Laura J. Vella

Packaging of prions into exosomes is associated with a novel pathway of PrP processing.

Prion diseases are fatal, transmissible neurodegenerative disorders associated with conversion of the host‐encoded prion protein (PrPC) into an abnormal pathogenic isoform (PrPSc). Following exposure to the infectious agent (PrPSc) in acquired disease, infection is propagated in lymphoid tissues prior to neuroinvasion and spread within the central nervous system. The mechanism of prion dissemination is perplexing due to the lack of plausible PrPSc‐containing mobile cells that could account for prion spread between infected and uninfected tissues. Evidence exists to demonstrate that the culture media of prion‐infected neuronal cells contain PrPSc and infectivity but the nature of the infectivity remains unknown. In this study we have identified PrPC and PrPSc in association with endogenously expressing PrP neuronal cell‐derived exosomes. The exosomes from our prion‐infected neuronal cell line were efficient initiators of prion propagation in uninfected recipient cells and to non‐neuronal cells. Moreover, our neuronal cell line was susceptible to infection by non‐neuronal cell‐derived exosome PrPSc. Importantly, these exosomes produced prion disease when inoculated into mice. Exosome‐associated PrP is packaged via a novel processing pathway that involves the N‐terminal modification of PrP and selection of distinct PrP glycoforms for incorporation into these vesicles. These data extend our understanding of the relationship between PrP and exosomes by showing that exosomes can establish infection in both neighbouring and distant cell types and highlight the potential contribution of differentially processed forms of PrP in disease distribution. These data suggest that exosomes represent a potent pool of prion infectivity and provide a mechanism for studying prion spread and PrP processing in cells endogenously expressing PrP. Copyright

The role of exosomes in the processing of proteins associated with neurodegenerative diseases

Exosomes are small membranous vesicles secreted by a number of cell types and can be isolated from conditioned cell media or bodily fluids such as urine and plasma. Exosome biogenesis involves the inward budding of multivesicular bodies (MVB) to form intraluminal vesicles (ILV). When fused with the plasma membrane, the MVB releases the vesicles into the extracellular environment as exosomes. Proposed functions of these vesicles include roles in cell–cell signalling, removal of unwanted proteins, and the transfer of pathogens between cells, such as HIV-1. Another such pathogen which exploits this pathway is the prion, the infectious particle responsible for the transmissible neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD) of humans or bovine spongiform encephalopathy (BSE) of cattle. Interestingly, this work is mirrored by studies on another protein involved in neurodegenerative disease, the amyloid precursor protein (APP) which is associated with Alzheimer’s disease (AD). Recent work has found APP proteolytic fragments in association with exosomes, suggesting a common pathway previously unknown for proteins associated with neurodegenerative diseases. This review will be discussing the current literature regarding the role of exosomes in secretion of the proteins, PrP and APP, and the subsequent implications for neurodegenerative disease.

Inhibition of γ-secretase causes increased secretion of amyloid precursor protein C-terminal fragments in association with exosomes

Alzheimers disease (AD) is the most common form of dementia and is associated with the deposition of the 39‐ to 43‐amino acid β‐amyloid peptide (Aβ) in the brain. C‐terminal fragments (CTFs) of amyloid precursor protein (APP) can accumulate in endosomally derived multivesicular bodies (MVBs). These intracellular structures contain intraluminal vesicles that are released from the cell as exosomes when the MVB fuses with the plasma membrane. Here we have investigated the role of exosomes in the processing of APP and show that these vesicles contain APP‐ CTFs, as well as Aβ. In addition, inhibition of γ‐secre‐ tase results in a significant increase in the amount of α‐ and β‐secretase cleavage, further increasing the amount of APP‐CTFs contained within these exosomes. We identify several key members of the secretase family of proteases (BACE, PS1, PS2, and ADAM10) to be localized in exosomes, suggesting they may be a previously unidentified site of APP cleavage. These results provide further evidence for a novel pathway in which APP fragments are released from cells and have implications for the analysis of APP processing and diagnostics for Alzheimers disease.—Sharples, R. A., Vella, L. J., Nisbet, R. M., Naylor, R., Perez, K., Barnham, K. J., Masters, C. L., Hill, A. F. Inhibition of γ‐secretase causes increased secretion of amyloid precursor protein C‐terminal fragments in association with exosomes. FASEB J. 22, 1469–1478 (2008)

Enrichment of prion protein in exosomes derived from ovine cerebral spinal fluid

Prion diseases are transmissible neurodegenerative disorders affecting humans and a wide variety of animal species including sheep and cattle. The transmissible agent, the prion, is an abnormally folded form (PrP(Sc)) of the host encoded cellular prion protein (PrP(C)). Distribution of the prion protein in the fluids of species susceptible to these diseases is of importance to human health and the iatrogenic spread of prion disease. Aside from blood which is confirmed to be a source of prion infectivity, it is currently unclear which other body fluids harbor a significant transmission risk. In the current study we examined two ovine fluids; pseudo-afferent lymph and cerebral spinal fluid (CSF), for the presence of exosomes and concurrent enrichment of the normal, cellular form of the prion protein (PrP(C)). Here we demonstrate the existence of exosomes in both pseudo-afferent lymph and CSF isolated from sheep. In the CSF derived exosomes we were able to show an enrichment of PrP(C) over unfractionated CSF. This experimental approach suggests that CSF derived exosomes could be used as a novel means of detecting abnormal forms of the prion protein and provide an in vivo link between these vesicles and prion disease pathogenesis.

EVpedia: a community web portal for extracellular vesicles research

MOTIVATION Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. RESULTS We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. AVAILABILITY AND IMPLEMENTATION The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.

C-Jun N-terminal kinase controls TDP-43 accumulation in stress granules induced by oxidative stress

BackgroundTDP-43 proteinopathies are characterized by loss of nuclear TDP-43 expression and formation of C-terminal TDP-43 fragmentation and accumulation in the cytoplasm. Recent studies have shown that TDP-43 can accumulate in RNA stress granules (SGs) in response to cell stresses and this could be associated with subsequent formation of TDP-43 ubiquinated protein aggregates. However, the initial mechanisms controlling endogenous TDP-43 accumulation in SGs during chronic disease are not understood. In this study we investigated the mechanism of TDP-43 processing and accumulation in SGs in SH-SY5Y neuronal-like cells exposed to chronic oxidative stress. Cell cultures were treated overnight with the mitochondrial inhibitor paraquat and examined for TDP-43 and SG processing.ResultsWe found that mild stress induced by paraquat led to formation of TDP-43 and HuR-positive SGs, a proportion of which were ubiquitinated. The co-localization of TDP-43 with SGs could be fully prevented by inhibition of c-Jun N-terminal kinase (JNK). JNK inhibition did not prevent formation of HuR-positive SGs and did not prevent diffuse TDP-43 accumulation in the cytosol. In contrast, ERK or p38 inhibition prevented formation of both TDP-43 and HuR-positive SGs. JNK inhibition also inhibited TDP-43 SG localization in cells acutely treated with sodium arsenite and reduced the number of aggregates per cell in cultures transfected with C-terminal TDP-43 162-414 and 219-414 constructs.ConclusionsOur studies are the first to demonstrate a critical role for kinase control of TDP-43 accumulation in SGs and may have important implications for development of treatments for FTD and ALS, targeting cell signal pathway control of TDP-43 aggregation.

MEK Inhibition, Alone or in Combination with BRAF Inhibition, Affects Multiple Functions of Isolated Normal Human Lymphocytes and Dendritic Cells

Vella and colleagues show that inhibition of BRAF (dabrafenib) had no effect on healthy donor T cells and monocyte-derived dendritic cells (MoDC), but that MEK inhibition (trametinib) suppressed T-cell proliferation, cytokine production, antigen-specific expansion, and MoDC cross-presentation. Combination therapy with BRAF and MEK inhibition is currently in clinical development for the treatment of BRAF-mutated malignant melanoma. BRAF inhibitors are associated with enhanced antigen-specific T-lymphocyte recognition in vivo. Consequently, BRAF inhibition has been proposed as proimmunogenic and there has been considerable enthusiasm for combining BRAF inhibition with immunotherapy. MEK inhibitors inhibit ERK phosphorylation regardless of BRAF mutational status and have been reported to impair T-lymphocyte and modulate dendritic cell function. In this study, we investigate the effects on isolated T lymphocytes and monocyte-derived dendritic cells (moDC) of a MEK (trametinib) and BRAF (dabrafenib) inhibitor combination currently being evaluated in a randomized controlled clinical trial. The effects of dabrafenib and trametinib, alone and in combination, were studied on isolated normal T lymphocytes and moDCs. Lymphocyte viability, together with functional assays including proliferation, cytokine production, and antigen-specific expansion, were assessed. MoDC phenotype in response to lipopolysaccharide stimulation was evaluated by flow cytometry, as were effects on antigen cross-presentation. Dabrafenib did not have an impact on T lymphocytes or moDCs, whereas trametinib alone or in combination with dabrafenib suppressed T-lymphocyte proliferation, cytokine production, and antigen-specific expansion. However, no significant decrease in CD4+ or CD8+ T-lymphocyte viability was observed following kinase inhibition. MoDC cross-presentation was suppressed in association with enhanced maturation following combined inhibition of MEK and BRAF. The results of this study demonstrate that MEK inhibition, alone or in combination with BRAF inhibition, can modulate immune cell function, and further studies in vivo will be required to evaluate the potential clinical impact of these findings. Cancer Immunol Res; 2(4); 351–60. ©2014 AACR.

Mouse-adapted sporadic human Creutzfeldt–Jakob disease prions propagate in cell culture

Cell based models used for the study of prion diseases have traditionally employed mouse-adapted strains of sheep scrapie prions. To date, attempts to generate human prion propagation in cell culture have been unsuccessful. Rabbit kidney epithelial cells (RK13) are permissive to infection with prions from a variety of species upon expression of cognate PrP transgenes. We explored RK13 cells expressing human PrP for their utility as a cell line capable of sustaining infection with human prions. RK13 cells processed exogenously expressed human PrP similarly to exogenously expressed mouse PrP but were not permissive to infection when exposed to sporadic Creutzfeldt-Jakob disease prions. Transmission of the same sporadic Creutzfeldt Jakob disease prions to wild-type mice generated a strain of mouse-adapted human prions, which efficiently propagated in RK13 cells expressing mouse PrP, demonstrating these cells are permissive to infection by mouse-adapted human prions. Our observations underscore the likelihood that, in contrast to prions derived from non-human mammals, additional unidentified cofactors or subcellular environment are critical for the generation of human 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.

The emerging role of exosomes in epithelial-mesenchymal-transition in cancer.

Metastasis in cancer consists of multiple steps, including epithelial–mesenchymal-transition (EMT), which is characterized by the loss of epithelial-like characteristics and the gain of mesenchymal-like attributes including cell migration and invasion. It is clear that the tumor microenvironment can promote the metastatic cascade and that intercellular communication is necessary for this to occur. Exosomes are small membranous vesicles secreted by most cell types into the extracellular environment and they are important communicators in the tumor microenvironment. They promote angiogenesis, invasion, and proliferation in recipient cells to support tumor growth and a prometastatic phenotype. Although it is clear that exosomes contribute to cancer cell plasticity, experimental evidence to define exosome induced plasticity as EMT is only just coming to light. This review will discuss recent research on exosomal regulation of the EMT process in the tumor microenvironment.