Endocytosis of Extracellular Vesicles and Release of Their Cargo from Endosomes

Extracellular vesicles (EVs), such as exosomes, can mediate long-distance communication between cells by delivering biomolecular cargo. It is speculated that EVs undergo back-fusion at multivesicular bodies (MVBs) in recipient cells to release their functional cargo. However, direct evidence is lacking. Tracing the cellular uptake of EVs with high resolution as well as acquiring direct evidence for the release of EV cargo is challenging mainly because of technical limitations. Here, we developed an analytical methodology, combining state-of-theart molecular tools and correlative light and electron microscopy, to identify the intracellular site for EV cargo release. GFP was loaded inside EVs through the expression of GFP-CD63, a fusion of GFP to the cytosolic tail of CD63, in EV producer cells. In addition, we genetically engineered a cell line which expresses anti-GFP fluobody that specifically recognizes the EV cargo (GFP). Incubation of anti-GFP fluobody- expressing cells with GFP-CD63 EVs resulted in the formation of fluobody punctae, designating cytosolic exposure of GFP. Endosomal damage was not observed in EV acceptor cells. Ultrastructural analysis of the underlying structures at GFP/ fluobody double-positive punctae demonstrated that EV cargo release occurs from endosomes/lysosomes. Finally, we show that neutralization of endosomal pH and cholesterol accumulation in endosomes leads to blockage of EV cargo exposure. In conclusion, we report that a fraction of internalized EVs fuse with the limiting membrane of endosomes/lysosomes in an acidification-dependent manner, which results in EV cargo exposure to the cell cytosol

DNAJB6b-enriched small extracellular vesicles decrease polyglutamine aggregation in in vitro and in vivo models of Huntington disease

Huntington disease (HD) is a devastating neurodegenerative disorder characterized by aggregation of huntingtin (HTT) protein containing expanded polyglutamine (polyQ) tracts. DNAJB6, a member of the DNAJ chaperone family, was reported to efficiently inhibit polyQ aggregation in vitro, in cell models, and in vivo in flies, xenopus, and mice. For the delivery of exogenous DNAJB6 to the brain, the DNAJB6 needs to be protected against (enzymatic) degradation and show good penetration into brain tissue. Here, we tested the potential of small extracellular vesicles (sEVs) derived from neural stem cells (NSCs) for delivery of DNAJB6 as anti-amyloidogenic cargo. Administration of sEVs isolated from DNAJB6-overexpressing cells to cells expressing expanded polyQ tracts suppressed HTT aggregation. Furthermore, intrathecal injection of DNAJB6-enriched sEVs into R6/2 transgenic HD mice significantly reduced mutant HTT aggregation in the brain. Taken together, our data suggest that sEV-mediated molecular chaperone delivery may hold potential to delay disease onset in HD

Heparan sulfate proteoglycan-mediated dynamin-dependent transport of neural stem cell exosomes in an in vitro blood-brain barrier model

Drug delivery to the brain is greatly hampered by the presence of the blood-brain barrier (BBB) which tightly regulates the passage of molecules from blood to brain and vice versa. Nanocarriers, in which drugs can be encapsulated, can move across the blood-brain barrier (BBB) via the process of transcytosis, thus showing promise to improve drug delivery to the brain. Here, we demonstrate the use of natural nanovesicles, that is, exosomes, derived from C17.2 neural stem cells (NSCs) to efficiently carry a protein cargo across an in vitro BBB model consisting of human brain microvascular endothelial cells. We show that the exosomes are primarily taken up in brain endothelial cells via endocytosis, while heparan sulfate proteoglycans (HSPGs) act as receptors. Taken together, our data support the view that NSC exosomes may act as biological nanocarriers for efficient passage across the BBB. Nanomedicines that target HSPGs may improve their binding to brain endothelial cells and, possibly, show subsequent transcytosis across the BBB

Erratum: Breaking free: endocytosis and endosomal escape of extracellular vesicles

The authors want to make the following corrections to this paper[1]. In the section “Intracellular trafficking of EVs”, the citations after the sentence “Using another cell-free assay, Morandi et al. provided interesting insights into the process of endosomal fusion in EV cargo release”and “Murphy et al. tackled this question by comparing an FDA-approved cutting-edge lipid nanoparticle (LNP) formulation with EVs in terms of uptake and cargo (specifically gRNA) delivery” were missed. Correctly modify as follows: Using another cell-free assay, Morandi et al. provided interesting insights into the process of endosomal fusion in EV cargo release[22,68,87]. Murphy et al. tackled this question by comparing an FDA-approved cutting-edge lipid nanoparticle (LNP) formulation with EVs in terms of uptake and cargo (specifically gRNA) delivery[135]. Corresponding subsequent citation numbers also need to be adjusted: reference 140 is changed to reference 135, reference 135 is changed to reference 136, reference 136 is changed to reference 137, reference 137 is changed to reference 138, reference 138 is changed to reference 139, and reference 139 is changed to reference 140. We apologize for any inconvenience caused and state that the scientific conclusions are unaffected. The original article has been updated

Preparation of chaperone-loaded neural stem cell-derived extracellular vesicles to reduce protein aggregation in Huntington’s disease cellular models

Summary: Here, we present a protocol using genetic engineering techniques to prepare small extracellular vesicles (sEVs) enriched in the chaperone protein DNAJB6. We describe steps to prepare cell lines overexpressing DNAJB6, followed by the isolation and characterization of sEVs from cell conditioned media. Further, we describe assays to examine effects of DNAJB6-loaded sEVs on protein aggregation in Huntington’s disease cellular models. The protocol can be readily repurposed to study protein aggregation in other neurodegenerative disorders or extended to other therapeutic proteins.For complete details on the use and execution of this protocol, please refer to Joshi et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Designing a sky-scanning Fabry–Perot interferometer system with a large size integrating sphere

Integrating spheres can be manufactured at sizes ranging from 1 mm to 3 m in diameter. It has been agreed widely by manufacturers that fitting a large-sized integrating sphere as per the customer requirement is a complex process. Thus, these spheres due to its large beam divergence from the exit port, have never been deployed for the calibration of aeronomy applications. In this article a optical system is described to integrate a large-sized integrating sphere with the Fabry–Perot etalon and thus devise a set-up to generate the concentric ring pattern and record it on the detector. The experimental set-up was verified by optical simulation using ZEMAX (OpticStudio) so as to validate the system’s performance in the laboratory. The important parameter finesse is calculated and its accuracy is found to be 80%

Membrane distillation for the concentration of raw cane-sugar syrup and membrane clarified sugarcane juice

Membrane Distillation (MD) is a process being investigated the world over as a low cost energy saving alternative to conventional separation processes such as distillation and reverse osmosis. MD has a wide range of benefits such as a 100% (theoretical) rejection of ionic species, macromolecules, colloids, cells and other non-volatiles, lower operating pressures, reduced chemical interaction between membrane and process solutions, less demanding membrane mechanical properties and reduced vapour spaces. This process is ideally suited for the concentration of aqueous streams such as fruit juice and sugar solutions. Clarified cane sugar solution (20°Brix) obtained from the sugar mill, immediately after the Dorr filtration was subjected to membrane distillation, whereby the sensible heat from this stream was used to remove water as vapour through a hydrophobic polypropylene membrane. The influence of cross-flow rate and continuous water addition on flux was evaluated

DNAJB6-Containing Extracellular Vesicles as Chaperone Delivery Systems: A Proteomic Analysis

Cell-derived extracellular vesicles (EVs) are effectors of cell-to-cell communication that are in the spotlight as promising candidates for in vivo drug delivery because of their ability to enter cells and deliver cargo. For example, proteins of interest can be loaded into EVs to mediate protein transfer into target cells. To determine causality between EV content and function, which is also important to assess the clinical safety of EVs, it is crucial to comprehensively characterize their complete molecular composition. Here, we investigated EVs loaded with the chaperone protein DNAJB6. Chaperone proteins assist in protein folding and have been suggested to alleviate protein aggregation diseases, such as Alzheimer’s disease and Huntington’s disease. We analyzed and compared the proteome of EVs isolated from wildtype HEK293T cells with that of EVs from HEK 293T cells overexpressing DNAJB6-WT or loss-of-function mutant DNAJB6-M3. Comprehensive analysis of proteomics data showed enhanced levels of DNAJB6 as well as protein-folding-related proteins in EVs derived from DNAJB6-overexpression cells. Interestingly, upregulation of a chaperone and its protein-folding-related proteins resulted in downregulation of another chaperone plus its related proteins, and vice versa. This implies the presence of compensatory mechanisms in the cellular expression of chaperones. Collectively, we provide the proteomic EV signatures underlying EV mediated DNAJB6 transmission by HEK293T cells, with the aim of establishing a causal relationship between EV protein content and EV function

DNAJB6b-enriched small extracellular vesicles decrease polyglutamine aggregation in in vitro and in vivo models of Huntington disease

Huntington disease (HD) is a devastating neurodegenerative disorder characterized by aggregation of huntingtin (HTT) protein containing expanded polyglutamine (polyQ) tracts. DNAJB6, a member of the DNAJ chaperone family, was reported to efficiently inhibit polyQ aggregation in vitro, in cell models, and in vivo in flies, xenopus, and mice. For the delivery of exogenous DNAJB6 to the brain, the DNAJB6 needs to be protected against (enzymatic) degradation and show good penetration into brain tissue. Here, we tested the potential of small extracellular vesicles (sEVs) derived from neural stem cells (NSCs) for delivery of DNAJB6 as anti-amyloidogenic cargo. Administration of sEVs isolated from DNAJB6-overexpressing cells to cells expressing expanded polyQ tracts suppressed HTT aggregation. Furthermore, intrathecal injection of DNAJB6-enriched sEVs into R6/2 transgenic HD mice significantly reduced mutant HTT aggregation in the brain. Taken together, our data suggest that sEV-mediated molecular chaperone delivery may hold potential to delay disease onset in HD