To date, the transsynaptic spread of tau pathology in Alzheimer’s disease has been studied using animal or in vitro models. In a study published in the Nature publication Laboratory Investigation, Miyoshi et al. (2021) describe a flow cytometry assay to quantify the depolarisation of isolated human synaptic neuronal terminals, known as synaptosomes. Synaptosomes are considered a relevant model system for studying human synaptic dysfunction in neurodegenerative diseases and were isolated from the human brains following autopsy (56 cases with varying diagnoses: mostly AD with additional conditions such as cerebral amyloid angiopathy and atherosclerosis, and three controls).
Using the ex vivo model, the group observed a depolarisation-induced release of EVs, along with tetraspanin EV markers. EVs released from neural synapses were characterised using several methods, including tunable resistive pulse sensing for size analysis (confirming EVs were in the size range consistent with exosomes), and Western blots which showed the tetraspanin CD63 and faint tau bands. Seeding activity of exosomal tau was enhanced by amyloid-β, as measured by a tau biosensor based on fluorescence resonance energy transfer (FRET). The authors concluded that the study is consistent with amyloid-β as the upstream initiating pathology and may provide support for tau-targeted therapeutic approaches and aggregation inhibitors – including a reduction of secretion of tau-bearing exosomal subpopulations.
For many cancers, including head and neck squamous cell carcinomas (HNSCC), microRNAs are among the many molecular alterations that have been described. Recently, researchers in Sau Paulo, Brazil, explored the potential effect of EVs originating from squamous cell carcinoma cells on the immune system response. EV size distribution was measured using TRPS, with further characterisation achieved through flow cytometry and transmission electron microscopy.
EVs were isolated from two distinct cancer cell lines and applied to dendritic cells derived from circulating monocytes (mono-DCs), with subsequent internalisation confirmed via confocal microscopy. EV internalisation appeared to disrupt mono-DC maturation; this is significant as dendritic cell maturation is a key aspect of the adaptive immune response, and impairment of dendritic cell function has been widely studied in cancer. By comparing EV-microRNA from (and between) these cell lines with microRNA in patient plasma, the group provided evidence supporting the hypothesis that EV-mediated microRNA transport is a cell signalling tool with immune modulatory effects in HNSCC.
As part of rigorous safety and immunogenic assessments of biopharmaceutical products, regulatory agencies often encourage the characterisation of sub-micron particles. As a method suited to the analysis of samples with heterogenous particle size distributions, TRPS capabilities were assessed by Bayer AG and the Ludwig Maximilian University of Munich, when applied to solutions of biopharmaceuticals. Samples were prepared by applying ‘stir-stress’ to IgG monoclonal antibodies (mAbs) to induce aggregation and were filtered through a 5 µm membrane filter to remove large aggregates that would block the nanopore. A spiking-in approach was applied to introduce an electrolyte concentration sufficient for stable measurement conditions. The authors confirmed the capability of TRPS for this application and suggested a novel approach to data evaluation for such proteinaceous samples.
Mutations in the GBA gene encoding glucocerebrosidase, a lysosomal enzyme with important functions in cellular housekeeping, are a prevalent but poorly understood genetic risk factor for Lewy body disorders (LBD). The relationship between GBA mutations and LBD was investigated through the lens of EV research, partly by comparing cerebrospinal fluid- and brain-derived EVs from both GBA mutation carriers and non-carriers. The study presents novel insights into LBD and present LBD EVs as promising sources as biomarkers; LBD EVs from both human CSF and frontal cortex were described as carrying a ‘pathological package’. Of note, LBD EVs were enriched in ceramides, potentially reflecting a loss of protein homeostasis (and control of de novo ceramide synthesis) in the endoplasmic reticulum. Brain- and CSF-derived EVs were isolated using Izon’s qEV isolation platform, followed by size and concentration analysis by tunable resistive pulse sensing.
A collaborative group of Australian-based researchers recently explored potential mechanisms of RNA loading into small EVs. A long noncoding RNA of retroviral origin, which was enriched by more than 200-fold in EVs compared to in parental cells, was identified following RNA-Seq analysis of small EV RNAs from mouse dendritic cells. It is thought that a repetitive 26-nucleotide motif within the long noncoding RNA may directly promotes the loading of RNA into small EVs. The enriched long non-coding RNA was subsequently confirmed in other cell types. EV size distribution was measured by Izon’s tunable resistive pulse sensing.