Characterization of two novel myelin proteins

Myelination is a key step in vertebrate evolution: oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS) tightly wrap their cytoplasmic processes around axons. This allows rapid, saltatory nerve impulse conduction while keeping axon diameters small, thus permitting compact, powerful nervous systems at relatively small metabolic cost. In addition, signalling pathways between myelinating cells and axons mediate mutual trophic support, axonal growth and morphology.

Although myelination has been studied intensely, many questions remain regarding the differentiation of myelinating glial cells, regulation of myelin specific gene expression, and neuro-glial interactions. To identify novel factors involved in myelin formation and function we performed a microarray screen, comparing gene expression levels in wild type zebrafish (zf) with those in the myelin-deficient colourlesst3/sox10 mutant. In my PhD project I characterized two novel transcripts identified in this screen: zwilling and claudin k.

For the zwilling transcript, I reconstructed a full length cDNA, which contains two short open-reading-frames coding for protein sequences that are highly similar and conserved in teleosts. I demonstrated the presence of these proteins via mass spectrometry in myelin membrane fractions. Both Zwilling proteins are highly basic and have no predicted structural motives. In this they are similar to myelin basic protein, one of the major myelin constituents. Myelin basic protein possesses an N-terminal myristyl anchor (an N-terminal myristoylation site is also predicted and conserved for the Zwilling proteins) and associates tightly to the myelin membrane and mediates compaction of individual lamellae.

Zebrafish Claudin k is a teleost-specific protein and is expressed exclusively in Schwann cells and oligodendrocytes. Claudins are essential components of tight junctions, which form selective permeability barriers across paracellular pathways and function as a “fence” between apical and basolateral membrane domains in epithelia. In myelin of mammals, Claudins are expressed in autotypic tight junctions of oligodendrocytes (Claudin 11/OSP) and Schwann cells (Claudin 19). They contribute to electrical insulation, presumably by regulating ion flow between interstitium and intramyelinic space. The fence function of Claudins may also contribute to compartmentalization of the myelin membrane into different domains. Using monoclonal and polyclonal antibodies, which I raised against peptides, I showed that Claudin k localizes to the Schmidt-Lantermann incisures, the paranodal loops, and the inner and outer mesaxons – similar to mammalian Claudin 11 and 19. While mammalian Claudin 11 and 19 are restricted to the CNS and PNS respectively, zebrafish possess not only Claudin k but also Claudin 11 and 19, all of which we find to be expressed in both CNS and PNS. That raises the question whether myelin Claudins in teleosts are redundant or evolved divergent functions. To address this question, I analyzed the phylogenetic relationship of Claudins and performed knock down experiments in zebrafish larvae.

To further establish zebrafish as a model organism for myelination, we have identified and cloned the claudin k promoter. It drives strong, specific expression of transgenic reporters in myelinating glia, in both the CNS and the PNS. Using the flexible Tol2 mediated Gal4-UAS system (Paquet, Bhat et al. 2009) with independent driver and responder constructs and optional fluorescent reporters, this promoter can be used to express proteins specifically in myelinating glia. We generated transgenic lines expressing membrane-bound eGFP, eGFP-Claudin k and Cherry-Claudin k fusion proteins. We selected two strong and specific membrane-bound eGFP lines labeling the oligodendrocyte and Schwann cell membrane. For eGFP-Claudin k fusion lines we verified the proper localization of the fusion protein to autotypic tight junctions. These lines will enable the monitoring of myelination and tight junction formation in vivo and can serve as a read out for screens affecting myelination and proper tight junction formation. For transgenic expression of Cherry-Claudin k fusion protein, we see partial localization to the tight junctions but a major proportion of the protein aggregates within the cell body and processes. Since myelin protein aggregation with subsequent ER stress, including the unfolded protein response, proteasomal degradation and autophagy, have been implicated in myelin disorders, such as Pelizaeus-Merzbacher, vanishing white matter, and Charcot-Marie-Tooth disease (Lin and Popko 2009), we propose this transgenic line to serve as a more general model for diseases caused by ER stress due to misfolded proteins in myelinating glia.