Extracellular vesicles (EVs) are communication channels between different cell types in the brain, between the brain and the periphery and vice-versa, playing a fundamental role in physiology and pathology. The evidence that EVs might be able to cross the blood-brain barrier (BBB) make them very promising candidates as nanocarriers to treat brain pathologies. EVs contain a cocktail of bioactive factors, yet their content and surface can be further engineered to enhance their biological activity, stability and targeting ability. Native and engineered EVs have been reported for the treatment of different brain pathologies, although issues related to their modest accumulation and limited local therapeutic effect in the brain still need to be addressed. In this review, we cover the therapeutic applications of native and bioengineered EVs for brain diseases. We also review recent data about the interaction between EVs and the BBB and discuss the challenges and opportunities in clinical translation of EVs as brain therapeutics.Copyright © 2021. Published by Elsevier B.V.

Extracellular vesicles (EVs) derived from neural stem cells (NSC-EVs), astrocytes (ADEVs), and microglia (MDEVs) have neuroregenerative properties. This review discusses the therapeutic efficacy of NSC-EVs, ADEVs, and MDEVs in traumatic brain injury (TBI) models. The translational value and future directions for such EV therapy are also deliberated. Studies have demonstrated that NSC-EV or ADEV therapy can mediate neuroprotective effects and improve motor and cognitive function after TBI. Furthermore, NSC-EVs or ADEVs generated after priming parental cells with growth factors or brain-injury extracts can mediate improved therapeutic benefits. However, the therapeutic effects of naïve MDEVs are yet to be tested rigorously in TBI models. Studies using activated MDEVs have reported both adverse and beneficial effects. NSC-EV, ADEV, or MDEV therapy for TBI is not ready for clinical translation. Rigorous testing of their efficacy for preventing chronic neuroinflammatory cascades and enduring motor and cognitive impairments after treatment in the acute phase of TBI, an exhaustive evaluation of their miRNA or protein cargo, and the effects of delayed EV administration post-TBI for reversing chronic neuroinflammation and enduring brain impairments, are needed. Moreover, the most beneficial route of administration for targeting EVs into different neural cells in the brain after TBI and the efficacy of well-characterized EVs from NSCs, astrocytes, or microglia derived from human pluripotent stem cells need to be evaluated. EV isolation methods for generating clinical-grade EVs must also be developed. Overall, NSC-EVs and ADEVs promise to mitigate TBI-induced brain dysfunction, but additional preclinical studies are needed before their clinical translation.© The Author(s) 2023. Published by Oxford University Press.

Recent studies suggest that astrocytes released a great quantity of extracellular vesicles (AEVs) to communicate with other brain cells. Under pathological conditions, AEVs are widely associated with the pathogenesis of neurobiological diseases by horizontally transferring pathogenic factors to neighboring cells or peripheral immune cells. Their beneficial role is also evident by the fact that they are involved in neuroprotection and neuroregeneration through alleviating apoptosis, maintaining neuronal function, and repairing neural injuries. The strong association of AEVswith neurological disorders makes AEVs a promising target for disease diagnosis, treatment, and prevention. The identification of disease-specific cargos in AEVs isolated from the patients' biofluids suggests AEVs as an attractive platform for biomarker development. Furthermore, the inhibition of inflammatory/toxic AEV release and the preservation of neuroprotective AEV release have been considered as potential therapeutic strategies in CNS disorder treatment and prevention, respectively. Here, we summarize the biological roles of AEVs as pathological contributors, protective/regenerative factors, and potential diagnostic biomarkers and therapeutic targets for neurological disorders, with a focus on recent progresses and emerging concepts.Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cognitive dysfunction and neuroinflammation are conspicuously observed in Gulf War Illness (GWI). We investigated whether brain inflammation in GWI is associated with activation of high mobility group box-1 (HMGB1) and complement-related proteins in neurons and astrocytes, and brain inflammation can be tracked through neuron-derived extracellular vesicles (NDEVs) and astrocyte-derived EVs (ADEVs) found in the circulating blood. We exposed animals to GWI-related chemicals pyridostigmine bromide, DEET and permethrin, and moderate stress for 28 days. We performed behavioral tests 10 months post-exposure and quantified activated microglia and reactive astrocytes in the cerebral cortex. Then, we measured the concentration of HMGB1, proinflammatory cytokines, and complement activation-related proteins in the cerebral cortex, and NDEVs and ADEVs in the circulating blood. Cognitive impairments persisted in GWI rats at 10 months post-exposure, which were associated with increased density of activated microglia and reactive astrocytes in the cerebral cortex. Moreover, the level of HMGB1 was elevated in the cerebral cortex with altered expression in the cytoplasm of neuronal soma and dendrites as well as the extracellular space. Also, higher levels of proinflammatory cytokines (TNFa, IL-1b, and IL-6), and complement activation-related proteins (C3 and TccC5b-9) were seen in the cerebral cortex. Remarkably, increased levels of HMGB1 and proinflammatory cytokines observed in the cerebral cortex of GWI rats could also be found in NDEVs isolated from the blood. Similarly, elevated levels of complement proteins seen in the cerebral cortex could be found in ADEVs. The results provide new evidence that persistent cognitive dysfunction and chronic neuroinflammation in a model of GWI are linked with elevated HMGB1 concentration and complement activation. Furthermore, the results demonstrated that multiple biomarkers of neuroinflammation could be tracked reliably via analyses of NDEVs and ADEVs in the circulating blood. Execution of such a liquid biopsy approach is especially useful in clinical trials for monitoring the remission, persistence or progression of brain inflammation in GWI patients with drug treatment.Published by Elsevier Inc.

Astrocytes fulfill neuronal trophic roles normally, but are transformed in Alzheimer disease (AD) into A1-type reactive astrocytes that may destroy neurons through unknown mechanisms.To investigate astrocyte inflammatory mechanisms, astrocyte-derived exosomes (ADEs) were isolated immunochemically from plasma samples of AD patients and matched controls for enzyme-linked immunosorbent assay quantification of complement proteins.ADE levels of C1q, C4b, C3d, factor B, factor D, Bb, C3b, and C5b-C9 terminal complement complex, but not mannose-binding lectin, normalized by the CD81 exosome marker were significantly higher for AD patients (n = 28) than age- and gender-matched controls (all p < 0.0001). ADE normalized levels of interleukin (IL)-6, tumor necrosis factor-α, and IL-1β were significantly higher for AD patients than controls, but there was greater overlap between the two groups than for complement proteins. Mean ADE levels of complement proteins for AD patients in a longitudinal study were significantly higher (n = 16, p < 0.0001) at the AD2 stage of moderate dementia than at the AD1 preclinical stage 5 to 12 years earlier, which were the same as for controls. ADE levels of complement regulatory proteins CD59, CD46, decay-accelerating factor (DAF), and complement receptor type 1, but not factor I, were significantly lower for AD patients than controls (p < 0.0001 for CD59 and DAF), were diminished by the AD1 stage, and were further decreased at the AD2 stage.ADE complement effector proteins in AD are produced by dysregulated systems, attain higher levels than in controls, and may potentially damage neurons in the late inflammatory phase of AD. Ann Neurol 2018;83:544-552.© 2018 American Neurological Association.

Synapse loss in Alzheimer's disease (AD) correlates with cognitive decline. Involvement of microglia and complement in AD has been attributed to neuroinflammation, prominent late in disease. Here we show in mouse models that complement and microglia mediate synaptic loss early in AD. C1q, the initiating protein of the classical complement cascade, is increased and associated with synapses before overt plaque deposition. Inhibition of C1q, C3, or the microglial complement receptor CR3 reduces the number of phagocytic microglia, as well as the extent of early synapse loss. C1q is necessary for the toxic effects of soluble β-amyloid (Aβ) oligomers on synapses and hippocampal long-term potentiation. Finally, microglia in adult brains engulf synaptic material in a CR3-dependent process when exposed to soluble Aβ oligomers. Together, these findings suggest that the complement-dependent pathway and microglia that prune excess synapses in development are inappropriately activated and mediate synapse loss in AD. Copyright © 2016, American Association for the Advancement of Science.

Our previous study showed that neuronal apoptosis was significantly increased upon treatment of conditioned medium (CM) from necroptotic astrocytes (NAS), leaving the underlying mechanism unclear. Considering the nutritive and supportive roles of astrocytes, we first examined the neurotrophic phenotype of necroptotic astrocytes with focus on glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), two important neurotrophic factors, and it was unexpectedly found that the expression of GDNF and BDNF were up-regulated in necroptotic astrocytes in vitro. A question was raised as to whether the functional secreted forms of neurotrophic factors were increased. Considering that extracellular vesicles (EVs) were carriers of secreted substances and their roles in cellular interaction, we isolated EVs from astrocytes and found EVs from normal and necroptotic astrocytes (EVs-NAS) had characteristics of exosomes. We then examined GDNF and BDNF in EVs-NAS, and BDNF was interestingly found as an immature form of pro-BDNF. The expression of pro-BDNF was found to be increased in EVs-NAS, and EVs-NAS had a negative effect on neuronal survival. To verify that whether pro-BDNF was involved in the detrimental effect of EVs-NAS, anti-pro-BDNF antibody was applied, and we found that neuronal apoptosis-induced by EVs-NAS could be significantly attenuated by blocking pro-BDNF, which suggested that necroptotic astrocytes induced neuronal apoptosis partially through EVs-derived pro-BDNF. The data expand our understanding in neurotrophic phenotype of necroptotic astrocytes, and may provide us new strategies targeting on EVs-NAS in treatment of neurological diseases.Copyright © 2021. Published by Elsevier Inc.

Alzheimer's disease (AD) is the most common neurodegenerative disturbances. Dysfunction of synaptic plasticity and decline in cognitive functions are the most prominent features of AD, but the mechanisms of pathogenesis have not been well elucidated. In this paper, transforming growth factor-β1 (TGF-β1) was found to be reduced in the hippocampus of AD mouse which was accompanied by impaired pine density, synaptic plasticity, and memory function. Hippocampal injection of TGF-β1 rescued the AD-induced memory function impairment. In addition, TGF-β1 ameliorated synaptic plasticity and increased synaptic plasticity-associated protein expression including Arc, NR2B, and PSD-95 in mouse model of AD. Furthermore, we demonstrated that Akt/Wnt/β-catenin pathway protein expression in the hippocampus was suppressed in a mouse model of AD and TGF-β1 significantly enhanced the phosphorylation Akt, GSK3β, and increased the nuclear β-catenin. These results indicate that TGF-β1activates PI3K/Akt/Wnt/β-catenin signaling in mouse model of AD, which is important for promoting synaptic plasticity related to memory function. More importantly, suppression of PI3K/Akt/Wnt/β-catenin pathway compromised the beneficial effects of TGFβ1 in Alzheimer's model. Hence, TGF-β1 shows protective effect on neurons, which might be through the PI3K/Akt/Wnt/β-catenin signaling pathway, serving as a potential target in AD pathology.

The homeostasis of metal ions, such as iron, copper, zinc and calcium, in the brain is crucial for maintaining normal physiological functions. Studies have shown that imbalance of these metal ions in the brain is closely related to the onset and progression of Alzheimer's disease (AD), the most common neurodegenerative disorder in the elderly.Erroneous deposition/distribution of the metal ions in different brain regions induces oxidative stress. The metal ions imbalance and oxidative stress together or independently promote amyloid-β (Aβ) overproduction by activating β- or γ-secretases and inhibiting α-secretase, it also causes tau hyperphosphorylation by activating protein kinases, such as glycogen synthase kinase-3β (GSK-3β), cyclin-dependent protein kinase-5 (CDK5), mitogen-activated protein kinases (MAPKs), etc., and inhibiting protein phosphatase 2A (PP2A). The metal ions imbalances can also directly or indirectly disrupt organelles, causing endoplasmic reticulum (ER) stress; mitochondrial and autophagic dysfunctions, which can cause or aggravate Aβ and tau aggregation/accumulation, and impair synaptic functions. Even worse, the metal ions imbalance-induced alterations can reversely exacerbate metal ions misdistribution and deposition. The vicious cycles between metal ions imbalances and Aβ/tau abnormalities will eventually lead to a chronic neurodegeneration and cognitive deficits, such as seen in AD patients.The metal ions imbalance induces Aβ and tau pathologies by directly or indirectly affecting multiple cellular/subcellular pathways, and the disrupted homeostasis can reversely aggravate the abnormalities of metal ions transportation/deposition. Therefore, adjusting metal balance by supplementing or chelating the metal ions may be potential in ameliorating AD pathologies, which provides new research directions for AD treatment.© The Author(s) 2020.

The prion protein (PrP) plays a key role in the pathogenesis of prion diseases. However, the normal function of the protein remains unclear. The cellular isoform (PrP(C)) is expressed most abundantly in the brain, but has also been detected in other non-neuronal tissues as diverse as lymphoid cells, lung, heart, kidney, gastrointestinal tract, muscle, and mammary glands. Cell biological studies of PrP contribute to our understanding of PrP(C) function. Like other membrane proteins, PrP(C) is post-translationally processed in the endoplasmic reticulum and Golgi on its way to the cell surface after synthesis. Cell surface PrP(C) constitutively cycles between the plasma membrane and early endosomes via a clathrin-dependent mechanism, a pathway consistent with a suggested role for PrP(C) in cellular trafficking of copper ions. Although PrP(-/-) mice have been reported to have only minor alterations in immune function, PrP(C) is up-regulated in T cell activation and may be expressed at higher levels by specialized classes of lymphocytes. Furthermore, antibody cross-linking of surface PrP(C) modulates T cell activation and leads to rearrangements of lipid raft constituents and increased phosphorylation of signaling proteins. These findings appear to indicate an important but, as yet, ill-defined role in T cell function. Recent work has suggested that PrP(C) is required for self-renewal of haematopoietic stem cells. PrP(C) is highly expressed in the central nervous system, and since this is the major site of prion pathology, most interest has focused on defining the role of PrP(C) in neurones. Although PrP(-/-) mice have a grossly normal neurological phenotype, even when neuronal PrP(C) is knocked out postnatally, they do have subtle abnormalities in synaptic transmission, hippocampal morphology, circadian rhythms, and cognition and seizure threshold. Other postulated neuronal roles for PrP(C) include copper-binding, as an anti- and conversely, pro-apoptotic protein, as a signaling molecule, and in supporting neuronal morphology and adhesion. The prion protein may also function as a metal binding protein such as copper, yielding cellular antioxidant capacity suggesting a role in the oxidative stress homeostasis. Finally, recent observations on the role of PrP(C) in long-term memory open a challenging field.

Prion protein (PrP) protects neural cells against oxidative stress, hypoxia, ischemia, and hypoglycemia. In the present study we confirm that cultured PrP-deficient neurons are more sensitive to oxidative stress than wild-type neurons and present the novel findings that wild-type, but not PrP-deficient astrocytes protect wild-type cerebellar neurons against oxidative stress and that exosomes released from stressed wild-type, but not from stressed PrP-deficient astrocytes reduce neuronal cell death induced by oxidative stress. We show that neuroprotection by exosomes of stressed astrocytes depends on exosomal PrP but not on neuronal PrP and that astrocyte-derived exosomal PrP enters into neurons, suggesting neuronal uptake of astrocyte-derived exosomes. Upon exposure of wild-type astrocytes to hypoxic or ischemic conditions PrP levels in exosomes were increased. By mass spectrometry and Western blot analysis, we detected increased levels of 37/67 kDa laminin receptor, apolipoprotein E and the ribosomal proteins S3 and P0, and decreased levels of clusterin/apolipoprotein J in exosomes from wild-type astrocytes exposed to oxygen/glucose deprivation relative to exosomes from astrocytes maintained under normoxic conditions. The levels of these proteins were not altered in exosomes from stressed PrP-deficient astrocytes relative to unstressed PrP-deficient astrocytes. These results indicate that PrP in astrocytes is a sensor for oxidative stress and mediates beneficial cellular responses, e.g. release of exosomes carrying PrP and other molecules, resulting in improved survival of neurons under hypoxic and ischemic conditions.© 2015 Wiley Periodicals, Inc.

Alzheimer disease (AD) is the most common form of neurodegenerative disease, estimated to contribute 60-70% of all cases of dementia worldwide. According to the prevailing amyloid cascade hypothesis, amyloid-β (Aβ) deposition in the brain is the initiating event in AD, although evidence is accumulating that this hypothesis is insufficient to explain many aspects of AD pathogenesis. The discovery of increased levels of inflammatory markers in patients with AD and the identification of AD risk genes associated with innate immune functions suggest that neuroinflammation has a prominent role in the pathogenesis of AD. In this Review, we discuss the interrelationships between neuroinflammation and amyloid and tau pathologies as well as the effect of neuroinflammation on the disease trajectory in AD. We specifically focus on microglia as major players in neuroinflammation and discuss the spatial and temporal variations in microglial phenotypes that are observed under different conditions. We also consider how these cells could be modulated as a therapeutic strategy for AD.

Multi-pathway approaches for the treatment of complex polygenic disorders are emerging as alternatives to classical monotarget therapies and microRNAs are of particular interest in that regard. MicroRNA research has come a long way from their initial discovery to the cumulative appreciation of their regulatory potential in healthy and diseased brain. However, systematic interrogation of putative therapeutic or toxic effects of microRNAs in (models of) Alzheimer's disease is currently missing and fundamental research findings are yet to be translated into clinical applications. Here, we review the literature to summarize the knowledge on microRNA regulation in Alzheimer's pathophysiology and to critically discuss whether and to what extent these increasing insights can be exploited for the development of microRNA-based therapeutics in the clinic.© 2021. The Author(s).

Current evidence indicates that extracellular vesicles (EVs) participate in intercellular signaling, and in the regulation and amplification of neuroinflammation. We have previously shown that ethanol activates glial cells through Toll-like receptor 4 (TLR4) by triggering neuroinflammation. Here, we evaluate if ethanol and the TLR4 response change the release and inflammatory content of astrocyte-derived EVs, and whether these vesicles are capable of communicating with neurons by spreading neuroinflammation.Cortical neurons and astrocytes in culture were used. EVs were isolated from the extracellular medium of the primary culture of the WT and TLR4-KO astrocytes treated with or without ethanol (40 mM) for 24 h. Flow cytometry, nanoparticle tracking analysis technology, combined with exosomal molecular markers (tetraspanins) along with electron microscopy, were used to characterize and quantify EVs. The content of EVs in inflammatory proteins, mRNA, and miRNAs was analyzed by Western blot and RT-PCR in both astrocyte-derived EVs and the neurons incubated or not with these EVs. Functional analyses of miRNAs were also performed.We show that ethanol increases the number of secreted nanovesicles and their content by raising the levels of both inflammatory-related proteins (TLR4, NFκB-p65, IL-1R, caspase-1, NLRP3) and by changing miRNAs (mir-146a, mir-182, and mir-200b) in the EVs from the WT-astrocytes compared with those from the untreated WT cells. No changes were observed in either the number of isolated EVs or their content between the untreated and ethanol-treated TLR4-KO astrocytes. We also show that astrocyte-derived EVs could be internalized by naïve cortical neurons to increase the neuronal levels of inflammatory protein (COX-2) and miRNAs (e.g., mir-146a) and to compromise their survival. The functional analysis of miRNAs revealed the regulatory role of the expressed miRNAs in some genes involved in several inflammatory pathways.These results suggest that astrocyte-derived EVs could act as cellular transmitters of inflammation signaling by spreading and amplifying the neuroinflammatory response induced by ethanol through TLR4 activation.

Extracellular vesicles (EVs) are small bilipid layer-enclosed vesicles that can be secreted by all tested types of brain cells. Being a key intercellular communicator, EVs have emerged as a key contributor to the pathogenesis of various neurodegenerative diseases (NDs) including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease through delivery of bioactive cargos within the central nervous system (CNS). Importantly, CNS cell-derived EVs can be purified via immunoprecipitation, and EV cargos with altered levels have been identified as potential biomarkers for the diagnosis and prognosis of NDs. Given the essential impact of EVs on the pathogenesis of NDs, pathological EVs have been considered as therapeutic targets and EVs with therapeutic effects have been utilized as potential therapeutic agents or drug delivery platforms for the treatment of NDs. In this review, we focus on recent research progress on the pathological roles of EVs released from CNS cells in the pathogenesis of NDs, summarize findings that identify CNS-derived EV cargos as potential biomarkers to diagnose NDs, and comprehensively discuss promising potential of EVs as therapeutic targets, agents, and drug delivery systems in treating NDs, together with current concerns and challenges for basic research and clinical applications of EVs regarding NDs.© 2022. The Author(s).

Extracellular vesicles (EVs) are cell-derived membrane vesicles virtually secreted by all cells, including brain cells. EVs are a major term that includes apoptotic bodies, microvesicles and exosomes. The release of EVs has been recognized as an important modulator in cross-talking between neurons, astrocytes, microglia and oligodendrocytes, not only in central nervous system (CNS) physiology but also in neurodegenerative and neuroinflammatory disease states as well as in brain tumors, such as glioma. EVs are able to cross the blood brain barrier (BBB), spread to body fluids and reach distant tissues. This prominent spreading ability has suggested that EVs can be exploited into several different clinical applications ranging from biomarkers to therapeutic carriers. Exosomes, the well-studied group of EVs, have been emerging as a promising tool for therapeutic delivery strategies due to their intrinsic features, such as the stability, biocompatibility and stealth capacity when circulating in bloodstream, the ability to overcome natural barriers and inherent targeting properties. Over the last years, it became apparent that EVs can be loaded with specific cargoes directly in isolated EVs or by modulation of producer cells. In addition, the engineering of its membrane for targeting purposes is expected to allow generating carriers with unprecedented abilities for delivery in specific organs or tissues. Nevertheless, some challenges remain regarding the loading and targeting of EVs for which more research is necessary, and will be discussed in this review. Recently-emerged promising derivations are also discussed, such as exosome associated with adeno-associated virus (AAV) vectors (vexosomes), enveloped protein nanocages (EPNs) and exosome-mimetic nanovesicles. This article provides an updated review of this fast-progressing field of EVs and their role in brain diseases, particularly focusing in their therapeutic applications.Copyright © 2017 Elsevier B.V. All rights reserved.

In multicellular organisms, distant cells can exchange information by sending out signals composed of single molecules or, as increasingly exemplified in the literature, via complex packets stuffed with a selection of proteins, lipids, and nucleic acids, called extracellular vesicles (EVs; also known as exosomes and microvesicles, among other names). This Review covers some of the most striking functions described for EV secretion but also presents the limitations on our knowledge of their physiological roles. While there are initial indications that EV-mediated pathways operate in vivo, the actual nature of the EVs involved in these effects still needs to be clarified. Here, we focus on the context of tumor cells and their microenvironment, but similar results and challenges apply to all patho/physiological systems in which EV-mediated communication is proposed to take place.Copyright © 2016 Elsevier Inc. All rights reserved.