Connexin43 promotes exocytosis of damaged lysosomes through actin remodelling.

A robust and efficient cellular response to lysosomal membrane damage prevents leakage from the lysosome lumen into the cytoplasm. This response is understood to happen through either lysosomal membrane repair or lysophagy. Here we report exocytosis as a third response mechanism to lysosomal damage, which is further potentiated when membrane repair or lysosomal degradation mechanisms are impaired. We show that Connexin43 (Cx43), a protein canonically associated with gap junctions, is recruited from the plasma membrane to damaged lysosomes, promoting their secretion and accelerating cell recovery. The effects of Cx43 on lysosome exocytosis are mediated by a reorganization of the actin cytoskeleton that increases plasma membrane fluidity and decreases cell stiffness. Furthermore, we demonstrate that Cx43 interacts with the actin nucleator Arp2, the activity of which was shown to be necessary for Cx43-mediated actin rearrangement and lysosomal exocytosis following damage. These results define a novel mechanism of lysosomal quality control whereby Cx43-mediated actin remodelling potentiates the secretion of damaged lysosomes.

Contribution of extracellular vesicles for the pathogenesis of retinal diseases: shedding light on blood-retinal barrier dysfunction.

Retinal degenerative diseases, including diabetic retinopathy (DR) and age-related macular degeneration (AMD), loom as threats to vision, causing detrimental effects on the structure and function of the retina. Central to understanding these diseases, is the compromised state of the blood-retinal barrier (BRB), an effective barrier that regulates the influx of immune and inflammatory components. Whether BRB breakdown initiates retinal distress, or is a consequence of disease progression, remains enigmatic. Nevertheless, it is an indication of retinal dysfunction and potential vision loss.The intricate intercellular dialogues among retinal cell populations remain unintelligible in the complex retinal milieu, under conditions of inflammation and oxidative stress. The retina, a specialized neural tissue, sustains a ceaseless demand for oxygen and nutrients from two vascular networks. The BRB orchestrates the exchange of molecules and fluids within this specialized region, comprising the inner BRB (iBRB) and the outer BRB (oBRB). Extracellular vesicles (EVs) are small membranous structures, and act as messengers facilitating intercellular communication in this milieu.EVs, both from retinal and peripheral immune cells, increase complexity to BRB dysfunction in DR and AMD. Laden with bioactive cargoes, these EVs can modulate the retinal microenvironment, influencing disease progression. Our review delves into the multifaceted role of EVs in retinal degenerative diseases, elucidating the molecular crosstalk they orchestrate, and their microRNA (miRNA) content. By shedding light on these nanoscale messengers, from their biogenesis, release, to interaction and uptake by target cells, we aim to deepen the comprehension of BRB dysfunction and explore their therapeutic potential, therefore increasing our understanding of DR and AMD pathophysiology.

Expression and Function of Connexins in Extracellular Vesicles.

Extracellular vesicles (EVs) are recognized as major vehicles for exchange of information across distant cells and tissues, which have been extensively explored for diagnosis and therapeutic purposes. The presence of multiple connexin (Cx) proteins has been described in EVs, where they might facilitate EV-cell communication. However, quantitative changes in Cx levels and functional assessment of Cx channels have only been established for Cx43. In present work, we provide a detailed description of the protocols we have optimized to assess the expression and permeability of Cx43 channels in EVs derived from cultured cells and human peripheral blood. Particularly, we include some modifications to improve quantitative analysis of EV-Cx43 by enzyme-linked immunosorbent assay (ELISA) and assessment of channel functionality by sucrose-density gradient ultracentrifugation, which can be easily adapted to other Cx family members, leveraging the development of diagnostic and therapeutic applications based on Cx-containing EVs.

Effect of Blueberry Supplementation on a Diet-Induced Rat Model of Prediabetes-Focus on Hepatic Lipid Deposition, Endoplasmic Stress Response and Autophagy.

Blueberries, red fruits enriched in polyphenols and fibers, are envisaged as a promising nutraceutical intervention in a plethora of metabolic diseases. Prediabetes, an intermediate state between normal glucose tolerance and type 2 diabetes, fuels the development of complications, including hepatic steatosis. In previous work, we have demonstrated that blueberry juice (BJ) supplementation benefits glycemic control and lipid profile, which was accompanied by an amelioration of hepatic mitochondrial bioenergetics. The purpose of this study is to clarify the impact of long-term BJ nutraceutical intervention on cellular mechanisms that govern hepatic lipid homeostasis, namely autophagy and endoplasmic reticulum (ER) stress, in a rat model of prediabetes. Two groups of male Wistar rats, 8-weeks old, were fed a prediabetes-inducing high-fat diet (HFD) and one group was fed a control diet (CD). From the timepoint where the prediabetic phenotype was achieved (week 16) until the end of the study (week 24), one of the HFD-fed groups was daily orally supplemented with 25 g/kg body weight (BW) of BJ (HFD + BJ). BW, caloric intake, glucose tolerance and insulin sensitivity were monitored throughout the study. The serum and hepatic lipid contents were quantified. Liver and interscapular brown and epidydimal white adipose tissue depots (iBAT and eWAT) were collected for histological analysis and to assess thermogenesis, ER stress and autophagy markers. The gut microbiota composition and the short-chain fatty acids (SCFAs) content were determined in colon fecal samples. BJ supplementation positively impacted glycemic control but was unable to prevent obesity and adiposity. BJ-treated animals presented a reduction in fecal SCFAs, increased markers of arrested iBAT thermogenesis and energy expenditure, together with an aggravation of HFD-induced lipotoxicity and hepatic steatosis, which were accompanied by the inhibition of autophagy and ER stress responses in the liver. In conclusion, despite the improvement of glucose tolerance, BJ supplementation promoted a major impact on lipid management mechanisms at liver and AT levels in prediabetic animals, which might affect disease course.

Loss of the lysosomal protein CLN3 modifies the lipid content of the nuclear envelope leading to DNA damage and activation of YAP1 pro-apoptotic signaling.

Batten disease is characterized by early-onset blindness, juvenile dementia and death during the second decade of life. The most common genetic causes are mutations in the CLN3 gene encoding a lysosomal protein. There are currently no therapies targeting the progression of the disease, mostly due to the lack of knowledge about the disease mechanisms. To gain insight into the impact of CLN3 loss on cellular signaling and organelle function, we generated CLN3 knock-out cells in a human cell line (CLN3-KO), and performed RNA sequencing to obtain the cellular transcriptome. Following a multi-dimensional transcriptome analysis, we identified the transcriptional regulator YAP1 as a major driver of the transcriptional changes observed in CLN3-KO cells. We further observed that YAP1 pro-apoptotic signaling is hyperactive as a consequence of CLN3 functional loss in retinal pigment epithelia cells, and in the hippocampus and thalamus of CLN3exΔ7/8 mice, an established model of Batten disease. Loss of CLN3 activates YAP1 by a cascade of events that starts with the inability of releasing glycerophosphodiesthers from CLN3-KO lysosomes, which leads to perturbations in the lipid content of the nuclear envelope and nuclear dysmorphism. This results in increased number of DNA lesions, activating the kinase c-Abl, which phosphorylates YAP1, stimulating its pro-apoptotic signaling. Altogether, our results highlight a novel organelle crosstalk paradigm in which lysosomal metabolites regulate nuclear envelope content, nuclear shape and DNA homeostasis. This novel molecular mechanism underlying the loss of CLN3 in mammalian cells and tissues may open new c-Abl-centric therapeutic strategies to target Batten disease.

Unveiling the antitumor mechanism of 7α-acetoxy-6ß-hydroxyroyleanone from Plectranthus hadiensis in glioblastoma.

ETHNOPHARMACOLOGICAL RELEVANCE: Glioblastoma (GB) is the most aggressive and prevalent glioma within the central nervous system. Despite considerable efforts, GB continues to exhibit a dismal 5-year survival rate (∼6%). This is largely attributed to unfavorable prognosis and lack of viable treatment options. Therefore, novel therapies centered around plant-derived compounds emerge as a compelling avenue to enhance patient survival and well-being. The South African species, Plectranthus hadiensis Schweinf. (P. hadiensis), a member of the Lamiaceae family, has a history of use in traditional medicine for treating a range of diseases, including respiratory, digestive, and liver disorders. This species exhibits diverse biological activities, such as anti-inflammatory and antitumoral properties, likely attributed to its rich composition of naturally occurring diterpenes, like the abietane diterpene, 7α-acetoxy-6ß-hydroxyroyleanone (Roy). Roy has demonstrated promising antitumor effects in various cancer cell lines, making it a compelling candidate for further investigation into its mechanisms against GB. AIM OF THE STUDY: This study aims to investigate the antitumor activity and potential mechanism of Roy, a natural lead compound, in GB cells. MATERIAL AND METHODS: Roy was isolated from the acetonic extract of P. hadiensis and its antitumor mechanism was assessed in a panel of human GB cell lines (U87, A172, H4, U373, and U118) to mimic tumor heterogeneity. Briefly, the impact of Roy treatment on the metabolic activity of cells was evaluated by Alamar Blue® assay, while cell death, cell cycle regulation, mitochondrial membrane potential, and activated caspase-3 activity were evaluated by flow cytometry. Measurement of mRNA levels of target genes was performed by qPCR, while protein expression was assessed by Western blotting. Cell uptake and impact on mitochondrial morphology were evaluated by confocal microscopy. RESULTS: Roy induced G2/M cell cycle arrest, mitochondrial fragmentation, and apoptosis by inhibiting the expression of anti-apoptotic proteins and increasing the levels of activated caspase-3. The concentrations of Roy needed to achieve significant inhibitory outcomes were notably lower (6-9 fold) than those of temozolomide (TMZ), the standard first-line treatment, for achieving comparable effects. In addition, at low concentrations (16 µM), Roy affected the metabolic activity of tumor cells while having no significant impact on non-tumoral cells (microglia and astrocytes). CONCLUSION: Overall, Roy demonstrated a robust antitumor activity against GB cells offering a promising avenue for the development of novel chemotherapeutic approaches.