Yun C. Yung

LPA Receptors: Subtypes and Biological Actions

Lysophosphatidic acid (LPA) is a small, ubiquitous phospholipid that acts as an extracellular signaling molecule by binding to and activating at least five known G protein-coupled receptors (GPCRs): LPA(1)-LPA(5). They are encoded by distinct genes named LPAR1-LPAR5 in humans and Lpar1-Lpar5 in mice. The biological roles of LPA are diverse and include developmental, physiological, and pathophysiological effects. This diversity is mediated by broad and overlapping expression patterns and multiple downstream signaling pathways activated by cognate LPA receptors. Studies using cloned receptors and genetic knockout mice have been instrumental in uncovering the significance of this signaling system, notably involving basic cellular processes as well as multiple organ systems such as the nervous system. This has further provided valuable proof-of-concept data to support LPA receptors and LPA metabolic enzymes as targets for the treatment of medically important diseases that include neuropsychiatric disorders, neuropathic pain, infertility, cardiovascular disease, inflammation, fibrosis, and cancer.

FTY720 (fingolimod) efficacy in an animal model of multiple sclerosis requires astrocyte sphingosine 1-phosphate receptor 1 (S1P1) modulation

Sphingosine 1-phosphate (S1P), a lysophospholipid, has gained relevance to multiple sclerosis through the discovery of FTY720 (fingolimod), recently approved as an oral treatment for relapsing forms of multiple sclerosis. Its mechanism of action is thought to be immunological through an active phosphorylated metabolite, FTY720-P, that resembles S1P and alters lymphocyte trafficking through receptor subtype S1P1. However, previously reported expression and in vitro studies of S1P receptors suggested that direct CNS effects of FTY720 might theoretically occur through receptor modulation on neurons and glia. To identify CNS cells functionally contributing to FTY720 activity, genetic approaches were combined with cellular and molecular analyses. These studies relied on the functional assessment, based on clinical score, of conditional null mouse mutants lacking S1P1 in CNS cell lineages and challenged by experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. All conditional null mutants displayed WT lymphocyte trafficking that responded normally to FTY720. In marked contrast, EAE was attenuated and FTY720 efficacy was lost in CNS mutants lacking S1P1 on GFAP-expressing astrocytes but not on neurons. In situ hybridization studies confirmed that astrocyte loss of S1P1 was the key alteration in functionally affected mutants. Reductions in EAE clinical scores were paralleled by reductions in demyelination, axonal loss, and astrogliosis. Receptor rescue and pharmacological experiments supported the loss of S1P1 on astrocytes through functional antagonism by FTY720-P as a primary FTY720 mechanism. These data identify nonimmunological CNS mechanisms of FTY720 efficacy and implicate S1P signaling pathways within the CNS as targets for multiple sclerosis therapies.

Constitutional Aneuploidy in the Normal Human Brain

The mouse brain contains genetically distinct cells that differ with respect to chromosome number manifested as aneuploidy (Rehen et al., 2001); however, the relevance to humans is not known. Here, using double-label fluorescence in situ hybridization for the autosome chromosome 21 (chromosome 21 point probes combined with chromosome 21 “paint” probes), along with immunocytochemistry and cell sorting, we present evidence for chromosome gain and loss in the human brain. Chromosome 21 aneuploid cells constitute ∼4% of the estimated one trillion cells in the human brain and include non-neuronal cells and postmitotic neurons identified by the neuronspecific nuclear protein marker. In comparison, human interphase lymphocytes present chromosome 21 aneuploidy rates of 0.6%. Together, these data demonstrate that human brain cells (both neurons and non-neuronal cells) can be aneuploid and that the resulting genetic mosaicism is a normal feature of the human CNS.

LPA receptor signaling: pharmacology, physiology, and pathophysiology

Lysophosphatidic acid (LPA) is a small ubiquitous lipid found in vertebrate and nonvertebrate organisms that mediates diverse biological actions and demonstrates medicinal relevance. LPA’s functional roles are driven by extracellular signaling through at least six 7-transmembrane G protein-coupled receptors. These receptors are named LPA1–6 and signal through numerous effector pathways activated by heterotrimeric G proteins, including Gi/o, G12/13, Gq, and Gs. LPA receptor-mediated effects have been described in numerous cell types and model systems, both in vitro and in vivo, through gain- and loss-of-function studies. These studies have revealed physiological and pathophysiological influences on virtually every organ system and developmental stage of an organism. These include the nervous, cardiovascular, reproductive, and pulmonary systems. Disturbances in normal LPA signaling may contribute to a range of diseases, including neurodevelopmental and neuropsychiatric disorders, pain, cardiovascular disease, bone disorders, fibrosis, cancer, infertility, and obesity. These studies underscore the potential of LPA receptor subtypes and related signaling mechanisms to provide novel therapeutic targets.

Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain

Single-nucleus gene expression Identifying the genes expressed at the level of a single cell nucleus can better help us understand the human brain. Blue et al. developed a single-nuclei sequencing technique, which they applied to cells in classically defined Brodmann areas from a postmortem brain. Clustering of gene expression showed concordance with the area of origin and defining 16 neuronal subtypes. Both excitatory and inhibitory neuronal subtypes show regional variations that define distinct cortical areas and exhibit how gene expression clusters may distinguish between distinct cortical areas. This method opens the door to widespread sampling of the genes expressed in a diseased brain and other tissues of interest. Science, this issue p. 1586 Individual neurons have specific transcriptomic signatures and transcriptomic heterogeneity. The human brain has enormously complex cellular diversity and connectivities fundamental to our neural functions, yet difficulties in interrogating individual neurons has impeded understanding of the underlying transcriptional landscape. We developed a scalable approach to sequence and quantify RNA molecules in isolated neuronal nuclei from a postmortem brain, generating 3227 sets of single-neuron data from six distinct regions of the cerebral cortex. Using an iterative clustering and classification approach, we identified 16 neuronal subtypes that were further annotated on the basis of known markers and cortical cytoarchitecture. These data demonstrate a robust and scalable method for identifying and categorizing single nuclear transcriptomes, revealing shared genes sufficient to distinguish previously unknown and orthologous neuronal subtypes as well as regional identity and transcriptomic heterogeneity within the human brain.

Characterizing transcriptional heterogeneity through pathway and gene set overdispersion analysis

The transcriptional state of a cell reflects a variety of biological factors, from cell-type-specific features to transient processes such as the cell cycle, all of which may be of interest. However, identifying such aspects from noisy single-cell RNA-seq data remains challenging. We developed pathway and gene set overdispersion analysis (PAGODA) to resolve multiple, potentially overlapping aspects of transcriptional heterogeneity by testing gene sets for coordinated variability among measured cells.

Lysophosphatidic Acid Signaling May Initiate Fetal Hydrocephalus

Blockade of lysophosphatidic acid signaling provides a new strategy for treating fetal hydrocephalus. Is the Cause of Hydrocephalus Blood Simple? Hydrocephalus or “water on the brain” is caused by accumulation of cerebrospinal fluid (CSF) in the cerebral ventricles during fetal development and is one of the most common neurological disorders of newborns, occurring in 1 in 1500 live births. One apparent cause of hydrocephalus is bleeding into the cerebral ventricles or brain tissue of the fetus, suggesting that factors or components in blood may trigger development of this severe neurological disorder. The most common treatment is surgical insertion of an intraventricular shunt that drains excess CSF from the cerebral ventricles, but this approach only relieves intracranial pressure and does not solve the root cause of the disorder. Yung et al. set out to investigate which factors in blood trigger hydrocephalus using an in vivo fetal mouse model that they developed. They identify a blood-borne lipid called lysophosphatidic acid (LPA) as a potential cause of hydrocephalus and show that when LPA is prevented from binding to its receptor LPA1 by a receptor antagonist, that hydrocephalus does not develop in fetal mice. The authors injected serum, plasma, or red blood cells into the cerebral ventricles of the brains of fetal mice in utero at 13.5 days of gestation. The animals were then assessed prenatally 1 or 5 days later or postnatally at several different time points. Injection of serum or plasma but not red blood cells induced CSF accumulation and hydrocephalus, with animals displaying enlarged heads, dilated ventricles, and thinning of the cortex. The investigators reasoned that LPA, a blood-borne lipid that is known to be important for the developing cerebral cortex, might be involved in the development of hydrocephalus. When they injected a solution containing LPA into the cerebral ventricles of fetal mice in utero, the mice did indeed develop severe hydrocephalus. The authors wondered how an increase in LPA might affect cortical development and lead to hydrocephalus. They show that injection of LPA resulted in altered adhesion and mislocalization of neural progenitor cells along the surface of the ventricles and that this mislocalization depended on expression of the LPA1 receptor by these cells. When the researchers repeated their experiments with fetal mice lacking the LPA1 receptor, they were unable to induce hydrocephalus. The key finding came with their demonstration that an LPA1 receptor antagonist blocked the ability of LPA to induce hydrocephalus in the fetal mice. These results suggest that LPA and its LPA1 receptor may be new therapeutic targets for developing drugs that could be used in conjunction with surgery to treat this debilitating neurological disease. Fetal hydrocephalus (FH), characterized by the accumulation of cerebrospinal fluid, an enlarged head, and neurological dysfunction, is one of the most common neurological disorders of newborns. Although the etiology of FH remains unclear, it is associated with intracranial hemorrhage. Here, we report that lysophosphatidic acid (LPA), a blood-borne lipid that activates signaling through heterotrimeric guanosine 5′-triphosphate–binding protein (G protein)–coupled receptors, provides a molecular explanation for FH associated with hemorrhage. A mouse model of intracranial hemorrhage in which the brains of mouse embryos were exposed to blood or LPA resulted in development of FH. FH development was dependent on the expression of the LPA1 receptor by neural progenitor cells. Administration of an LPA1 receptor antagonist blocked development of FH. These findings implicate the LPA signaling pathway in the etiology of FH and suggest new potential targets for developing new treatments for FH.

Aneuploid mosaicism in the developing and adult cerebellar cortex

Neuroprogenitor cells (NPCs) in several telencephalic proliferative regions of the mammalian brain, including the embryonic cerebral cortex and postnatal subventricular zone (SVZ), display cell division “defects” in normal cells that result in aneuploid adult progeny. Here, we identify the developing cerebellum as a major, nontelencephalic proliferative region of the vertebrate central nervous system (CNS) that also produces aneuploid NPCs and nonmitotic cells. Mitotic NPCs assessed by metaphase chromosome analyses revealed that 15.3% and 20.8% of cerebellar NPCs are aneuploid at P0 and P7, respectively. By using immunofluorescent analysis of cerebellar NPCs, we show that chromosome segregation defects contribute to the generation of cells with an aneuploid genomic complement. Nonmitotic cells were assessed by fluorescence‐activated cell sorting (FACS) coupled with fluorescence in situ hybridization (FISH), which revealed neuronal and nonneuronal aneuploid populations in both the adult mouse and human cerebellum. Taken together, these results demonstrate that the prevalence of neural aneuploidy includes nontelencephalic portions of the neuraxis and suggest that the generation and maintenance of aneuploid cells is a widespread, if not universal, property of central nervous system development and organization. J. Comp. Neurol. 507:1944–1951, 2008.

Lysophosphatidic Acid Signaling in the Nervous System

The brain is composed of many lipids with varied forms that serve not only as structural components but also as essential signaling molecules. Lysophosphatidic acid (LPA) is an important bioactive lipid species that is part of the lysophospholipid (LP) family. LPA is primarily derived from membrane phospholipids and signals through six cognate G protein-coupled receptors (GPCRs), LPA1-6. These receptors are expressed on most cell types within central and peripheral nervous tissues and have been functionally linked to many neural processes and pathways. This Review covers a current understanding of LPA signaling in the nervous system, with particular focus on the relevance of LPA to both physiological and diseased states.

Neuronal DNA content variation (DCV) with regional and individual differences in the human brain

It is widely assumed that the human brain contains genetically identical cells through which postgenomic mechanisms contribute to its enormous diversity and complexity. The relatively recent identification of neural cells throughout the neuraxis showing somatically generated mosaic aneuploidy indicates that the vertebrate brain can be genomically heterogeneous (Rehen et al. [2001] Proc. Natl. Acad. Sci. U. S. A. 98:13361–13366; Rehen et al. [2005] J. Neurosci. 25:2176–2180; Yurov et al. [2007] PLoS ONE:e558; Westra et al. [2008] J. Comp. Neurol. 507:1944–1951). The extent of human neural aneuploidy is currently unknown because of technically limited sample sizes, but is reported to be small (Iourov et al. [2006] Int. Rev. Cytol. 249:143–191). During efforts to interrogate larger cell populations by using DNA content analyses, a surprising result was obtained: human frontal cortex brain cells were found to display “DNA content variation (DCV)” characterized by an increased range of DNA content both in cell populations and within single cells. On average, DNA content increased by ∼250 megabases, often representing a substantial fraction of cells within a given sample. DCV within individual human brains showed regional variation, with increased prevalence in the frontal cortex and less variation in the cerebellum. Further, DCV varied between individual brains. These results identify DCV as a new feature of the human brain, encompassing and further extending genomic alterations produced by aneuploidy, which may contribute to neural diversity in normal and pathophysiological states, altered functions of normal and disease‐linked genes, and differences among individuals. J. Comp. Neurol. 518:3981–4000, 2010.