Insight into the Role of Extracellular Vesicles in Lysosomal Storage Disorders

Extracellular vesicles (EVs) have received increasing attention over the last two decades. Initially, they were considered as just a garbage disposal tool; however, it has progressively become clear that their protein, nucleic acid (namely miRNA and mRNA), and lipid contents have signaling functions. Besides, it has been established that cells release different types of vesicular structures for which characterization is still in its infancy. Many stress conditions, such as hypoxia, senescence, and oncogene activation have been associated with the release of higher levels of EVs. Further, evidence has shown that autophagic–lysosomal pathway abnormalities also affect EV release. In fact, in neurodegenerative diseases characterized by the accumulation of toxic proteins, although it has not become clear to what extent the intracellular storage of undigested materials itself has beneficial/adverse effects, these proteins have also been shown to be released extracellularly via EVs. Lysosomal storage disorders (LSDs) are characterized by accumulation of undigested substrates within the endosomal–lysosomal system, due either to genetic mutations in lysosomal proteins or to treatment with pharmacological agents. Here, we review studies investigating the role of lysosomal and autophagic dysfunction on the release of EVs, with a focus on studies exploring the release of EVs in LSD models of both genetic and pharmacological origin. A better knowledge of EV-releasing pathways activated in lysosomal stress conditions will provide information on the role of EVs in both alleviating intracellular storage of undigested materials and spreading the pathology to the neighboring tissue

Lysosomal Exocytosis, Exosome Release and Secretory Autophagy: The Autophagic- and Endo-Lysosomal Systems Go Extracellular

Beyond the consolidated role in degrading and recycling cellular waste, the autophagic- and endo-lysosomal systems play a crucial role in extracellular release pathways. Lysosomal exocytosis is a process leading to the secretion of lysosomal content upon lysosome fusion with plasma membrane and is an important mechanism of cellular clearance, necessary to maintain cell fitness. Exosomes are a class of extracellular vesicles originating from the inward budding of the membrane of late endosomes, which may not fuse with lysosomes but be released extracellularly upon exocytosis. In addition to garbage disposal tools, they are now considered a cell-to-cell communication mechanism. Autophagy is a cellular process leading to sequestration of cytosolic cargoes for their degradation within lysosomes. However, the autophagic machinery is also involved in unconventional protein secretion and autophagy-dependent secretion, which are fundamental mechanisms for toxic protein disposal, immune signalling and pathogen surveillance. These cellular processes underline the crosstalk between the autophagic and the endosomal system and indicate an intersection between degradative and secretory functions. Further, they suggest that the molecular mechanisms underlying fusion, either with lysosomes or plasma membrane, are key determinants to maintain cell homeostasis upon stressing stimuli. When they fail, the accumulation of undigested substrates leads to pathological consequences, as indicated by the involvement of autophagic and lysosomal alteration in human diseases, namely lysosomal storage disorders, age-related neurodegenerative diseases and cancer. In this paper, we reviewed the current knowledge on the functional role of extracellular release pathways involving lysosomes and the autophagic- and endo-lysosomal systems, evaluating their implication in health and disease

Drug-Induced Lysosomal Impairment Is Associated with the Release of Extracellular Vesicles Carrying Autophagy Markers

Amiodarone is a cationic amphiphilic drug used as an antiarrhythmic agent. It induces phospholipidosis, i.e., the accumulation of phospholipids within organelles of the endosomal–lysosomal system. Extracellular vesicles (EVs) are membrane-enclosed structures released by any type of cell and retrieved in every fluid of the body. EVs have been initially identified as a system to dispose cell waste, but they are also considered to be an additional manner to transmit intercellular signals. To understand the role of EVs in drug-induced phospholipidosis, we investigated EVs release in amiodarone-treated HEK-293 cells engineered to produce fluorescently labelled EVs. We observed that amiodarone induces the release of a higher number of EVs, mostly of a large/medium size. EVs released upon amiodarone treatment do not display significant morphological changes or altered size distribution, but they show a dose-dependent increase in autophagy associated markers, indicating a higher release of EVs with an autophagosome-like phenotype. Large/medium EVs also show a higher content of phospholipids. Drugs inducing lysosomal impairment such as chloroquine and bafilomycin A1 similarly prompt a higher release of EVs enriched in autophagy markers. This result suggests a mechanism associated with amiodarone-induced lysosomal impairment more than a connection with the accumulation of specific undigested substrates. Moreover, the implementation of the lysosomal function by overexpressing TFEB, a master gene regulator of lysosomal biogenesis, prevents the amiodarone-induced release of EVs, suggesting that this could be a feasible target to attenuate drug-induced abnormalities

Evidence of Lysosomal β-Hexosaminidase Enzymatic Activity Associated with Extracellular Vesicles: Potential Applications for the Correction of Sandhoff Disease

Extracellular vesicles (EVs) can be isolated from biological fluids and cell culture medium. Their nanometric dimension, relative stability, and biocompatibility have raised considerable interest for their therapeutic use as delivery vehicles of macromolecules, namely nucleic acids and proteins. Deficiency in lysosomal enzymes and associated proteins is at the basis of a group of genetic diseases known as lysosomal storage disorders (LSDs), characterized by the accumulation of undigested substrates into lysosomes. Among them, GM2 gangliosidoses are due to a deficiency in the activity of lysosomal enzyme β-hexosaminidase, leading to the accumulation of the GM2 ganglioside and severe neurological symptoms. Current therapeutic approaches, including enzyme replacement therapy (ERT), have proven unable to significantly treat these conditions. Here, we provide evidence that the lysosomal β-hexosaminidase enzyme is associated with EVs released by HEK cells and that the EV-associated activity can be increased by overexpressing the α-subunit of β-hexosaminidase. The delivery of EVs to β-hexosaminidase-deficient fibroblasts results in a partial cross-correction of the enzymatic defect. Overall findings indicate that EVs could be a source of β-hexosaminidase that is potentially exploitable for developing therapeutic approaches for currently untreatable LSDs