Würzburg, Univ., Diss., 2008. / Zsfassung in dt. Sprache.

Universiẗat, Diss., 2004--München.

Myelination drastically speeds up action potential propagation along axons, which is fundamental for the correct function of neuronal circuits. However, axon-oligodendrocyte interactions regulating the onset of myelin formation remain unclear. I sought to determine how reticulospinal axons control myelination, as they are the first myelinated in the zebrafish spinal cord. I genetically manipulated zebrafish in order to either remove such axons from a region of the spinal cord, or to increase their number, and characterized oligodendrocyte-lineage cells following this axonal loss- or gain-of-function. In kinesin-binding protein (kbp) mutants, reticulospinal hindbrain neurons start axonogenesis but axons fail to grow along the entire spinal cord as in wildtype, providing an axon-deficient posterior spinal cord and an intact anterior region. I found that early stages of oligodendrocyte development, such as the specification of oligodendrocyte precursors, their distribution and migration were not affected in the posterior spinal cord of these mutants. However, both the proliferation and the survival of late precursors were impaired, resulting in a significant reduction of mature oligodendrocytes in the posterior region of mutants at the onset of myelination. Since the anterior spinal cord of mutants is indistinguishable from wildtype, these results demonstrate that reticulospinal axons provide a mitogenic and a survival signal to a subset of developing OPCs, enabling their differentiation and lineage progression. I then found that the absence of reticulospinal axons did not affect the timing of oligodendrocyte differentiation, which matured on time, suggesting that this follows an intrinsic timer, as previous studies suggested. Oligodendrocytes also did not myelinate incorrect axonal targets, but instead adapted to the reduced axonal surface by elaborating fewer myelin sheaths. Additionally, oligodendrocytes made shorter sheaths, and also incorrectly ensheathed neuron somas in the mutant spinal cord, suggesting that either kbp function or a precise amount of axonal surface are required to prevent ectopic myelination of somas and to promote the longitudinal growth of myelin sheaths. In wildtype animals, the two reticulospinal Mauthner axons are the very first myelinated in the spinal cord. In animals where Notch1a function is temporarily abrogated or hoxb1 genes are temporarily upregulated, supernumerary Mauthner neurons are generated. I found that these extra axons are robustly myelinated, with no impairment of myelination of adjacent axons. Surprisingly, the number of oligodendrocytes was not altered, but I found that each individual oligodendrocyte elaborated more myelin sheaths, whose total length was also longer than in wildtypes. Additionally, dorsal oligodendrocytes, which normally myelinate only small-calibre dorsal axons, readily extended processes ventrally to myelinate the supernumerary large-calibre Mauthner axons, in addition to small-calibre axons. These results suggest that oligodendrocytes are plastic and are not destined to myelinate a particular type of axon, and conversely, that axonal signals that induce myelination are similar for different axons. The long-standing observation that oligodendrocytes tend to myelinate either few large axons or many small axons thus reflects local interactions of oligodendrocyte processes with the nearby axons, rather than different subtypes of oligodendrocytes specified by an intrinsic programme of differentiation. Collectively, this work shows that axons extensively influence both oligodendrocyte lineage progression and oligodendrocyte myelinating potential in vivo.

No description available.

No description available.

Myelin ist eine Membran von entscheidender Bedeutung. In Nervensystem von Wirbeltieren isoliert es die Axone und fördert so die schnelle Weiterleitung von Nervenimpulsen. Um als Isolator zu wirken, muss sich Myelin zu einer stabilen, Lipid-reichen Struktur zusammenlagern. Während die biochemische Zusammensetzung von Myelin gut beschrieben ist, sind Mechanismen, welche diese einzigartige Zusammensetzung regulieren, schlecht verstanden. In dieser Arbeit zeigen wir, dass Oligodendrozyten eine Art molekulares Sieb verwenden, um Membranproteine aus kompakten Myelin-Domain herrauszufiltern. Das Myelin Basische Protein (MBP) bildet eine Größen-abhängige Barriere und kontrolliert den Zugang von Proteinen in das kompakten Myelin, basierend auf der Größe ihrer zytoplasmatischen Domänen. Nur Proteine mit einer cytosolischen Domäne von weniger als 30 Aminosäuren können diese die Permeabilitätsbarriere überwinden. Im Hinblick auf den zugrundeliegenden Mechanismus zeigen wir, dass MBP nach Bindung an die innere Membran der Myelin-Doppelschicht mit sich selbst assoziiert und eine Phasentrennung auftritt. Diese Selbstorganisation erfordert hydrophobe Wechselwirkungen zwischen den Phenylalanin-Reste von MBP. Ein Ersetzen der Phenylalanin-Reste durch Serine hemmt die Selbst-Assoziation der MBP-Moleküle, ohne jedoch die Membranbindung zu beeinflussen. Wir zeigen weiter, dass die Selbst-Assoziation der MBP-Moleküle während der Entwicklung für die Verdrängung von sperrigen Proteinen aus den kompakten Myelin-Membranen benötigt wird. Dieses System könnte sich in Oligodendrozyten entwickelt haben, um eine anisotrope Membran-Organisation zu bilden, welche den Aufbau der der isolierenden, lipidreichen Membranen ermöglicht.

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Abstract The function of the nervous system is based on the targeted transmission of electrical impulses assuring the coordinated function of tissues and organs. Myelination of the neuronal axons allows the fast saltatory conduction by producing repetitive sites where sodium channels cluster on the axolemma. In the peripheral nervous system (PNS), myelin is formed by differentiation of the Schwann cell plasma membrane. The cells form myelin by first wrapping consecutive layers of the plasma membrane around the axons and then excluding almost all of the cytoplasm from the structure, forming compacted and non-compacted membrane compartments. The myelin-associated glycoprotein (MAG) is located in all of the non-compacted regions of the PNS myelin sheath. Its two isoforms, L-MAG and S-MAG, differ only by the carboxy-terminal tails of their respective cytoplasmic domains. Both of the MAG isoforms are found in PNS myelin. They are differentially expressed during development and, until now, it has been thought that L-MAG is not present in the mature PNS myelin sheaths of murines. This study shows that both MAG isoforms can be found in different non-compacted membrane compartments in the mature PNS myelin sheaths in dorsal root ganglia (DRG)/Schwann cell cocultures. Early myelin development and myelin maturation were analyzed by means of a study of the expression of two early myelin markers, MAG and galactosyl cerebrosides (Gal-CB), believed to play roles in both myelin formation and maintenance. In order to allow the exploitation of the full potential of the DRG/Schwann cell coculture model through the use of mouse mutants, a coculture method was developed in which mouse DRGs and Schwann cells are able, for the first time, to produce significant amounts of myelin. To further explore the role of MAG in myelin maintenance and stability, the stability of purified MAG was studied through extensive degradation experiments.

The sites of biosynthesis and incorporation of the abundant CNS myelin proteins 2' , 3' -cyclic nucleotide-3'-phosphodiesterase (CNPase) and P2 protein into the growing myelin membrane were investigated. Cell-free translation systems programmed with mRNA from rat brain, rabbit spinal cord, free and bound polysomes and purified myelin demonstrated conclusively that both CNPase and P2 are synthesized on free polysomes like the myelin basic proteins (MBPs) but unlike the proteolipid protein (PLP), the major intrinsic membrane protein of CNS myelin, which is known to be synthesized at the oligodendrocyte endoplasmic reticulum on bound polysomes (Colman et al., 1982) . These observations were supported by labelling studies on rats in vivo during the period of maximal myelin deposition. Newly synthesized CNPase associated with the myelin membrane very rapidly after labelling (~2 minutes) and this is consistent with the view that there is only a brief delay between synthesis and incorporation into their target membrane for extrinsic-type plasma membrane proteins. An RNA fraction isolated from purified CNS myelin was not enriched in mRNAs coding for CNPase and P2 but a considerable enrichment of mRNAs coding for MBPs was observed. This phenomenon has important implications for the cell biology of myelination since it suggests that although MBPs, CNPase and P2 are all basic extrinsic membrane proteins, and synthesized on free polysomes, different mechanisms for their transport to the myelin membrane exist. The addition of dog pancreatic microsomes (DPM) during translation showed no membrane association for CNPase however, at least 50% of MBPs were observed to non-specifically associate with these membranes. When newly synthesized MBP and P2 were incubated post-translationally with DPM or rabbit spinal cord myelin P2 only associated with myelin whereas MBP showed an equal affinity for both types of membranes. The segregation of MBP free polysomes at the myelin membrane during synthesis ensures that the nascent MBP polypeptides associate with the correct membrane. Recent evidence has shown that the free polysome-mRNA complex is bound to the cytoskeleton during protein synthesis. After extensive characterization of the purified rat brain oligodendrocyte and myelin-associated cytoskeletons it was shown that the synthesis of MBPs and CNPase only occurs from mRNA that is associated with the cytoskeleton and not when it is part of the cytoplasmic mRNA pool. Lipid analysis of the purified rat brain myelin-associated cytoskeleton revealed the presence of tightly bound lipid with a considerable enrichment of cerebroside and sphingomyelin (the latter at the expense of phosphatidylethanolamine). These studies on the cytoskeletal involvement in myelinogenesis suggest that extrinsic CNS myelin proteins are synthesized on the cytoskeleton and that post-translational cytoskeletal transport of these proteins to the growing myelin membrane may take place.

No description available.