Continued dysfunction of capillary pericytes promotes no-reflow after experimental stroke in vivo.

Incomplete reperfusion of the microvasculature ('no-reflow') after ischaemic stroke damages salvageable brain tissue. Previous ex vivo studies suggest pericytes are vulnerable to ischaemia and may exacerbate no-reflow, but the viability of pericytes and their association with no-reflow remains under-explored in vivo. Using longitudinal in vivo two-photon single-cell imaging over 7 days, we showed that 87% of pericytes constrict during cerebral ischaemia and remain constricted post reperfusion, and 50% of the pericyte population are acutely damaged. Moreover, we revealed ischaemic pericytes to be fundamentally implicated in capillary no-reflow by limiting and arresting blood flow within the first 24 h post stroke. Despite sustaining acute membrane damage, we observed that over half of all cortical pericytes survived ischaemia and responded to vasoactive stimuli, upregulated unique transcriptomic profiles and replicated. Finally, we demonstrated the delayed recovery of capillary diameter by ischaemic pericytes after reperfusion predicted vessel reconstriction in the subacute phase of stroke. Cumulatively, these findings demonstrate that surviving cortical pericytes remain both viable and promising therapeutic targets to counteract no-reflow after ischaemic stroke.

Spatial Transcriptomics-correlated Electron Microscopy maps transcriptional and ultrastructural responses to brain injury.

Understanding the complexity of cellular function within a tissue necessitates the combination of multiple phenotypic readouts. Here, we developed a method that links spatially-resolved gene expression of single cells with their ultrastructural morphology by integrating multiplexed error-robust fluorescence in situ hybridization (MERFISH) and large area volume electron microscopy (EM) on adjacent tissue sections. Using this method, we characterized in situ ultrastructural and transcriptional responses of glial cells and infiltrating T-cells after demyelinating brain injury in male mice. We identified a population of lipid-loaded "foamy" microglia located in the center of remyelinating lesion, as well as rare interferon-responsive microglia, oligodendrocytes, and astrocytes that co-localized with T-cells. We validated our findings using immunocytochemistry and lipid staining-coupled single-cell RNA sequencing. Finally, by integrating these datasets, we detected correlations between full-transcriptome gene expression and ultrastructural features of microglia. Our results offer an integrative view of the spatial, ultrastructural, and transcriptional reorganization of single cells after demyelinating brain injury.

Pathways linking aging and atheroprotection in Mif-deficient atherosclerotic mice.

Atherosclerosis is a chronic inflammatory condition of our arteries and the main underlying pathology of myocardial infarction and stroke. The pathogenesis is age-dependent, but the links between disease progression, age, and atherogenic cytokines and chemokines are incompletely understood. Here, we studied the chemokine-like inflammatory cytokine macrophage migration inhibitory factor (MIF) in atherogenic Apoe-/- mice across different stages of aging and cholesterol-rich high-fat diet (HFD). MIF promotes atherosclerosis by mediating leukocyte recruitment, lesional inflammation, and suppressing atheroprotective B cells. However, links between MIF and advanced atherosclerosis across aging have not been systematically explored. We compared effects of global Mif-gene deficiency in 30-, 42-, and 48-week-old Apoe-/- mice on HFD for 24, 36, or 42 weeks, respectively, and in 52-week-old mice on a 6-week HFD. Mif-deficient mice exhibited reduced atherosclerotic lesions in the 30/24- and 42/36-week-old groups, but atheroprotection, which in the applied Apoe-/- model was limited to lesions in the brachiocephalic artery and abdominal aorta, was not detected in the 48/42- and 52/6-week-old groups. This suggested that atheroprotection afforded by global Mif-gene deletion differs across aging stages and atherogenic diet duration. To characterize this phenotype and study the underlying mechanisms, we determined immune cells in the periphery and vascular lesions, obtained a multiplex cytokine/chemokine profile, and compared the transcriptome between the age-related phenotypes. We found that Mif deficiency promotes lesional macrophage and T-cell counts in younger but not aged mice, with subgroup analysis pointing toward a role for Trem2+ macrophages. The transcriptomic analysis identified pronounced MIF- and aging-dependent changes in pathways predominantly related to lipid synthesis and metabolism, lipid storage, and brown fat cell differentiation, as well as immunity, and atherosclerosis-relevant enriched genes such as Plin1, Ldlr, Cpne7, or Il34, hinting toward effects on lesional lipids, foamy macrophages, and immune cells. Moreover, Mif-deficient aged mice exhibited a distinct plasma cytokine/chemokine signature consistent with the notion that mediators known to drive inflamm'aging are either not downregulated or even upregulated in Mif-deficient aged mice compared with the corresponding younger ones. Lastly, Mif deficiency favored formation of lymphocyte-rich peri-adventitial leukocyte clusters. While the causative contributions of these mechanistic pillars and their interplay will be subject to future scrutiny, our study suggests that atheroprotection due to global Mif-gene deficiency in atherogenic Apoe-/- mice is reduced upon advanced aging and identifies previously unrecognized cellular and molecular targets that could explain this phenotype shift. These observations enhance our understanding of inflamm'aging and MIF pathways in atherosclerosis and may have implications for translational MIF-directed strategies.

High-Resolution RNA Sequencing from PFA-Fixed Microscopy Sections.

Obtaining high-quality RNA sequencing results from archived biological tissues, such as paraformaldehyde (PFA)-fixed sections for microscopy, is challenging due to the incompatibility of current high-throughput RNA sequencing methods. Here, we present a low-input method for RNA sequencing from archived PFA-fixed sections. Using this method, we routinely obtain high-quality sequencing results from archived mouse brain sections that are prepared for imaging without any special care for avoiding RNA degradation. The PFA cross-linking locks and protects RNA from degradation but cross-linking is also hard to reverse. For this goal, we developed an effective decrosslinking protocol based on Proteinase K activity to retrieve PFA-cross-linked mRNAs which was followed up by a Smart-seq2 library preparation protocol. Our protocol enables spatially defined transcriptomic analysis of archived sections and allows the genomic analysis of PFA-fixed samples. Furthermore, our protocol inactivates pathogenic samples and allows working under regular biosafety levels.

T cells modulate the microglial response to brain ischemia.

Neuroinflammation after stroke is characterized by the activation of resident microglia and the invasion of circulating leukocytes into the brain. Although lymphocytes infiltrate the brain in small number, they have been consistently demonstrated to be the most potent leukocyte subpopulation contributing to secondary inflammatory brain injury. However, the exact mechanism of how this minimal number of lymphocytes can profoundly affect stroke outcome is still largely elusive. Here, using a mouse model for ischemic stroke, we demonstrated that early activation of microglia in response to stroke is differentially regulated by distinct T cell subpopulations - with TH1 cells inducing a type I INF signaling in microglia and regulatory T cells (TREG) cells promoting microglial genes associated with chemotaxis. Acute treatment with engineered T cells overexpressing IL-10 administered into the cisterna magna after stroke induces a switch of microglial gene expression to a profile associated with pro-regenerative functions. Whereas microglia polarization by T cell subsets did not affect the acute development of the infarct volume, these findings substantiate the role of T cells in stroke by polarizing the microglial phenotype. Targeting T cell-microglia interactions can have direct translational relevance for further development of immune-targeted therapies for stroke and other neuroinflammatory conditions.

Microglia states and nomenclature: A field at its crossroads.

Microglial research has advanced considerably in recent decades yet has been constrained by a rolling series of dichotomies such as "resting versus activated" and "M1 versus M2." This dualistic classification of good or bad microglia is inconsistent with the wide repertoire of microglial states and functions in development, plasticity, aging, and diseases that were elucidated in recent years. New designations continuously arising in an attempt to describe the different microglial states, notably defined using transcriptomics and proteomics, may easily lead to a misleading, although unintentional, coupling of categories and functions. To address these issues, we assembled a group of multidisciplinary experts to discuss our current understanding of microglial states as a dynamic concept and the importance of addressing microglial function. Here, we provide a conceptual framework and recommendations on the use of microglial nomenclature for researchers, reviewers, and editors, which will serve as the foundations for a future white paper.

CD8+ T cells induce interferon-responsive oligodendrocytes and microglia in white matter aging.

A hallmark of nervous system aging is a decline of white matter volume and function, but the underlying mechanisms leading to white matter pathology are unknown. In the present study, we found age-related alterations of oligodendrocyte cell state with a reduction in total oligodendrocyte density in aging murine white matter. Using single-cell RNA-sequencing, we identified interferon (IFN)-responsive oligodendrocytes, which localize in proximity to CD8+ T cells in aging white matter. Absence of functional lymphocytes decreased the number of IFN-responsive oligodendrocytes and rescued oligodendrocyte loss, whereas T-cell checkpoint inhibition worsened the aging response. In addition, we identified a subpopulation of lymphocyte-dependent, IFN-responsive microglia in the vicinity of the CD8+ T cells in aging white matter. In summary, we provide evidence that CD8+ T-cell-induced, IFN-responsive oligodendrocytes and microglia are important modifiers of white matter aging.

Heterocomplexes between the atypical chemokine MIF and the CXC-motif chemokine CXCL4L1 regulate inflammation and thrombus formation.

To fulfil its orchestration of immune cell trafficking, a network of chemokines and receptors developed that capitalizes on specificity, redundancy, and functional selectivity. The discovery of heteromeric interactions in the chemokine interactome has expanded the complexity within this network. Moreover, some inflammatory mediators, not structurally linked to classical chemokines, bind to chemokine receptors and behave as atypical chemokines (ACKs). We identified macrophage migration inhibitory factor (MIF) as an ACK that binds to chemokine receptors CXCR2 and CXCR4 to promote atherogenic leukocyte recruitment. Here, we hypothesized that chemokine-chemokine interactions extend to ACKs and that MIF forms heterocomplexes with classical chemokines. We tested this hypothesis by using an unbiased chemokine protein array. Platelet chemokine CXCL4L1 (but not its variant CXCL4 or the CXCR2/CXCR4 ligands CXCL8 or CXCL12) was identified as a candidate interactor. MIF/CXCL4L1 complexation was verified by co-immunoprecipitation, surface plasmon-resonance analysis, and microscale thermophoresis, also establishing high-affinity binding. We next determined whether heterocomplex formation modulates inflammatory/atherogenic activities of MIF. Complex formation was observed to inhibit MIF-elicited T-cell chemotaxis as assessed by transwell migration assay and in a 3D-matrix-based live cell-imaging set-up. Heterocomplexation also blocked MIF-triggered migration of microglia in cortical cultures in situ, as well as MIF-mediated monocyte adhesion on aortic endothelial cell monolayers under flow stress conditions. Of note, CXCL4L1 blocked binding of Alexa-MIF to a soluble surrogate of CXCR4 and co-incubation with CXCL4L1 attenuated MIF responses in HEK293-CXCR4 transfectants, indicating that complex formation interferes with MIF/CXCR4 pathways. Because MIF and CXCL4L1 are platelet-derived products, we finally tested their role in platelet activation. Multi-photon microscopy, FLIM-FRET, and proximity-ligation assay visualized heterocomplexes in platelet aggregates and in clinical human thrombus sections obtained from peripheral artery disease (PAD) in patients undergoing thrombectomy. Moreover, heterocomplexes inhibited MIF-stimulated thrombus formation under flow and skewed the lamellipodia phenotype of adhering platelets. Our study establishes a novel molecular interaction that adds to the complexity of the chemokine interactome and chemokine/receptor-network. MIF/CXCL4L1, or more generally, ACK/CXC-motif chemokine heterocomplexes may be target structures that can be exploited to modulate inflammation and thrombosis.

ADAM10 and ADAM17 promote SARS-CoV-2 cell entry and spike protein-mediated lung cell fusion.

The severe-acute-respiratory-syndrome-coronavirus-2 (SARS-CoV-2) is the causative agent of COVID-19, but host cell factors contributing to COVID-19 pathogenesis remain only partly understood. We identify the host metalloprotease ADAM17 as a facilitator of SARS-CoV-2 cell entry and the metalloprotease ADAM10 as a host factor required for lung cell syncytia formation, a hallmark of COVID-19 pathology. ADAM10 and ADAM17, which are broadly expressed in the human lung, cleave the SARS-CoV-2 spike protein (S) in vitro, indicating that ADAM10 and ADAM17 contribute to the priming of S, an essential step for viral entry and cell fusion. ADAM protease-targeted inhibitors severely impair lung cell infection by the SARS-CoV-2 variants of concern alpha, beta, delta, and omicron and also reduce SARS-CoV-2 infection of primary human lung cells in a TMPRSS2 protease-independent manner. Our study establishes ADAM10 and ADAM17 as host cell factors for viral entry and syncytia formation and defines both proteases as potential targets for antiviral drug development.

TREM2-dependent lipid droplet biogenesis in phagocytes is required for remyelination.

Upon demyelinating injury, microglia orchestrate a regenerative response that promotes myelin repair, thereby restoring rapid signal propagation and protecting axons from further damage. Whereas the essential phagocytic function of microglia for remyelination is well known, the underlying metabolic pathways required for myelin debris clearance are poorly understood. Here, we show that cholesterol esterification in male mouse microglia/macrophages is a necessary adaptive response to myelin debris uptake and required for the generation of lipid droplets upon demyelinating injury. When lipid droplet biogenesis is defective, innate immune cells do not resolve, and the regenerative response fails. We found that triggering receptor expressed on myeloid cells 2 (TREM2)-deficient mice are unable to adapt to excess cholesterol exposure, form fewer lipid droplets, and build up endoplasmic reticulum (ER) stress. Alleviating ER stress in TREM2-deficient mice restores lipid droplet biogenesis and resolves the innate immune response. Thus, we conclude that TREM2-dependent formation of lipid droplets constitute a protective response required for remyelination to occur.

Dissociation of microdissected mouse brain tissue for artifact free single-cell RNA sequencing.

Single-cell RNA sequencing (scRNA-seq) provides the transcriptome of individual cells and addresses previously intractable problems including the central nervous system's transcriptional responses during health and disease. However, dissociating brain cells is challenging and induces artificial transcriptional responses. Here, we describe an enzymatic dissociation method for mouse brain that prevents dissociation artifacts and lowers technical variations with standardized steps. We tested this protocol on microdissected brain tissue of 3-week- to 24-month-old mice and obtained high-quality scRNA-seq results. For complete details on the use and execution of this protocol, please refer to Safaiyan et al. (2021).

White matter aging drives microglial diversity.

Aging results in gray and white matter degeneration, but the specific microglial responses are unknown. Using single-cell RNA sequencing from white and gray matter separately, we identified white matter-associated microglia (WAMs), which share parts of the disease-associated microglia (DAM) gene signature and are characterized by activation of genes implicated in phagocytic activity and lipid metabolism. WAMs depend on triggering receptor expressed on myeloid cells 2 (TREM2) signaling and are aging dependent. In the aged brain, WAMs form independent of apolipoprotein E (APOE), in contrast to mouse models of Alzheimer's disease, in which microglia with the WAM gene signature are generated prematurely and in an APOE-dependent pathway similar to DAMs. Within the white matter, microglia frequently cluster in nodules, where they are engaged in clearing degenerated myelin. Thus, WAMs may represent a potentially protective response required to clear degenerated myelin accumulating during white matter aging and disease.

Designed CXCR4 mimic acts as a soluble chemokine receptor that blocks atherogenic inflammation by agonist-specific targeting.

Targeting a specific chemokine/receptor axis in atherosclerosis remains challenging. Soluble receptor-based strategies are not established for chemokine receptors due to their discontinuous architecture. Macrophage migration-inhibitory factor (MIF) is an atypical chemokine that promotes atherosclerosis through CXC-motif chemokine receptor-4 (CXCR4). However, CXCR4/CXCL12 interactions also mediate atheroprotection. Here, we show that constrained 31-residue-peptides ('msR4Ms') designed to mimic the CXCR4-binding site to MIF, selectively bind MIF with nanomolar affinity and block MIF/CXCR4 without affecting CXCL12/CXCR4. We identify msR4M-L1, which blocks MIF- but not CXCL12-elicited CXCR4 vascular cell activities. Its potency compares well with established MIF inhibitors, whereas msR4M-L1 does not interfere with cardioprotective MIF/CD74 signaling. In vivo-administered msR4M-L1 enriches in atherosclerotic plaques, blocks arterial leukocyte adhesion, and inhibits atherosclerosis and inflammation in hyperlipidemic Apoe-/- mice in vivo. Finally, msR4M-L1 binds to MIF in plaques from human carotid-endarterectomy specimens. Together, we establish an engineered GPCR-ectodomain-based mimicry principle that differentiates between disease-exacerbating and -protective pathways and chemokine-selectively interferes with atherosclerosis.

Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity.

The causative agent of coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For many viruses, tissue tropism is determined by the availability of virus receptors and entry cofactors on the surface of host cells. In this study, we found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1. A SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells facing the nasal cavity. Our data provide insight into SARS-CoV-2 cell infectivity and define a potential target for antiviral intervention.

Continuous and Discrete Neuron Types of the Adult Murine Striatum.

The mammalian striatum is involved in many complex behaviors and yet is composed largely of a single neuron class: the spiny projection neuron (SPN). It is unclear to what extent the functional specialization of the striatum is due to the molecular specialization of SPN subtypes. We sought to define the molecular and anatomical diversity of adult SPNs using single-cell RNA sequencing (scRNA-seq) and quantitative RNA in situ hybridization (ISH). We computationally distinguished discrete versus continuous heterogeneity in scRNA-seq data and found that SPNs in the striatum can be classified into four major discrete types with no implied spatial relationship between them. Within these discrete types, we find continuous heterogeneity encoding spatial gradients of gene expression and defining anatomical location in a combinatorial mechanism. Our results suggest that neuronal circuitry has a substructure at far higher resolution than is typically interrogated, which is defined by the precise identity and location of a neuron.

The Multitasking Potential of Alarmins and Atypical Chemokines.

When the human genome was sequenced, it came as a surprise that it contains "only" 21,306 protein-coding genes. However, complexity and diversity are multiplied by alternative splicing, non-protein-coding transcripts, or post-translational modifications (PTMs) on proteome level. Here, we discuss how the multi-tasking potential of proteins can substantially enhance the complexity of the proteome further, while at the same time offering mechanisms for the fine-regulation of cell responses. Discoveries over the past two decades have led to the identification of "surprising" and previously unrecognized functionalities of long known cytokines, inflammatory mediators, and intracellular proteins that have established novel molecular networks in physiology, inflammation, and cardiovascular disease. In this mini-review, we focus on alarmins and atypical chemokines such as high-mobility group box protein-1 (HMGB-1) and macrophage migration-inhibitory factor (MIF)-type proteins that are prototypical examples of these classes, featuring a remarkable multitasking potential that allows for an elaborate fine-tuning of molecular networks in the extra- and intracellular space that may eventually give rise to novel "task"-based precision medicine intervention strategies.

Autism-associated neuroligin-4 mutation selectively impairs glycinergic synaptic transmission in mouse brainstem synapses.

In human patients, loss-of-function mutations of the postsynaptic cell-adhesion molecule neuroligin-4 were repeatedly identified as monogenetic causes of autism. In mice, neuroligin-4 deletions caused autism-related behavioral impairments and subtle changes in synaptic transmission, and neuroligin-4 was found, at least in part, at glycinergic synapses. However, low expression levels precluded a comprehensive analysis of neuroligin-4 localization, and overexpression of neuroligin-4 puzzlingly impaired excitatory but not inhibitory synaptic function. As a result, the function of neuroligin-4 remains unclear, as does its relation to other neuroligins. To clarify these issues, we systematically examined the function of neuroligin-4, focusing on excitatory and inhibitory inputs to defined projection neurons of the mouse brainstem as central model synapses. We show that loss of neuroligin-4 causes a profound impairment of glycinergic but not glutamatergic synaptic transmission and a decrease in glycinergic synapse numbers. Thus, neuroligin-4 is essential for the organization and/or maintenance of glycinergic synapses.

High fidelity hypothermic preservation of primary tissues in organ transplant preservative for single cell transcriptome analysis.

BACKGROUND: High-fidelity preservation strategies for primary tissues are in great demand in the single cell RNAseq community. A reliable method would greatly expand the scope of feasible multi-site collaborations and maximize the utilization of technical expertise. When choosing a method, standardizability and fidelity are important factors to consider due to the susceptibility of single-cell RNAseq analysis to technical noise. Existing approaches such as cryopreservation and chemical fixation are less than ideal for failing to satisfy either or both of these standards. RESULTS: Here we propose a new strategy that leverages preservation schemes developed for organ transplantation. We evaluated the strategy by storing intact mouse kidneys in organ transplant preservative solution at hypothermic temperature for up to 4 days (6 h, 1, 2, 3, and 4 days), and comparing the quality of preserved and fresh samples using FACS and single cell RNAseq. We demonstrate that the strategy effectively maintained cell viability, transcriptome integrity, cell population heterogeneity, and transcriptome landscape stability for samples after up to 3 days of preservation. The strategy also facilitated the definition of the diverse spectrum of kidney resident immune cells, to our knowledge the first time at single cell resolution. CONCLUSIONS: Hypothermic storage of intact primary tissues in organ transplant preservative maintains the quality and stability of the transcriptome of cells for single cell RNAseq analysis. The strategy is readily generalizable to primary specimens from other tissue types for single cell RNAseq analysis.

Conditional Deletion of All Neurexins Defines Diversity of Essential Synaptic Organizer Functions for Neurexins.

Neurexins are recognized as key organizers of synapses that are essential for normal brain function. However, it is unclear whether neurexins are fundamental building blocks of all synapses with similar overall functions or context-dependent specifiers of synapse properties. To address this question, we produced triple cKO (conditional knockout) mice that allow ablating all neurexin expression in mice. Using neuron-specific manipulations combined with immunocytochemistry, paired recordings, and two-photon Ca2+ imaging, we analyzed excitatory synapses formed by climbing fibers on Purkinje cells in cerebellum and inhibitory synapses formed by parvalbumin- or somatostatin-positive interneurons on pyramidal layer 5 neurons in the medial prefrontal cortex. After pan-neurexin deletions, we observed in these synapses severe but dramatically different synaptic phenotypes that ranged from major impairments in their distribution and function (climbing-fiber synapses) to large decreases in synapse numbers (parvalbumin-positive synapses) and severe alterations in action potential-induced presynaptic Ca2+ transients (somatostatin-positive synapses). Thus, neurexins function primarily as context-dependent specifiers of synapses.