Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes.

Microglia, the resident immune cells of the brain, rapidly change states in response to their environment, but we lack molecular and functional signatures of different microglial populations. Here, we analyzed the RNA expression patterns of more than 76,000 individual microglia in mice during development, in old age, and after brain injury. Our analysis uncovered at least nine transcriptionally distinct microglial states, which expressed unique sets of genes and were localized in the brain using specific markers. The greatest microglial heterogeneity was found at young ages; however, several states-including chemokine-enriched inflammatory microglia-persisted throughout the lifespan or increased in the aged brain. Multiple reactive microglial subtypes were also found following demyelinating injury in mice, at least one of which was also found in human multiple sclerosis lesions. These distinct microglia signatures can be used to better understand microglia function and to identify and manipulate specific subpopulations in health and disease

Dissection of artifactual and confounding glial signatures by single-cell sequencing of mouse and human brain

A key aspect of nearly all single-cell sequencing experiments is dissociation of intact tissues into single-cell suspensions. While many protocols have been optimized for optimal cell yield, they have often overlooked the effects that dissociation can have on ex vivo gene expression. Here, we demonstrate that use of enzymatic dissociation on brain tissue induces an aberrant ex vivo gene expression signature, most prominently in microglia, which is prevalent in published literature and can substantially confound downstream analyses. To address this issue, we present a rigorously validated protocol that preserves both in vivo transcriptional profiles and cell-type diversity and yield across tissue types and species. We also identify a similar signature in postmortem human brain single-nucleus RNA-sequencing datasets, and show that this signature is induced in freshly isolated human tissue by exposure to elevated temperatures ex vivo. Together, our results provide a methodological solution for preventing artifactual gene expression changes during fresh tissue digestion and a reference for future deeper analysis of the potential confounding states present in postmortem human samples

Novel modes of communication between neuronal activity and microglial process dynamics

Microglia are morphologically dynamic cells that survey neuronal dendrites and rapidly respond to ATP. However, the role of ATP in mediating neuron-microglia communication remains to be determined. We therefore investigated the question whether high neuronal activity would evoke ATP release and thereby trigger a change in microglial process dynamics. To address this we used acute hippocampal brain slices and two-photon laser scanning microscopy and we developed a novel method for fixation and immunolabeling of microglia processes. We discovered that multiple brief applications of NMDA triggered a transient outgrowth of microglia processes similar to application of ATP. The outgrowth was reversible and repeatable, indicating that it was not due to excitotoxic damage. ATP release, secondary to NMDAR activation, was the key mediator as blocking purinergic receptors abolished outgrowth. Hemichannel opening is a well-defined mechanism for ATP release, but outgrowth still occurred in the absence of the hemichannel protein pannexin 1 and in the presence of the hemichannel blocker carbenoxolone. Utilizing whole cell patch clamping we demonstrated that activation of dendritic NMDAR on single neurons was sufficient to trigger microglia process outgrowth. These results suggest that dendritic neuronal NMDAR activation triggers ATP release via a hemichannel-independent mechanism. It is well established that high neuronal activity leads to a reduction in extracellular Ca²+ which causes opening of astrocytic Cx43 hemichannels and subsequent ATP release. We therefore investigated whether hemichannel opening could trigger a change in microglial process dynamics. Indeed, removal of extracellular Ca²+ triggered a microglial response, which we refer to as microglial process focalization because it was distinctively different from the NMDA-evoked process outgrowth. This focalization was also mediated by ATP as it was blocked by selective blockade of microglial purinergic receptors and we observed a strong inverse relationship between the concentration of extracellular Ca²+ and microglial responses. Carbenoxolone, which did not block NMDA-evoked process outgrowth, resulted in a dose-dependent block of microglial process focalization which is consistent with the mechanism of ATP release being opening of Cx43 hemichannels. Taken together, our data provide novel insight into how high neuronal activity triggers release of ATP as a mechanism for enhancing neuron-microglia communication.Medicine, Faculty ofGraduat

The Function of the human interferon-β1a glycan determined in vivo

Recombinant human interferon-β (rhIFN-β) is the leading therapeutic intervention shown to change the cause of relapsing-remitting multiple sclerosis, and both a nonglycosylated and a significantly more active glycosylated variant of rhIFN-β are used in treatment. This study investigates the function of the rhIFN-β1a glycan moiety and its individual carbohydrate residues, using the myxovirus resistance (Mx) mRNA as a biomarker in Mx-congenic mice. We showed that the Mx mRNA level in blood leukocytes peaked 3 h after s.c. administration of rhIFN-β1a. In addition, a clear dose-response relationship was confirmed, and the Mx response was shown to be receptor-mediated. Using specific glycosidases, different glycosylation analogs of rhIFN-β1a were obtained, and their activities were determined. The glycosylated rhIFN-β1a showed significantly higher activity than its deglycosylated counterpart, due to a protein stabilization/solubilization effect of the glycan. It is interesting to note that the terminating sialic acids were essential for these effects. Conclusively, the structure/bioactivity relationship of rhIFN-β1a was determined in vivo, and it provided a novel insight into the role of the rhIFN-β1a glycan and its carbohydrate residues. The possibilities of improving the pharmacological properties of rhIFN-β1a using glycoengineering are discussed.10 page(s

FEAST: A flow cytometry-based toolkit for interrogating microglial engulfment of synaptic and myelin proteins

Abstract Although engulfment is a hallmark of microglia function, fully validated platforms that facilitate high-throughput quantification of this process are lacking. Here, we present FEAST (Flow cytometric Engulfment Assay for Specific Target proteins), which enables interrogation of in vivo engulfment of synaptic material by brain resident macrophages at single-cell resolution. We optimize FEAST for two different analyses: quantification of fluorescent material inside live cells and of engulfed endogenous proteins within fixed cells. To overcome false-positive engulfment signals, we introduce an approach suitable for interrogating engulfment in microglia from perfusion-fixed tissue. As a proof-of-concept for the specificity and versatility of FEAST, we examine the engulfment of synaptic proteins after optic nerve crush and of myelin in two mouse models of demyelination (treatment with cuprizone and injections of lysolecithin). We find that microglia, but not brain-border associated macrophages, engulf in these contexts. Our work underscores how FEAST can be utilized to gain critical insight into functional neuro-immune interactions that shape development, homeostasis, and disease