Biology subjects

Antas, P.

Publications and source records attributed to Antas, P..

5 recordsLinked to original sources

bioRxiv2024

Induction of the ISR by AB5 subtilase cytotoxin drives IFN-I expression in pDCs via STING activation

We demonstrate that exposure to the AB5 subtilase cytotoxin (SubAB) induces the unfolded protein response (UPR) in human peripheral blood mononuclear cells, concomitant with a pro-inflammatory response across distinct cell subsets. Notably, SubAB selectively induces type-I interferon (IFN) expression in plasmacytoid dendritic cells, acting synergistically with Toll-like receptor 7 stimulation. The induction of type-I IFN in response to SubAB relies on stimulator of interferon genes (STING) activation, coupled with protein synthesis inhibition mediated by protein kinase R-like endoplasmic reticulum kinase and phosphorylation of the eukaryotic translation initiation factor 2 subunit-alpha. By impeding mRNA translation through the integrated stress response, SubAB precipitates the downregulation of the negative innate signaling feedback regulator Tax1-binding protein 1. This downregulation is necessary to unleash TANK-binding kinase 1 signaling associated with STING activation. These findings shed new light on how UPR-inducing conditions may regulate the immune system during infection or pathogenesis.

immunology↗

bioRxiv2024

Enhancing lysosome function via mTOR/TFEB activation reduces lipofuscin-like granules in early Age-related Macular Degeneration

Age-related macular degeneration (AMD) is the most common blinding disease in the western world and is currently incurable. Although the exact causes of AMD are not clear, the primary origin of pathology appears to be in the retinal pigment epithelium (RPE). RPE is responsible for the daily digestion of photoreceptor outer segments (POS), which imposes a heavy continuous burden on the lysosomal network. POS feeding assay in vitro suggested that the accumulation of autofluorescence granules (AFG), similar to lipofuscin in vivo, derives from lysosomal dysfunction. Here we show that synchronous phagocytosis of POS leads to early transient mTOR activation followed by inhibition in late phagosome maturation. One of its substrates, the transcription factor EB (TFEB) increases during phagosome maturation albeit mostly in its inactive phosphorylated form. We questioned whether induction of the mTOR/TFEB axis could improve digestion of POS and hence reduce AFG load. Treatment of POS-fed cells with rapamycin, an mTORC1 inhibitor after the appearance of AFG results in 30% reduction of AFG load. This effect is dependent on active lysosomal enzymes and induction of active dephosphorylated TFEB with consequent activation of GADD34 and lysosomal biogenesis. As a proof of concept, we show that overexpressing a constitutively active form of unphosphorylated TFEB dramatically reduces POS-dependent AFG accumulation. Overall, this study suggests that viral or pharmacological approaches activating the mTOR/TFEB axis in the RPE could be beneficial as cell-protective treatment of early/intermediate cases of AMD, acting to delay progression of the disease.

molecular biology↗

bioRxiv2024

VPS34-IN1 inhibits cap-mediated translation and synergizes with STING to drive type-I IFN expression in human plasmacytoid DCs

Inhibition of the phosphatidylinositol kinase vacuolar protein sorting 34 (VPS34) with the pharmacological compound VPS34-IN1 has a range of effects on the dynamics of endosomes. While VPS34 inhibition has been suggested as a potential therapeutic approach for treating certain cancers, our findings indicate that it has minimal cytotoxic effects on leukemic blastic plasmacytoid dendritic cell neoplasms (BPDCN). VPS34-IN1, however, interferes with plasmacytoid dendritic cells (pDCs) function by blocking the recruitment of serum and glucocorticoid-regulated kinase 3 (SGK3) to endosomes, which is shown to be necessary for Toll-like receptor 7 (TLR7) signaling. In a contrasting parallel, VPS34-IN1 triggers the activation of the stimulator of interferon genes (STING) and significantly enhances pDCs response to the STING agonist 23-cyclic guanosine monophosphate-adenosine monophosphate (23-cGAMP). This cooperative action with VPS34-IN1 leads to strongly increased expression of type-I interferons (IFNs), associated with an alteration of STING degradation and importantly, inhibition of cap-mediated mRNA translation. Inhibition of protein synthesis by VPS34-IN1 appears to be central to this synergy with STING activation, notably by compromising the expression of IFIT1/ISG56, a negative regulator of innate signaling. Thus, despite their limited toxicity towards different cancer lines, inhibitors targeting VPS34 and SGK3 may present promising compounds for controlling the expression of type-I IFNs in response to various microbial stimuli and pathological contexts. One-sentence summaryPharmacological inhibition of VPS34 affects multiple signaling pathways downstream of innate immunity receptors and consequently can inhibit or potentiate type-I Interferon induction according to the danger or microbial stimuli received by plasmacytoid DCs.

immunology↗

bioRxiv2023

3D human retinal organoid model for the study of early diabetic retinopathy

Diabetic retinopathy (DR) is a significant complication of diabetes and a primary cause of visual impairment among working-age individuals. DR is a degenerative condition in which hyperglycaemia results in morphological and functional changes in certain retinal cells. Existing treatments mainly address the advanced stages of the disease, which involve vascular defects such as macular edema or neovascularization. However, it is now known that retinal neurodegeneration and inflammation precede these vascular changes. Therefore, there is a pressing need to identify new therapeutic approaches that target the early stages of DR and prevent its progression. In the last decade, the development of reliable in vitro models resembling the complexity of the retinal tissue has significantly improved. Namely, three-dimensional (3D) retinal organoids derived from human induced-pluripotent stem cells (hiPSCs) recapitulate the cellular organization and complexity of the human retina. Here, we used hiPSCs-derived retinal organoids to generate a model of early DR. In this model, we observe well-established molecular and cellular features of early DR: i) loss of retinal ganglion and amacrine cells; ii) glial reactivity and inflammation, with increased expression of the vascular endothelial-derived growth factor (VEGF) and interleukin-1{beta} (IL-1{beta}), and monocyte chemoattractant protein-1 (MCP-1) secretion; iii) increased levels of reactive oxygen species accompanied by activation of key enzymes involved in antioxidative stress response. The data provided highlight the utility of retinal organoid technology in modelling early-stage DR. This offers new avenues for the development of targeted therapeutic interventions on neurodegeneration and inflammation in the initial phase of DR, potentially slowing the diseases progression.

neuroscience↗

bioRxiv2021

FORMATION OF LIPOFUSCIN-LIKE AUTOFLUORESCENT GRANULES IN THE RETINAL PIGMENT EPITHELIUM REQUIRES LYSOSOME DYSFUNCTION

PurposeWe aim to characterize the pathways required for autofluorescent granule (AFG) formation by retinal pigment epithelium (RPE) cells using cultured monolayers. MethodsWe fed RPE monolayers in culture with a single pulse of photoreceptor outer segments (POS). After 24h the cells started accumulating AFGs similar to lipofuscin in vivo. Using this model, we used a variety of light and electron microscopical techniques, flow cytometry and western blot to analyze the formation of AFGs. We also generated a mutant RPE line lacking Cathepsin D by gene editing. ResultsAFGs appear to derive from incompletely digested POS-containing phagosomes and are surrounded after 72h by a single membrane containing lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AFG formation but that impairment of lysosomal pH or catalytic activity, particularly Cathepsin D activity, enhances AF accumulation. ConclusionsWe conclude that lysosomal dysfunction results in incomplete POS degradation and AFG accumulation.

cell biology↗