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.

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.