Extracellular vesicles set the stage for brain plasticity and recovery by multimodal signalling.

Extracellular vesicles (EVs) are extremely versatile naturally occurring membrane particles that convey complex signals between cells. EVs of different cellular sources are capable of inducing striking therapeutic responses in neurological disease models. Differently from pharmacological compounds that act by modulating defined signalling pathways, EV-based therapeutics possess multiple abilities via a variety of effectors, thus allowing the modulation of complex disease processes that may have very potent effects on brain tissue recovery. When applied in vivo in experimental models of neurological diseases, EV-based therapeutics have revealed remarkable effects on immune responses, cell metabolism and neuronal plasticity. This multimodal modulation of neuroimmune networks by EVs profoundly influences disease processes in a highly synergistic and context-dependent way. Ultimately, the EV-mediated restoration of cellular functions helps to set the stage for neurological recovery. With this review we first outline the current understanding of the mechanisms of action of EVs, describing how EVs released from various cellular sources identify their cellular targets and convey signals to recipient cells. Then, mechanisms of action applicable to key neurological conditions such as stroke, multiple sclerosis and neurodegenerative diseases are presented. Pathways that deserve attention in specific disease contexts are discussed. We subsequently showcase considerations about EV biodistribution and delineate genetic engineering strategies aiming at enhancing brain uptake and signalling. By sketching a broad view of EV-orchestrated brain plasticity and recovery, we finally define possible future clinical EV applications and propose necessary information to be provided ahead of clinical trials. Our goal is to provide a steppingstone that can be used to critically discuss EVs as next generation therapeutics for brain diseases.

Phase I clinical trial of intracerebroventricular transplantation of allogeneic neural stem cells in people with progressive multiple sclerosis.

We report the analysis of 1 year of data from the first cohort of 15 patients enrolled in an open-label, first-in-human, dose-escalation phase I study (ClinicalTrials.gov: NCT03282760, EudraCT2015-004855-37) to determine the feasibility, safety, and tolerability of the transplantation of allogeneic human neural stem/progenitor cells (hNSCs) for the treatment of secondary progressive multiple sclerosis. Participants were treated with hNSCs delivered via intracerebroventricular injection in combination with an immunosuppressive regimen. No treatment-related deaths nor serious adverse events (AEs) were observed. All participants displayed stability of clinical and laboratory outcomes, as well as lesion load and brain activity (MRI), compared with the study entry. Longitudinal metabolomics and lipidomics of biological fluids identified time- and dose-dependent responses with increased levels of acyl-carnitines and fatty acids in the cerebrospinal fluid (CSF). The absence of AEs and the stability of functional and structural outcomes are reassuring and represent a milestone for the safe translation of stem cells into regenerative medicines.

Time-resolved single-cell RNAseq profiling identifies a novel Fabp5+ subpopulation of inflammatory myeloid cells with delayed cytotoxic profile in chronic spinal cord injury.

Traumatic spinal cord injuries (SCI) are a group of highly debilitating pathologies affecting thousands annually, and adversely affecting quality of life. Currently, no fully restorative therapies exist, and SCI still results in significant personal, societal and financial burdens. Inflammation plays a major role in the evolution of SCI, with myeloid cells, including bone marrow derived macrophages (BMDMs) and microglia (MG) being primary drivers of both early secondary pathogenesis and delayed wound healing events. The precise role of myeloid cell subsets is unclear as upon crossing the blood-spinal cord barrier, infiltrating bone marrow derived macrophages (BMDMs) may take on the morphology of resident microglia, and upregulate canonical microglia markers, thus making the two populations difficult to distinguish. Here, we used time-resolved scRNAseq and transgenic fate-mapping to chart the transcriptional profiles of tissue-resident and -infiltrating myeloid cells in a mouse model of thoracic contusion SCI. Our work identifies a novel subpopulation of foam cell-like inflammatory myeloid cells with increased expression of Fatty Acid Binding Protein 5 (Fabp5) and comprise both tissue-resident and -infiltrating cells. Fabp5+ inflammatory myeloid cells display a delayed cytotoxic profile that is predominant at the lesion epicentre and extends into the chronic phase of SCI.

Oligodendroglia-to-neuron material transfer lights up the mouse CNS.

Intercellular material transfer in the central nervous system (CNS) supports neuronal survival and activity. Mayrhofer et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20221632) characterize extensive regionally coordinated transfer of oligodendroglial ribosomal and nuclear material toward neurons, linked to satellite oligodendrocyte-neuron pairs in the mouse CNS.

Spatio-temporally deciphering peripheral nerve regeneration in vivo after extracellular vesicle therapy under NIR-II fluorescence imaging.

Extracellular vesicles (EVs) show potential as a therapeutic tool for peripheral nerve injury (PNI), promoting neurological regeneration. However, there are limited data on the in vivo spatio-temporal trafficking and biodistribution of EVs. In this study, we introduce a new non-invasive near-infrared fluorescence imaging strategy based on glucose-conjugated quantum dot (QDs-Glu) labeling to target and track EVs in a sciatic nerve injury rat model in real-time. Our results demonstrate that the injected EVs migrated from the uninjured site to the injured site of the nerve, with an increase in fluorescence signals detected from 4 to 7 days post-injection, indicating the release of contents from the EVs with therapeutic effects. Immunofluorescence and behavioral tests revealed that the EV therapy promoted nerve regeneration and functional recovery at 28 days post-injection. We also found a relationship between functional recovery and the NIR-II fluorescence intensity change pattern, providing novel evidence for the therapeutic effects of EV therapy using real-time NIR-II imaging at the live animal level. This approach initiates a new path for monitoring EVs in treating PNI under in vivo NIR-II imaging, enhancing our understanding of the efficacy of EV therapy on peripheral nerve regeneration and its mechanisms.

Targeting succinate metabolism to decrease brain injury upon mechanical thrombectomy treatment of ischemic stroke.

Current treatments for acute ischemic stroke aim to reinstate a normal perfusion in the ischemic territory but can also cause significant ischemia-reperfusion (IR) injury. Previous data in experimental models of stroke show that ischemia leads to the accumulation of succinate, and, upon reperfusion, the accumulated succinate is rapidly oxidized by succinate dehydrogenase (SDH) to drive superoxide production at mitochondrial complex I. Despite this process initiating IR injury and causing further tissue damage, the potential of targeting succinate metabolism to minimize IR injury remains unexplored. Using both quantitative and untargeted high-resolution metabolomics, we show a time-dependent accumulation of succinate in both human and mouse brain exposed to ischemia ex vivo. In a mouse model of ischemic stroke/mechanical thrombectomy mass spectrometry imaging (MSI) shows that succinate accumulation is confined to the ischemic region, and that the accumulated succinate is rapidly oxidized upon reperfusion. Targeting succinate oxidation by systemic infusion of the SDH inhibitor malonate upon reperfusion leads to a dose-dependent decrease in acute brain injury. Together these findings support targeting succinate metabolism upon reperfusion to decrease IR injury as a valuable adjunct to mechanical thrombectomy in ischemic stroke.

Cell culture-derived extracellular vesicles: Considerations for reporting cell culturing parameters.

Cell culture-conditioned medium (CCM) is a valuable source of extracellular vesicles (EVs) for basic scientific, therapeutic and diagnostic applications. Cell culturing parameters affect the biochemical composition, release and possibly the function of CCM-derived EVs (CCM-EV). The CCM-EV task force of the Rigor and Standardization Subcommittee of the International Society for Extracellular Vesicles aims to identify relevant cell culturing parameters, describe their effects based on current knowledge, recommend reporting parameters and identify outstanding questions. While some recommendations are valid for all cell types, cell-specific recommendations may need to be established for non-mammalian sources, such as bacteria, yeast and plant cells. Current progress towards these goals is summarized in this perspective paper, along with a checklist to facilitate transparent reporting of cell culturing parameters to improve the reproducibility of CCM-EV research.

Bioinformatics analysis identified apolipoprotein E as a hub gene regulating neuroinflammation in macrophages and microglia following spinal cord injury.

Macrophages and microglia play important roles in chronic neuroinflammation following spinal cord injury (SCI). Although macrophages and microglia have similar functions, their phagocytic and homeostatic abilities differ. It is difficult to distinguish between these two populations in vivo, but single-cell analysis can improve our understanding of their identity and heterogeneity. We conducted bioinformatics analysis of the single-cell RNA sequencing dataset GSE159638, identifying apolipoprotein E (APOE) as a hub gene in both macrophages and microglia in the subacute and chronic phases of SCI. We then validated these transcriptomic changes in a mouse model of cervical spinal cord hemi-contusion and observed myelin uptake, lipid droplets, and lysosome accumulation in macrophages and microglia following SCI. Finally, we observed that knocking out APOE aggravated neurological dysfunction, increased neuroinflammation, and exacerbated the loss of white matter. Targeting APOE and the related cholesterol efflux represents a promising strategy for reducing neuroinflammation and promoting recovery following SCI.

Small Extracellular Vesicles Secreted by Nigrostriatal Astrocytes Rescue Cell Death and Preserve Mitochondrial Function in Parkinson's Disease.

Extracellular vesicles (EVs) are emerging as powerful players in cell-to-cell communication both in healthy and diseased brain. In Parkinson's disease (PD)-characterized by selective dopaminergic neuron death in ventral midbrain (VMB) and degeneration of their terminals in striatum (STR)-astrocytes exert dual harmful/protective functions, with mechanisms not fully elucidated. Here, this study shows that astrocytes from the VMB-, STR-, and VMB/STR-depleted brains release a population of small EVs  in a region-specific manner. Interestingly, VMB-astrocytes secreted the highest rate of EVs, which is further exclusively increased in response to CCL3, a chemokine that promotes robust dopaminergic neuroprotection in different PD models. The neuroprotective potential of nigrostriatal astrocyte-EVs is investigated in differentiated versus undifferentiated SH-SY5Y cells exposed to oxidative stress and mitochondrial toxicity. EVs from both VMB- and STR-astrocytes counteract H2 O2 -induced caspase-3 activation specifically in differentiated cells, with EVs from CCL3-treated astrocytes showing a higher protective effect. High resolution respirometry further reveals that nigrostriatal astrocyte-EVs rescue neuronal mitochondrial complex I function impaired by the neurotoxin MPP+ . Notably, only EVs from VMB-astrocyte fully restore ATP production, again specifically in differentiated SH-SY5Y. These results highlight a regional diversity in the nigrostriatal system for the secretion and activities of astrocyte-EVs, with neuroprotective implications for PD.

Soluble factors influencing the neural stem cell niche in brain physiology, inflammation, and aging.

Within the adult central nervous system (CNS) of most mammals resides a resident stem cell population, known as neural stem cells (NSCs). NSCs are located within specific niches of the CNS and maintain a self-renewal and proliferative capacity to generate new neurons, astrocytes, and oligodendrocytes throughout adulthood. The NSC niches are dynamic and active environments that are within proximity to the systemic circulation and the cerebrospinal fluid (CSF). Therefore, NSCs respond not only to factors present in the local microenvironment of the niche but also to factors present in the systemic macroenvironment. The factors can be soluble forms such as cytokines and chemokines located in the circulation or directly from local cells, such as microglia and astrocytes. Additionally, recent evidence points towards physiological aging and its association with a progressive loss of function and a decline in the self-renewal and regenerative capacities of CNS NSCs, which can be further exacerbated by changes in the local and systemic milieu. This review will highlight the main intrinsic and extrinsic regulators of neural stem cell function under homeostatic and inflammatory conditions including those trafficked within extracellular membrane vesicles. Further, discussion will center around how intrinsic and extrinsic factors impact normal homeostatic functions within the adult brain and in aging.

The role of neural stem cells in regulating glial scar formation and repair.

Glial scars are a common pathological occurrence in a variety of central nervous system (CNS) diseases and injuries. They are caused after severe damage and consist of reactive glia that form a barrier around the damaged tissue that leads to a non-permissive microenvironment which prevents proper endogenous regeneration. While there are a number of therapies that are able to address some components of disease, there are none that provide regenerative properties. Within the past decade, neural stem cells (NSCs) have been heavily studied due to their potent anti-inflammatory and reparative capabilities in disease and injury. Exogenously applied NSCs have been found to aid in glial scar healing by reducing inflammation and providing cell replacement. However, endogenous NSCs have also been found to contribute to the reactive environment by different means. Further understanding how NSCs can be leveraged to aid in the resolution of the glial scar is imperative in the use of these cells as regenerative therapies. To do so, humanised 3D model systems have been developed to study the development and maintenance of the glial scar. Herein, we explore the current work on endogenous and exogenous NSCs in the glial scar as well as the novel 3D stem cell-based technologies being used to model this pathology in a dish.

Therapeutically expanded human regulatory T-cells are super-suppressive due to HIF1A induced expression of CD73.

The adoptive transfer of regulatory T-cells (Tregs) is a promising therapeutic approach in transplantation and autoimmunity. However, because large cell numbers are needed to achieve a therapeutic effect, in vitro expansion is required. By comparing their function, phenotype and transcriptomic profile against ex vivo Tregs, we demonstrate that expanded human Tregs switch their metabolism to aerobic glycolysis and show enhanced suppressive function through hypoxia-inducible factor 1-alpha (HIF1A) driven acquisition of CD73 expression. In conjunction with CD39, CD73 expression enables expanded Tregs to convert ATP to immunosuppressive adenosine. We conclude that for maximum therapeutic benefit, Treg expansion protocols should be optimised for CD39/CD73 co-expression.

Metabolic Control of Smoldering Neuroinflammation.

Compelling evidence exists that patients with chronic neurological conditions, which includes progressive multiple sclerosis, display pathological changes in neural metabolism and mitochondrial function. However, it is unknown if a similar degree of metabolic dysfunction occurs also in non-neural cells in the central nervous system. Specifically, it remains to be clarified (i) the full extent of metabolic changes in tissue-resident microglia and infiltrating macrophages after prolonged neuroinflammation (e.g., at the level of chronic active lesions), and (ii) whether these alterations underlie a unique pathogenic phenotype that is amenable for therapeutic targeting. Herein, we discuss how cell metabolism and mitochondrial function govern the function of chronic active microglia and macrophages brain infiltrates and identify new metabolic targets for therapeutic approaches aimed at reducing smoldering neuroinflammation.

Stem Cell Therapies for Progressive Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by demyelination and axonal degeneration. MS patients typically present with a relapsing-remitting (RR) disease course, manifesting as sporadic attacks of neurological symptoms including ataxia, fatigue, and sensory impairment. While there are several effective disease-modifying therapies able to address the inflammatory relapses associated with RRMS, most patients will inevitably advance to a progressive disease course marked by a gradual and irreversible accrual of disabilities. Therapeutic intervention in progressive MS (PMS) suffers from a lack of well-characterized biological targets and, hence, a dearth of successful drugs. The few medications approved for the treatment of PMS are typically limited in their efficacy to active forms of the disease, have little impact on slowing degeneration, and fail to promote repair. In looking to address these unmet needs, the multifactorial therapeutic benefits of stem cell therapies are particularly compelling. Ostensibly providing neurotrophic support, immunomodulation and cell replacement, stem cell transplantation holds substantial promise in combatting the complex pathology of chronic neuroinflammation. Herein, we explore the current state of preclinical and clinical evidence supporting the use of stem cells in treating PMS and we discuss prospective hurdles impeding their translation into revolutionary regenerative medicines.

Physiological and Pathological Factors Affecting Drug Delivery to the Brain by Nanoparticles.

The prevalence of neurological/neurodegenerative diseases, such as Alzheimer's disease is known to be increasing due to an aging population and is anticipated to further grow in the decades ahead. The treatment of brain diseases is challenging partly due to the inaccessibility of therapeutic agents to the brain. An increasingly important observation is that the physiology of the brain alters during many brain diseases, and aging adds even more to the complexity of the disease. There is a notion that the permeability of the blood-brain barrier (BBB) increases with aging or disease, however, the body has a defense mechanism that still retains the separation of the brain from harmful chemicals in the blood. This makes drug delivery to the diseased brain, even more challenging and complex task. Here, the physiological changes to the diseased brain and aged brain are covered in the context of drug delivery to the brain using nanoparticles. Also, recent and novel approaches are discussed for the delivery of therapeutic agents to the diseased brain using nanoparticle based or magnetic resonance imaging guided systems. Furthermore, the complement activation, toxicity, and immunogenicity of brain targeting nanoparticles as well as novel in vitro BBB models are discussed.

Neural stem cells traffic functional mitochondria via extracellular vesicles.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.

NSCs: Sentinel Cells of the Brain.

Adult neural stem cells (NSCs) have the ability to oscillate between activated and dormant states in response to extrinsic regulators. In this issue of Cell Stem Cell, Belenguer et al. (2020) identify a direct role for systemic TNF-α, which acts through its receptors TNFRII and TNFRI as a regulator of NSC activation and a return to quiescence, respectively.