Sarah D. Ackerman

The adhesion G protein-coupled receptor GPR56 is a cell-autonomous regulator of oligodendrocyte development

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.

The adhesion GPCR Gpr56 regulates oligodendrocyte development via interactions with Gα12/13 and RhoA

In the vertebrate central nervous system, myelinating oligodendrocytes are postmitotic and derive from proliferative oligodendrocyte precursor cells (OPCs). The molecular mechanisms that govern oligodendrocyte development are incompletely understood, but recent studies implicate the adhesion class of G protein-coupled receptors (aGPCRs) as important regulators of myelination. Here, we use zebrafish and mouse models to dissect the function of the aGPCR Gpr56 in oligodendrocyte development. We show that gpr56 is expressed during early stages of oligodendrocyte development. Additionally, we observe a significant reduction of mature oligodendrocyte number and of myelinated axons in gpr56 zebrafish mutants. This reduction results from decreased OPC proliferation, rather than increased cell death or altered neural precursor differentiation potential. Finally, we show that these functions are mediated by Gα12/13 proteins and Rho activation. Together, our data establish Gpr56 as a regulator of oligodendrocyte development.

The scales and tales of myelination: using zebrafish and mouse to study myelinating glia

Myelin, the lipid-rich sheath that insulates axons to facilitate rapid conduction of action potentials, is an evolutionary innovation of the jawed-vertebrate lineage. Research efforts aimed at understanding the molecular mechanisms governing myelination have primarily focused on rodent models; however, with the advent of the zebrafish model system in the late twentieth century, the use of this genetically tractable, yet simpler vertebrate for studying myelination has steadily increased. In this review, we compare myelinating glial cell biology during development and regeneration in zebrafish and mouse and enumerate the advantages and disadvantages of using each model to study myelination. This article is part of a Special Issue entitled SI: Myelin Evolution.

Dynein/dynactin is necessary for anterograde transport of Mbp mRNA in oligodendrocytes and for myelination in vivo

Significance Oligodendrocytes in the brain insulate neuronal axons in layers of fatty myelin to facilitate fast electrical signaling. Myelin basic protein (MBP), an important myelin component, is transported as mRNA away from the cell body before being translated into protein. In zebrafish, the anterograde motor kinesin transports mbp mRNA away from the cell body. We now identify myelination defects in zebrafish caused by a mutation in the retrograde motor complex dynein/dynactin, which normally transports cargos back toward the cell body. However, this mutant displays defects in anterograde mbp mRNA transport. We confirm in mammalian oligodendrocyte cultures that drug inhibition of dynein arrests transport in both directions and decreases MBP protein levels. Thus, dynein/dynactin is paradoxically required for anterograde mbp mRNA transport. Oligodendrocytes in the central nervous system produce myelin, a lipid-rich, multilamellar sheath that surrounds axons and promotes the rapid propagation of action potentials. A critical component of myelin is myelin basic protein (MBP), expression of which requires anterograde mRNA transport followed by local translation at the developing myelin sheath. Although the anterograde motor kinesin KIF1B is involved in mbp mRNA transport in zebrafish, it is not entirely clear how mbp transport is regulated. From a forward genetic screen for myelination defects in zebrafish, we identified a mutation in actr10, which encodes the Arp11 subunit of dynactin, a critical activator of the retrograde motor dynein. Both the actr10 mutation and pharmacological dynein inhibition in zebrafish result in failure to properly distribute mbp mRNA in oligodendrocytes, indicating a paradoxical role for the retrograde dynein/dynactin complex in anterograde mbp mRNA transport. To address the molecular mechanism underlying this observation, we biochemically isolated reporter-tagged Mbp mRNA granules from primary cultured mammalian oligodendrocytes to show that they indeed associate with the retrograde motor complex. Next, we used live-cell imaging to show that acute pharmacological dynein inhibition quickly arrests Mbp mRNA transport in both directions. Chronic pharmacological dynein inhibition also abrogates Mbp mRNA distribution and dramatically decreases MBP protein levels. Thus, these cell culture and whole animal studies demonstrate a role for the retrograde dynein/dynactin motor complex in anterograde mbp mRNA transport and myelination in vivo.

GPR56/ADG RG1 regulates development and maintenance of peripheral myelin

Myelin is a multilamellar sheath generated by specialized glia called Schwann cells (SCs) in the peripheral nervous system (PNS), which serves to protect and insulate axons for rapid neuronal signaling. In zebrafish and rodent models, we identify GPR56/ADGRG1 as a conserved regulator of PNS development and health. We demonstrate that, during SC development, GPR56-dependent RhoA signaling promotes timely radial sorting of axons. In the mature PNS, GPR56 is localized to distinct SC cytoplasmic domains, is required to establish proper myelin thickness, and facilitates organization of the myelin sheath. Furthermore, we define plectin—a scaffolding protein previously linked to SC domain organization, myelin maintenance, and a series of disorders termed “plectinopathies”—as a novel interacting partner of GPR56. Finally, we show that Gpr56 mutants develop progressive neuropathy-like symptoms, suggesting an underlying mechanism for peripheral defects in some human patients with GPR56 mutations. In sum, we define Gpr56 as a new regulator in the development and maintenance of peripheral myelin.

Whole Genome Sequencing-Based Mapping and Candidate Identification of Mutations from Fixed Zebrafish Tissue

As forward genetic screens in zebrafish become more common, the number of mutants that cannot be identified by gross morphology or through transgenic approaches, such as many nervous system defects, has also increased. Screening for these difficult-to-visualize phenotypes demands techniques such as whole-mount in situ hybridization (WISH) or antibody staining, which require tissue fixation. To date, fixed tissue has not been amenable for generating libraries for whole genome sequencing (WGS). Here, we describe a method for using genomic DNA from fixed tissue and a bioinformatics suite for WGS-based mapping of zebrafish mutants. We tested our protocol using two known zebrafish mutant alleles, gpr126st49 and egr2bfh227, both of which cause myelin defects. As further proof of concept we mapped a novel mutation, stl64, identified in a zebrafish WISH screen for myelination defects. We linked stl64 to chromosome 1 and identified a candidate nonsense mutation in the F-box and WD repeat domain containing 7 (fbxw7) gene. Importantly, stl64 mutants phenocopy previously described fbxw7vu56 mutants, and knockdown of fbxw7 in wild-type animals produced similar defects, demonstrating that stl64 disrupts fbxw7. Together, these data show that our mapping protocol can map and identify causative lesions in mutant screens that require tissue fixation for phenotypic analysis.

Microglial transglutaminase-2 drives myelination and myelin repair via GPR56/ADGRG1 in oligodendrocyte precursor cells

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.

Fbxw7 is a critical regulator of Schwann cell myelinating potential

Myelin insulates and protects axons in vertebrate nervous systems. In the central nervous system (CNS), oligodendrocytes (OLs) make numerous myelin sheaths on multiple axons, whereas in the peripheral nervous system (PNS) myelinating Schwann cells (SCs) make just one myelin sheath on a single axon. Why the myelinating potentials of OLs and SCs are so fundamentally different is unclear. Here, we find that loss of Fbxw7, an E3 ubiquitin ligase component, enhances the myelinating potential of SCs. Fbxw7 mutant SCs are seen myelinating multiple axons in a fashion reminiscent of OLs as well as aberrantly myelinating large axons while simultaneously ensheathing small unmyelinated axons - typically distinct roles of myelinating SCs and non-myelinating Remak SCs, respectively. We found that several of the Fbxw7 mutant phenotypes, including the ability to generate thicker myelin sheaths, were due to dysregulation of mTOR. However, the remarkable ability of mutant SCs to either myelinate multiple axons or myelinate some axons while simultaneously encompassing other unmyelinated axons is independent of mTOR signaling. This indicates distinct roles for Fbxw7 in regulating multiple aspects of SC behavior and that novel Fbxw7-regulated mechanisms control modes of myelination previously thought to fundamentally distinguish myelinating SCs from non-myelinating SCs and OLs. Our data reveal unexpected plasticity in the myelinating potential of SCs, which may have important implications for our understanding of both PNS and CNS myelination and myelin repair.

Mutations in dock1 disrupt early Schwann cell development

BackgroundIn the peripheral nervous system (PNS), specialized glial cells called Schwann cells produce myelin, a lipid-rich insulating sheath that surrounds axons and promotes rapid action potential propagation. During development, Schwann cells must undergo extensive cytoskeletal rearrangements in order to become mature, myelinating Schwann cells. The intracellular mechanisms that drive Schwann cell development, myelination, and accompanying cell shape changes are poorly understood.MethodsThrough a forward genetic screen in zebrafish, we identified a mutation in the atypical guanine nucleotide exchange factor, dock1, that results in decreased myelination of peripheral axons. Rescue experiments and complementation tests with newly engineered alleles confirmed that mutations in dock1 cause defects in myelination of the PNS. Whole mount in situ hybridization, transmission electron microscopy, and live imaging were used to fully define mutant phenotypes.ResultsWe show that Schwann cells in dock1 mutants can appropriately migrate and are not decreased in number, but exhibit delayed radial sorting and decreased myelination during early stages of development.ConclusionsTogether, our results demonstrate that mutations in dock1 result in defects in Schwann cell development and myelination. Specifically, loss of dock1 delays radial sorting and myelination of peripheral axons in zebrafish.

Dynein/dynactin regulate anterograde Mbp mRNA transport in oligodendrocytes to promote myelination

Loss-of-function mutations in PYCR2 cause hypomyelinating leukodystrophy 10 (HLD10) (MIM: 616420) characterized by postnatal degenerative microcephaly, severely delayed psychomotor development, and hypomyelination. Here, we report 3 additional patients with a novel germline homozygous missense G249V, which lies at the PYCR2 dimer interface and lowers its enzymatic activity. PYCR2 is a mitochondrial enzyme essential for proline synthesis but its function in the brain is not yet understood. We generated Pycr2-/mice that partly recapitulate the human phenotype by displaying a failure to thrive, reduced lifespan, postnatal neurological defects and hypomyelination. Consistent with this, we show that PYCR2 is broadly expressed in mouse brain tissues and its loss leads to neuronal and oligodendrocytes maturation defects. To further delineate the function of PYCR2, metabolites related to amino-acid biosynthesis and key neurotransmitters were quantified directly in mouse brain. Unexpectedly, the only difference detected was a robust increase in glycine levels. Intriguing, hyperglycinemia is also a hallmark of glycine encephalopathy, a genetic disorder which causes severe neurological symptoms. Furthermore, we demonstrate in mouse brain and human cells that PYCR2 regulates the level of SHMT2, the mitochondrial enzyme responsible for glycine synthesis. Our findings reveal that PYCR2 is an enzyme with moonlighting potential, essential to control white matter and neuronal postnatal development and identify the SHMT2 pathway as a potential target to treat neurological disorder due to the loss of PYCR2.