Extracellular vesicles (EVs) play a key role in propagating the spread of disease-related proteins in neurodegenerative diseases – that was one of the key messages delivered during ISEV2021’s first plenary session by Andrew Hill of La Trobe University, Australia. The presentation titled ‘Extracellular vesicles and their role in neurodegenerative diseases’ covered a series of landmark studies investigating EVs and their role in spreading disease-associated proteins, and the diagnostic potential of EV miRNA profiles for the diagnosis of neurodegenerative diseases.
EVs as traffickers of neurodegenerative disease-associated proteins
The accumulation of protein misfolding is a common hallmark of neurodegenerative diseases, and prion diseases are no exception. Prion diseases are characterised by the presence of abnormal, pathogenic agents that can induce abnormal folding of specific normal cellular proteins called prion proteins. The identification of prion propagation mechanisms is an important aspect of understanding prion diseases, as PRPsc (the disease-associated conformation) can trigger healthy prion proteins (PRP) to fold abnormally. While transmission is difficult to study, in vitro studies have suggested direct cell-to-cell contact<super-script>1<super-script> and tunnelling nanotubes<super-script>2<super-script> as potential mechanisms for prion propagation.
Among the studies highlighted in Hill’s ISEV talk was one from 2005 showing EVs (reported in the paper as exosomes) could carry both PrP and PrPsc.<super-script>3<super-script> Hill described several approaches used to study the propagation of prion proteins, including the use of cell cultures exposed to a brain homogenate taken from a mouse that has been infected with mouse prions. Following exposure, cells take up the infection and begin to spread it between themselves. As the normal form of the prion protein is susceptible to digestion by protease K, western blots could be used to distinguish between normal and disease-associated forms of the protein.
Evidence supporting a role for EVs in propagating the spread of disease-associated prion proteins has been drawn from a number of studies highlighted during the presentation. For example, prion disease can be induced in mice by administering EVs isolated from injected fibroblast and neuronal cell lines. While direct cell-to-cell contact is an effective mechanism for propagating PrPsc, it is not a requirement; healthy cells co-cultured with infected cells in a Transwell system become infected with PrPsc – in the absence of direct cell-to-cell contact.<super-script>4<super-script>
Hill also highlighted studies from 2015 and 2016 which examined how manipulating the release of EVs impacted prion transmission between cells. Indeed, a strong relationship was identified; the suppression of EV release almost abolished infectivity,<super-script>5<super-script> while the stimulation of EV release increased the intercellular transfer of prions.<super-script>4<super-script>
Hill then shared unpublished findings (Coleman et al.) indicating that EVs are more infectious than cell extracts (presumably referring to PRPsc-containing EVs and cells), and that EV membranes enhance prion infection efficiency. The importance of EV membrane integrity in protein misfolding has been reported previously in studies of α-synuclein; Ugalde et al.<super-script>6,7<super-script> used a protein misfolding cyclic amplification (PMCA) assay to show that EV-driven modulation of protein misfolding is dependent on EV membrane integrity.
Looking back on the decade: EV miRNA profiling in prion disease
In the second part of the talk, Hill explored his group’s work on EV-RNA as potential diagnostic tools for neurodegenerative diseases. Highlights included:
- The identification of nine EV-miRNAs (reported as exosomal miRNA) that are differentially expressed in prion-infected cells, compared to non-infected cells<super-script>8<super-script>
- Use of a panel of 16 differentially expressed EV-miRNAs to ‘predict’ Alzheimer’s disease (AD) in serum samples of healthy individuals and in people who had been diagnosed with AD. The panel was used in a blind analysis to correctly diagnose 13 of 15 AD patients (87% sensitivity). Of 35 healthy controls, 27 were correctly confirmed as AD-negative (77% specificity). It is possible that a higher specificity may be realised eventually; PET imaging data revealed that five of the 35 healthy controls (who were falsely categorised as AD-positive) had a high amyloid-β burden; if they were to develop AD at a later date, the panel’s specificity would be increased to 91%.
- The report of an improved method for isolating exosomes from brain tissue. Improving the sample purity of brain-derived exosomes is a critical step towards: identifying brain-specific disease-associated miRNA for subsequent identification in the circulation, and identifying pre-clinical EV miRNA signatures for diagnostic purposes.<super-script>10<super-script>
- The identification of EV-associated proteins with direct links to amyloid lateral sclerosis.<super-script>11<super-script>
- Recent findings from studies of an animal model of prion disease, which led to the development of a potential EV-miRNA diagnostic panel. Identified EV-miRNAs were known to target pathways relevant to prion disease and were validated in human clinical serum samples from patients who died of prion disease (sensitivity 86%, specificity 89%).<super-script>12<super-script>
Sample purity key to developing EV-related biomarkers for neurodegenerative disease
Growing evidence points towards EVs as propagators of disease-associated proteins in neurodegenerative diseases, with EV-associated RNA showing potential as diagnostic tools. Although there is little certainty about molecular changes and any causative relationships with disease progression, the discovery of relevant biomarkers is a source of optimism for many affected by neurodegenerative diseases.<super-script>13<super-script>
As highlighted by Hill in this plenary presentation, progress towards a ‘brain liquid biopsy’ may have wider implications when applied to other diseases. From a technical standpoint, this work also highlights the importance of sample purity in enabling the development of highly sensitive and specific diagnostic tools for clinical use.
