Biology subjects

Agostinis, P.

Publications and source records attributed to Agostinis, P..

14 recordsLinked to original sources

bioRxiv2026

Macrophage-instructed GSDME couples glioblastoma cell-state plasticity with inflammatory cell death

Glioblastoma (GBM) cell states reflect spatial microenvironmental interactions. Here, using COMET spatial proteomics and RNAscope across multiregional human GBM tissue, spanning tumor cores with pseudopalisading regions, infiltrative margins and peripheral regions, together with multiplex spatial profiling of 202 specimens from 33 patients with matched primary and recurrent tumors, we identify full-length gasdermin E (GSDME-FL) as a macrophage-instructed regulator of malignant cell plasticity. Integration with single-cell transcriptomics and functional perturbation shows that GSDME-high tumor cells localize to macrophage-rich perivascular niches and are associated with delayed recurrence and longer patient survival. Mechanistically, macrophage-derived S100A4 activates EGFR-Sp1 signaling to induce GSDME-FL in neighboring GBM cells. GSDME-FL restrains hypoxia-associated mesenchymal plasticity and shapes macrophage-induced tumor state transitions independently of caspase activation. During immunogenic cell death (ICD), cleaved GSDME promotes pre-lytic swelling, early ATP efflux, and the release of canonical ICD-associated cytokines and chemokines. NLRP3 signaling further supports ATP release and licenses macrophage phagocytosis of dying GBM cells, whereas NINJ1-dependent membrane rupture enables terminal HMGB1 release. Vaccination with ICD-treated glioma cells elicits tumor rejection in a prophylactic intracranial challenge model. Notably, co-expression of GSDME and NINJ1 in macrophage-rich perivascular niches provides a spatial correlate of pathway convergence in patient tumors. Thus, our findings define how a spatial macrophage niche induces GSDME-FL, thereby coupling malignant cell state plasticity to the inflammatory properties of GBM cell death.

cancer biology↗

bioRxiv2026

A druggable ATP13A3-antizyme switch controls adaptive polyamine uptake in cancer

Cellular polyamine depletion is a promising anticancer strategy, but compensatory polyamine uptake limits efficacy when synthesis is blocked by DFMO (difluoromethylornithine), a clinically approved inhibitor of ornithine decarboxylase. The transporter and feedback mechanism driving this adaptive response have remained unclear. Despite their similar biochemical properties, we identify ATP13A3, rather than ATP13A2, as the principal DFMO-responsive polyamine importer, suggesting that these isoforms regulate distinct polyamine fluxes. Mechanistically, the polyamine sensor antizyme not only restrains polyamine biosynthesis but also selectively inhibits ATP13A3-mediated uptake, a brake that is relieved upon DFMO treatment. This regulatory circuit exposes distinct polyamine-acquisition states across cancers, defining synthesis- and/or uptake-biased subtypes that can shift during disease progression. Melanoma metastasis and vemurafenib resistance evolve toward increased ATP13A3-dependent uptake. The polyamine uptake branch controlled by ATP13A3-antizyme regulation can be pharmacologically blocked by AMXT 1501, which directly inhibits ATP13A3. Together, our findings explain DFMO adaptation through ATP13A3-antizyme control and establish ATP13A3 as a targetable node for polyamine depletion strategies in multiple cancers, supporting ongoing clinical evaluation of combined DFMO/AMXT 1501 therapy.

cell biology↗

bioRxiv2026

Autophagy in lymphatic endothelial cells promotes lung metastasis

Macroautophagy/autophagy is the main lysosomal pathway for the degradation and recycling of cytoplasmic cargo, with emerging roles in endothelial cell (EC) biology. While autophagy has been extensively studied in blood ECs, its function in lymphatic ECs (LECs) remains unexplored. Given the central role of the lymphatic system in antitumor immunity and metastatic spread, we investigated how LEC autophagy affects metastatic lung colonization. In line with previous reports showing that autophagy regulates the availability of the egress signal sphingosine 1 phosphate (S1P) in secondary lymphoid organs (SLOs), lungs of non-tumor-bearing mice with LEC-specific genetic deletion of Atg5 (ATG5LEC-KO mice) exhibited reduced lymphocyte infiltration. Remarkably, in tumor-bearing mice, either genetic loss of LEC-autophagy or pharmacological blockade of S1P lyase by 4-deoxypyridoxine (DOP) suppressed lung metastasis. Pulmonary immune profiling revealed that while DOP enhanced effector T cell activity despite lower numbers, LEC-autophagy-deficient mice markedly increased the B and CD8 T Cell abundance coupled with profound reduction of VEGFR3 expression in the lung lymphatic vasculature. Together, these findings uncover an autophagy-dependent remodeling of the lymphatic routes and immune niches that fosters metastatic seeding and growth in the lung.

cancer biology↗

bioRxiv2025

Autophagy maintains HEV identity and function during inflammation

High endothelial venules (HEVs) play a crucial role in adaptive immune responses in secondary and tertiary lymphoid organs. They are uniquely equipped with high levels of peripheral node addressins (PNAd), harboring carbohydrate structures that serve as L-Selectin ligands to efficiently facilitate lymphocyte homing. During inflammation, the HEV network expands in SLOs, increasing lymphocyte infiltration, but the underlying mechanisms maintaining HEVs remain underexplored. Here, we report that autophagy is essential for HEV function and expansion. Using single-cell transcriptomics, intravital imaging, and an inducible HEV tracer system in mice, we demonstrate that autophagy deficiency compromises LT{beta}R-signaling and the Unfolded Protein Response in HEVs, leading to disrupted PNAd production, dedifferentiation, and reduced lymphocyte homing. Autophagy deficiency and LT{beta}R blockade impaired HEV function and reduced skin inflammation in psoriasis-bearing mice by limiting immune infiltration and cytokine release. Our work uncovers an unprecedented role of autophagy in safeguarding HEV identity and function during inflammation. HighlightsO_LIHigh endothelial venules (HEVs) exhibit heightened autophagy in comparison to non-HEV blood endothelial cells, which further increases during inflammation. C_LIO_LIAutophagy is pivotal in maintaining HEV fate and function, specifically during inflammation, via LT{beta}R signaling and the Unfolded Protein Response (UPR), ensuring the proper production of peripheral node addressins (PNAd) that serve as L-selectin ligands for the efficient influx of naive lymphocytes. C_LIO_LIBlocking autophagy in HEVs leads to disrupted PNAd production, HEV flattening and dedifferentiation, and reduced lymphocyte homing. C_LIO_LIGenetic and pharmacological perturbation of HEV function in the lymph nodes and skin lesions of psoriasis-bearing mice impaired neutrophil and lymphocyte recruitment, as well as cytokine secretion, thereby alleviating skin inflammation. C_LI

cell biology↗

bioRxiv2024

A deep phenotyping study in mouse and iPSC models to understand the role of oligodendroglia in optic neuropathy in Wolfram syndrome

Wolfram syndrome (WS) is a rare childhood disease characterized by diabetes mellitus, diabetes insipidus, blindness, deafness, neurodegeneration and eventually early death, due to autosomal recessive mutations in the WFS1 (and WFS2) gene. While it is categorized as a neurodegenerative disease, it is increasingly becoming clear that other cell types besides neurons may be affected and contribute to the pathogenesis. MRI studies in patients and phenotyping studies in WS rodent models indicate white matter/myelin loss, implicating a role for oligodendroglia in WS-associated neurodegeneration. In this study, we sought to determine if oligodendroglia are affected in WS and whether their dysfunction may be the primary cause of the observed optic neuropathy and brain neurodegeneration. We demonstrate that 7.5-month-old Wfs1{Delta}exon8 mice display signs of abnormal myelination and a reduced number of oligodendrocyte precursor cells (OPCs) as well as abnormal axonal conduction in the optic nerve. An MRI study of the brain furthermore revealed grey and white matter loss in the cerebellum, brainstem, and superior colliculus, as is seen in WS patients. To further dissect the role of oligodendroglia in WS, we performed a transcriptomics study of WS patient iPSC-derived OPCs and pre-myelinating oligodendrocytes. Transcriptional changes compared to isogenic control cells were found for genes with a role in ER function. However, a deep phenotyping study of these WS patient iPSC-derived oligodendroglia unveiled normal differentiation, mitochondria-associated endoplasmic reticulum (ER) membrane interactions and mitochondrial function, and no overt signs of ER stress. Overall, the current study indicates that oligodendroglia functions are largely preserved in the WS mouse and patient iPSC-derived models used in this study. These findings do not support a major defect in oligodendroglia function as the primary cause of WS, and warrant further investigation of neurons and neuron-oligodendroglia interactions as a target for future neuroprotective or -restorative treatments for WS.

neuroscience↗

bioRxiv2023

Simultaneous detection of membrane contact dynamics and associated Ca2+ signals by reversible chemogenetic reporters

Membrane contact sites (MCSs) are hubs allowing various cell organelles to coordinate their activities. The dynamic nature of these sites and their small size hinder analysis by current imaging techniques. To overcome these limitations, we here designed a series of reversible chemogenetic reporters incorporating improved, low-affinity variants of splitFAST, and studied the dynamics of different MCSs at high spatiotemporal resolution, both in vitro and in vivo. We demonstrated that these versatile reporters suit different experimental setups well, allowing one to address challenging biological questions. Using these novel probes, we identified a hitherto unknown pathway, in which calcium (Ca2+) signalling dynamically regulates endoplasmic reticulum-mitochondria juxtaposition, characterizing the underlying mechanism. Finally, by integrating Ca2+-sensing capabilities into the splitFAST technology, we introduced PRINCESS (PRobe for INterorganelle Ca2+-Exchange Sites based on SplitFAST), an unprecedented class of reporters to simultaneously detect MCSs and measure the associated Ca2+ dynamics using a single biosensor.

cell biology↗

bioRxiv2023

Missense mutations in CMS22 patients reveal that PREPL has both enzymatic and non-enzymatic functions

Congenital myasthenic syndrome-22 (CMS22) is a rare genetic disorder caused by mutations in the prolyl endopeptidase-like (PREPL) gene. Since previous reports only described patients with deletions and nonsense mutations in PREPL, nothing is known about the effect of missense mutations in the pathology of CMS22. In this study, we have characterized missense mutations in PREPL in three CMS22 patients, all with hallmark phenotypes. Biochemical evaluation revealed that these missense mutations do not impair hydrolase activity, thereby challenging the conventional diagnostic criteria. Structural analysis shows that the mutations affect regions most likely involved in intra-protein or protein-protein interactions. Indeed, binding to a selected group of known interactors was differentially reduced for the three mutants. The importance of non-hydrolytic functions of PREPL was investigated in catalytically inactive PREPL p.Ser559Ala cell lines which showed that hydrolytic activity of PREPL is needed for normal mitochondrial function but not for regulating AP1-mediated transport in the trans-Golgi network. In conclusion, these studies show that CMS22 can be caused not only by deletion and truncation of PREPL but also by missense mutations that do not necessarily result in a loss of hydrolytic activity of PREPL. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/572145v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@13dc0bborg.highwire.dtl.DTLVardef@13865d6org.highwire.dtl.DTLVardef@1a5ed3borg.highwire.dtl.DTLVardef@79fdf5_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2023

Similar metabolic pathways are affected in both Congenital Myasthenic Syndrome 22 and Prader Willi Syndrome

Loss of prolyl endopeptidase-like (PREPL) encoding a serine hydrolase with (thio)esterase activity leads to the recessive metabolic disorder Congenital Myasthenic Syndrome-22 (CMS22). It is characterized by severe neonatal hypotonia, feeding problems, growth retardation, and hyperphagia leading to rapid weight gain later in childhood. The phenotypic similarities with Prader-Willi syndrome (PWS) are striking, suggesting that similar pathways are affected. The aim of this study was to identify changes in the hypothalamic-pituitary axis in mouse models for both disorders and to examine mitochondrial function in skin fibroblasts of patients and knockout cell lines. We have demonstrated that Prepl is downregulated in the brains of neonatal PWS-IC-p/+m mice. In addition, the hypothalamic-pituitary axis is similarly affected in both Prepl-/- and PWS-IC-p/+m mice resulting in defective orexigenic signaling and growth retardation. Furthermore, we demonstrated that mitochondrial function is altered in PREPL knockout HEK293T cells and can be rescued with the supplementation of coenzyme Q10. Finally, PREPL-deficient and PWS patient skin fibroblasts display defective mitochondrial bioenergetics. The mitochondrial dysfunction in PWS fibroblasts can be rescued by overexpression of PREPL. In conclusion, we provide the first molecular links between CMS22 and PWS, raising the possibility that PREPL substrates might become therapeutic targets for treating both disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/566752v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@308afforg.highwire.dtl.DTLVardef@f5a8feorg.highwire.dtl.DTLVardef@29d9eforg.highwire.dtl.DTLVardef@1a3061a_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Aberrant N-glycosylation in pathogenic variants of adenosine deaminase 2 underlying human ADA2 deficiency

Human deficiency of adenosine deaminase 2 (DADA2) is an autoinflammatory disease caused by pathogenic variants in ADA2 that lead to impaired deaminase activity. Recently, a lysosomal function of ADA2 has been proposed but an intracellular form of the protein has not yet been characterized. Here, we analyze protein expression of mutant ADA2 in human monocyte-derived macrophages from 10 DADA2 patients. We identify an intracellular low-molecular-weight (LMW) form of ADA2 that undergoes glycan trimming by -mannosidases and is absent in DADA2 macrophages. Subcellular fractionation and immunofluorescence microscopy demonstrate that LMW-ADA2 is localized in the lysosomes. By overexpression of 34 ADA2 variants in HEK293T and U-937 cells, we show that absence of LMW-ADA2 strongly correlates with reduced deaminase activity and predicts variant pathogenicity. In conclusion, we describe a previously unreported intracellular hypoglycosylated form of ADA2 and establish the absence of this LMW-ADA2 as a cellular characteristic of DADA2. Thereby, we introduce a protein correlate of the recently described lysosomal form of ADA2. SummaryEhlers et al. demonstrate that mutant ADA2 fails to undergo glycan processing beyond the ER leading to the absence of intracellular low-molecular-weight (LMW) ADA2 in DADA2 patients macrophages. LMW-ADA2 localizes to the lysosomes and can be generated from extracellular wild-type ADA2.

immunology↗

bioRxiv2023

An autophagy program that promotes T cell egress from the lymph node controls responses to immune checkpoint blockade

Lymphatic endothelial cells (LECs) lining the lymphatic vessels of the lymph node (LN) parenchyma orchestrate leukocyte trafficking and peripheral T cell dynamics. T cell responses to immunotherapy largely rely on peripheral T cell recruitment in tumors. Yet, a systematic and molecular understanding of how LECs within the LNs control T cell dynamics under steady state and tumor-bearing conditions is lacking. Using intravital and high-resolution imaging combined with immune phenotyping, we show that LEC-specific deletion of the essential autophagy gene Atg5 alters intranodal positioning of lymphocytes and accrues their persistence in the LNs, by increasing the availability of the main egress signal S1P. Single-cell RNA-sequencing of tumor-draining LNs from WT and ATG5LEC-KO mice unveils that loss of ATG5 remodels niche-specific LEC phenotypes, involved in molecular pathways regulating lymphocyte trafficking and LEC-T cell interactions. Functionally, loss of LEC-autophagy prevents recruitment of tumor-infiltrating T cells and NK cells and abrogates tumor regression in response to anti-PD-1 or anti-CTLA4-based immunotherapy. Thus, a unique LEC-autophagy program boosts immune-checkpoint responses by guiding systemic T cell dynamics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/549282v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1a4bfe1org.highwire.dtl.DTLVardef@1317fe2org.highwire.dtl.DTLVardef@c2fa20org.highwire.dtl.DTLVardef@b35dba_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

TUMOR ENDOTHELIAL CELL AUTOPHAGY IS A KEY VASCULAR-IMMUNE CHECKPOINT IN MELANOMA

Tumor endothelial cells (TECs) actively repress inflammatory responses and maintain an immune-excluded tumor phenotype. However, the molecular mechanisms that sustain TEC-mediated immunosuppression remain largely elusive. Here, we show that autophagy ablation in TECs boosts antitumor immunity by supporting infiltration and effector function of T cells, thereby restricting melanoma growth. In melanoma-bearing mice, loss of TEC autophagy leads to the transcriptional expression of an immunostimulatory/inflammatory TEC phenotype driven by heightened NF-kB and STING signaling. In line, single-cell transcriptomic datasets from melanoma patients disclose an enriched InflammatoryHigh/AutophagyLow TEC phenotype in correlation with clinical responses to immunotherapy. Congruently, patients responding to immunotherapy exhibit an increased presence of inflamed vessels, interfacing with infiltrating CD8+ T cells. Mechanistically, STING-dependent immunity in TECs is not critical for the immunomodulatory effects of autophagy ablation, since NF-kB-driven inflammation remains functional in STING/ATG5 double knockout TECs. Hence, autophagy is a principal tumor vascular anti-inflammatory mechanism dampening melanoma antitumor immunity.

cancer biology↗

bioRxiv2020

The IRE1 Inhibitor KIRA6 Curtails The Inflammatory Trait Of Immunogenic Anticancer Treatments By Targeting HSP60 Independent Of IRE1

Cellular stress evoked by immunogenic anticancer treatments engaging the unfolded protein response (UPR) can elicit inflammation with conflicting therapeutic outcomes. To define cell-autonomous mechanisms coupling the UPR to molecular mediators of inflammation, we profiled the transcriptome of cancer cells responding to immunogenic or weakly immunogenic-treatments. Bioinformatics-driven pathway analysis indicated that immunogenic treatments instigated NF-{kappa}B/AP-1-inflammatory pathways, which were abolished by the IRE1-kinase inhibitor KIRA6. Cell-free fractions of chemotherapy and KIRA6 co-treated cancer cells were deprived of pro-inflammatory/chemoattractant factors and failed to mobilize innate immune cells. Strikingly, these potent KIRA6 anti-inflammatory effects were found to be independent of IRE1. Generation of a KIRA6-clickable photoaffinity probe, mass spectrometry and co-immunoprecipitation analysis identified cytosolic HSP60 as a KIRA6 off-target in the NF-{kappa}B pathway. In sum, our study unravels that inflammation evoked by immunogenic treatments is curtailed by KIRA6 independently of IRE1 and further suggests great caution in interpreting the anti-inflammatory action of IRE1 chemical inhibitors.

cancer biology↗

bioRxiv2020

Downregulation of miR-17-92 cluster by PERK fine-tunes unfolded protein response mediated apoptosis

An important event in the unfolded protein response (UPR) is the activation of the endoplasmic reticulum kinase PERK (EIF2AK3). The PERK signalling branch first mediates a prosurvival response, which switches into a proapoptotic response upon prolonged ER stress. However, the molecular mechanisms of PERK-mediated cell death are not well understood. Here we show that expression of the primary miR-17-92 transcript and mature miRNAs belonging to miR-17-92 cluster is decreased during UPR. We found that activity of miR-17-92 promoter reporter was reduced during UPR in a PERK-dependent manner. We show that activity of miR-17-92 promoter is repressed by ectopic expression of ATF4 and NRF2. The promoter deletion analysis and ChIP assays mapped the region responding to UPR-mediated repression to site in the proximal region of the miR-17-92 promoter. Hypericin-mediated photo-oxidative ER damage reduced the expression of miRNAs belonging to miR-17-92 cluster in wild-type but not in PERK-deficient cells. Importantly, ER stress-induced apoptosis was inhibited upon miR-17-92 overexpression in SH-SY5Y and H9c2 cells. Our results reveal a novel function for NRF2, where repression of miR-17-92 cluster by NRF2 plays an important role in ER stress-mediated apoptosis. The data presented here provides mechanistic details how sustained PERK signalling via NRF2 mediated repression of miR-17-92 cluster can potentiate cell death.

cell biology↗

bioRxiv2018

Parl Deficiency In Mouse Causes Coenzyme Q Depletion, Complex III Defects, And Leigh-Like Syndrome

The mitochondrial intramembrane rhomboid protease Parl has been implicated in diverse functions in vitro, but its physiological role in vivo remains unclear. Here we show that Parl ablation in mouse causes a striking necrotizing encephalomyelopathy similar to Leigh syndrome, a mitochondrial disease characterized by disrupted energy production. Mice with conditional Parl deficiency in the nervous system, but not in muscle, develop a similar phenotype as germline Parl knockouts demonstrating the vital role of Parl in neurological homeostasis. Genetic modification of two major Parl substrates, Pink1 and Pgam5, do not modify this severe neurological phenotype. Parl-/- brain mitochondria are affected by defects in Complex III activity and in coenzyme Q biosynthesis. Parl is necessary for the stable expression of Ttc19, required for Complex III activity, and of Coq4, essential in coenzyme Q biosynthesis. Thus, Parl plays a previously overseen constitutive role in the maintenance of the respiratory chain in the nervous system, and its deficiency causes progressive mitochondrial dysfunction and Leigh-like syndrome.

molecular biology↗