Shane A. Liddelow

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.

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.

Barrier mechanisms in the developing brain.

The adult brain functions within a well-controlled stable environment, the properties of which are determined by cellular exchange mechanisms superimposed on the diffusion restraint provided by tight junctions at interfaces between blood, brain and cerebrospinal fluid (CSF). These interfaces are referred to as “the” blood–brain barrier. It is widely believed that in embryos and newborns, this barrier is immature or “leaky,” rendering the developing brain more vulnerable to drugs or toxins entering the fetal circulation from the mother. New evidence shows that many adult mechanisms, including functionally effective tight junctions are present in embryonic brain and some transporters are more active during development than in the adult. Additionally, some mechanisms present in embryos are not present in adults, e.g., specific transport of plasma proteins across the blood–CSF barrier and embryo-specific intercellular junctions between neuroependymal cells lining the ventricles. However developing cerebral vessels appear to be more fragile than in the adult. Together these properties may render developing brains more vulnerable to drugs, toxins, and pathological conditions, contributing to cerebral damage and later neurological disorders. In addition, after birth loss of protection by efflux transporters in placenta may also render the neonatal brain more vulnerable than in the fetus.

The blood-CSF barrier explained: when development is not immaturity

It is often suggested that during development the brain barriers are immature. This argument stems from teleological interpretations and experimental observations of the high protein concentrations in fetal cerebrospinal fluid (CSF) and decreases in apparent permeability of passive markers during development. We argue that the developmental blood–CSF barrier restricts the passage of lipid‐insoluble molecules by the same mechanism as in the adult (tight junctions) rendering the paracellular pathway an unlikely route of entry. Instead, we suggest that both protein and passive markers are transferred across the epithelium through a transcellular route. We propose that changes in volume of distribution can largely explain the decrease in apparent permeability for passive markers and that developmentally regulated cellular transfer explains changes in CSF protein concentrations. The blood–CSF tight junctions are functionally mature from very early in development, and it appears that transfer from blood into embryonic brain occurs predominately via CSF rather than the vasculature. BioEssays 30:237–248, 2008.

ApoE4 markedly exacerbates tau-mediated neurodegeneration in a mouse model of tauopathy

APOE4 is the strongest genetic risk factor for late-onset Alzheimer disease. ApoE4 increases brain amyloid-β pathology relative to other ApoE isoforms. However, whether APOE independently influences tau pathology, the other major proteinopathy of Alzheimer disease and other tauopathies, or tau-mediated neurodegeneration, is not clear. By generating P301S tau transgenic mice on either a human ApoE knock-in (KI) or ApoE knockout (KO) background, here we show that P301S/E4 mice have significantly higher tau levels in the brain and a greater extent of somatodendritic tau redistribution by three months of age compared with P301S/E2, P301S/E3, and P301S/EKO mice. By nine months of age, P301S mice with different ApoE genotypes display distinct phosphorylated tau protein (p-tau) staining patterns. P301S/E4 mice develop markedly more brain atrophy and neuroinflammation than P301S/E2 and P301S/E3 mice, whereas P301S/EKO mice are largely protected from these changes. In vitro, E4-expressing microglia exhibit higher innate immune reactivity after lipopolysaccharide treatment. Co-culturing P301S tau-expressing neurons with E4-expressing mixed glia results in a significantly higher level of tumour-necrosis factor-α (TNF-α) secretion and markedly reduced neuronal viability compared with neuron/E2 and neuron/E3 co-cultures. Neurons co-cultured with EKO glia showed the greatest viability with the lowest level of secreted TNF-α. Treatment of P301S neurons with recombinant ApoE (E2, E3, E4) also leads to some neuronal damage and death compared with the absence of ApoE, with ApoE4 exacerbating the effect. In individuals with a sporadic primary tauopathy, the presence of an ε4 allele is associated with more severe regional neurodegeneration. In individuals who are positive for amyloid-β pathology with symptomatic Alzheimer disease who usually have tau pathology, ε4-carriers demonstrate greater rates of disease progression. Our results demonstrate that ApoE affects tau pathogenesis, neuroinflammation, and tau-mediated neurodegeneration independently of amyloid-β pathology. ApoE4 exerts a ‘toxic’ gain of function whereas the absence of ApoE is protective.

Transporters of the blood–brain and blood–CSF interfaces in development and in the adult☆

The protective barriers of the brain provide a complex series of physical and chemical obstacles to movement of macromolecules from the periphery into the central nervous system. Studies on these barriers have been focused on two main research areas: (i) anatomical and physiological descriptions of their properties, including during development where functioning barriers are likely to be important for normal neuronal growth; and (ii), investigations of these barriers during disease and attempts at overcoming their defenses in order to deliver drugs to the central nervous system. Both fields are now advanced by the application of molecular gene expression studies of cerebral endothelia (blood vasculature, site of the blood-brain barrier) and choroid plexus epithelia (site of the blood-cerebrospinal fluid barrier) from developing and adult brains, particularly with respect to solute-linked carriers and other transporters. These new techniques provide a wealth of information on the changing nature of transporters at barrier interfaces during normal development and following disease. This review outlines published findings from transcriptome and qPCR studies of expression of genes coding for transporters in these barriers, with a focus on developing brain. The findings clearly support earlier published physiological data describing specific transport mechanisms across barrier interfaces both in the adult and in particular in the developing brain.

Efflux mechanisms at the developing brain barriers: ABC-transporters in the fetal and postnatal rat

Proteins of the ATP-binding cassette (ABC) family, present at the blood-brain barrier interfaces, have been shown to reduce the entry of compounds from blood into the brain by active efflux. Their substrates are diverse including many drugs and toxins and therefore provide an important mechanism for brain neuroprotection. However, knowledge of their presence and function in the developing brain is very limited. We have used qPCR and immunocytochemistry to determine gene expression and localisation of four main barrier ABC-transporters (pgp/ABCB1, MRP1/ABCC1, MRP4/ABCC4 and BCRP/ABCG2) in the fetal and neonatal rat brain cerebral blood vessels (site of blood-brain barrier) and choroid plexus (site of blood-CSF barrier). The study shows that ABC-transporters localise to the brain barriers even at early fetal stages and although pgp expression was lower in the fetus, the other transporters were expressed at comparable levels in fetal and adult brains suggesting direct neuroprotection of the brain in addition to that provided by the placenta. BCRP was expressed at higher levels in developing choroid plexus and was only detected at fetal stages on the blood-facing side of epithelial cells indicating a particular role of this transporter for early brain efflux mechanisms.

Blood–CSF barrier function in the rat embryo

Blood–cerebrospinal fluid (CSF) barrier function and expansion of the ventricular system were investigated in embryonic rats (E12–18). Permeability markers (sucrose and inulin) were injected intraperitoneally and concentrations measured in plasma and CSF at two sites (lateral and 4th ventricles) after 1 h. Total protein concentrations were also measured. CSF/plasma concentration ratios for endogenous protein were stable at ∼ 20% at E14–18 and subsequently declined. In contrast, ratios for sucrose (100%) and inulin (40%) were highest at the earliest ages studied (E13–14) and then decreased substantially. Between E13 and E16 the volume of the lateral ventricles increased over three‐fold. Decreasing CSF/plasma concentration ratios for small, passively diffusing molecules during embryonic development may not reflect changes in permeability. Instead, increasing volume of distribution appears to be important in this decline. The intracellular presence of a small marker (3000 Da biotin–dextranamine) in plexus epithelial cells following intraperitoneal injection indicates a transcellular route of transfer. Ultrastructural evidence confirmed that choroid plexus tight junctions are impermeable to small molecules at least as early as E15, indicating the blood–CSF barrier is morphologically and functionally mature early in embryonic development. Comparison of two albumins (human and bovine) showed that transfer of human albumin (surrogate for endogenous protein) was 4–5 times greater than bovine, indicating selective blood‐to‐CSF transfer. The number of plexus epithelial cells immunopositive for endogenous plasma protein increased in parallel with increases in total protein content of the expanding ventricular system. Results suggest that different transcellular mechanisms for protein and small molecule transfer are operating across the embryonic blood–CSF interface.

Molecular Characterisation of Transport Mechanisms at the Developing Mouse Blood–CSF Interface: A Transcriptome Approach

Exchange mechanisms across the blood–cerebrospinal fluid (CSF) barrier in the choroid plexuses within the cerebral ventricles control access of molecules to the central nervous system, especially in early development when the brain is poorly vascularised. However, little is known about their molecular or developmental characteristics. We examined the transcriptome of lateral ventricular choroid plexus in embryonic day 15 (E15) and adult mice. Numerous genes identified in the adult were expressed at similar levels at E15, indicating substantial plexus maturity early in development. Some genes coding for key functions (intercellular/tight junctions, influx/efflux transporters) changed expression during development and their expression patterns are discussed in the context of available physiological/permeability results in the developing brain. Three genes: Secreted protein acidic and rich in cysteine (Sparc), Glycophorin A (Gypa) and C (Gypc), were identified as those whose gene products are candidates to target plasma proteins to choroid plexus cells. These were investigated using quantitative- and single-cell-PCR on plexus epithelial cells that were albumin- or total plasma protein-immunopositive. Results showed a significant degree of concordance between plasma protein/albumin immunoreactivity and expression of the putative transporters. Immunohistochemistry identified SPARC and GYPA in choroid plexus epithelial cells in the embryo with a subcellular distribution that was consistent with transport of albumin from blood to cerebrospinal fluid. In adult plexus this pattern of immunostaining was absent. We propose a model of the cellular mechanism in which SPARC and GYPA, together with identified vesicle-associated membrane proteins (VAMPs) may act as receptors/transporters in developmentally regulated transfer of plasma proteins at the blood–CSF interface.