Mechanisms of axonal support by oligodendrocyte-derived extracellular vesicles.

Extracellular vesicles (EVs) have recently emerged as versatile elements of cell communication in the nervous system, mediating tissue homeostasis. EVs influence the physiology of their target cells via horizontal transfer of molecular cargo between cells and by triggering signalling pathways. In this Review, we discuss recent work revealing that EVs mediate interactions between oligodendrocytes and neurons, which are relevant for maintaining the structural integrity of axons. In response to neuronal activity, myelinating oligodendrocytes release EVs, which are internalized by neurons and provide axons with key factors that improve axonal transport, stress resistance and energy homeostasis. Glia-to-neuron transfer of EVs is thus a crucial facet of axonal preservation. When glial support is impaired, axonal integrity is progressively lost, as observed in myelin-related disorders, other neurodegenerative diseases and with normal ageing. We highlight the mechanisms that oligodendroglial EVs use to sustain axonal integrity and function.

The power of imaging to understand extracellular vesicle biology in vivo.

Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.

Assessment of technical and clinical utility of a bead-based flow cytometry platform for multiparametric phenotyping of CNS-derived extracellular vesicles.

BACKGROUND: Extracellular vesicles (EVs) originating from the central nervous system (CNS) can enter the blood stream and carry molecules characteristic of disease states. Therefore, circulating CNS-derived EVs have the potential to serve as liquid-biopsy markers for early diagnosis and follow-up of neurodegenerative diseases and brain tumors. Monitoring and profiling of CNS-derived EVs using multiparametric analysis would be a major advance for biomarker as well as basic research. Here, we explored the performance of a multiplex bead-based flow-cytometry assay (EV Neuro) for semi-quantitative detection of CNS-derived EVs in body fluids. METHODS: EVs were separated from culture of glioblastoma cell lines (LN18, LN229, NCH82) and primary human astrocytes and measured at different input amounts in the MACSPlex EV Kit Neuro, human. In addition, EVs were separated from blood samples of small cohorts of glioblastoma (GB), multiple sclerosis (MS) and Alzheimer's disease patients as well as healthy controls (HC) and subjected to the EV Neuro assay. To determine statistically significant differences between relative marker signal intensities, an unpaired samples t-test or Wilcoxon rank sum test were computed. Data were subjected to tSNE, heatmap clustering, and correlation analysis to further explore the relationships between disease state and EV Neuro data. RESULTS: Glioblastoma cell lines and primary human astrocytes showed distinct EV profiles. Signal intensities were increasing with higher EV input. Data normalization improved identification of markers that deviate from a common profile. Overall, patient blood-derived EV marker profiles were constant, but individual EV populations were significantly increased in disease compared to healthy controls, e.g. CD36+EVs in glioblastoma and GALC+EVs in multiple sclerosis. tSNE and heatmap clustering analysis separated GB patients from HC, but not MS patients from HC. Correlation analysis revealed a potential association of CD107a+EVs with neurofilament levels in blood of MS patients and HC. CONCLUSIONS: The semi-quantitative EV Neuro assay demonstrated its utility for EV profiling in complex samples. However, reliable statistical results in biomarker studies require large sample cohorts and high effect sizes. Nonetheless, this exploratory trial confirmed the feasibility of discovering EV-associated biomarkers and monitoring circulating EV profiles in CNS diseases using the EV Neuro assay. Video Abstract.

Extracellular vesicles (EVs) are tiny particles released by cells, carrying unique biomolecules specific to their cell of origin. EVs from the central nervous system (CNS) can reach the blood, where they could serve as liquid-biopsy markers for diagnosing brain diseases like neurodegenerative disorders and tumors. This study evaluated a flow cytometry platform (here termed EV Neuro assay), which can detect multiple EV-associated markers simultaneously, to assess its potential for identifying CNS-derived EVs and disease-specific markers in complex samples including the blood. The study compared different sample materials and methods for isolating EVs. We found distinct EV profiles in EVs derived from glioblastoma and human astrocytes, with signal intensities increasing as more EVs were present. Analyzing serum or plasma from patients with brain diseases and healthy individuals, we observed that EV marker intensities were varying between individuals. Importantly, data normalization improved the identification of disease-specific markers, such as CD36⁺EVs in glioblastoma and GALC⁺EVs in multiple sclerosis, which were significantly higher in disease compared to healthy controls. Advanced clustering analysis techniques effectively distinguished glioblastoma patients from controls. Furthermore, a potential correlation between CD107a⁺EVs and neurofilament levels in multiple sclerosis patients was discovered. Overall, the semi-quantitative EV Neuro assay proved useful for profiling EVs in complex samples. However, for more reliable results in biomarker studies, larger sample cohorts and higher effect sizes are necessary. Nonetheless, this initial trial confirmed the potential of the EV Neuro assay for discovering disease-associated EV markers and monitoring circulating EV profiles in CNS diseases.

Oligodendrocytes support axonal transport and maintenance via exosome secretion.

Neurons extend long axons that require maintenance and are susceptible to degeneration. Long-term integrity of axons depends on intrinsic mechanisms including axonal transport and extrinsic support from adjacent glial cells. The mechanisms of support provided by myelinating oligodendrocytes to underlying axons are only partly understood. Oligodendrocytes release extracellular vesicles (EVs) with properties of exosomes, which upon delivery to neurons improve neuronal viability in vitro. Here, we show that oligodendroglial exosome secretion is impaired in 2 mouse mutants exhibiting secondary axonal degeneration due to oligodendrocyte-specific gene defects. Wild-type oligodendroglial exosomes support neurons by improving the metabolic state and promoting axonal transport in nutrient-deprived neurons. Mutant oligodendrocytes release fewer exosomes, which share a common signature of underrepresented proteins. Notably, mutant exosomes lack the ability to support nutrient-deprived neurons and to promote axonal transport. Together, these findings indicate that glia-to-neuron exosome transfer promotes neuronal long-term maintenance by facilitating axonal transport, providing a novel mechanistic link between myelin diseases and secondary loss of axonal integrity.

Dual role of the RNA helicase DDX5 in post-transcriptional regulation of myelin basic protein in oligodendrocytes.

In the central nervous system, oligodendroglial expression of myelin basic protein (MBP) is crucial for the assembly and structure of the myelin sheath. MBP synthesis is tightly regulated in space and time, particularly at the post-transcriptional level. We have identified the DEAD-box RNA helicase DDX5 (also known as p68) in a complex with Mbp mRNA in oligodendroglial cells. Expression of DDX5 is highest in progenitor cells and immature oligodendrocytes, where it localizes to heterogeneous populations of cytoplasmic ribonucleoprotein (RNP) complexes associated with Mbp mRNA in the cell body and processes. Manipulation of the amount of DDX5 protein inversely affects the level of MBP. We present evidence that DDX5 is involved in post-transcriptional regulation of MBP protein synthesis, with implications for oligodendroglial development. In addition, knockdown of DDX5 results in an increased abundance of MBP isoforms containing exon 2 in immature oligodendrocytes, most likely by regulating alternative splicing of Mbp Our findings contribute to the understanding of the complex nature of MBP post-transcriptional control in immature oligodendrocytes where DDX5 appears to affect the abundance of MBP proteins via distinct but converging mechanisms.

Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte-neuron communication.

Reciprocal interactions between neurons and oligodendrocytes are not only crucial for myelination, but also for long-term survival of axons. Degeneration of axons occurs in several human myelin diseases, however the molecular mechanisms of axon-glia communication maintaining axon integrity are poorly understood. Here, we describe the signal-mediated transfer of exosomes from oligodendrocytes to neurons. These endosome-derived vesicles are secreted by oligodendrocytes and carry specific protein and RNA cargo. We show that activity-dependent release of the neurotransmitter glutamate triggers oligodendroglial exosome secretion mediated by Ca²âº entry through oligodendroglial NMDA and AMPA receptors. In turn, neurons internalize the released exosomes by endocytosis. Injection of oligodendroglia-derived exosomes into the mouse brain results in functional retrieval of exosome cargo in neurons. Supply of cultured neurons with oligodendroglial exosomes improves neuronal viability under conditions of cell stress. These findings indicate that oligodendroglial exosomes participate in a novel mode of bidirectional neuron-glia communication contributing to neuronal integrity.

Emerging roles of extracellular vesicles in the nervous system.

Information exchange executed by extracellular vesicles, including exosomes, is a newly described form of intercellular communication important in the development and physiology of neural systems. These vesicles can be released from cells, are packed with information including signaling proteins and both coding and regulatory RNAs, and can be taken up by target cells, thereby facilitating the transfer of multilevel information. Recent studies demonstrate their critical role in physiological processes, including nerve regeneration, synaptic function, and behavior. These vesicles also have a sinister role in the propagation of toxic amyloid proteins in neurodegenerative conditions, including prion diseases and Alzheimer's and Parkinson's diseases, in inducing neuroinflammation by exchange of information between the neurons and glia, as well as in aiding tumor progression in the brain by subversion of normal cells. This article provides a summary of topics covered in a symposium and is not meant to be a comprehensive review of the subject.

Vesiclepedia: a compendium for extracellular vesicles with continuous community annotation.

Extracellular vesicles (EVs) are membraneous vesicles released by a variety of cells into their microenvironment. Recent studies have elucidated the role of EVs in intercellular communication, pathogenesis, drug, vaccine and gene-vector delivery, and as possible reservoirs of biomarkers. These findings have generated immense interest, along with an exponential increase in molecular data pertaining to EVs. Here, we describe Vesiclepedia, a manually curated compendium of molecular data (lipid, RNA, and protein) identified in different classes of EVs from more than 300 independent studies published over the past several years. Even though databases are indispensable resources for the scientific community, recent studies have shown that more than 50% of the databases are not regularly updated. In addition, more than 20% of the database links are inactive. To prevent such database and link decay, we have initiated a continuous community annotation project with the active involvement of EV researchers. The EV research community can set a gold standard in data sharing with Vesiclepedia, which could evolve as a primary resource for the field.

Release of bulk cell free DNA during physical exercise occurs independent of extracellular vesicles.

PURPOSE: Strenuous exercise induces a rapid and transient elevation of cell free DNA (cfDNA) concentration in blood plasma. The detection of cfDNA in the presence of plasma nucleases could indicate an association of cfDNA with protective vesicular structures. Several cell types release extracellular vesicles (EVs), including exosomes and shedding microvesicles, which are known to mediate the exchange of proteins and nucleic acids (largely RNA) between cells. Here, we assessed whether EVs play a role in the exercise-dependent release of cfDNA in blood plasma. METHODS: Venous blood collected from healthy volunteers before and after incremental treadmill exercise was separated into vesicular (EV) and soluble fractions. Nuclear and mitochondrial DNA content in plasma supernatants and EV fractions was determined by quantitative real-time PCR (qPCR). RESULTS: We show that the majority of cfDNA is located in the plasma supernatants. Only minute amounts of DNA were observed in the EV-associated fractions including microvesicles and exosomes. Nuclear and mitochondrial DNA species differ in terms of their quantities in the several plasma fractions. CONCLUSIONS: Our results indicate that cfDNA liberated in response to acute physical exercise is not released by vesicular means and circulates in a soluble form in blood plasma which could indicate different biological functions exerted by cfDNA and EVs. The different nature of DNA species in plasma has major implications for the preparation of plasma and other bodily fluids prior to analysis.

Heterogeneous nuclear ribonucleoprotein (hnRNP) F is a novel component of oligodendroglial RNA transport granules contributing to regulation of myelin basic protein (MBP) synthesis.

Myelin basic protein (MBP) is a major component of central nervous system (CNS) myelin. The absence of MBP results in the loss of almost all compact myelin in the CNS. MBP mRNA is sorted into RNA granules that are transported to the periphery of oligodendrocytes in a translationally inactive state. A central mediator of this transport process is the trans-acting factor heterogeneous nuclear ribonucleoprotein (hnRNP) A2 that binds to the cis-acting A2-response element in the 3'UTR of MBP mRNA. Recently, we found that activation of the Src family nonreceptor tyrosine kinase Fyn in oligodendrocytes leads to phosphorylation of hnRNP A2 and to increased translation of MBP mRNA. Here, we identify the RNA-binding protein hnRNP F as a novel component of MBP mRNA transport granules. It is associated with hnRNP A2 and MBP mRNA in cytoplasmic granular structures and is involved in post-transcriptional regulation of MBP expression. Fyn kinase activity results in phosphorylation of hnRNP F in the cytoplasm and its release from MBP mRNA and RNA granules. Our results define hnRNP F as a regulatory element of MBP expression in oligodendrocytes and imply an important function of hnRNP F in the control of myelin synthesis.

A critical role for the cholesterol-associated proteolipids PLP and M6B in myelination of the central nervous system.

The formation of central nervous system myelin by oligodendrocytes requires sterol synthesis and is associated with a significant enrichment of cholesterol in the myelin membrane. However, it is unknown how oligodendrocytes concentrate cholesterol above the level found in nonmyelin membranes. Here, we demonstrate a critical role for proteolipids in cholesterol accumulation. Mice lacking the most abundant myelin protein, proteolipid protein (PLP), are fully myelinated, but PLP-deficient myelin exhibits a reduced cholesterol content. We therefore hypothesized that "high cholesterol" is not essential in the myelin sheath itself but is required for an earlier step of myelin biogenesis that is fully compensated for in the absence of PLP. We also found that a PLP-homolog, glycoprotein M6B, is a myelin component of low abundance. By targeting the Gpm6b-gene and crossbreeding, we found that single-mutant mice lacking either PLP or M6B are fully myelinated, while double mutants remain severely hypomyelinated, with enhanced neurodegeneration and premature death. As both PLP and M6B bind membrane cholesterol and associate with the same cholesterol-rich oligodendroglial membrane microdomains, we suggest a model in which proteolipids facilitate myelination by sequestering cholesterol. While either proteolipid can maintain a threshold level of cholesterol in the secretory pathway that allows myelin biogenesis, lack of both proteolipids results in a severe molecular imbalance of prospective myelin membrane. However, M6B is not efficiently sorted into mature myelin, in which it is 200-fold less abundant than PLP. Thus, only PLP contributes to the high cholesterol content of myelin by association and co-transport.

Uptake of extracellular vesicles into immune cells is enhanced by the protein corona.

The influence of a protein corona on the uptake of nanoparticles in cells has been demonstrated in various publications over the last years. Extracellular vesicles (EVs), can be seen as natural nanoparticles. However, EVs are produced under different cell culture conditions and little is known about the protein corona forming on EVs and its influence on their uptake by target cells. Here, we use a proteomic approach in order to analyze the protein composition of the EVs themselves and the protein composition of a human blood plasma protein corona around EVs. Moreover, we analyze the influence of the protein corona on EV uptake into human monocytes and compare it with the influence on the uptake of engineered liposomes. We show that the presence of a protein corona increases the uptake of EVs in human monocytes. While for liposomes this seems to be triggered by the presence of immunoglobulins in the protein corona, for EVs blocking the Fc receptors on monocytes did not show an influence of uptake. Therefore, other mechanisms of docking to the cell membrane and uptake are most like involved, demonstrating a clear difference between EVs and liposomes as technically produced nanocarriers.

Transport of the major myelin proteolipid protein is directed by VAMP3 and VAMP7.

CNS myelination by oligodendrocytes requires directed transport of myelin membrane components and a timely and spatially controlled membrane expansion. In this study, we show the functional involvement of the R-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE) proteins VAMP3/cellubrevin and VAMP7/TI-VAMP in myelin membrane trafficking. VAMP3 and VAMP7 colocalize with the major myelin proteolipid protein (PLP) in recycling endosomes and late endosomes/lysosomes, respectively. Interference with VAMP3 or VAMP7 function using small interfering RNA-mediated silencing and exogenous expression of dominant-negative proteins diminished transport of PLP to the oligodendroglial cell surface. In addition, the association of PLP with myelin-like membranes produced by oligodendrocytes cocultured with cortical neurons was reduced. We furthermore identified Syntaxin-4 and Syntaxin-3 as prime acceptor Q-SNAREs of VAMP3 and VAMP7, respectively. Analysis of VAMP3-deficient mice revealed no myelination defects. Interestingly, AP-3δ-deficient mocha mice, which suffer from impaired secretion of lysosome-related organelles and missorting of VAMP7, exhibit a mild dysmyelination characterized by reduced levels of select myelin proteins, including PLP. We conclude that PLP reaches the cell surface via at least two trafficking pathways with distinct regulations: (1) VAMP3 mediates fusion of recycling endosome-derived vesicles with the oligodendroglial plasma membrane in the course of the secretory pathway; (2) VAMP7 controls exocytosis of PLP from late endosomal/lysosomal organelles as part of a transcytosis pathway. Our in vivo data suggest that exocytosis of lysosome-related organelles controlled by VAMP7 contributes to myelin biogenesis by delivering cargo to the myelin membrane.

From axon-glial signalling to myelination: the integrating role of oligodendroglial Fyn kinase.

Central nervous system myelination requires recognition and signalling processes between neuronal axons and oligodendrocytes. Complex cellular rearrangements occur in myelination-competent oligodendrocytes requiring spatio-temporal control mechanisms. Although the molecular repertoire is becoming increasingly transparent, the signalling mechanisms governing myelination initiation are only poorly understood. The non-receptor tyrosine kinase Fyn has been implicated in axon-glial signal transduction and in several cellular processes required for oligodendrocyte maturation and myelination. Here, we review oligodendroglial Fyn signalling and discuss the role of Fyn in axon-glia interaction mediating myelination.

Extracellular Vesicles at CNS barriers: Mode of action.

The exchange of molecules between the brain and periphery is limited by cellular barriers such as the blood-brain barrier (BBB) and the blood-CSF barrier (BCB). Extracellular vesicles (EVs) secreted by brain cells or circulating in the blood stream interact with these barriers and provide a pathway for brain-periphery communication. This review briefly summarizes the main current concepts of EVs signaling over the BBB/BCB. EVs can either be released by barrier cells upon stimulation, act on barrier cells modulating barrier properties, or cross the barrier transferring cargo between the circulation and the brain. The mechanisms of EV signaling and passage over the BBB are increasingly being explored, with inflammation being a main driver. EVs acting at or through the barriers possess wide-ranging effects on brain-periphery communication in both healthy and pathological states. A deeper understanding of the mechanisms of action is important for translation into biomedical applications for brain diseases.

Progressive axonopathy when oligodendrocytes lack the myelin protein CMTM5.

Oligodendrocytes facilitate rapid impulse propagation along the axons they myelinate and support their long-term integrity. However, the functional relevance of many myelin proteins has remained unknown. Here, we find that expression of the tetraspan-transmembrane protein CMTM5 (chemokine-like factor-like MARVEL-transmembrane domain containing protein 5) is highly enriched in oligodendrocytes and central nervous system (CNS) myelin. Genetic disruption of the Cmtm5 gene in oligodendrocytes of mice does not impair the development or ultrastructure of CNS myelin. However, oligodendroglial Cmtm5 deficiency causes an early-onset progressive axonopathy, which we also observe in global and tamoxifen-induced oligodendroglial Cmtm5 mutants. Presence of the WldS mutation ameliorates the axonopathy, implying a Wallerian degeneration-like pathomechanism. These results indicate that CMTM5 is involved in the function of oligodendrocytes to maintain axonal integrity rather than myelin biogenesis.

Superfood for axons: Glial exosomes boost axonal energetics by delivery of SIRT2.

Axon integrity depends on support by glia facilitating axonal maintenance and energy homeostasis, but the molecular mechanisms are not well understood. In this issue of Neuron, Chamberlain et al. (2021) provide evidence that oligodendrocyte-to-axon transfer of SIRT2 via extracellular vesicles (exosomes) enables deacetylation of mitochondrial proteins, enhancing axonal energy production.

Extracellular Vesicles in neural cell interaction and CNS homeostasis.

Central nervous system (CNS) homeostasis critically depends on the interaction between neurons and glia cells. Extracellular vesicles (EVs) recently emerged as versatile messengers in CNS cell communication. EVs are released by neurons and glia in activity-dependent manner and address multiple target cells within and outside the nervous system. Here, we summarize the recent advances in understanding the physiological roles of EVs in the nervous system and their ability to deliver signals across the CNS barriers. In addition to the disposal of cellular components via EVs and clearance by phagocytic cells, EVs are involved in plasticity-associated processes, mediate trophic support and neuroprotection, promote axonal maintenance, and modulate neuroinflammation. While individual functional components of the EV cargo are becoming progressively identified, the role of neural EVs as compound multimodal signaling entities remains to be elucidated. Novel transgenic models and imaging technologies allow EV tracking in vivo and provide further insight into EV targeting and their mode of action. Overall, EVs represent key players in the maintenance of CNS homeostasis essential for the lifelong performance of neural networks and thus provide a wide spectrum of biomedical applications.

Kinetics and Topology of DNA Associated with Circulating Extracellular Vesicles Released during Exercise.

Although it is widely accepted that cancer-derived extracellular vesicles (EVs) carry DNA cargo, the association of cell-free circulating DNA (cfDNA) and EVs in plasma of healthy humans remains elusive. Using a physiological exercise model, where EVs and cfDNA are synchronously released, we aimed to characterize the kinetics and localization of DNA associated with EVs. EVs were separated from human plasma using size exclusion chromatography or immuno-affinity capture for CD9+, CD63+, and CD81+ EVs. DNA was quantified with an ultra-sensitive qPCR assay targeting repetitive LINE elements, with or without DNase digestion. This model shows that a minute part of circulating cell-free DNA is associated with EVs. During rest and following exercise, only 0.12% of the total cfDNA occurs in association with CD9+/CD63+/CD81+EVs. DNase digestion experiments indicate that the largest part of EV associated DNA is sensitive to DNase digestion and only ~20% are protected within the lumen of the separated EVs. A single bout of running or cycling exercise increases the levels of EVs, cfDNA, and EV-associated DNA. While EV surface DNA is increasing, DNAse-resistant DNA remains at resting levels, indicating that EVs released during exercise (ExerVs) do not contain DNA. Consequently, DNA is largely associated with the outer surface of circulating EVs. ExerVs recruit cfDNA to their corona, but do not carry DNA in their lumen.