Biology subjects

Vella, L. J.

Publications and source records attributed to Vella, L. J..

5 recordsLinked to original sources

bioRxiv2024

Proteome of human glioblastoma and meningioma tissue small extracellular vesicles

Small extracellular vesicles have gained attention in neuroscience due to their role in cell-to- cell communication and their potential diagnostic and therapeutic applications. Despite progress in the field, there remains a gap in our understanding of the composition and function of extracellular vesicles with regards to brain tumours. Previous studies have primarily evaluated extracellular vesicles obtained from patient fluids or cell culture medium, rather than directly from tumour tissue. Here we successfully isolated small extracellular vesicles from surgical tissue biopsies of glioblastomas or meningiomas, marking, marking the first report of in situ extracellular vesicle isolation from brain tumours. The protein content of the tumour tissue and their extracellular vesicles was characterized using tandem mass spectrometric proteomics, revealing proteins exclusively detected or enriched in extracellular vesicles relative to the tumour tissue. While our study confirmed proteins previously identified in glioblastoma and meningioma extracellular vesicles from various sources, it also identified novel proteins and pathways associated with extracellular vesicles from these tumour types. This study underscores the benefit of analysing in situ extracellular vesicles derived directly from brain tissue for insights into tumour biology and highlights the need for further research comparing extracellular vesicles from various types and grades of brain tumours.

biochemistry↗

bioRxiv2024

Multi-omics characterization of highly enriched human plasma extracellular vesicles.

Extracellular vesicles (EVs) in blood plasma offer a valuable reservoir of intracellular cellular cargo, making them a promising source of liquid based biomarkers. The molecular cargo of small EVs (sEVs) is of particular interest because some EV subtypes encapsulate cargo from organelles including mitochondria, endosomes, and the autophagy pathways, which are implicated in multiple diseases. However, the complexity of plasma, with its abundance of non-EV particles and plasma proteins, presents challenges for their molecular characterization using mass spectrometry based omics technologies. Here, we optimised a rigorous method to isolate sEVs from human plasma based on both density and size. Following this, we analysed the protein and lipid content of sEVs from multiple individuals. We demonstrate the advantage of obtaining highly enriched sEVs from plasma for enhancing the detection of protein networks associated with mitochondria and the endosomal network, and also tissue types including the central nervous system. Some of the EV associated proteins reported here have not been detected in plasma, nor plasma sEVs, previously. We show that sphingomyelin lipids are the most abundant lipids in plasma sEVs (33.7 mol% total lipids) and provide the first report on cholesterol ester content. We demonstrate a 16-fold decrease in cholesterol ester lipids in sEVs compared to platelet free plasma and suggest that cholesterol ester content could serve as a valuable measure for assessing the effectiveness of plasma separation protocols or kits in enriching for sEVs. Our study highlights the benefit of reducing co-isolates from plasma sEV preparations to enable the detection of proteins and lipids with potential biomarker utility, and underscores the need for ongoing development of improved high throughput sEV isolation technologies.

biochemistry↗

bioRxiv2023

Delivery of mRNA encoding an anti-Tau monoclonal antibody and engineered scFv intrabody results in functional antibody expression in SH-SY5Y cells

Monoclonal antibodies have emerged as a leading therapeutic agent for the treatment of disease, including Alzheimers disease. In the last year, two anti-amyloid monoclonal antibodies, lecanemab and aducanumab, have been approved in the USA for the treatment of Alzheimers disease, whilst several tau-targeting monoclonal antibodies are currently in clinical trials. Such antibodies, however, are expensive and timely to produce and require frequent dosing regimens to ensure disease-modifying effects. Synthetic in vitro-transcribed mRNA encoding antibodies for endogenous protein expression holds the potential to overcome many of the limitations associated with protein antibody production. Here, we have generated synthetic in vitro-transcribed mRNA encoding a tau specific monoclonal antibody as a full-sized IgG and as a single chain variable fragment (scFv). In vitro transfection of human neuroblastoma SH-SY5Y cells demonstrated the ability of the synthetic mRNA to be translated into functional tau-specific antibodies. Furthermore, we show that the translation of the tau-specific scFv as an intrabody results in the specific engagement of intracellular tau. This work highlights the utility of mRNA for the delivery of antibody therapeutics, including intrabodies, for the targeting of tau in tauopathies.

neuroscience↗

bioRxiv2020

Characterization of Brain-Derived Extracellular Vesicle Lipids in Alzheimer's Disease

Lipid dyshomeostasis is associated with the most common form of dementia, Alzheimers disease (AD). Substantial progress has been made in identifying positron emission tomography (PET) and cerebrospinal fluid (CSF) biomarkers for AD, but they have limited use as front-line, non-invasive diagnostic tools. Small extracellular vesicles (EVs) are released by all cell types and contain an enriched subset of their parental cell molecular composition, including lipids. EVs are released from the brain into the periphery, providing a potential source of tissue and disease specific lipid biomarkers. However, the EV lipidome of the central nervous system (CNS) is currently unknown and the potential of brain-derived EVs (BDEVs) to inform on lipid dyshomeostasis in AD remains unclear. The aim of this study was to reveal the lipid composition of BDEVs in human frontal cortex tissue, and to determine whether BDEVs in AD have altered lipid profiles compared to age-matched neurological controls (NC). Here, using semi-quantitative mass spectrometry, we describe the BDEV lipidome, covering 4 lipid categories, 17 lipid classes and 692 lipid molecules. Frontal cortex-derived BDEVs were enriched in glycerophosphoserine (PS) lipids, a characteristic of small EVs. Here we report that BDEVs are enriched in ether-containing PS lipids. A novel finding that further establishes ether lipids as a feature of EVs. While no significant changes were detected in the frontal cortex in AD, the lipid profile of the BDEVs from this tissue exhibited disease related differences. AD BDEVs had altered glycerophospholipid (GP) and sphingolipid (SP) levels, specifically increased plasmalogen glycerophosphoethanolamine (PE-P) and decreased polyunsaturated fatty acyl containing lipids (PUFAs), and altered amide-linked acyl chain content in sphingomyelin (SM) and ceramide (Cer) lipids relative to vesicles from neurological control subjects. The most prominent alteration being a two-fold decrease in lipid species containing docosahexaenoic acid (DHA). The in-depth lipidome analysis provided in this study highlights the advantage of EVs over more complex tissues for improved detection of dysregulated lipids that may serve as potential biomarkers in the periphery.

molecular biology↗

bioRxiv2020

Influence of species and processing parameters on recovery and content of brain tissue-derived extracellular vesicles

Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque, and mouse. Effects of tissue perfusion and a model of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with those of macaque and, especially, mouse, suggesting opportunities for optimization. Where possible, perfusion should be performed in animal models. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Finally, different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.

neuroscience↗