A novel myelin protein zero transgenic zebrafish designed for rapid readout of in vivo myelination.

Demyelination occurs following many neurological insults, most notably in multiple sclerosis (MS). Therapeutics that promote remyelination could slow the neurological decline associated with chronic demyelination; however, in vivo testing of candidate small molecule drugs and signaling cascades known to impact myelination is expensive and labor intensive. Here, we describe the development of a novel zebrafish line which uses the putative promoter of Myelin Protein Zero (mpz), a major structural protein in myelin, to drive expression of Enhanced Green Fluorescent Protein (mEGFP) specifically in the processes and nascent internodes of myelinating glia. We observe that changes in fluorescence intensity in Tg(mpz:mEGFP) larvae are a reliable surrogate for changes in myelin membrane production per se in live larvae following bath application of drugs. These changes in fluorescence are strongly predictive of changes in myelin-specific mRNAs [mpz, 36K and myelin basic protein (mbp)] and protein production (Mbp). Finally, we observe that certain drugs alter nascent internode number and length, impacting the overall amount of myelin membrane synthesized and a number of axons myelinated without significantly changing the number of myelinating oligodendrocytes. These studies demonstrate that the Tg(mpz:mEGFP) reporter line responds effectively to positive and negative small molecule regulators of myelination, and could be useful for identifying candidate drugs that specifically target myelin membrane production in vivo. Combined with high throughput cell-based screening of large chemical libraries and automated imaging systems, this transgenic line is useful for rapid large scale whole animal screening to identify novel myelinating small molecule compounds in vivo.

Zebrafish as a model to investigate CNS myelination.

Myelin plays a critical role in proper neuronal function by providing trophic and metabolic support to axons and facilitating energy-efficient saltatory conduction. Myelination is influenced by numerous molecules including growth factors, hormones, transmembrane receptors and extracellular molecules, which activate signaling cascades that drive cellular maturation. Key signaling molecules and downstream signaling cascades controlling myelination have been identified in cell culture systems. However, in vitro systems are not able to faithfully replicate the complex in vivo signaling environment that occurs during development or following injury. Currently, it remains time-consuming and expensive to investigate myelination in vivo in rodents, the most widely used model for studying mammalian myelination. As such, there is a need for alternative in vivo myelination models, particularly ones that can test molecular mechanisms without removing oligodendrocyte lineage cells from their native signaling environment or disrupting intercellular interactions with other cell types present during myelination. Here, we review the ever-increasing role of zebrafish in studies uncovering novel mechanisms controlling vertebrate myelination. These innovative studies range from observations of the behavior of single cells during in vivo myelination as well as mutagenesis- and pharmacology-based screens in whole animals. Additionally, we discuss recent efforts to develop novel models of demyelination and oligodendrocyte cell death in adult zebrafish for the study of cellular behavior in real time during repair and regeneration of damaged nervous systems.

Digestion products of the PH20 hyaluronidase inhibit remyelination.

OBJECTIVE: Oligodendrocyte progenitor cells (OPCs) recruited to demyelinating lesions often fail to mature into oligodendrocytes (OLs) that remyelinate spared axons. The glycosaminoglycan hyaluronan (HA) accumulates in demyelinating lesions and has been implicated in the failure of OPC maturation and remyelination. We tested the hypothesis that OPCs in demyelinating lesions express a specific hyaluronidase, and that digestion products of this enzyme inhibit OPC maturation. METHODS: Mouse OPCs grown in vitro were analyzed for hyaluronidase expression and activity. Gain of function studies were used to define the hyaluronidases that blocked OPC maturation. Mouse and human demyelinating lesions were assessed for hyaluronidase expression. Digestion products from different hyaluronidases and a hyaluronidase inhibitor were tested for their effects on OPC maturation and functional remyelination in vivo. RESULTS: OPCs demonstrated hyaluronidase activity in vitro and expressed multiple hyaluronidases, including HYAL1, HYAL2, and PH20. HA digestion by PH20 but not other hyaluronidases inhibited OPC maturation into OLs. In contrast, inhibiting HA synthesis did not influence OPC maturation. PH20 expression was elevated in OPCs and reactive astrocytes in both rodent and human demyelinating lesions. HA digestion products generated by the PH20 hyaluronidase but not another hyaluronidase inhibited remyelination following lysolecithin-induced demyelination. Inhibition of hyaluronidase activity lead to increased OPC maturation and promoted increased conduction velocities through lesions. INTERPRETATION: We determined that PH20 is elevated in demyelinating lesions and that increased PH20 expression is sufficient to inhibit OPC maturation and remyelination. Pharmacological inhibition of PH20 may therefore be an effective way to promote remyelination in multiple sclerosis and related conditions.

Hyaluronan anchored to activated CD44 on central nervous system vascular endothelial cells promotes lymphocyte extravasation in experimental autoimmune encephalomyelitis.

The extravasation of lymphocytes across central nervous system (CNS) vascular endothelium is a key step in inflammatory demyelinating diseases including multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The glycosaminoglycan hyaluronan (HA) and its receptor, CD44, have been implicated in this process but their precise roles are unclear. We find that CD44(-/-) mice have a delayed onset of EAE compared with wild type animals. Using an in vitro lymphocyte rolling assay, we find that fewer slow rolling (<1 µm/s) wild type (WT) activated lymphocytes interact with CD44(-/-) brain vascular endothelial cells (ECs) than with WT ECs. We also find that CD44(-/-) ECs fail to anchor HA to their surfaces, and that slow rolling lymphocyte interactions with WT ECs are inhibited when the ECs are treated with a pegylated form of the PH20 hyaluronidase (PEG-PH20). Subcutaneous injection of PEG-PH20 delays the onset of EAE symptoms by ~1 day and transiently ameliorates symptoms for 2 days following disease onset. These improved symptoms correspond histologically to degradation of HA in the lumen of CNS blood vessels, decreased demyelination, and impaired CD4(+) T-cell extravasation. Collectively these data suggest that HA tethered to CD44 on CNS ECs is critical for the extravasation of activated T cells into the CNS providing new insight into the mechanisms promoting inflammatory demyelinating disease.

Mass-related traumatic tissue displacement and behavior: a screen for treatments that reduce [corrected] harm to bystander cells and recovery of function.

In this study, we focused on a preclinical model of brain compression injury that has relevance to pathological conditions such as tumor, hematoma, blood clot, and intracerebral bony fragment. We investigated behavioral impairment as a result of rapid-onset small mass, and the factors involved in lesion formation and neuroplasticity. An epidural bead implantation method was adopted. Two sizes (1.5 mm and 2.0 mm thick) of hemisphere-shaped beads were used. The beads were implanted into various locations over the sensorimotor cortex (SMC--anterior, middle and posterior). The effects of early versus delayed bead removal were examined to model clinical neurosurgical or other treatment procedures. Forelimb and hind-limb behavioral deficits and recovery were observed, and histological changes were quantified to determine brain reaction to focal compression. Our results showed that the behavioral deficits of compression were influenced by the location, timing of compression release, and magnitude of compression. Even persistent compression by the thicker bead (2.0 mm) caused only minor behavioral deficits, followed by fast recovery within a week in most animals, suggesting a mild lesion pattern for this model. Brain tissue was compressed into a deformed shape under pressure with slight tissue damage, evidenced by pathological evaluation on hematoxylin and eosin (H&E)- and TUNEL-stained sections. Detectable but not severe behavioral dysfunction exhibited by this model makes it particularly suitable for direct assessment of adverse effects of interventions on neuroplasticity after brain compression injury. This model may permit development of treatment strategies to alleviate brain mass effects, without disrupting neuroplasticity.

Neural stem cell niches: roles for the hyaluronan-based extracellular matrix.

Neural stem/progenitor cells capable of differentiating into the neurons and glial cells that populate the mammalian central nervous system (CNS) persist in specific neural stem cell niches that regulate stem cell proliferation, survival and differentiation. There is growing evidence that the extracellular matrix within neural stem cell niches is required for neural stem cell maintenance. Here, we review findings supporting a pivotal role for the glycosaminoglycan hyaluronan (HA) and its transmembrane receptors in neural stem/progenitor cell proliferation, differentiation and maturation. We also outline findings supporting changing roles for HA as cells become committed to distinct lineages in the brain and spinal cord.

An organotypic slice culture model of chronic white matter injury with maturation arrest of oligodendrocyte progenitors.

BACKGROUND: CNS myelination disturbances commonly occur in chronic white matter lesions in neurodevelopmental and adult neurological disorders. Recent studies support that myelination failure can involve a disrupted cellular repair mechanism where oligodendrocyte (OL) progenitor cells (OPCs) proliferate in lesions with diffuse astrogliosis, but fail to fully differentiate to mature myelinating OLs. There are no in vitro models that reproduce these features of myelination failure. RESULTS: Forebrain coronal slices from postnatal day (P) 0.5/1 rat pups were cultured for 1, 5, or 9 days in vitro (DIV). Slices rapidly exhibited diffuse astrogliosis and accumulation of the extracellular matrix glycosaminoglycan hyaluronan (HA), an inhibitor of OPC differentiation and re-myelination. At 1 DIV ~1.5% of Olig2+ OLs displayed caspase-3 activation, which increased to ~11.5% by 9 DIV. At 1 DIV the density of PDGFRα+ and PDGFRα+/Ki67+ OPCs were significantly elevated compared to 0 DIV (P < 0.01). Despite this proliferative response, at 9 DIV ~60% of white matter OLs were late progenitors (preOLs), compared to ~7% in the postnatal day 10 rat (P < 0.0001), consistent with preOL maturation arrest. Addition of HA to slices significantly decreased the density of MBP+ OLs at 9 DIV compared to controls (217 ± 16 vs. 328 ± 17 cells/mm2, respectively; P = 0.0003), supporting an inhibitory role of HA in OL lineage progression in chronic lesions. CONCLUSIONS: Diffuse white matter astrogliosis and early OPC proliferation with impaired OL maturation were reproduced in this model of myelination failure. This system may be used to define mechanisms of OPC maturation arrest and myelination failure related to astrogliosis and HA accumulation.