GPR56, an orphan G protein-coupled receptor (GPCR) from the family of adhesion GPCRs, plays an indispensable role in cortical development and lamination. Mutations in the GPR56 gene cause a malformed cerebral cortex in both humans and mice that resembles cobblestone lissencephaly, which is characterized by overmigration of neurons beyond the pial basement membrane. However, the molecular mechanisms through which GPR56 regulates cortical development remain elusive due to the unknown status of its ligand. Here we identify collagen, type III, alpha-1 (gene symbol Col3a1) as the ligand of GPR56 through an in vitro biotinylation/ proteomics approach. Further studies demonstrated that Col3a1 null mutant mice exhibit overmigration of neurons beyond the pial basement membrane and a cobblestone-like cortical malformation similar to the phenotype seen in Gpr56 null mutant mice. Functional studies suggest that the interaction of collagen III with its receptor GPR56 inhibits neural migration in vitro. As for intracellular signaling, GPR56 couples to the Gα 12/13 family of G proteins and activates RhoA pathway upon ligand binding. Thus, collagen III regulates the proper lamination of the cerebral cortex by acting as the major ligand of GPR56 in the developing brain.brain malformation | meningeal fibroblasts D uring cerebral cortical development, most cortical neurons are born deep in specialized proliferative regions near the lining of the lateral ventricles and migrate out to take up residence in the surface layer of the brain, eventually forming a highly folded sheet of six neuronal layers (1-5). Disruptions in normal neuronal migration and positioning lead to cortical disorders, one of which is cobblestone lissencephaly (6). Cobblestone lissencephaly is typically seen in three distinct human congenital muscular dystrophy syndromes: muscle-eye-brain disease, Fukuyama-type congenital muscular dystrophy, and Walker-Warburg syndrome (6). Mutant mice with deletions in some members of the integrin pathway molecules and the extracellular matrix (ECM) constituents also exhibit cortical migration defects with deficiencies in basal lamina integrity and cortical ectopias, features that resemble the human cobblestone malformation (7-12). The suggested mechanism leading to cobblestone lissencephaly has been a defective pial basement membrane (BM) (6). However, recent literature demonstrates that abnormal neuronal migration may account partially for the improper formation of the cobblestone-like cortex (13-16).GPR56 is an orphan G protein-coupled receptor (GPCR) from the family of adhesion GPCRs. Mutations in GPR56 cause a specific human brain malformation called bilateral frontoparietal polymicrogyria (BFPP), an autosomal recessively inherited developmental disorder of the brain (17)(18)(19)(20). Magnetic resonance imaging of BFPP brains shows numerous (poly) small (micro) gyri, which extend diffusely across the frontal and parietal lobes with a decreasing anterior-to-posterior gradient of severity (17)(18)(19)(20). Further studies in...

SUMMARY Myelin ensheathes axons to allow rapid propagation of action potentials and proper nervous system function. In the peripheral nervous system, Schwann cells (SCs) radially sort axons into a 1:1 relationship before wrapping an axonal segment to form myelin. SC myelination requires the adhesion G protein-coupled receptor GPR126, which undergoes autoproteolytic cleavage into an N-terminal fragment (NTF) and a 7-transmembrane-containing C-terminal fragment (CTF). Here, we show that GPR126 has domain-specific functions in SC development whereby the NTF is necessary and sufficient for axon sorting while the CTF promotes wrapping through cAMP elevation. These biphasic roles of GPR126 are governed by interactions with Laminin-211, which we define as a novel ligand for GPR126 that modulates receptor signaling via a tethered agonist. Our work suggests a model in which Laminin-211 mediates GPR126-induced cAMP levels to control early and late stages of SC development.

The AICD (amyloid precursor protein [APP] intracellular domain) and C31, the caspase-cleaved C-terminal fragment of APP, have been found in the brains of patients with Alzheimer's disease (AD). Here, we demonstrate for the first time that the C-terminal fragments of APP (AICD [C57, C59] and C31) exert neurotoxicity on differentiated PC 12 cells and rat primary cortical neurons by inducing the expression of glycogen synthase kinase 3beta, forming a ternary complex with Fe65 and CP2/LSF/LBP1 in the nucleus, whereas deletion mutants and a point mutant with Y682G of the YENPTY domain, a Fe65 binding domain, do not. Moreover, expression of APP770 and Swedish mutant form of APP increased the levels of C-terminal fragments of APP (APP-CTs) in neuronal cells and also induced the up-regulation of glycogen synthase kinase-3beta at both the mRNA and the protein levels. In addition, we show that CP2/LSF/LBP1 binding site (nt +0 to approximately +10) in human glycogen synthase kinase 3beta promoter region is essential for the induction of the gene transcription by APP-CTs. The neurotoxicities induced by APP-CTs (AICD and C31) were accompanied by an increase in the active form of glycogen synthase kinase-3beta, and by the induction of tau phosphorylation and a reduction in nuclear beta-catenin levels, and led to apoptosis.

Understanding the structural organization of organs and organisms at the cellular level is a fundamental challenge in biology. This task has been approached by reconstructing three-dimensional structure from images taken from serially sectioned tissues, which is not only labor-intensive and time-consuming but also error-prone. Recent advances in tissue clearing techniques allow visualization of cellular structures and neural networks inside of unsectioned whole tissues or the entire body. However, currently available protocols require long process times. Here, we present the rapid and highly reproducible ACT-PRESTO (active clarity technique-pressure related efficient and stable transfer of macromolecules into organs) method that clears tissues or the whole body within 1 day while preserving tissue architecture and protein-based signals derived from endogenous fluorescent proteins. Moreover, ACT-PRESTO is compatible with conventional immunolabeling methods and expedites antibody penetration into thick specimens by applying pressure. The speed and consistency of this method will allow high-content mapping and analysis of normal and pathological features in intact organs and bodies.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)␤ superfamily of ligands that regulate many crucial aspects of embryonic development and organogenesis. Unlike other TGF␤ ligands, co-receptors for BMP ligands have not been described. Here we show that DRAGON, a glycosylphosphatidylinositol-anchored member of the repulsive guidance molecule family, which is expressed early in the developing nervous system, enhances BMP but not TGF␤ signaling. DRAGON binds directly to BMP2 and BMP4 but not to BMP7 or other TGF␤ ligands. The enhancing action of DRAGON on BMP signaling is also reduced by administration of Noggin, a soluble BMP antagonist, indicating that the action of DRAGON is ligand-dependent. DRAGON associates directly with BMP type I (ALK2, ALK3, and ALK6) and type II (ActRII and ActRIIB) receptors, and its signaling is reduced by dominant negative Smad1 and ALK3 or -6 receptors. In the Xenopus embryo, DRAGON both reduces the threshold of the ability of Smad1 to induce mesodermal and endodermal markers and alters neuronal and neural crest patterning. The direct interaction of DRAGON with BMP ligands and receptors indicates that it is a BMP co-receptor that potentiates BMP signaling. Transforming growth factor beta (TGF␤)1 superfamily ligands that include the TGF␤, bone morphogenetic protein (BMP), growth and differentiation factor, and nodal-related families play a pleiotropic role in vertebrate development by influencing cell specification, differentiation, proliferation, patterning, and migration (1, 2). These functions require the tight control of ligand production, ensuring a highly ordered spatiotemporal distribution and specific activation, via receptor complexes, of particular intracellular signaling pathways. The TGF␤/activin/nodal ligand subfamily contributes to the specification of endoderm and mesoderm in pregastrula embryos and at gastrula stages, to dorsal mesoderm formation and anterior-posterior patterning (3, 4). Later, TGF␤ ligands influence the body axis and patterning of the nervous system (5). BMPs, a second major ligand subfamily, contribute to the ventralization of germ layers in the early embryo and suppress the default neural cell fate of the ectoderm (6). BMPs also participate later in development in the formation and patterning of the neural crest, heart, blood, kidney, limb, muscle, and skeletal system (7).Signal transduction in the BMP subfamily is initiated by ligand binding to a receptor complex composed of two type I and two type II receptors. Three different BMP type I receptors (activin receptor-like kinase ALK2, ALK3, and ALK6) and three BMP type II receptors (BMP type II receptor (BMPRII), activin type IIA receptor (ActRIIA), activin type IIB receptor (ActRIIB)), each with intracellular serine/threonine kinase domains, have been identified (8). Ligand binding induces phosphorylation of the type I receptor by the type II receptor, which leads to phosphorylation of cytoplasmic receptor-activated Smads. The BMP subfamily signals through one set...

Mutations in GPR56, a member of the adhesion G protein-coupled receptor family, cause a human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). Magnetic resonance imaging (MRI) of BFPP brains reveals myelination defects in addition to brain malformation. However, the cellular role of GPR56 in oligodendrocyte development remains unknown. Here, we demonstrate that loss of Gpr56 leads to hypomyelination of the central nervous system in mice. GPR56 levels are abundant throughout early stages of oligodendrocyte development, but are downregulated in myelinating oligodendrocytes. Gpr56-knockout mice manifest with decreased oligodendrocyte precursor cell (OPC) proliferation and diminished levels of active RhoA, leading to fewer mature oligodendrocytes and a reduced number of myelinated axons in the corpus callosum and optic nerves. Conditional ablation of Gpr56 in OPCs leads to a reduced number of mature oligodendrocytes as seen in constitutive knockout of Gpr56. Together, our data define GPR56 as a cell-autonomous regulator of oligodendrocyte development.

DRG11, a transcription factor expressed in embryonic dorsal root ganglion (DRG) and dorsal horn neurons, has a role in the development of sensory circuits. We have used a genomic binding strategy to screen for the promoter region of genes regulated by DRG11. One gene with a promoter region binding to the DNA binding domain of DRG11 encodes a novel membrane-associated [glycosylphosphatidylinositol (GPI)-anchored] protein that we call DRAGON. DRAGON expression is transcriptionally regulated by DRG11, and it is coexpressed with DRG11 in embryonic DRG and spinal cord. DRAGON expression in these areas is reduced in DRG11 null mutants. DRAGON is expressed, however, in the neural tube before DRG11, and unlike DRG11 it is expressed in the brain and therefore must be regulated by other transcriptional regulatory elements. DRAGON shares high sequence homology with two other GPI-anchored membrane proteins: the mouse ortholog of chick repulsive guidance molecule (mRGM), which is expressed in the mouse nervous system in areas complementary to DRAGON, and DRAGON-like muscle (DL-M), the expression of which is restricted to skeletal and cardiac muscle. A comparative genomic analysis indicates that the family of RGM-related genes-mRGM, DRAGON, and DL-M-are highly conserved among mammals, zebrafish, chick, and Caenorhabditis elegans but not Drosophila. DRAGON, RGM, and DL-M mRNA expression in the zebrafish embryo is similar to that in the mouse. Neuronal cell adhesion assays indicate that DRAGON promotes and mRGM reduces adhesion of mouse DRG neurons. We show that DRAGON interacts with itself homophilically. The dynamic expression, ordered spatial localization, and adhesive properties of the RGM-related family of membrane-associated proteins are compatible with specific roles in development.

In the central nervous system (CNS), myelin formation and repair are regulated by oligodendrocyte (OL) lineage cells, which sense and integrate signals from their environment, including from other glial cells and the extracellular matrix (ECM). The signaling pathways that coordinate this complex communication, however, remain poorly understood. The adhesion G protein-coupled receptor ADGRG1 (also known as GPR56) is an evolutionarily conserved regulator of OL development in humans, mice, and zebrafish, although its activating ligand for OL lineage cells is unknown. Here, we report that microglia-derived transglutaminase-2 (TG2) signals to ADGRG1 on OL precursor cells (OPCs) in the presence of the ECM protein laminin and that TG2/laminin-dependent activation of ADGRG1 promotes OPC proliferation. Signaling by TG2/laminin to ADGRG1 on OPCs additionally improves remyelination in two murine models of demyelination. These findings identify a novel glia-to-glia signaling pathway that promotes myelin formation and repair, and suggest new strategies to enhance remyelination.