Importin 13-dependent axon diameter growth regulates conduction speeds along myelinated CNS axons.

Axon diameter influences the conduction properties of myelinated axons, both directly, and indirectly through effects on myelin. However, we have limited understanding of mechanisms controlling axon diameter growth in the central nervous system, preventing systematic dissection of how manipulating diameter affects myelination and conduction along individual axons. Here we establish zebrafish to study axon diameter. We find that importin 13b is required for axon diameter growth, but does not affect cell body size or axon length. Using neuron-specific ipo13b mutants, we assess how reduced axon diameter affects myelination and conduction, and find no changes to myelin thickness, precision of action potential propagation, or ability to sustain high frequency firing. However, increases in conduction speed that occur along single myelinated axons with development are tightly linked to their growth in diameter. This suggests that axon diameter growth is a major driver of increases in conduction speeds along myelinated axons over time.

Neuronal activity disrupts myelinated axon integrity in the absence of NKCC1b.

Through a genetic screen in zebrafish, we identified a mutant with disruption to myelin in both the CNS and PNS caused by a mutation in a previously uncharacterized gene, slc12a2b, predicted to encode a Na+, K+, and Cl- (NKCC) cotransporter, NKCC1b. slc12a2b/NKCC1b mutants exhibited a severe and progressive pathology in the PNS, characterized by dysmyelination and swelling of the periaxonal space at the axon-myelin interface. Cell-type-specific loss of slc12a2b/NKCC1b in either neurons or myelinating Schwann cells recapitulated these pathologies. Given that NKCC1 is critical for ion homeostasis, we asked whether the disruption to myelinated axons in slc12a2b/NKCC1b mutants is affected by neuronal activity. Strikingly, we found that blocking neuronal activity completely prevented and could even rescue the pathology in slc12a2b/NKCC1b mutants. Together, our data indicate that NKCC1b is required to maintain neuronal activity-related solute homeostasis at the axon-myelin interface, and the integrity of myelinated axons.

The oligodendrocyte-specific antibody 'CC1' binds Quaking 7.

The mouse monoclonal antibody marketed as anti-adenomatous polyposis coli clone CC1, often referred to as CC1, is the antibody most commonly used to specifically label mature oligodendrocytes without labeling myelin. Previous studies have shown that despite being raised against adenomatous polyposis coli, this antibody binds another unknown antigen. We show that the CC1 antibody binds Quaking 7, an RNA-binding protein that is highly up-regulated in myelinating oligodendrocytes in the central nervous system. The monoclonal antibody anti-adenomatous polyposis coli (APC) clone CC1, is the antibody most commonly used to specifically label the cell bodies of mature oligodendrocytes. Despite being raised against APC, previous studies showed this antibody binds another unknown antigen. We show that the CC1 antibody binds Quaking (QKI) 7, an RNA-binding protein which is highly up-regulated in myelinating oligodendrocytes.

Imaging Myelination In Vivo Using Transparent Animal Models.

Myelination by oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system is essential for nervous system function and health. Despite its importance, we have a relatively poor understanding of the molecular and cellular mechanisms that regulate myelination in the living animal, particularly in the CNS. This is partly due to the fact that myelination commences around birth in mammals, by which time the CNS is complex and largely inaccessible, and thus very difficult to image live in its intact form. As a consequence, in recent years much effort has been invested in the use of smaller, simpler, transparent model organisms to investigate mechanisms of myelination in vivo. Although the majority of such studies have employed zebrafish, the Xenopus tadpole also represents an important complementary system with advantages for investigating myelin biology in vivo. Here we review how the natural features of zebrafish embryos and larvae and Xenopus tadpoles make them ideal systems for experimentally interrogating myelination by live imaging. We outline common transgenic technologies used to generate zebrafish and Xenopus that express fluorescent reporters, which can be used to image myelination. We also provide an extensive overview of the imaging modalities most commonly employed to date to image the nervous system in these transparent systems, and also emerging technologies that we anticipate will become widely used in studies of zebrafish and Xenopus myelination in the near future.

Complete Loss of Netrin-1 Results in Embryonic Lethality and Severe Axon Guidance Defects without Increased Neural Cell Death.

Netrin-1 regulates cell migration and adhesion during the development of the nervous system, vasculature, lung, pancreas, muscle, and mammary gland. It is also proposed to function as a dependence ligand that inhibits apoptosis; however, studies disagree regarding whether netrin-1 loss-of-function mice exhibit increased cell death. Furthermore, previously studied netrin-1 loss-of-function gene-trap mice express a netrin-1-ß-galactosidase protein chimera with potential for toxic gain-of-function effects, as well as a small amount of wild-type netrin-1 protein. To unambiguously assess loss of function, we generated netrin-1 floxed and netrin-1 null mouse lines. Netrin-1(-/-) mice die earlier and exhibit more severe axon guidance defects than netrin-1 gene-trap mice, revealing that complete loss of function is more severe than previously reported. Netrin-1(-/-) embryos also exhibit increased expression of the netrin receptors DCC and neogenin that are proposed dependence receptors; however, increased apoptosis was not detected, inconsistent with netrin-1 being an essential dependence receptor ligand in the embryonic spinal cord.

Netrin 1 regulates blood-brain barrier function and neuroinflammation.

Blood-brain barrier function is driven by the influence of astrocyte-secreted factors. During neuroinflammatory responses the blood-brain barrier is compromised resulting in central nervous system damage and exacerbated pathology. Here, we identified endothelial netrin 1 induction as a vascular response to astrocyte-derived sonic hedgehog that promotes autocrine barrier properties during homeostasis and increases with inflammation. Netrin 1 supports blood-brain barrier integrity by upregulating endothelial junctional protein expression, while netrin 1 knockout mice display disorganized tight junction protein expression and barrier breakdown. Upon inflammatory conditions, blood-brain barrier endothelial cells significantly upregulated netrin 1 levels in vitro and in situ, which prevented junctional breach and endothelial cell activation. Finally, netrin 1 treatment during experimental autoimmune encephalomyelitis significantly reduced blood-brain barrier disruption and decreased clinical and pathological indices of disease severity. Our results demonstrate that netrin 1 is an important regulator of blood-brain barrier maintenance that protects the central nervous system against inflammatory conditions such as multiple sclerosis and experimental autoimmune encephalomyelitis.

Progressive disorganization of paranodal junctions and compact myelin due to loss of DCC expression by oligodendrocytes.

Paranodal axoglial junctions are critical for maintaining the segregation of axonal domains along myelinated axons; however, the proteins required to organize and maintain this structure are not fully understood. Netrin-1 and its receptor Deleted in Colorectal Cancer (DCC) are proteins enriched at paranodes that are expressed by neurons and oligodendrocytes. To identify the specific function of DCC expressed by oligodendrocytes in vivo, we selectively eliminated DCC from mature myelinating oligodendrocytes using an inducible cre regulated by the proteolipid protein promoter. We demonstrate that DCC deletion results in progressive disruption of the organization of axonal domains, myelin ultrastructure, and myelin protein composition. Conditional DCC knock-out mice develop balance and coordination deficits and exhibit decreased conduction velocity. We conclude that DCC expression by oligodendrocytes is required for the maintenance and stability of myelin in vivo, which is essential for proper signal conduction in the CNS.

Heterogeneity of oligodendrocyte progenitor cells in adult human brain.

OBJECTIVE: Remyelination in multiple sclerosis has been attributed to the presence of oligodendrocyte progenitor cells (OPCs) in brain parenchyma. However, the precise identity of these progenitors is poorly defined. Here, we characterized populations of OPCs in the adult human brain and examined their myelination capacity and profile of miRNAs. Comparisons were made with fetal OPCs and mature oligodendrocytes. METHODS: We isolated human adult and fetal (early-to-mid second trimester) OPCs from surgically resected brain tissues using O4-, A2B5-, and MOG-directed fluorescence activated cell sorting and transplanted them into dysmyelinated shiverer slices to examine their myelination capacity. We used qRT-PCR to analyze expression of selective miRNAs implicated in OPC biology. RESULTS: Three subsets of putative OPCs were identified in adult brains: (1) A2B5(+), (2) O4(low), and (3) A2B5(+)O4(high)MOG(+) progenitors. In comparison, fetal brains contained (1) A2B5(+), (2) O4(+), and (3) A2B5(+)O4(+) progenitors, but no MOG(+) cells. We demonstrate that like fetal OPCs, adult OPCs have the capacity to ensheathe cerebellar axons. However, adult OPCs exhibit low to undetectable expression of miRNAs that were highly expressed in O4-expressing fetal OPCs. Adult OPCs also express different miRNAs compared to mature oligodendrocytes. INTERPRETATION: We conclude that phenotypically distinct subsets of OPCs are present in adult human brain and these OPCs show differential miRNA expression compared to fetal OPCs and mature oligodendrocytes. These suggest that remyelination in adult brain may involve multiple populations of progenitors within the brain and that OPC differentiation in adulthood may be differentially regulated compared to development.

Full-length and fragmented netrin-1 in multiple sclerosis plaques are inhibitors of oligodendrocyte precursor cell migration.

Oligodendrocytes exhibit a limited capacity to remyelinate in multiple sclerosis. Factors present in multiple sclerosis lesions are thought to inhibit oligodendrocyte precursor cell migration, limiting their recruitment to axons requiring remyelination; however, few inhibitors have been identified. A candidate inhibitor is netrin-1, a secreted protein that repels migrating oligodendrocyte precursor cells during neural development and is expressed by myelinating oligodendrocytes in the mature rodent central nervous system. Herein, we examined the distribution of netrin-1 in adult human white matter and multiple sclerosis lesions. We detected full-length netrin-1 protein and shorter netrin-1 fragments in samples of normal white matter and of multiple sclerosis lesions from adult human brain. We demonstrate that peptides corresponding to amino terminal domains VI and V of netrin-1 repel migrating oligodendrocyte precursor cells, but lack the chemoattractant activity of full-length netrin-1. Furthermore, recombinant domains VI-V of netrin-1 disrupt the chemoattractant activity of full-length netrin-1, consistent with a competitive mechanism of action. These findings indicate that full-length and fragmented forms of netrin-1, found in multiple sclerosis lesions, have the capacity to inhibit oligodendrocyte precursor migration, identifying netrin-1 as a potential target for therapies that promote remyelination.

Oligodendrocyte precursor cell transplantation into organotypic cerebellar shiverer slices: a model to study myelination and myelin maintenance.

Current in vitro models to investigate the consequence of oligodendrocyte-specific loss-of-function mutations on myelination are primarily limited to co-culture experiments, which do not accurately recapitulate the complex in vivo environment. Here, we describe the development of an in vitro model of myelination and myelin maintenance in which oligodendrocyte precursor cells are transplanted into organotypic cerebellar slice cultures derived from dysmyelinated shiverer mice. Compared to neuron-oligodendrocyte co-cultures, organotypic slices more closely mimic the environment in vivo, while utilizing a genetic background that allows for straight-forward identification of myelin generated by transplanted cells. We show at the ultrastructural level that the myelin generated by wild-type transplanted oligodendrocytes is compact and terminates in cytoplasmic loops that form paranodal junctions with the axon. This myelination results in the appropriate sequestering of axonal proteins into specialized domains surrounding the nodes of Ranvier. We also demonstrate the applicability of this approach for xenograft transplantation of oligodendrocyte precursor cells derived from rat or human sources. This method provides a time-efficient and cost-effective adjunct to conditional knockout mouse lines or in vivo transplantation models to study oligodendrocyte-specific loss-of-function mutations. Furthermore, the approach can be readily used to assess the effect of pharmacological manipulations on myelin, providing a tool to better understand myelination and develop effective therapeutic strategies to treat myelin-related diseases.

Regulation of miRNA 219 and miRNA Clusters 338 and 17-92 in Oligodendrocytes.

MicroRNAs (miRs) regulate diverse molecular and cellular processes including oligodendrocyte (OL) precursor cell (OPC) proliferation and differentiation in rodents. However, the role of miRs in human OPCs is poorly understood. To identify miRs that may regulate these processes in humans, we isolated OL lineage cells from human white matter and analyzed their miR profile. Using endpoint RT-PCR assays and quantitative real-time PCR, we demonstrate that miR-219, miR-338, and miR-17-92 are enriched in human white matter and expressed in acutely isolated human OLs. In addition, we report the expression of closely related miRs (miR-219-1-3p, miR-219-2-3p, miR-1250, miR-657, miR-3065-5p, miR-3065-3p) in both rodent and human OLs. Our findings demonstrate that miRs implicated in rodent OPC proliferation and differentiation are regulated in human OLs and may regulate myelination program in humans. Thus, these miRs should be recognized as potential therapeutic targets in demyelinating disorders.

A central role for RhoA during oligodendroglial maturation in the switch from netrin-1-mediated chemorepulsion to process elaboration.

The guidance cue netrin-1 and its receptor Deleted in Colorectal Cancer play distinct roles during different stages of oligodendrocyte development. A gradient of netrin-1 repels migrating oligodendrocyte precursor cells (OPCs) in the embryonic spinal cord by promoting process collapse, but later in development netrin-1 increases oligodendrocyte process extension and branching. Here we investigate the intracellular mechanism that governs this switch in response to netrin-1, and focus on the role of the GTPase RhoA and its effector Rho Kinase (ROCK) downstream of netrin-1 in OPCs and maturing oligodendrocytes. In OPCs, we show that netrin-1 induces a sustained increase in RhoA activity that requires Deleted in Colorectal Cancer function. Furthermore, we demonstrate that activation of RhoA and ROCK is required for the reduction in OPC process length triggered by netrin-1, and for the chemorepellent response made by OPCs to netrin-1. Unlike OPCs, application of netrin-1 to oligodendrocytes decreases RhoA activity. We demonstrate that inactivation of RhoA is essential for netrin-1 to increase oligodendrocyte process branching. We conclude that netrin-1 induces distinct morphological responses in OPCs and oligodendrocytes through differential regulation of RhoA activity.