Extracellular Vesicles and Neurodegenerative Diseases

Extracellular vesicles (EVs) include exosomes and microvesicles and have been shown to have roles in the CNS ranging from the removal of unwanted biomolecules to intercellular communication to the spread of pathogenic proteins associated with neurodegenerative diseases. EVs carry protein, lipid, and genetic cargo, and research over more than a decade has shown that they contain the misfolded forms of proteins associated with Alzheimer's, Parkinson's, and the prion diseases. Altered genetic cargo, usually in the form of miRNAs, have also been identified in EVs patients with these diseases, suggesting that EVs may be a source of disease biomarkers. Whether EVs play a key role in the pathogenesis of neurological diseases remains to be firmly established because most current research is performed using cell culture and transgenic animal models. If EVs are identified as a key pathological contributor to neurological conditions, they will form a novel target for therapeutic intervention. This Dual Perspectives article will discuss the current understanding of the role of EVs in neurological diseases and raise some of the limitations of our current understandings of this field.</p

Exosomes in the pathology of neurodegenerative diseases

More than 30 years ago, two unexpected findings were discovered that challenged conventional thinking in biology. The first was the identification of a misfolded protein with transmissible properties associated with a group of neurodegenerative diseases known as transmissible spongiform encephalopathies. The second was the discovery of a new pathway used for the extracellular release of biomolecules, including extracellular vesicles called exosomes. Two decades later, the convergence of these pathways was shown when exosomes were found to play a significant role in both the transmission and propagation of protein aggregates in disease. Recent research hasnowrevealed that the majority of proteins involved in neurodegenerative diseases are transported in exosomes, and that external stresses due to age-related impairment of protein quality control mechanisms can promote the transcellular flux of these proteins in exosomes. Significantly, exosomes provide an environment that can induce the conformational conversion of native proteins into aggregates that can be transmitted to otherwise aggregate-free cells in the brain. Here we review the current roles of exosomes in the pathology of neurodegenerative diseases

Therapeutically harnessing extracellular vesicles

The field of extracellular vesicle (EV) research has developed rapidly over the last decade from the study of fundamental biology to a subject of significant clinical relevance. The potential of harnessing EVs in the diagnosis and treatment of diseases — including cancer and neurological and cardiovascular disorders — is now being recognized. Accordingly, the applications of EVs as therapeutic targets, biomarkers, novel drug delivery agents and standalone therapeutics are being actively explored. This Review provides a brief overview of the characteristics and physiological functions of the various classes of EV, focusing on their association with disease and emerging strategies for their therapeutic exploitation.</p

Stimulating the Release of Exosomes Increases the Intercellular Transfer of Prions

Exosomes are small extracellular vesicles releasedby cells and play important roles in intercellular communication and pathogen transfer. Exosomes have been implicated in several neurodegenerative diseases, including prion disease and Alzheimer disease. Prion disease arises upon misfolding of the normal cellular prion protein, PrPC, into the disease-associated isoform, PrPSc. The disease has a unique transmissible etiology, and exosomes represent a novel and efficient method for prion transmission. The precise mechanism by which prions are transmitted from cell to cell remains to be fully elucidated, although three hypotheses have been proposed: direct cell-cell contact, tunneling nanotubes, and exosomes. Given the reported presence of exosomes inbiological fluids and in the lipid and nucleic acid contents of exosomes, these vesicles represent an ideal mechanism for encapsulating prions and potential cofactors to facilitate prion transmission. This study investigates the relationship between exosome release and intercellular prion dissemination. Stimulation of exosome release through treatment with an ionophore, monensin, revealed a corresponding increasein in tercellular transferofprion infectivity. Conversely, inhibition of exosome release using GW4869 to target the neutral sphingomyelinase pathway induced a decrease in intercellular prion transmission. Further examination of the effect of monensin on PrP conversion revealed that monensin also alters the conformational stability of PrPC, leading to increased generation of proteinase K-resistant prion protein. The findings presented here provide support for a positive relationship between exosome release and intercellular transfer of prion infectivity, highlighting an integral role for exosomes in facilitating the unique transmissible nature of prions

Focus on extracellular vesicles: exosomes and their role in protein trafficking and biomarker potential in Alzheimer's and Parkinson's disease

Growing evidence indicates that small extracellular vesicles, called exosomes, are prominent mediators of neurodegenerative diseases such as prion, Alzheimer’s and Parkinson’s disease. Exosomes contain neurodegenerative disease associated proteins such as the prion protein, β-amyloid and α-synuclein. Only demonstrated so far in vivo with prion disease, exosomes are hypothesised to also facilitate the spread of β-amyloid and α-synuclein from their cells of origin to the extracellular environment. In the current review, we will discuss the role of exosomes in Alzheimer’s and Parkinson’s disease including their possible contribution to disease propagation and pathology and highlight their utility as a diagnostic in neurodegenerative disease

Neuroinflammatory Modulation of Extracellular Vesicle Biogenesis and Cargo Loading

Increasing evidence suggests neuroinflammation is a highly coordinated response involving multiple cell types and utilising several different forms of cellular communication. In addition to the well documented cytokine and chemokine messengers, extracellular vesicles (EVs) have emerged as key regulators of the inflammatory response. EVs act as vectors of intercellular communication, capable of travelling between different cells and tissues to deliver selectively packaged protein, miRNA, and lipids from the parent cell. During neuroinflammation, EVs transmit specific inflammatory mediators, particularly from microglia, to promote inflammatory resolution. This mini-review will highlight the novel neuroinflammatory mechanisms contributing to the biogenesis and selective packaging of EVs

Methods for loading therapeutics into extracellular vesicles and generating extracellular vesicles mimetic-nanovesicles

Extracellular vesicles (EVs) are membrane bound vesicles released into the extracellular environment by eukaryotic and prokaryotic cells. EVs are enriched in active biomolecules and they can horizontally transfer cargo to recipient cells. In recent years EVs have demonstrated promising clinical applications due to their theragnostic potential. Although EVs have promising therapeutic potential, there are several challenges associated with using EVs before transition from the laboratory to clinical use. Some of these challenges include issues around low yield, isolation and purification methodologies, and efficient engineering (loading) of EVs with therapeutic cargo. Also, to achieve higher therapeutic efficiency, EV architecture and cargo may need to be manipulated prior to clinical application. Some of these issues have been addressed by developing biomimetic EVs. EV mimetic-nanovesicles (M-NVs) are a type of artificial EVs which can be generated from all cell types with comparable characteristics as EVs for an alternative therapeutic modality. In this review, we will discuss current techniques for modifying EVs and methodology used to generate and customize EV mimetic-nanovesicles.</p

Extracellular Vesicles Isolated from the Brains of rTg4510 Mice Seed Tau Protein Aggregation in a Threshold-dependent Manner

The microtubule-associated protein tau has a critical role in Alzheimer disease and related tauopathies. There is accumulating evidence that tau aggregates spread and replicate in a prion-like manner, with the uptake of pathological tau seeds causing misfolding and aggregation of monomeric tau in recipient cells. Here we focused on small extracellular vesicles enriched for exosomes that were isolated from the brains of tau transgenic rTg4510 and control mice. We found that these extracellular vesicles contained tau, although the levels were significantly higher in transgenic mice that have a pronounced tau pathology. Tau in the vesicles was differentially phosphorylated, although to a lower degree than in the brain cells from which they were derived. Several phospho-epitopes (AT8, AT100, and AT180) thought to be critical for tau pathology were undetected in extracellular vesicles. Despite this, when assayed with FRET tau biosensor cells, extracellular vesicles derived from transgenic mice were capable of seeding tau aggregation in a threshold-dependent manner. We also observed that the dye used to label extracellular vesicle membranes was still present during nucleation and formation of tau inclusions, suggesting either a role for membranes in the seeding or in the process of degradation. Together, we clearly demonstrate that extracellular vesicles can transmit tau pathology. This indicates a role for extracellular vesicles in the transmission and spreading of tau pathology. The characteristics of tau in extracellular vesicles and the seeding threshold we identified may explain why tau pathology develops very slowly in neurodegenerative diseases such as Alzheimer disease

Novel miR-29b target regulation patterns are revealed in two different cell lines

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene or protein expression by targeting mRNAs and triggering either translational repression or mRNA degradation. Distinct expression levels of miRNAs, including miR-29b, have been detected in various biological fluids and tissues from a large variety of disease models. However, how miRNAs “react” and function in different cellular environments is still largely unknown. In this study, the regulation patterns of miR-29b between human and mouse cell lines were compared for the first time. CRISPR/Cas9 gene editing was used to stably knockdown miR-29b in human cancer HeLa cells and mouse fibroblast NIH/3T3 cells with minimum off-targets. Genome editing revealed mir-29b-1, other than mir-29b-2, to be the main source of generating mature miR-29b. The editing of miR-29b decreased expression levels of its family members miR-29a/c via changing the tertiary structures of surrounding nucleotides. Comparing transcriptome profiles of human and mouse cell lines, miR-29b displayed common regulation pathways involving distinct downstream targets in macromolecular complex assembly, cell cycle regulation, and Wnt and PI3K-Akt signalling pathways; miR-29b also demonstrated specific functions reflecting cell characteristics, including fibrosis and neuronal regulations in NIH/3T3 cells and tumorigenesis and cellular senescence in HeLa cells.</p

Planktonic and Biofilm-Derived Pseudomonas aeruginosa Outer Membrane Vesicles Facilitate Horizontal Gene Transfer of Plasmid DNA

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria package various cargo, including DNA that can be transferred to other bacteria or to host cells. OMV-Associated DNA has been implicated in mediating horizontal gene transfer (HGT) between bacteria, which includes the dissemination of antibiotic resistance genes within and between bacterial species. Despite the known ability of OMVs to mediate HGT, the mechanisms of DNA packaging into OMVs remain poorly characterized, as does the effect of bacterial growth conditions on the DNA cargo composition of OMVs and their subsequent abilities to mediate HGT. In this study, we examined the DNA content of OMVs produced by the opportunistic pathogen Pseudomonas aeruginosa grown in either planktonic or biofilm conditions. Analysis of planktonic growth-derived OMVs revealed their ability to package and protect plasmid DNA from DNase degradation and to transfer plasmid-encoded antibiotic resistance genes to recipient, antibioticsensitive P. aeruginosa bacteria at a greater efficiency than transformation with plasmid alone. Comparisons of planktonic and biofilm-derived P. aeruginosa OMVs demonstrated that biofilm-derived OMVs were smaller but were associated with more plasmid DNA than planktonic-derived OMVs. Additionally, biofilm-derived P. aeruginosa OMVs were more efficient in the transformation of competent P. aeruginosa bacteria, compared to transformations with an equivalent number of planktonic-derived OMVs. The findings of this study highlight the importance of bacterial growth conditions for the packaging of DNA within P. aeruginosa OMVs and their ability to facilitate HGT, thus contributing to the spread of antibiotic resistance genes between P. aeruginosa bacteria. IMPORTANCE Bacterial membrane vesicles (BMVs) mediate interbacterial communication, and their ability to package DNA specifically contributes to biofilm formation, antibiotic resistance, and HGT between bacteria. However, the ability of P. aeruginosa OMVs to mediate HGT has not yet been demonstrated. Here, we reveal that P. aeruginosa planktonic and biofilm-derived OMVs can deliver plasmid-encoded antibiotic resistance to recipient P. aeruginosa. Additionally, we demonstrated that P. aeruginosa biofilmderived OMVs were associated with more plasmid DNA compared to planktonic-derived OMVs and were more efficient in the transfer of plasmid DNA to recipient bacteria. Overall, this demonstrated the ability of P. aeruginosa OMVs to facilitate the dissemination of antibiotic resistance genes, thereby enabling the survival of susceptible bacteria during antibiotic treatment. Investigating the roles of biofilm-derived BMVs may contribute to furthering our understanding of the role of BMVs in HGT and the spread of antibiotic resistance in the environment.</p