Richard Daneman

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.

Pericytes are required for blood-brain barrier integrity during embryogenesis

Vascular endothelial cells in the central nervous system (CNS) form a barrier that restricts the movement of molecules and ions between the blood and the brain. This blood–brain barrier (BBB) is crucial to ensure proper neuronal function and protect the CNS from injury and disease. Transplantation studies have demonstrated that the BBB is not intrinsic to the endothelial cells, but is induced by interactions with the neural cells. Owing to the close spatial relationship between astrocytes and endothelial cells, it has been hypothesized that astrocytes induce this critical barrier postnatally, but the timing of BBB formation has been controversial. Here we demonstrate that the barrier is formed during embryogenesis as endothelial cells invade the CNS and pericytes are recruited to the nascent vessels, over a week before astrocyte generation. Analysing mice with null and hypomorphic alleles of Pdgfrb, which have defects in pericyte generation, we demonstrate that pericytes are necessary for the formation of the BBB, and that absolute pericyte coverage determines relative vascular permeability. We demonstrate that pericytes regulate functional aspects of the BBB, including the formation of tight junctions and vesicle trafficking in CNS endothelial cells. Pericytes do not induce BBB-specific gene expression in CNS endothelial cells, but inhibit the expression of molecules that increase vascular permeability and CNS immune cell infiltration. These data indicate that pericyte–endothelial cell interactions are critical to regulate the BBB during development, and disruption of these interactions may lead to BBB dysfunction and neuroinflammation during CNS injury and disease.

A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila

In Drosophila, enhancer trap strategies allow rapid access to expression patterns, molecular data, and mutations in trapped genes. However, they do not give any information at the protein level, e.g., about the protein subcellular localization. Using the green fluorescent protein (GFP) as a mobile artificial exon carried by a transposable P-element, we have developed a protein trap system. We screened for individual flies, in which GFP tags full-length endogenous proteins expressed from their endogenous locus, allowing us to observe their cellular and subcellular distribution. GFP fusions are targeted to virtually any compartment of the cell. In the case of insertions in previously known genes, we observe that the subcellular localization of the fusion protein corresponds to the described distribution of the endogenous protein. The artificial GFP exon does not disturb upstream and downstream splicing events. Many insertions correspond to genes not predicted by the Drosophila Genome Project. Our results show the feasibility of a protein trap in Drosophila. GFP reveals in real time the dynamics of proteins distribution in the whole, live organism and provides useful markers for a number of cellular structures and compartments.

Wnt/β-catenin signaling is required for CNS, but not non-CNS, angiogenesis

Despite the importance of CNS blood vessels, the molecular mechanisms that regulate CNS angiogenesis and blood−brain barrier (BBB) formation are largely unknown. Here we analyze the role of Wnt/β-catenin signaling in regulating the formation of CNS blood vessels. First, through the analysis of TOP-Gal Wnt reporter mice, we identify that canonical Wnt/β-catenin signaling is specifically activated in CNS, but not non-CNS, blood vessels during development. This activation correlates with the expression of different Wnt ligands by neural progenitor cells in distinct locations throughout the CNS, including Wnt7a and Wnt7b in ventral regions and Wnt1, Wnt3, Wnt3a, and Wnt4 in dorsal regions. Blockade of Wnt/β-catenin signaling in vivo specifically disrupts CNS, but not non-CNS, angiogenesis. These defects include reduction in vessel number, loss of capillary beds, and the formation of hemorrhagic vascular malformations that remain adherent to the meninges. Furthermore, we demonstrate that Wnt/β-catenin signaling regulates the expression of the BBB-specific glucose transporter glut-1. Taken together these experiments reveal an essential role for Wnt/β-catenin signaling in driving CNS-specific angiogenesis and provide molecular evidence that angiogenesis and BBB formation are in part linked.

The Mouse Blood-Brain Barrier Transcriptome: A New Resource for Understanding the Development and Function of Brain Endothelial Cells

The blood-brain barrier (BBB) maintains brain homeostasis and limits the entry of toxins and pathogens into the brain. Despite its importance, little is known about the molecular mechanisms regulating the development and function of this crucial barrier. In this study we have developed methods to highly purify and gene profile endothelial cells from different tissues, and by comparing the transcriptional profile of brain endothelial cells with those purified from the liver and lung, we have generated a comprehensive resource of transcripts that are enriched in the BBB forming endothelial cells of the brain. Through this comparison we have identified novel tight junction proteins, transporters, metabolic enzymes, signaling components, and unknown transcripts whose expression is enriched in central nervous system (CNS) endothelial cells. This analysis has identified that RXRalpha signaling cascade is specifically enriched at the BBB, implicating this pathway in regulating this vital barrier. This dataset provides a resource for understanding CNS endothelial cells and their interaction with neural and hematogenous cells.

The Blood-Brain Barrier in Health and Disease

The blood–brain barrier (BBB) is a term used to describe a series of properties possessed by the vasculature of the central nervous system (CNS) that tightly regulate the movement of ions, molecules, and cells between the blood and the CNS. This barrier is crucial to provide the appropriate environment to allow for proper neural function, as well as protect the CNS from injury and disease. In this review, I discuss the cellular and molecular composition of the BBB and how the development and function of the BBB is regulated by interactions with the CNS microenvironment. I further discuss what is known about BBB dysfunction during CNS injury and disease, as well as methodology used to deliver drugs across the BBB to the CNS. ANN NEUROL 2012;72:648–672

The Blood–Brain Barrier

Blood vessels are critical to deliver oxygen and nutrients to all of the tissues and organs throughout the body. The blood vessels that vascularize the central nervous system (CNS) possess unique properties, termed the blood-brain barrier, which allow these vessels to tightly regulate the movement of ions, molecules, and cells between the blood and the brain. This precise control of CNS homeostasis allows for proper neuronal function and also protects the neural tissue from toxins and pathogens, and alterations of these barrier properties are an important component of pathology and progression of different neurological diseases. The physiological barrier is coordinated by a series of physical, transport, and metabolic properties possessed by the endothelial cells (ECs) that form the walls of the blood vessels, and these properties are regulated by interactions with different vascular, immune, and neural cells. Understanding how these different cell populations interact to regulate the barrier properties is essential for understanding how the brain functions during health and disease.

Oligodendrocyte-Encoded HIF Function Couples Postnatal Myelination and White Matter Angiogenesis

Myelin sheaths provide critical functional and trophic support for axons in white matter tracts of the brain. Oligodendrocyte precursor cells (OPCs) have extraordinary metabolic requirements during development as they differentiate to produce multiple myelin segments, implying that they must first secure adequate access to blood supply. However, mechanisms that coordinate myelination and angiogenesis are unclear. Here, we show that oxygen tension, mediated by OPC-encoded hypoxia-inducible factor (HIF) function, is an essential regulator of postnatal myelination. Constitutive HIF1/2α stabilization resulted in OPC maturation arrest through autocrine activation of canonical Wnt7a/7b. Surprisingly, such OPCs also show paracrine activity that induces excessive postnatal white matter angiogenesis in vivo and directly stimulates endothelial cell proliferation in vitro. Conversely, OPC-specific HIF1/2α loss of function leads to insufficient angiogenesis in corpus callosum and catastrophic axon loss. These findings indicate that OPC-intrinsic HIF signaling couples postnatal white matter angiogenesis, axon integrity, and the onset of myelination in mammalian forebrain.

Blood-brain barrier permeability is increased after acute adult stroke but not neonatal stroke in the rat

The immaturity of the CNS at birth greatly affects injury after stroke but the contribution of the blood–brain barrier (BBB) to the differential response to stroke in adults and neonates is poorly understood. We asked whether the structure and function of the BBB is disrupted differently in neonatal and adult rats by transient middle cerebral artery occlusion. In adult rats, albumin leakage into injured regions was markedly increased during 2–24 h reperfusion but leakage remained low in the neonates. Functional assays employing intravascular tracers in the neonates showed that BBB permeability to both large (70 kDa dextran) and small (3 kDa dextran), gadolinium (III)-diethyltriaminepentaacetic acid tracers remained largely undisturbed 24 h after reperfusion. The profoundly different functional integrity of the BBB was associated with the largely nonoverlapping patterns of regulated genes in endothelial cells purified from injured and uninjured adult and neonatal brain at 24 h (endothelial transcriptome, 31,042 total probe sets). Within significantly regulated 1266 probe sets in injured adults and 361 probe sets in neonates, changes in the gene expression of the basal lamina components, adhesion molecules, the tight junction protein occludin, and matrix metalloproteinase-9 were among the key differences. The protein expression of collagen-IV, laminin, claudin-5, occludin, and zonula occludens protein 1 was also better preserved in neonatal rats. Neutrophil infiltration remained low in acutely injured neonates but neutralization of cytokine-induced neutrophil chemoattractant-1 in the systemic circulation enhanced neutrophil infiltration, BBB permeability, and injury. The markedly more integrant BBB in neonatal brain than in adult brain after acute stroke may have major implications for the treatment of neonatal stroke.

A Novel Purification Method for CNS Projection Neurons Leads to the Identification of Brain Vascular Cells as a Source of Trophic Support for Corticospinal Motor Neurons

One of the difficulties in studying cellular interactions in the CNS is the lack of effective methods to purify specific neuronal populations of interest. We report the development of a novel purification scheme, cholera toxin β (CTB) immunopanning, in which a particular CNS neuron population is selectively labeled via retrograde axonal transport of the cell-surface epitope CTB, and then purified via immobilization with anti-CTB antibody. We have demonstrated the usefulness and versatility of this method by purifying both retinal ganglion cells and corticospinal motor neurons (CSMNs). Genomic expression analyses of purified CSMNs revealed that they express significant levels of many receptors for growth factors produced by brain endothelial cells; three of these factors, CXCL12, pleiotrophin, and IGF2 significantly enhanced purified CSMN survival, similar to previously characterized CSMN trophic factors BDNF and IGF1. In addition, endothelial cell conditioned medium significantly promoted CSMN neurite outgrowth. These findings demonstrate a useful method for the purification of several different types of CNS projection neurons, which in principle should work in many mammalian species, and provide evidence that endothelial-derived factors may represent an overlooked source of trophic support for neurons in the brain.