Mariko L. Bennett

An RNA-Sequencing Transcriptome and Splicing Database of Glia, Neurons, and Vascular Cells of the Cerebral Cortex

The major cell classes of the brain differ in their developmental processes, metabolism, signaling, and function. To better understand the functions and interactions of the cell types that comprise these classes, we acutely purified representative populations of neurons, astrocytes, oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia, endothelial cells, and pericytes from mouse cerebral cortex. We generated a transcriptome database for these eight cell types by RNA sequencing and used a sensitive algorithm to detect alternative splicing events in each cell type. Bioinformatic analyses identified thousands of new cell type-enriched genes and splicing isoforms that will provide novel markers for cell identification, tools for genetic manipulation, and insights into the biology of the brain. For example, our data provide clues as to how neurons and astrocytes differ in their ability to dynamically regulate glycolytic flux and lactate generation attributable to unique splicing of PKM2, the gene encoding the glycolytic enzyme pyruvate kinase. This dataset will provide a powerful new resource for understanding the development and function of the brain. To ensure the widespread distribution of these datasets, we have created a user-friendly website (http://web.stanford.edu/group/barres_lab/brain_rnaseq.html) that provides a platform for analyzing and comparing transciption and alternative splicing profiles for various cell classes in the brain.

Neurotoxic reactive astrocytes are induced by activated microglia.

Reactive astrocytes are strongly induced by central nervous system (CNS) injury and disease, but their role is poorly understood. Here we show that a subtype of reactive astrocytes, which we termed A1, is induced by classically activated neuroinflammatory microglia. We show that activated microglia induce A1 astrocytes by secreting Il-1α, TNF and C1q, and that these cytokines together are necessary and sufficient to induce A1 astrocytes. A1 astrocytes lose the ability to promote neuronal survival, outgrowth, synaptogenesis and phagocytosis, and induce the death of neurons and oligodendrocytes. Death of axotomized CNS neurons in vivo is prevented when the formation of A1 astrocytes is blocked. Finally, we show that A1 astrocytes are abundant in various human neurodegenerative diseases including Alzheimer’s, Huntington’s and Parkinson’s disease, amyotrophic lateral sclerosis and multiple sclerosis. Taken together these findings help to explain why CNS neurons die after axotomy, strongly suggest that A1 astrocytes contribute to the death of neurons and oligodendrocytes in neurodegenerative disorders, and provide opportunities for the development of new treatments for these diseases.

Microglia: scapegoat, saboteur, or something else?

Microglia are resident immune cells in the brain and spinal cord. These cells provide immune surveillance and are mobilized in response to disparate diseases and injuries. Although microglial activation is often considered neurotoxic, microglia are essential defenders against many neurodegenerative diseases. It also seems increasingly likely that microglial dysfunction can underlie certain neurological diseases without an obvious immune component.

New tools for studying microglia in the mouse and human CNS

Significance Microglia are the tissue resident macrophages of the brain and spinal cord, implicated in important developmental, homeostatic, and disease processes, although our understanding of their roles is complicated by an inability to distinguish microglia from related cell types. Although they share many features with other macrophages, microglia have distinct developmental origins and functions. Here we validate a stable and robustly expressed microglial marker for both mouse and human, transmembrane protein 119 (Tmem119). We use custom-made antibodies against Tmem119 to perform deep RNA sequencing of developing microglia, and demonstrate that microglia mature by the second postnatal week in mice. The antibodies, cell isolation methods, and RNAseq profiles presented here will greatly facilitate our understanding of microglial function in health and disease. The specific function of microglia, the tissue resident macrophages of the brain and spinal cord, has been difficult to ascertain because of a lack of tools to distinguish microglia from other immune cells, thereby limiting specific immunostaining, purification, and manipulation. Because of their unique developmental origins and predicted functions, the distinction of microglia from other myeloid cells is critically important for understanding brain development and disease; better tools would greatly facilitate studies of microglia function in the developing, adult, and injured CNS. Here, we identify transmembrane protein 119 (Tmem119), a cell-surface protein of unknown function, as a highly expressed microglia-specific marker in both mouse and human. We developed monoclonal antibodies to its intracellular and extracellular domains that enable the immunostaining of microglia in histological sections in healthy and diseased brains, as well as isolation of pure nonactivated microglia by FACS. Using our antibodies, we provide, to our knowledge, the first RNAseq profiles of highly pure mouse microglia during development and after an immune challenge. We used these to demonstrate that mouse microglia mature by the second postnatal week and to predict novel microglial functions. Together, we anticipate these resources will be valuable for the future study and understanding of microglia in health and disease.

Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors

In the developing central nervous system (CNS), the control of synapse number and function is critical to the formation of neural circuits. We previously demonstrated that astrocyte-secreted factors powerfully induce the formation of functional excitatory synapses between CNS neurons. Astrocyte-secreted thrombospondins induce the formation of structural synapses, but these synapses are postsynaptically silent. Here we use biochemical fractionation of astrocyte-conditioned medium to identify glypican 4 (Gpc4) and glypican 6 (Gpc6) as astrocyte-secreted signals sufficient to induce functional synapses between purified retinal ganglion cell neurons, and show that depletion of these molecules from astrocyte-conditioned medium significantly reduces its ability to induce postsynaptic activity. Application of Gpc4 to purified neurons is sufficient to increase the frequency and amplitude of glutamatergic synaptic events. This is achieved by increasing the surface level and clustering, but not overall cellular protein level, of the GluA1 subunit of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) glutamate receptor (AMPAR). Gpc4 and Gpc6 are expressed by astrocytes in vivo in the developing CNS, with Gpc4 expression enriched in the hippocampus and Gpc6 enriched in the cerebellum. Finally, we demonstrate that Gpc4-deficient mice have defective synapse formation, with decreased amplitude of excitatory synaptic currents in the developing hippocampus and reduced recruitment of AMPARs to synapses. These data identify glypicans as a family of novel astrocyte-derived molecules that are necessary and sufficient to promote glutamate receptor clustering and receptivity and to induce the formation of postsynaptically functioning CNS synapses.

A Combination of Ontogeny and CNS Environment Establishes Microglial Identity

Microglia, the brains resident macrophages, are dynamic CNS custodians with surprising origins in the extra-embryonic yolk sac. The consequences of their distinct ontogeny are unknown but critical to understanding and treating brain diseases. We created a brain macrophage transplantation system to disentangle how environment and ontogeny specify microglial identity. We find that donor cells extensively engraft in the CNS of microglia-deficient mice, and even after exposure to a cell culture environment, microglia fully regain their identity when returned to the CNS. Though transplanted macrophages from multiple tissues can express microglial genes in the brain, only those of yolk-sac origin fully attain microglial identity. Transplanted macrophages of inappropriate origin, including primary human cells in a humanized host, express disease-associated genes and specific ontogeny markers. Through brain macrophage transplantation, we discover new principles of microglial identity that have broad applications to the study of disease and development of myeloid cell therapies.

Single-Cell RNA Sequencing of Lymph Node Stromal Cells Reveals Niche-Associated Heterogeneity

SUMMARY Stromal cells (SCs) establish the compartmentalization of lymphoid tissues critical to the immune response. However, the full diversity of lymph node (LN) SCs remains undefined. Using droplet‐based single‐cell RNA sequencing, we identified nine peripheral LN non‐endothelial SC clusters. Included are the established subsets, Ccl19hi T‐zone reticular cells (TRCs), marginal reticular cells, follicular dendritic cells (FDCs), and perivascular cells. We also identified Ccl19lo TRCs, likely including cholesterol‐25‐hydroxylase+ cells located at the T‐zone perimeter, Cxcl9+ TRCs in the T‐zone and interfollicular region, CD34+ SCs in the capsule and medullary vessel adventitia, indolethylamine N‐methyltransferase+ SCs in the medullary cords, and Nr4a1+ SCs in several niches. These data help define how transcriptionally distinct LN SCs support niche‐restricted immune functions and provide evidence that many SCs are in an activated state. Graphical Abstract Figure. No Caption available. HighlightsSingle‐cell RNA sequencing of lymph node stromal cells reveals nine clustersKnown subsets TRCs, MRCs, FDCs, and perivascular cells identifiedFive additional stromal cell clusters identified and anatomical locations determinedTwo clusters, Cxcl9+ TRCs and Nr4a1+ SCs, are defined by activation signatures &NA; Lymph node stromal cells support diverse processes, but bulk assessments obscure their niche‐specific functions. Rodda et al. identify transcriptional profiles for nine lymph node stromal cell clusters using single‐cell RNA sequencing, validate subset markers in situ, and suggest niche‐restricted functions.

Developmental heterogeneity of microglia and brain myeloid cells revealed by deep single-cell RNA sequencing

Microglia are increasingly recognized for their major contributions during brain development and neurodegenerative disease. It is currently unknown if these functions are carried out by subsets of microglia during different stages of development and adulthood or within specific brain regions. Here, we performed deep single-cell RNA sequencing (scRNA-seq) of microglia and related myeloid cells sorted from various regions of embryonic, postnatal, and adult mouse brains. We found that the majority of adult microglia with homeostatic signatures are remarkably similar in transcriptomes, regardless of brain region. By contrast, postnatal microglia represent a more heterogeneous population. We discovered that postnatal white matter-associated microglia (WAM) are strikingly different from microglia in other regions and express genes enriched in degenerative disease-associated microglia. These postnatal WAM have distinct amoeboid morphology, are metabolically active, and phagocytose newly formed oligodendrocytes. This scRNA-seq atlas will be a valuable resource for dissecting innate immune functions in health and disease. Highlights Myeloid scRNA-seq atlas across brain regions and developmental stages Limited transcriptomic heterogeneity of homeostatic microglia in the adult brain Phase-specific gene sets of proliferating microglia along cell cycle pseudotime Phagocytic postnatal white matter-associated microglia sharing DAM gene signatures

A genetically distinct microglial subset promotes myelination

Microglia are brain‐resident macrophages with important, but insufficiently understood functions in development, health, and disease. In a new exciting study, Wlodarczyk and colleagues uncover a transient subset of CD11c+ microglia that regulate CNS myelination via IGF‐1 expression. These findings represent not only the first evidence for a microglial role in myelinogenesis, but the first for a functionally distinct, genetically defined subpopulation of microglia.