMSO controls (9.2% and 7.1%, respectively). VUS c.724A>T cells, with rapamycin increasing autophagic cells (9.74% vs. 6.5%; p=0.0152). In CRISPR-edited cells, all treatments increased the number of autophagic cells compared to the heterozygous cells: c.1008T>G (rapamycin 27.1% vs. 16.7%, p<0.001; metformin 27.2% vs. 17.6%, p<0.001) and c.4375C>T (rapamycin 21.3% vs. 13.1%, p=0.0021; metformin 21.5% vs. 13.6%, p=0.0029). Editing also restored metformin responsiveness in VUS cells (12.5% vs. 8.4%; p=0.0055). Immunochemistry confirmed increased total LC3II and decreased p-S6K across treated cells compared to the control (DMSO). ConclusionThese findings demonstrate that TSC2 haploinsufficiency functionally impairs autophagy prior to second-hit loss. Metformin effectively restores autophagy with phenotypical changes of mTORC1 blockade, highlighting an accessible translational strategy to restore and induce autophagy in TSC cells.

MTCH2 (mitochondrial carrier homolog 2) is a noncanonical member of the solute carrier family with five transmembrane (TM) helices, localized to the mitochondrial outer membrane. MTCH2's atypical topology creates a membrane-accessible hydrophilic groove, predicted to be necessary for its protein insertase and lipid scramblase activities. MTCH2 is linked to lipid metabolism and obesity and is required for starvation-induced mitochondrial hyperfusion. Here, we show that MTCH2 is a stable component of a complex containing the Armadillo (ARM) repeat-containing protein, ARMC1, and the DnaJ/Hsp40 chaperone protein, DNAJC11. Protein crosslinking, protein structural modeling, and molecular dynamics simulations demonstrate that the ARMC1 alpha-helical C-terminal domain (CTD) inserts into and stably interacts with the MTCH2 hydrophilic groove and blocks its lipid scramblase activity. We observe that starvation-induced mitochondrial fatty acid oxidation (FAO) is negatively regulated by the ARMC1-MTCH2 interaction. In ARMC1-deficient cells, FAO is stimulated compared to wild-type cells and lipid droplet abundance is significantly reduced. The altered lipid phenotype of ARMC1-/- cells is strictly dependent on MTCH2 and is reversed by ARMC1 expression in a manner dependent on its CTD. Beyond this metabolic axis, we also identify a function for ARMC1 in regulating lysosomal distribution and autophagic flux that is independent of its CTD and interaction with MTCH2. Thus, our data support a model in which the MTCH2-ARMC1 interaction functions as a metabolic switch during starvation to regulate the balance between fat storage and fat burning.

BackgroundEfferocytosis, the phagocytic clearance of apoptotic and damaged cells, promotes inflammation resolution and tissue repair following ischemic stroke. This study investigated temporal changes in efferocytosis and phagocytosis-related transcriptional programs during acute experimental stroke, examined the effects of aging on these responses, and assessed whether similar immune signatures are present in human ischemic stroke. MethodsPublicly available transcriptomic datasets from murine transient middle cerebral artery occlusion (tMCAO; GSE104036 and GSE112348), permanent middle cerebral artery occlusion (pMCAO; GSE137482), and human peripheral blood after ischemic stroke (GSE16561) were analyzed using OmicSoft/Ingenuity-style pathway analysis. Functional validation included in vivo assessment of efferocytosis after tMCAO and in vitro phagocytosis assays using bone marrow-derived macrophages from young and aged mice. ResultsBoth acute tMCAO models exhibited robust inflammatory activation together with sustained activation of phagocyte-related pathways during the first 24 hours after stroke. Human peripheral blood demonstrated similar inflammatory and phagocytic signatures, supporting translational relevance. Increased efferocytosis at 24 hours after tMCAO was associated with neuroprotection. Although both young and aged mice activated phagocytosis-related pathways after pMCAO, aged mice showed reduced phagosome formation. Consistent with these findings, macrophages from aged mice exhibited enhanced inflammatory responses and impaired uptake of apoptotic cells. ConclusionsA conserved post-stroke immune response characterized by inflammatory activation and phagocyte-mediated clearance was identified across murine and human datasets. Efficient efferocytosis was associated with neuroprotection, whereas aging impaired apoptotic cell clearance and promoted a pro-inflammatory macrophage phenotype, highlighting efferocytosis as a potential therapeutic target for ischemic stroke.

Innate immune cells sense pathogenic bacteria like Legionella pneumophila through patterns such as the protein flagellin, a critical component of the bacterial motility apparatus. Recognition of cytosolic flagellin in mouse immune cells is well understood and mediated by the receptors, neuronal apoptosis inhibitory protein (Naip) 5 or Naip6, which activate the Nlrc4 inflammasome multi-protein complex for initiating cell death or interleukin-1 family cytokine release. However, the role of human NAIP as a cytosolic flagellin sensor remains controversial. Using a multipronged approach, we demonstrate that in a reconstituted cell system human NLRC4 engaged Legionella FlaA flagellin directly (i.e. without the need for hNAIP), whereas human NAIP did not interact with FlaA. Ectopic cytosolic FlaA expression also induced NLRC4 oligomerization, a prerequisite for inflammasome activation, in the absence of NAIP. Unexpectedly, the presence of NAIP diminished the binding of NLRC4 to flagellins and subsequent interleukin-1{beta} release. Interestingly, in resting THP-1 cells, NAIP stably interacted with NLRC4, and during infection or stimulation with FlaA pro-inflammatory responses in THP-1 cells were predominantly NLRC4-dependent. Our data highlight NLRC4 as a putative direct sensor of cytosolic flagellins in the human system and NAIP as a potential negative regulator of flagellin sensing.

Selective autophagy is a host defense mechanism against Mycobacterium tuberculosis (Mtb) that restricts bacterial growth by targeting ubiquitin-coated bacilli for lysosomal degradation via autophagy receptors. Optineurin is a selective autophagy receptor that targets pathogens and modulates immune signaling; however, its precise structural mechanism during Mtb infection remains poorly defined. Here, we show that while Optineurin deficiency spares the global host transcriptomic response to infection, it collapses the host endolysosomal network, reducing LAMP1+ and LysoTracker+ reserves by half. Multi-dose bafilomycin A1 flux assays demonstrated that this structural depletion selectively blocks the dynamic, directional trafficking and functional delivery of autophagosomes to the pathogen, significantly reducing Mtb-DQ-BSA colocalization. Genetic complementation restored bacterial restriction in a manner dependent on three phosphosites (Ser187, Ser530, and the uncharacterized Ser556). In the context of reduced autophagic containment and increased Mtb replication, Optineurin deficiency accelerated necrotic-like host cell death. In vivo, Optineurin deficiency enhanced bacterial replication and impaired the Type I interferon response during acute Mtb infection but did not affect long-term survival. Together, these findings identify Optineurin as a critical regulator of autophagic flux, host cell death, and Type I interferon responses that limit early Mtb pathogenesis.

Pathogenic variants in RUBCN, encoding the Run domain Beclin-1 interacting and cysteine-rich domain-containing protein (Rubicon) have been implicated in autosomal recessive spinocerebellar ataxia 15 (SCAR15). However, the molecular mechanisms underlying disease pathogenesis remain poorly understood. Here, we report 18 individuals from 15 unrelated families harbouring biallelic RUBCN variants, who present with an aggressive neurodevelopmental disorder variably characterized by seizures, developmental delay, intellectual disability and movement abnormalities that cause regression, progressive brain atrophy and neurodegenerative features. Through functional characterization, we demonstrate that a subset of disease-associated putative truncating variants disrupt autophagy regulation. In Caenorhabditis elegans models, loss-of-function RUBCN variants result in an increased autophagic flux and impaired neuronal function, recapitulating key features in humans. Correspondingly, cellular assays reveal that nonsense and frameshift RUBCN variants lead to defective autophagy inhibition, underscoring a crucial role for RUBCN as a key negative autophagy regulator. Molecular dynamics simulations rank the eleven missense variants by structural effect, with p.Arg813Trp alone altering the target protein at both the local and the regional level and lying within the RAB7A-binding module that the truncating alleles remove altogether. Our findings establish and expand the RUBCN-related disorders as a clinically and molecularly distinct subset of autophagy-related diseases. By delineating both the genetic landscape and cellular consequences of Rubicon dysfunction, this study enhances our understanding of autophagy-related neurodevelopmental disorders and provides a foundation for future therapeutic investigations.

The ubiquitin-proteasome system (UPS) serves as the primary proteolytic machinery in eukaryotes, governing intracellular protein turnover to maintain proteome homeostasis. In plants, the HECT-type UPL3/4 ubiquitin ligases play vital roles in developmental and immune signaling. After ubiquitination by pathway-specific E3 ligases, substrates are physically relayed to proteasome-associated UPL3/4 ligases for further modification, which is necessary for their proteasome-mediated degradation. In this study, we investigated if the cellular influence of UPL3/4 extends beyond their direct role in substrate degradation. We discovered that UPL3/4 govern the ubiquitination not only of a broad array of immune-related substrates, but also of many UPS components, including E3 ligases. UPL3 physically interacts with PUB22, a pathway-specific U-box E3 ligase that negatively regulates immunity. PUB22 is controlled by a phospho-switch that converts it from an instable autoubiquitinated state to a stable phosphorylated E3 ligase that marks substrates for degradation. Remarkably, UPL3 only interacted with unphosphorylated PUB22 and facilitated its autoubiquitination-mediated degradation, thereby promoting the accumulation of PUB22 substrates. Moreover, the compromised immune phenotypes of upl3 upl4 mutant plants were largely dependent on PUB22 and its close paralogues. Thus, UPL3/4 control the stability of immune-related substrates not only through direct ubiquitination, but also indirectly by promoting autoubiquitination of PUB22 ligase and its paralogues. Controlling the stability of autoubiquitinating E3 ligases may be a universal mechanism whereby HECT-type ligases and the proteasomes they associated with, orchestrate cellular proteostasis in eukaryotes. Significance StatementThe ubiquitin-proteasome system (UPS) governs intracellular protein turnover to maintain proteome homeostasis in eukaryotes. Proteasome-associate HECT-type ubiquitin ligases play an important role in processing and degrading substrates delivered to the proteasome by pathway-specific E3 ligases. Here, we discover that in plants, HECT-type ligases not only promote the degradation of substrates, they also modify the E3 ligases that target these substrates to the proteasome. Specifically, HECT-type ligases facilitated or expanded the autoubiquitination of immune-suppressive E3 ligases, resulting in their proteasome-mediated degradation and onset of immunity. Our discoveries suggest that during plant immunity, HECT-type ligases and the proteasomes they associate with, control cellular proteostasis by governing the stabilities of both E3 ligases and their substrates.

Autophagy dysfunction and neuroinflammation are central to Alzheimer's disease (AD), yet how extracellular amyloid-{beta} (A{beta}) couples to impaired autophagic flux and heightened neuroinflammation remains unknown. Here, we identify the TAM receptor AXL as a molecular transducer that couples A{beta} sensing to the regulation of autophagy and neuroinflammation in astrocytes. A{beta} induces {gamma}-secretase-dependent cleavage of AXL, generating a nuclear intracellular domain (AXL-ICD) that forms phase-separated condensates and activates autophagy gene transcription through SIRT2-mediated recruitment of the RUVBL1/2-INO80 chromatin-remodeling complex. This axis is activated in astrocytes of postmortem AD brains. Concurrently, AXL-ICD binds to the SIRT2 catalytic domain and suppresses its deacetylase activity, increasing -tubulin acetylation and altering microtubule dynamics. While moderate AXL-ICD levels promote autophagic flux, excessive elevation paradoxically triggers microtubule hyperstabilization, thereby impairing autophagosome-lysosome fusion and causing pathological accumulation of autophagosomes and H2O2. The inhibitory peptide AxSBiP disrupts the AXL-ICD/SIRT2 interaction, restores autophagic flux, reduces plaque burden, and normalizes A{beta}-induced H2O2 production and astrogliosis in APP/PS1 mice. We propose the AXL-ICD/SIRT2 axis as an effective therapeutic target to reduce A{beta} burden and neuroinflammation in AD

Mitochondria integrate bioenergetic metabolism, redox control, genome maintenance, and stress signaling across all eukaryotes. Although plant and human mitochondria diverged substantially during evolution, both systems retain systems-level principles for sensing mitochondrial dysfunction and communicating stress signals to the nucleus. Here, we develop an integrative comparative in silico framework to evaluate whether plant mitochondrial stress signaling can provide a useful conceptual model for interpreting human mitochondrial disease vulnerability. Core Arabidopsis thaliana regulators representing alternative respiration, mitochondrial retrograde signaling, translational stress control, and genome surveillance were compared with functionally analogous human regulators involved in integrated stress response (ISR) signaling, mitochondrial DNA maintenance, and mitochondrial disease phenotypes. Domain architecture, protein-protein interaction topology, enrichment profiles, disease-gene associations, and promoter motif architecture were integrated to assess cross-kingdom convergence at the level of stress-response organization rather than direct orthologs. The plant network formed a compact AOX-NAC-centered stress module associated with respiratory flexibility and retrograde signaling, whereas the human network displayed expanded ISR and mtDNA maintenance modules enriched for mitochondrial disease associations. Promoter motif analyses further indicated lineage-specific transcription factor signatures but broadly comparable stress-responsive regulatory logic. Collectively, these results support the concept that plant mitochondrial stress systems represent simplified resilience-oriented architectures that can help generate experimentally testable hypotheses about failure points in human mitochondrial stress responses.

Skeletal muscle aging is characterized by the deterioration of muscle function, which can lead to negative quality-of-life outcomes including frailty and sarcopenia. While understanding the mechanisms of this process is increasingly important as the global population ages, previous molecular studies of skeletal muscle aging have been limited by statistical power and cell type resolution. In this study, we analyzed single-nucleus gene expression and chromatin accessibility data from 287 human skeletal muscle samples from individuals aged 20-79 years to explore sex- and cell type- specific aging effects. Across 467,126 nuclei from 13 cell types, we identify 384 age-associated genes and 4,061 age-associated chromatin regions. These age-associated molecular features are enriched for functional pathways, including metabolic processes, cell-to-cell communication, and senescence Kyoto Encyclopedia of Genes and Genomes KEGG terms. Age-associated closing chromatin was more common across fiber types and sexes than opening chromatin, and was enriched in active enhancer regions while depleted for active transcription start sites. We observe enrichment for specific transcription factor motifs in closing chromatin, including those of glucocorticoid and androgen receptors, both of which play a key role in the maintenance of healthy skeletal muscle. Together, these findings identify an age-associated regulatory shift, largely invisible in matched transcriptomic data, characterized by closing chromatin which reduces accessibility to hormone receptor binding sites and enhancer regions in the muscle fiber epigenome.

Lysosomes make key contributions to the maintenance of cellular proteostasis, and their functional compromise has been linked to aging and neurodegenerative disease. A defining characteristic of lysosomes is their relative acidity compared to other subcellular compartments, a quality that enables the efficient breakdown of macromolecules. Evidence suggests that neuronal lysosomal pH becomes dysregulated with aging and neurodegenerative disease, yet the mechanisms by which lysosomal pH is maintained remain incompletely understood. To better understand neuronal lysosomal pH regulation, we conducted a genome-wide CRISPRi-based screen in iPSC-derived iNeurons for modifiers of lysosomal pH. We validated several previously known regulators of lysosomal pH and identified novel pathways capable of modifying lysosomal pH, including protein UFMylation and mitochondrial homeostasis. We demonstrate that loss of the lysosomal cationic amino acid exporter, PQLC2, prevents lysosomal acidification in a manner independent of amino acid transport. A novel, tauopathy-associated mutation in PQLC2 impairs lysosomal acidification and drives tau accumulation. Together, this study reveals novel genes that modify lysosomal pH and highlights potential new targets for ameliorating age-related lysosome dysfunction.

Aphids use stylet mouthparts to probe plant tissues and deliver oral secretions into host cells, modulating immunity to establish long-term phloem feeding sites in sieve elements. This feeding behaviour make aphids efficient vectors of plant pathogens. We previously showed that Myzus persicae cathepsin B (CathB) effectors promote aphid colonization, accumulate in dynamic cytoplasmic processing bodies (p-bodies), bind the key Arabidopsis thaliana immune regulator EDS1, and recruit EDS1 together with its signalling partners PAD4 and ADR1 to p-bodies. However, the extent to which CathB effectors suppress EDS1-regulated immunity remains unclear. Here, we show that CathB6-expressing Arabidopsis lines exhibit substantial transcriptomic overlap with the eds1-2 mutant, consistent with CathB6 suppression of EDS1-dependent immunity. However, CathB6 also induces broader transcriptional reprogramming beyond that explained by loss of EDS1 function. A yeast two-hybrid screen identified the transcription factors GLK1 and GLK2, as well as several MORF proteins, as CathB6 interactors. CathB6-expressing plants recapitulated GLK- and MORF-regulated transcriptional changes, suppressing GUN1-associated activity while maintaining GLK-mediated expression of photosynthesis-associated nuclear genes (PhANGs). We found that GLKs promotes CathB6 nuclear accumulation and reduces its p-body association. TurboID proximity labelling further linked CathB6 to PhANG-associated proteins, known MORF2- and GUN1-interacting factors, p-body components, and actin-tubulin/myosin machinery, the latter being consistent with the highly dynamic behaviour of CathB6-associated p-bodies. Together, these data indicate that, beyond suppressing EDS1-mediated defenses, CathB6 interferes with the MORF2-GUN1-GLK signalling pathway, modulating plant defense responses while sustaining GLK-mediated PhANG expression to maintain cellular homeostasis during aphid attack. SIGNIFICANCE STATEMENTAphids damage crops and spread plant diseases while feeding. We discovered how a protein in aphid saliva, CathB6, helps these pests establish themselves on plants. CathB6 weakens a major immune pathway and alters communication between chloroplasts and the cell nucleus. This allows the aphid to reduce plant defences while maintaining photosynthesis and normal cell function. These findings reveal a sophisticated survival strategy and may identify ways to develop crops that are more resistant to aphids.

The cell-intrinsic complement system has emerged as an important orchestrator of a variety of cell-physiological processes, with complement components interacting with intracellular effector systems to regulate cellular responses to pathogens or noxious stimuli. For instance, intracellular C5 signaling through a mitochondrial C5a receptor (C5aR1) controls IL-1{beta} production in human monocytes and macrophages. Here, we investigated whether cell-intrinsic C3 similarly regulates inflammatory responses in macrophages. In LPS-stimulated C3 knockout THP-1-derived macrophages, interferon (IFN)-{beta} production was increased, accompanied by elevated expression of interferon-stimulated genes and enhanced secretion of IFN-induced cytokines and chemokines. C3-deficient cells showed increased phosphorylation of IRF3 at Ser396 and a stabilization of the interaction between IRF3 and TBK1, along with enhanced IRF3 dimerization and nuclear translocation. TBK1 phosphorylation was unaffected, indicating that C3 limits IRF3-TBK1 complex formation rather than upstream TBK1 activation. Small-molecule inhibitors of complement factors B and D restored full-length C3 abundance in LPS-stimulated primary human macrophages, consistent with inhibition of the C3 convertase. It also reduced LPS-induced IFN-{beta} production in primary human macrophages and THP-1 cells, suggesting that full-length, uncleaved C3 suppresses IFN-{beta} production. Collectively, these findings identify cell-intrinsic C3 as a suppressor of IFN-{beta} production in human macrophages, highlighting the importance of the cell-intrinsic complement system in fine-tuning inflammatory responses to pathogens.