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SummaryFailed regeneration of myelin around neuronal axons following central nervous system damage contributes to nerve dysfunction and clinical decline in various neurological conditions, for which there is an unmet therapeutic demand1,2. Here, we show that interaction between glial cells – astrocytes and mature myelin-forming oligodendrocytes – is a critical determinant of remyelination. Astrocytes support the survival of regenerating oligodendrocytes, via downregulation of the Nrf2 pathway associated with increased astrocytic cholesterol biosynthesis pathway activation. Remyelination fails following sustained astrocytic Nrf2 activation yet is restored by either cholesterol biosynthesis/efflux stimulation, or Nrf2 inhibition using the existing therapeutic Luteolin. We identify that astrocyte-oligodendrocyte interaction regulates remyelination, and reveal a drug strategy for central nervous system regeneration centred on targeting this interaction.

RationaleNeutrophils are important in the pathophysiology of COVID19 but the molecular changes contributing to altered neutrophil phenotypes following SARS-CoV-2 infection are not fully understood.ObjectivesTo use quantitative mass spectrometry-based proteomics to explore neutrophil phenotypes following acute SARS-CoV-2 infection and during recovery.MethodsProspective observational study of hospitalised patients with PCR-confirmed SARS-CoV-2 infection (May 2020-December 2020). Patients were enrolled within 96 hours of admission, with longitudinal sampling up to 29 days. Control groups comprised non-COVID19 acute lower respiratory tract infection (LRTI) and age-matched non-infected controls. Neutrophils isolated from peripheral blood were processed for mass spectrometry. COVID19 severity and recovery were defined using the WHO ordinal scale.Measurements and Main Results84 COVID19 patients were included and compared to 91 LRTI patients and 42 controls. 5,800 neutrophil proteins were identified and 1,748 proteins were significantly different (q-valueConclusionsSARS-CoV-2 infection results in the sustained presence of recirculating neutrophils with distinct metabolic profiles and altered capacities to respond to migratory signals and cues from other immune cells, pathogens or cytokines.Scientific Knowledge on the SubjectInflammation is the primary driver of morbidity and mortality in severe COVID19. Type I interferon responses, T-cell exhaustion, cytokine storm, emergency myelopoiesis, myeloid compartment dysregulation and procoagulant pathway activation are well established contributors to COVID19 disease severity. Neutrophils play an important role in COVID19, with elevated neutrophil-to-lymphocyte ratios and the emergence of a circulating immature neutrophil population in individuals with severe symptoms. Neutrophil infiltration in the lungs coupled with the release of neutrophil extracellular traps has also been reported in severe and fatal COVID19. The aim of this study was to quantitatively map the proteomes of peripheral blood neutrophils from a cohort of hospitalised COVID19 patients to understand how SARS-CoV-2 infection changes neutrophil phenotypes and functional capacity.What this study adds to the fieldHigh-resolution mass spectrometry was used to characterise the proteomes of peripheral blood neutrophils from >200 individuals at different stages of disease. This work has comprehensively mapped neutrophil molecular changes associated with mild and severe COVID19 and identified significant quantitative changes in more than 1700 proteins in neutrophils from patients hospitalised with COVID19 versus patients with non-COVID19 acute respiratory infections. The study identifies neutrophil protein signatures associated with COVID19 disease severity. The data also show that alterations in neutrophil proteomes can persist in fully recovered patients and identify distinct neutrophil proteomes in recovered versus non recovered patients. Our study provides novel insights into neutrophil responses during acute COVID19 and reveals that altered neutrophil phenotypes persist in convalescent COVID19 patients.

Using high resolution quantitative mass spectrometry we have generated in-depth proteome maps of human and mouseex vivoandin vitromicroglia. We reveal a tenfold difference in protein content ofex vivoandin vitrocells and fundamental differences in protein expression related to protein synthesis, metabolism, microglia markers and environmental sensors. While human and mouse microglia are different in their proteomes, the species differences are smaller than the changes induced by cell culture. Remarkably, xenografting human microglia derived from human stem cells into mouse brain restores thein vivo-like microglia proteomic signature including reduction in the total amount of protein, energy demand and protein synthesis, and re-expression of homeostatic microglia markers. We identify more than 9000 microglia proteins and discuss how they relate to microglia function. This data provides a vital proteomic resource for understandingin vitroandex vivomicroglia phenotypes.

Microglia are resident macrophages of the CNS that are involved in its development, homeostasis and response to infection and damage. Microglial activation is a common feature of neurological disorders, and although in some instances this activation can be damaging, protective and regenerative functions of microglia have been revealed. The most prominent example of the regenerative functions is a role for microglia in supporting regeneration of myelin after injury, a process that is critical for axonal health and relevant to numerous disorders in which loss of myelin integrity is a prevalent feature, such as multiple sclerosis, Alzheimer disease and motor neuron disease. Although drugs that are intended to promote remyelination are entering clinical trials, the mechanisms by which remyelination is controlled and how microglia are involved are not completely understood. In this Review, we discuss work that has identified novel regulators of microglial activation — including molecular drivers, population heterogeneity and turnover — that might influence their pro-remyelination capacity. We also discuss therapeutic targeting of microglia as a potential approach to promoting remyelination.

Microglia are resident tissue macrophages of the central nervous system (CNS) that arise from erythromyeloid progenitors during embryonic development. They play essential roles in CNS development, homeostasis and response to disease. Since microglia are difficult to procure from the human brain, several protocols have been developed to generate microglia-like cells from human induced pluripotent stem cells (hiPSCs). However, some concerns remain over the purity and quality of in vitro generated microglia. Here, we describe a new protocol that does not require co-culture with neural cells and yields cultures of 100% P2Y12+ 95% TMEM119+ ramified human microglia-like cells (hiPSC-MG). In the presence of neural precursor cell-conditioned media, hiPSC-MG expressed high levels of human microglia signature genes, including SALL1, CSF1R, P2RY12, TMEM119, TREM2, HEXB and SIGLEC11, as revealed by whole-transcriptome analysis. Stimulation of hiPSC-MG with lipopolysaccharide resulted in downregulation of P2Y12 expression, induction of IL1B mRNA expression and increase in cell capacitance. HiPSC-MG were phagocytically active and maintained their cell identity after transplantation into murine brain slices and human brain spheroids. Together, our new protocol for the generation of microglia-like cells from human iPSCs will facilitate the study of human microglial function in health and disease.

Microglia are the resident macrophages of the central nervous system (CNS), implicated in developmental processes, homeostasis, and responses to injury. Derived from the yolk sac during development, microglia self-renew, self-regulate their numbers during homeostatic conditions, and show a robust proliferative capacity even in adulthood. Together with monocyte-derived macrophages (MDM), microglia coordinate the regeneration of CNS myelin around axons, termed remyelination. Gene expression analyses and experimental modelling have identified pro-remyelination roles for microglia/MDM in clearance of myelin debris, secretion of growth factors, and remodelling of the extracellular matrix. Further investigations into the molecular mechanisms controlling these regenerative functions will reveal novel therapeutic strategies to enhance remyelination, by harnessing the beneficial effects of the innate immune response to injury.

Failed regeneration of central nervous system myelin contributes to clinical decline in neuroinflammatory and neurodegenerative diseases, for which there is an unmet therapeutic need. Here, we reveal that efficient remyelination requires death of pro-inflammatory microglia followed by repopulation to a pro-regenerative state. We propose that impaired microglia death and/or repopulation may underpin dysregulated microglia activation in neurological diseases, and reveal novel therapeutic targets to promote white matter regeneration.

Remyelination is an example of central nervous system (CNS) regeneration, whereby myelin is restored around demyelinated axons, re-establishing saltatory conduction and trophic/ metabolic support. In progressive multiple sclerosis, remyelination is limited or fails altogether which is considered to contribute to axonal damage/ loss and consequent disability. Macrophages have critical roles in both CNS damage and regeneration, such as remyelination. This diverse range in functions reflects the ability of macrophages to acquire tissue microenvironment-specific activation states. This activation is dynamically regulated during efficient regeneration, with a switch from pro-inflammatory to inflammation-resolution/ pro-regenerative phenotypes. Although some molecules and pathways have been implicated in the dynamic activation of macrophages, such as NFκB, the cellular and molecular mechanisms underpinning plasticity of macrophage activation are unclear. Identifying mechanisms regulating macrophage activation to pro-regenerative phenotypes may lead to novel therapeutic strategies to promote remyelination in multiple sclerosis.

Medulloblastoma is the most common malignant paediatric brain tumour. Standard treatment includes a combination of surgery, craniospinal radiotherapy and adjuvant chemotherapy. The chemotherapy agent Vincristine causes potent inhibition of tumour growth, however, high levels of toxicity and intrinsic drug resistance mechanisms, limit its clinical use. Jerantinine is a novel aspidosperma indole alkaloid that, like Vincristine, works by destabilising microtubule growth and hence blocks cell division. Here, we tested the hypothesis that Jerantinine would be an effective alternative to Vincristine in medulloblastoma. Jerantinine, like Vincristine, caused a dose-dependent decrease in cell viability and colony formation in medulloblastoma cell lines. Cytotoxicity of Vincristine could be potentiated with the ABCB1 inhibitor Verapamil, however there was no potentiation of Jerantinine’s activity indicating that Jerantinine functions independently of intrinsic ABCB1 expression and is able to circumvent this drug resistance mechanism. The effect of Jerantinine treatment was also assessed in 3D spheroid culture. Spheroid viability decreased with increasing concentrations of Jerantinine, as measured using the CellTiter-Glo(®) 3D cell viability assay. Automated imaging analysis (Celigo(®) cytometer) also showed an increase in cell death at a range of Jerantinine concentrations over 48 hours. Confocal microscopy showed comparable effects of Jerantinine and Vincristine in medulloblastoma cell lines, including cytoskeletal defects, inhibition of tubulin polymerisation and defective cell division. However unlike Vincristine, Jerantinine caused no visible disruption to cellular morphology of neural stem cells. These results suggest that Jerantinine is an effective, tumour-specific alternative to Vincristine in the treatment of paediatric medulloblastoma.

In the original publication, sixth author's surname was incorrectly published as "Llyod" instead of "Lloyd". The correct name should read as "Amy Lloyd".