Autophagy

Exocyst was initially uncovered in yeast genetic sec-screen as a tethering complex for exocytotic vesicles and this function was later found to be evolutionarily conserved in other eukaryotes including plants. Later however, a surprising engagement of the exocyst complex in autophagy was observed in animals, plants and recently also in yeast. Using the genetic approach we observed EXO70E2 exocyst complex subunit engagement in the defence response to Pseudomonas syringae attack linked to the autophagy pathway. CRISPR/CAS LOF mutant of EXO70E2 is more sensitive to Pseudomonas infection (both virulent as well as T3SS mutant) and autophagy flux monitored by NBR1 antibody is compromised in comparison to WT. We conclude that the plant exocyst complex linked to the EXO70E2 subunit participates in defence against Pseudomonas bacteria in conjunction with the autophagy pathway. HighlightArabidopsis exocyst subunit EXO70E2 affects selective autophagic flux monitored by NBR1 and is participating in defense against Pseudomonas syringae infection.

Autophagy is a catabolic process that degrades damaged organelles and aggregation-prone proteins and plays key roles during development and in maintaining cellular homeostasis. It can be induced by stress including starvation, oxidative stress, or accumulation of misfolded proteins. Autophagy declines with age and there is great interest in manipulating autophagy to improve neurodegenerative diseases, as its stimulation shows promise to improve diseases including Huntington, Alzheimer, and Parkinson. Endosomal microautophagy (e-MI) is a type of autophagy in which cytosolic proteins are delivered to late endosomes and degraded upon incorporation into intraluminal vesicles of multivesicular bodies. Here, we report that the actin nucleation-promoting factors (NPFs) known to activate the Arp2/3 complex to promote branched actin assembly can alter the dynamics of e-MI. We found that upon stress exposure, overexpression of the NPFs WASp, Wash, or SCAR results in an expedited induction of e-MI. Strikingly, Wash is uniquely required for physiological e-MI induction implying that NPFs are not functionally redundant for e-MI. We show that the WASH complex regulates e-MI on late endosomes acting via Arp2/3 and thus likely branched actin. Surprisingly, the regulation of e-MI by Wash is independent of retromer that is known to recruit Wash to early endosomes for its role in recycling of membrane proteins and rather reflects a novel degradative aspect of Wash function. Taken together, we identified a novel function of NPFs as upstream regulators of e-MI that could be used to activate e-MI ectopically to improve aggregate clearance during neurodegeneration.

Mucolipidosis type IV is a lysosomal storage disease that is characterized by delayed psychomotor development and retinal degeneration due to cell death, in addition to other symptoms that are due to aberrant functions of live tissues. Caenorhabditis elegans CUP-5 is the orthologue of human TRPML1, the protein that is dysfunctional in Mucolipidosis type IV patients. Mirroring Mucolipidosis type IV pathology, loss of C. elegans CUP-5 results in developing intestinal cell death in embryos leading to embryonic lethality, while other tissues in adults lacking CUP-5 are alive but dysfunctional. We had previously shown that ESCRT-Associated proteins and the ATP-Binding Cassette Transporter MRP-4 are necessary for acquiring aberrant and poorly functional lysosomes in the absence of CUP-5. In this study, we show that the aberrant lysosomes permeabilize or rupture, thus releasing lysosomal degradative enzymes that kill cells in the absence of CUP-5. We also show that the autophagy-related protein ATG-9 mediates, in an autophagy-independent manner, this lysosomal permeabilization. We finally propose phenotypic and biochemical models linking CUP-5 to lysosomal defects and cell death.

Elevated temperatures compromise plant immunity and increase susceptibility to bacterial pathogens through extensive reprogramming of hormone signaling pathways. Although autophagy contributes to both stress adaptation and pathogen defense, its role in hormone-dependent immune regulation under warm conditions remains unclear. Here, we investigated the contribution of NBR1 (NEIGHBOR OF BRCA1 GENE 1)-mediated selective autophagy to Arabidopsis immunity against Pseudomonas cannabina pv. alisalensis at elevated temperature. Bacterial infection under warming enhanced autophagic flux and promoted NBR1 turnover, indicating increased autophagic activity. Analysis of atg5 and nbr1 mutants, and NBR1-overexpressing lines, demonstrated that both core autophagy and NBR1-mediated selective autophagy contribute to bacterial immunity under warm conditions. Hormone and gene expression analyses indicated that NBR1 negatively regulates abscisic acid (ABA)-associated transcriptional responses during infection, while salicylic acid signaling was largely unaffected. Mechanistically, NBR1 physically associated with the ABA-responsive transcription factor ABI5 (ABA INSENSITIVE 5) and promoted its autophagy-dependent turnover in planta. ABI5 turnover was strongly reduced under warm conditions, leading to its accumulation in nbr1 and atg5 plants. Consistent with a functional role for ABI5 in this phenotype, genetic disruption of ABI5 largely reversed the increased susceptibility of nbr1 mutants at elevated temperature, whereas ABI5 overexpression increased susceptibility to bacterial infection. Together, our results identify NBR1-mediated selective autophagy as a regulatory mechanism that restrains ABA-associated susceptibility through the autophagy-dependent turnover of ABI5. These findings reveal a previously unrecognized connection between selective autophagy and ABA-dependent immune regulation and identify NBR1-mediated ABI5 turnover as a temperature-dependent mechanism that prevents stronger bacterial susceptibility under warm conditions.

Pancreatic ductal adenocarcinoma (PDAC) is dependent on autophagy for growth. Chloroquine/Hydroxychloroquine (CQ/HCQ), the sole FDA-approved autophagy inhibitors, have shown limited clinical efficacy as cancer therapies. To identify approaches to improve PDAC response to CQ, we performed a CQ-anchored, CRISPR-Cas9 mediated loss-of-function screen. We identified that the loss of genes encoding proteins upstream in the autophagy pathway enhanced CQ-mediated growth suppression. This indicated that simultaneous targeting of two distinct nodes of the same pathway, vertical inhibition, may be a more effective strategy than single node inhibition. We demonstrated that genetic loss or pharmacological inhibition of VPS34, a protein necessary for autophagosome nucleation, sensitized PDAC cells to inhibitors of the terminal stage of the autophagy pathway, including CQ and an inhibitor of PIKfyve. We extended this concept to the initiation complex and demonstrated that ULK1/2 inhibition synergized with CQ and PIKfyve inhibition to impair PDAC cell growth and increase apoptosis. Anticipating mechanisms of resistance to vertical autophagy inhibition, we performed reverse-phase protein array profiling and identified that vertical inhibition of the autophagy pathway resulted in enhanced activation of the PI3K-AKT-mTORC1 signaling pathway. Increased mTORC1 signaling resulted in heightened sensitivity to bi-steric mTORC1 inhibition in both cell line and organoid models of PDAC. This study identifies novel anti-autophagy inhibitor combinations that may improve the clinical efficacy of autophagy inhibition for PDAC treatment. IMPLICATIONSVertical inhibition of the autophagy pathway reduces pancreatic cancer cell growth, increases apoptosis, and enhances sensitivity to mTORC1 inhibition; thereby representing a novel therapeutic strategy for autophagy-driven pancreatic cancer.

The endoplasmic reticulum (ER) and its muscle-specialized form, the sarcoplasmic reticulum (SR), are crucial organelles in muscle cells, involved notably in protein synthesis, calcium regulation and muscle contraction. A well-known process involved in ER remodeling and homeostasis is ER-phagy, also called reticulophagy, a selective form of autophagic process in which ER-phagy receptors mediate the delivery of ER portions to lysosomes for degradation. SH3KBP1 is an adaptor protein involved in membrane trafficking. Recently, it was shown to control ER morphology and SR formation in striated skeletal muscle. In this study, we demonstrate that SH3KBP1 can bind to LC3B and CKAP4 proteins, bridging ER to autophagosome membranes, and is degraded by autophagy, in developing muscle fibers. Moreover, SH3KBP1 down-regulation impacts basal autophagy efficiency and ER-phagy stimulation; it also impairs the turnover of numerous ER-resident proteins. Our work highlights a new role for SH3KBP1 as a soluble ER-phagy receptor in striated skeletal muscle.

Gasdermins are pore-forming effectors that mediate pyroptosis, an inflammatory form of programmed cell death characterized by membrane permeabilization and the release of intracellular contents. Phylogenetically, gasdermin members can be broadly divided into two major branches, the GSDME/PJVK branch and the GSDMA/B/C/D branch. Whereas the GSDME mediated pyroptosis was traced back to metazoans, the functional origins of GSDMA/B/C/D branch remain poorly understood. As a basal representative of the GSDMA-D lineage in cartilaginous fish, Callorhinchus milii GSDMA/B (CmiGSDMA/B) provides essential information for the ancestral state of this branch. Here, we functionally characterized CmiGSDMA/B and identified CmiCASP1 as its upstream protease. Mechanistically, Lipopolysaccharide (LPS) activates CmiCASP1 via its CARD domain, leading to cleavage of CmiGSDMA/B into two functionally distinct products, N241 and N288. N241 binds cell membrane to drive pyroptosis, whereas N288 suppresses N241-triggered cell death. Interestingly, N241 exhibits bactericidal activity against Gram-negative bacteria in vitro, suggesting that antimicrobial activity may have been an early feature of the GSDMA-D lineage. Collectively, these findings provide insight into a non-canonical, LPS-responsive, caspase-driven pyroptosis pathway in cartilaginous fish and reveal the dual-fragment antagonistic regulation within this branch.

Motor Neurone Disease (MND) is characterised by progressive degeneration of upper and lower motor neurons, accompanied by cytoplasmic mislocalisation and hyperphosphorylation of TDP-43, hallmarks that implicate failure of endolysosomal proteostasis. The Endosomal Sorting Complexes Required for Transport (ESCRT) pathway governs multivesicular body (MVB) formation, lysosomal cargo delivery, and autophagosome closure, yet its expression profile in human MND tissue and mechanistic contribution to disease pathology have not been established. Here, we report subunit-specific dysregulation of ESCRT proteins in postmortem motor cortex and spinal cord from MND patients: CHMP2B (ESCRT-III) is significantly upregulated in both regions, whilst TSG101 (ESCRT-I) and VPS37A (ESCRT-I) are significantly downregulated in motor cortex, indicating a region-specific remodelling of the ESCRT network. In a tunicamycin-induced ER stress model using NSC-34 motor neuron-like cells and primary cortical neurons, TSG101 overexpression reduced total and phosphorylated TDP-43, suppressed mTOR signalling, and restored autophagic flux, whereas TSG101 knockdown exacerbated TDP-43 accumulation and cytoplasmic mislocalisation. CHMP2B modulation selectively regulated TDP-43 phosphorylation without altering total TDP-43 levels, consistent with a casein kinase 1-dependent mechanism operating independently of bulk autophagy. Both TSG101 and VPS4a were required to maintain neuronal CD9 tetraspanin localisation to early endosomes; their depletion redirected CD9 to late endosomal and lysosomal compartments under ER stress. Extracellular vesicle characterisation revealed a functional divergence: TSG101 is required for general exosome biogenesis, whereas VPS4a ATPase activity specifically mediates loading of pathological TDP-43 cargo into EVs. Dynamic light scattering confirmed that ER stress and ESCRT modulation produce distinct, condition-specific alterations in EV size and polydispersity. These findings establish ESCRT dysfunction as a multifaceted contributor to MND pathogenesis and identify TSG101, CHMP2B, and VPS4a as mechanistically distinct therapeutic targets warranting preclinical validation.

Lipid droplets (LDs) are conserved organelles that buffer lipid storage and stress, yet their dynamics and functions in neurons remain largely unknown. Here, we report activity-dependent dynamics of neuronal LDs, visualized by a novel, genetically encoded LD reporter (termed LipiDew), in both cultured neurons and mouse motor cortex. Using LipiDew, we found that various paradigms of neuronal activation induced predominant and transient formation of LDs in neurites. Disruption of autophagic LD degradation (lipophagy) resulted in abnormal lipid accumulation in dendritic spines and shafts, promoted recruitment of synaptic scaffolding proteins to LDs, and altered intracellular calcium kinetics in neurons. In addition, mice with neuron-specific genetic impairment of lipophagy showed motor function defects. Together, these findings identify activity-dependent LD formation and lipophagic clearance in neuronal compartments as a crucial regulatory mechanism of synaptic integrity and neuronal function.

Lipid-processing microglia are among the microglial states most strongly associated with Alzheimers disease (AD) pathology, yet whether this association reproduces across independent cohorts, what transcriptional programs define the state, and which upstream signals and small molecules can modulate it remain unsolved. We address these questions through a cross-cohort analysis of one such substate (MG4) by integrating differential expression, transcription factor activity inference, gene set enrichment, and cell-cell communication across five independent single-nucleus RNA sequencing cohorts (ntotal = 140 donors), with paired single-nucleus ATAC sequencing in one multi-omic cohort for epigenomic corroboration. A held-out cohort (n = 150 donors) supported donor-level regression of MG4 proportion on ligand expression, and two spatial transcriptomics datasets (ntotal = 30 donors) related ligand expression to MG4 identity in neighboring spots. MG4 was reproducibly enriched in AD across all five cohorts (pooled log2 fold change = 0.90, p = 3.0 x 10-4). Expression-based inference and motif accessibility jointly nominated MITF and BACH1 as regulators of a program led by V-ATPase-driven lysosomal acidification and cholesterol efflux, a lysosomal-biogenesis signature distinct from the catabolic DAM and lipid-storage LDAM programs, with AD-specific upregulation of energy metabolism. FGF1 and TGFB2 were the most supported candidate upstream ligands, each significant in donor-level regression with further spatial evidence. Computational drug repurposing nominated ten blood-brain barrier-penetrant compounds as perturbational probes. Together, these results advance a described disease-associated microglial state into a reproducible, mechanistically framed regulatory model, providing candidate regulators, upstream ligands, and pharmacological probes for functional validation.

Metabolic pathways are increasingly recognized as tractable targets in aging and disease. Building on prior work demonstrating that supplementation with low-molecular weight metabolites (amino acids, vitamins, and their intermediates) can extend lifespan in Caenorhabditis elegans, we focused on pantothenate (vitamin B5), which is dysregulated in sarcopenic muscle and in several neurodegenerative and metabolic disorders. Pantothenate is the obligate precursor of coenzyme A through a short, highly conserved biosynthetic pathway in which loss-of-function mutations can cause neurodegeneration with brain iron accumulation. In C. elegans, the longevity curtailing transcription factor DAF-16/FOXO has a conserved binding element in the promoter region of pnk-1, encoding the first enzyme (PNK-1) in the coenzyme A pathway, and pnk-1 is markedly upregulated in long-lived daf-2 (insulin/-like receptor) mutants, implicating coenzyme A metabolism in longevity. Here, we demonstrate that CoA levels naturally increase during early life and decrease towards older age in C. elegans. Dietary pantothenate supplementation increases coenzyme A levels with minimal effects on lifespan but systemic effects on lipid metabolism, mitochondrial dynamics, and muscle structure under basal conditions. Under DAF-16-associated stress conditions, including heat and oxidative stress, pnk-1 expression is upregulated and pantothenate supplementation robustly extends lifespan and improves mobility. Finally, we demonstrate dysregulation of daf-16 and pnk-1 expression in amyotrophic lateral sclerosis (ALS) models, in which pantothenate supplementation confers both lifespan extension and cholinergic neuroprotection.

Mitochondrial homeostasis is maintained by multiple quality control pathways, including mitophagy, which targets dysfunctional mitochondria for degradation. During receptor-mediated mitophagy, the outer membrane proteins BNIP3 and NIX directly recruit autophagy machinery to the mitochondrial surface, though their precise regulation is still unclear. In recent years, new BNIP3- and NIX-interacting proteins have been identified that influence mitophagic flux. PPTC7 and FBXL4 target BNIP3 and NIX for proteasomal turnover to keep levels of the receptors low, whereas TMEM11 is proposed to spatially control mitophagy by interacting with receptors at active mitophagy sites. However, it is unclear how each of these interactions is controlled and how they interplay with each other. Here, we identify a repressor of mitophagy, ARMC1, which forms a complex with TMEM11, BNIP3, and NIX. During mitophagy activation, ARMC1 dissociates from the complex, freeing the receptors to initiate mitophagy. We find that TMEM11 then acts in an antagonistic relationship with PPTC7, protecting the receptors from proteasomal degradation. Our data are consistent with a two-stage model. At steady state, a population of sentinel receptors is repressed and primed to respond to mitochondrial dysfunction. Once mitophagy is activated, TMEM11 protects BNIP3 and NIX, ensuring a sustained mitophagic response. Our findings provide a framework for understanding how two key regulatory pathways intersect to modulate receptor-mediated mitophagy.

Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme that commonly exhibits altered expression in human diseases such as cancers and neurodegeneration, making it a valuable disease biomarker and putative therapeutic target. However, any treatment targeting LDHA will also disrupt normal metabolism, underscoring the need to investigate physiological consequences of inhibiting this enzyme. We previously established the fruit fly Drosophila melanogaster as a genetic model for studying LDH function in the context of growth, metabolism, and development. Here we expand upon those studies by investigating a serendipitous observation that Ldh mutant larvae exhibit diet-dependent lethality. Using a multiomic approach, we discovered this diet-dependent phenotype is independent of nutritional composition. Instead, Ldh mutant larvae are exercise intolerant and display reduced mobility, rendering mutant larvae sensitive to food consistency. Moreover, tissue-specific analysis reveals that LDH activity within muscle and peripheral glia are essential for larval viability raised on solid food. Intriguingly, these phenotypes mirror the pathophysiology of LDHA deficiency (Glycogen Storage Disease Type XI; GSD Type XI) in humans, where mild symptoms are exacerbated by physical exertion and environmental stress. Together, our findings further highlight the value of using Drosophila to explore the developmental and physiological consequences of Ldh inhibition.

Cellular senescence is a heterogeneous and evolving process involved in development, tissue repair, aging, and age-related diseases. Although senescence burden in tissues has been widely studied, its spatial organization remains poorly understood, particularly in vivo. Senescence encompasses a spectrum of distinct states, with cells differing in molecular signatures, secretory activity, persistence, and interactions with their microenvironment depending on the inducing stimulus and tissue context. This heterogeneity suggests that spatial organization may reflect underlying processes such as tissue repair, regeneration, or maladaptive remodeling, providing insight into senescence function and its pathological roles. Here, we propose a quantitative, multi-scale framework to characterize the spatial organization of senescent cell populations in post-infarction mouse hearts. By combining a senescence-signature scoring strategy with spatial statistical methods and functional data analysis, we assess whether senescent cells exhibit clustered or dispersed patterns, and how these spatial distributions evolve over time following infarction. This approach aims to provide new insights into the spatiotemporal dynamics of senescence in vivo and to identify spatial features that may inform therapeutic strategies targeting age-related and tissue repair-associated pathologies.

Cytoskeletal acetylation and autophagy are fundamental drivers of neuronal plasticity, yet how these pathways are coordinated across subcellular compartments remains unknown. Here, we identify LUZP1 as a signaling organizer that couples cytoskeletal acetylation to autophagy in hippocampal neurons. LUZP1 deficiency impaired neurite outgrowth and dendritic spine maturation while promoting ciliary elongation; these phenotypes were partially mirrored in neuron-specific Luzp1 knockout mice, which also showed altered locomotor behavior. Mechanistically, LUZP1 promoted neurite extension by enhancing ATAT1-dependent -tubulin acetylation while driving spine maturation and limiting ciliary growth by restraining HDAC6-dependent cortactin (CTTN) deacetylation. An acetylation-mimetic CTTN mutant rescued both spine and ciliary defects caused by LUZP1 deficiency. In parallel, blocking autophagy-dependent OFD1 degradation attenuated ciliary elongation, linking CTTN deacetylation to increased autophagy under LUZP1-deficient conditions. Finally, activated CaMKII associated with LUZP1 and selectively enhanced its interaction with HDAC6 and CTTN, coupling neuronal activity to cytoskeletal remodeling. Together, these findings identify a CaMKII-LUZP1 pathway that integrates cytoskeletal acetylation with autophagy to coordinate neuronal morphogenesis and ciliary homeostasis.

Acute ischemic stroke (AIS) induces profound systemic immune alterations that contribute to infection susceptibility. Here, we identify lipocalin-2 (LCN-2) as a rapidly induced and conserved regulator of stroke-associated immunosuppression. Using 3-MACE-Seq, cytokine profiling, and immunofluorescence in C57BL/6J mice subjected to transient middle cerebral artery occlusion (tMCAO), we show that LCN-2 is strongly upregulated in splenic red pulp macrophages (RPMs) within 24 hours and again 7 days post-tMCAO. LCN-2-expressing RPMs form immunological synapses with CD3+ T cells, thereby impacting T cell trafficking. Recombinant LCN-2 directly reprogrammed T cells and monocytes toward hyporesponsive, tolerogenic phenotypes by suppressing inflammatory cytokines, impairing chemotaxis, enhancing phagocytosis, and uncoupling oxidative burst. Human spleens likewise displayed LCN-2-expressing CD68+ RPMs, and LCN-2 preconditioning of monocytes reproduced reduced HLA-DR, CD80, CD206, and ROS with increased uptake of E. coli bioparticles. These findings identify LCN-2 signaling as a central orchestrator of stroke-induced peripheral immunoreprogramming and a potential therapeutic target to mitigate post-stroke immunodepression. SummaryAcute ischemic stroke induces LCN-2 in splenic red pulp macrophages, which reprogram T cells and monocytes toward tolerogenic, hyporesponsive states. Mouse and human data identify LCN-2 as a driver of peripheral immunodepression and a potential target to reduce infection risk. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/733904v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@149bd46org.highwire.dtl.DTLVardef@2974aaorg.highwire.dtl.DTLVardef@1aa7a52org.highwire.dtl.DTLVardef@144dbe5_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

Human endogenous retroviruses (HERVs) are mobile genetic sequences derived from ancient retroviral infections. While typically silenced, their reactivation has been implicated in gene dysregulation, aging, and immune-related transcriptional pathogenesis of some neurodegenerative diseases. Parkinson's disease (PD) is the second most common neurodegenerative disorder, yet its etiology and HERV reactivation remain poorly understood. This study investigates locus-specific HERV expression in early-stage PD, including genetic and non-genetic cases (all PD), idiopathic PD without a known genetic cause (iPD), and PD driven by leucine-rich repeat kinase 2 mutations (LRRK2 PD). We analyzed RNA-seq whole-blood samples from 492 individuals (358 all PD, 256 were iPD, 63 LRRK2 PD, and 134 healthy controls (HC)). We identified 20 significantly dysregulated HERV loci in all PD versus HC. Five HERV loci were shared with iPD analysis, and one HERV locus was shared with LRRK2 PD. Notably, these shared loci included HERV-H and ERVLE elements, indicating robust disease-associated retroviral signals independent of disease subtype. We found that genes proximal to these HERVs revealed pathways implicated in PD pathogenesis. Immune cell deconvolution showed increased neutrophil abundance and decreased resting CD4+ memory T cells proportions across the PD cohorts when compared to HC, consistent with neutrophil-lymphocyte ratio observed in previous peripheral immunity studies. Transcriptomic HERV alterations are present in whole blood across PD populations and are associated with dysregulation of fundamental cellular pathways and peripheral immune remodeling. Our findings motivate experimental validation of locus-specific HERV expression as a candidate blood-based signature with potential to inform PD neuroinflammatory and neurodegenerative processes.

Progressive intercellular spreading of -synuclein (S) is implicated in pathology initiation and propagation of synucleinopathies. However, how recipient neurons respond to incoming S and whether these responses contribute to disease-associated early metabolic events, remains unknown. Here, using extracellular monomeric S to model the earliest cellular response to spreading, we found that internalized S accumulates at tri-organelle contact sites linking mitochondria, endoplasmic reticulum, and endo/lysosomal compartments. At these interfaces, S stabilizes generally dynamic contacts and constrains their remodeling, thereby rewiring organelle communication. These effects require the acidic S C-terminus and are not recapitulated by intracellular S overexpression. Proteomic profiling of S-associated mitochondria identified a contact site-enriched but quality-control-deficient state. Functionally, spreading S impairs neuron-astrocyte mitochondrial quality control (MQC) by reducing neuronal mitochondria transfer to astrocytes, while enhancing mitochondrial import. Our findings establish organelle contact sites as critical target of spreading S, through which rewired organelle communication impairs MQC and neuron-astrocyte crosstalk.

Effective lysosome function is essential for health, and declines with ageing and disease. Upon lysosome damage, cells mount a complex stress response to restore homeostasis. Membrane ATG8ylation plays a central role, orchestrating lysosome repair, replacement and removal, either directly at the damaged membrane, via CASM (conjugation of ATG8s to single membranes), or at nascent autophagosomes, during lysophagy. Here, we identify a novel role for CASM in driving an antioxidant response to lysosome stress. Following damage, CASM regulates both lysosome ubiquitination and p62 recruitment. Membrane-associated p62 undergoes S349-phosphorylation, which permits KEAP1 sequestration, thereby releasing master transcription factor, NRF2. Liberated NRF2 translocates to the nucleus and promotes transcription of antioxidant and detoxifying genes, co-ordinating a cytoprotective response, in a CASM-dependent manner. These findings position CASM as a major upstream response to lysosome stress and uncover the p62/KEAP1/NRF2 axis as a novel effector pathway, termed SOLAR (SQSTM1/p62 Oligomer-mediated lysosome antioxidant response).

Macrophages destroy pathogens by engulfing them into phagosomes that mature into degradative phagolysosomes via lysosome fusion. Leishmania parasites subvert this antimicrobial pathway to establish intracellular infection and cause leishmaniasis. We previously identified the SNARE protein syntaxin-2 (Stx2) as a promoter of phagolysosome biogenesis that simultaneously limits particle binding and uptake. Consistent with this dual role, Stx2-depleted macrophages (Stx2-KD) show enhanced binding and internalization of Leishmania major. Stx2-KD macrophages also sustain higher intracellular parasite loads. We find that L. major actively targets macrophage Stx2 by selectively depleting Stx2 from phagosomes through its virulence metalloprotease GP63. Phagosomes containing GP63-deficient L. major retain Stx2 and acquire increased levels of lysosomal hydrolases and v-ATPase, restoring degradative capacity. In BALB/c mice, L. major infection markedly reduces Stx2 in infected tissues in a GP63-dependent manner. Collectively, our findings identify GP63-mediated Stx2 depletion as a key virulence strategy of L. major, positioning the GP63-Stx2 axis as a promising therapeutic target for leishmaniasis.