Extracellular vesicles and circulating nucleic acids最新文献

Aims: Blood biomarkers can improve drug development for Alzheimer's disease (AD) and its treatment. Neuron-derived extracellular vesicles (NDEVs) in plasma offer a minimally invasive platform for developing novel biomarkers that may be used to monitor the diverse pathogenic processes involved in AD. However, NDEVs comprise only a minor fraction of circulating extracellular vesicles (EVs). Most published studies have leveraged the L1 cell adhesion molecule (L1CAM) for NDEV immunocapture. We aimed to develop and optimize an alternative, highly specific immunoaffinity method to enrich blood NDEVs for biomarker development.

Methods: After screening multiple neuronal antigens, we achieved NDEV capture with high affinity and specificity using antibodies against Growth-Associated Protein (GAP) 43 and Neuroligin 3 (NLGN3). The EV identity of the captured material was confirmed by electron microscopy, western blotting, and proteomics. The specificity for neuronal origin was demonstrated by showing enrichment for neuronal markers (proteins, mRNA) and recovery of spiked neuronal EVs. We performed NDEV isolation retrospectively from plasma samples from two cohorts of early AD patients (N = 19 and N = 40) and controls (N = 20 and N = 19) and measured p181-Tau, amyloid-beta (Aβ) 42, brain-derived neurotrophic factor (BDNF), precursor brain-derived neurotrophic factor (proBDNF), glutamate receptor 2 (GluR2), postsynaptic density protein (PSD) 95, GAP43, and syntaxin-1.

Results: p181-Tau, Aβ42, and NRGN were elevated in AD samples, whereas proBDNF, GluR2, PSD95, GAP43, and Syntaxin-1 were reduced. Differences for p181-Tau, proBDNF, and GluR2 survived multiple-comparison correction and were correlated with cognitive scores. A model incorporating biomarkers correctly classified 94.7% of AD participants and 61.5% of control participants. The observed differences in NDEVs-associated biomarkers are consistent with previous findings.

Conclusion: NDEV isolation by GAP43 and NLGN3 immunocapture offers a robust novel platform for biomarker development in AD, suitable for large-scale validation.

Intercellular communication between diverse cell types is crucial for the maintenance of the central nervous system, and exosomes have been shown to play an important role in this process. Exosomes are small extracellular vesicles (EVs) that are released by all cell types and carry cargoes that can elicit downstream effects in recipient cells. Exosomal communication in the central nervous system has been implicated in many neurodegenerative diseases, ranging from Alzheimer's disease to major depressive disorder. Though there remain many unknowns in the field of EV biology, in vitro experiments can provide many insights into their potential roles in health and disease. In this review, we discuss the findings of many in vitro EV experiments, with a focus on the potential roles in regulating cell viability, inflammation, oxidative stress, and neurite integrity in the central nervous system.

Extracellular vesicles (EVs), or exosomes, are naturally occurring nano- and micro-sized membrane vesicles playing an essential role in cell-to-cell communication. There is a recent increasing interest in harnessing the therapeutic potential of these natural nanoparticles to develop cell-free regenerative medicine and manufacture highly biocompatible and targeted drug and gene delivery vectors, amongst other applications. In the context of developing novel and effective EV-based therapy, imaging tools are of paramount importance as they can be used to not only elucidate the underlying mechanisms but also provide the basis for optimization and clinical translation. In this review, recent efforts and knowledge advances on EV-based therapies have been briefly introduced, followed by an outline of currently available labeling strategies by which EVs can be conjugated with various imaging agents and/or therapeutic drugs and genes. A comprehensive review of prevailing EV imaging technologies is then presented along with examples and applications, with emphasis on imaging probes and agents, corresponding labeling methods, and the pros and cons of each imaging modality. Finally, the potential of theranostic EVs as a powerful new weapon in the arsenal of regenerative medicine and nanomedicine is summarized and envisioned.

Prenatal testing is important for the early detection and diagnosis of rare genetic conditions with life-changing implications for the patient and their family. Gaining access to the fetal genotype can be achieved using gold-standard invasive sampling methods, such as amniocentesis and chorionic villus sampling, but these carry a small risk of miscarriage. Non-invasive prenatal diagnosis (NIPD) for select rare monogenic conditions has been in clinical service in England since 2012 and has revolutionised the field of prenatal diagnostics by reducing the number of women undergoing invasive sampling procedures. Fetal-derived genomic material is present in a highly fragmented form amongst the maternal cell-free DNA (cfDNA) in circulation, with sequence coverage across the entire fetal genome. Cell-free fetal DNA (cffDNA) is the foundation for NIPD, and several technologies have been clinically implemented for the detection of paternally inherited and de novo pathogenic variants. Conversely, a low abundance of cffDNA within a high background of maternal cfDNA makes assigning maternally inherited variants to the fetal fraction a significantly more challenging task. Research is ongoing to expand available tests for maternal inheritance to include a broader range of monogenic conditions, as well as to uncover novel diagnostic avenues. This review covers the scope of technologies currently clinically available for NIPD of monogenic conditions and those still in the research pipeline towards implementation in the future.

Tau propagation, pathogenesis, and neurotoxicity are hallmarks of neurodegenerative diseases that result in cognitive impairment. Tau accumulates in Alzheimer's disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy, and related tauopathies. Knowledge of the mechanisms for tau propagation in neurodegeneration is necessary for understanding the development of dementia. Exosomes, known as extracellular vesicles (EVs), have emerged as participants in promoting tau propagation. Recent findings show that EVs generated by neurons expressing familial mutations of tauopathies of FTDP-17 (P301L and V337M) (mTau) and presenilin (A246E) (mPS1) in AD induce tau propagation and accumulation after injection into rodent brain. To gain knowledge of the proteome cargoes of the mTau and mPS1 EVs that promote tau pathogenesis, this review compares the proteomes of these EVs, which results in important new questions concerning EV mechanisms of tau pathogenesis. Proteomics data show that EVs produced by mTau- and mPS1-expressing iPSC neurons share proteins involved in exocytosis and vesicle secretion and, notably, these EVs also possess differences in protein components of vesicle-mediated transport, extracellular functions, and cell adhesion. It will be important for future studies to gain an understanding of the breadth of familial genetic mutations of tau, presenilin, and other genes in promoting EV initiation of tau propagation and pathogenesis. Furthermore, elucidation of EV cargo components that mediate tau propagation will have potential as biomarkers and therapeutic strategies to ameliorate dementia of tauopathies.

Aim: Receptor activator of nuclear factor-kappa B (RANK)-containing extracellular vesicles (EVs) bind RANK-Ligand (RANKL) on osteoblasts, and thereby simultaneously inhibit bone resorption and promote bone formation. Because of this, they are attractive candidates for therapeutic bone anabolic agents. Previously, RANK was detected in 1 in every 36 EVs from osteoclasts by immunogold electron microscopy. Here, we have sought to characterize the subpopulation of EVs from osteoclasts that contains RANK in more detail.

Methods: The tetraspanins CD9 and CD81 were localized in osteoclasts by immunofluorescence. EVs were visualized by transmission electron microscopy. A Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and immunoaffinity isolations examined whether RANK is enriched in specific types of EVs.

Results: Immunofluorescence showed CD9 was mostly on or near the plasma membrane of osteoclasts. In contrast, CD81 was localized deeper in the osteoclast's cytosolic vesicular network. By interferometry, both CD9 and CD81 positive EVs from osteoclasts were small (56-83 nm in diameter), consistent with electron microscopy. The CD9 and CD81 EV populations were mostly distinct, and only 22% of the EVs contained both markers. RANK was detected by SP-IRIS in 2%-4% of the CD9-containing EVs, but not in CD81-positive EVs, from mature osteoclasts. Immunomagnetic isolation of CD9-containing EVs from conditioned media of osteoclasts removed most of the RANK. A trace amount of RANK was isolated with CD81.

Conclusion: RANK was enriched in a subset of the CD9-positive EVs. The current study provides the first report of selective localization of RANK in subsets of EVs.

Extracellular vesicles (EVs) are a heterogeneous population of stable lipid membrane particles that play a critical role in the regulation of numerous physiological and pathological processes. EV cargo, which includes lipids, proteins, and RNAs including miRNAs, is affected by the metabolic status of the parental cell. Concordantly, abnormalities in the autophagic-endolysosomal pathway, as seen in lysosomal storage disorders (LSDs), can affect EV release as well as EV cargo. LSDs are a group of over 70 inheritable diseases, characterized by lysosomal dysfunction and gradual accumulation of undigested molecules. LSDs are caused by single gene mutations that lead to a deficiency of a lysosomal protein or lipid. Lysosomal dysfunction sets off a cascade of alterations in the endolysosomal pathway that can affect autophagy and alter calcium homeostasis, leading to energy imbalance, oxidative stress, and apoptosis. The pathophysiology of these diseases is very heterogenous, complex, and currently incompletely understood. LSDs lead to progressive multisystemic symptoms that often include neurological deficits. In this review, a kaleidoscopic overview will be given on the roles of EVs in LSDs, from their contribution to pathology and diagnostics to their role as drug delivery vehicles. Furthermore, EV cargo and surface engineering strategies will be discussed to show the potential of EVs in future LSD treatment, both in the context of enzyme replacement therapy, as well as future gene editing strategies like CRISPR/Cas. The use of engineered EVs as drug delivery vehicles may mask therapeutic cargo from the immune system and protect it from degradation, improving circulation time and targeted delivery.

Analysis of cell-free DNA (cfDNA) in the blood has shown promise for monitoring a variety of biological processes. Plasma cfDNA is a mixture comprising DNA molecules released from various bodily tissues, mediated by characteristic DNA fragmentations occurring during cell death. Fragmentation of cfDNA is non-random and contains tissue-of-origin information, which has been demonstrated in circulating fetal, tumoral, and transplanted organ-derived cfDNA molecules. Many studies have elucidated a plurality of fragmentomic markers for noninvasive prenatal, cancer, and organ transplantation assessment, such as fragment sizes, fragment ends, end motifs, and nucleosome footprints. Recently, researchers have further revealed the large population of previously unidentified long cfDNA molecules (kilobases in size) in the plasma DNA pool. This review focuses on the emerging biological properties of cfDNA, together with a discussion on its potential clinical implications.

The discovery and development of extracellular vesicles in tissue engineering have shown great potential for tissue regenerative therapies. However, their vesicle nature requires dosage-dependent administration and efficient interactions with recipient cells. Researchers have resorted to biomaterials for localized and sustained delivery of extracellular vesicles to the targeted cells, but not much emphasis has been paid on the design of the materials, which deeply impacts their molecular interactions with the loaded extracellular vesicles and subsequent delivery. Therefore, we present in this review a comprehensive survey of extracellular vesicle delivery systems from the viewpoint of material design at the molecular level. We start with general requirements of the materials and delve into different properties of delivery systems as a result of different designs, from material selections to processing strategies. Based on these differences, we analyzed the performance of extracellular vesicle delivery and tissue regeneration in representative studies. In light of the current missing links within the relationship of material structures, physicochemical properties and delivery performances, we provide perspectives on the interactions of materials and extracellular vesicles and the possible extension of materials. This review aims to be a strategic enlightenment for the future design of extracellular vesicle delivery systems to facilitate their translation from basic science to clinical applications.