Tetraspanin2 (Tspan2) is a member of the tetraspan/transmembrane4 superfamily restricted to the nervous system. The abundance of Tspan2 is low in physiological condition but increases greatly in myelin of PLP-deficient mice which may indicate its’ compensatory functions. Our experiments show that Tspan2 has no effect on delay of myelination at P14; young and old mice lacking Tspan2 has the same g-ratio as wild type. However, the quantity of unhealthy axons and axonal swellings are higher in aged animals lacking both PLP and Tspan2 than in single PLP knockout. Furthermore,the absence of PLP causes the decrease in the number of axons and rise in axon diameter; lack of
Tspan2 alone leads to decrease in quantity of 0.4-0.7 µm diameter axons in 40 weeks old mice. These findings point to a possible auxiliary role of Tspan2 in support of axonal transport and long-term axon preservation in PLP null condition.
ABSTRACT
Tetraspanin2 (Tspan2) is a member of the tetraspan/transmembrane4 superfamily restricted to the nervous system. The abundance of Tspan2 is low in physiological condition but increases greatly in myelin ofPLP-deficient mice which may indicate its’ compensatory functions. Our experiments show that Tspan2 has no effect on delay of myelination at P14; young and old mice lacking Tspan2 has the same g-ratio as wild type. However, the quantity of unhealthy axons and axonal swellings are higher in aged animals lacking both PLP and Tspan2 than in single PLP knockout. Furthermore, the absence of PLP causes the decrease in the number of axons and rise in axon diameter; lack of Tspan2 alone leads to decrease in quantity of 0.4-0.7 pm diameter axons in 40 weeks old mice. These findings point to a possible auxiliary role of Tspan2 in support of axonal transport and longterm axon preservation in PLP"M// condition.
INTRODUCTION
Myelination is the process of formation of multilayered myelin sheaths around the axons, mediated by oligodendrocytes in the CNS and Schwann cells in the PNS. Well known is the function of myelin in electrical insulation for rapid propagation of action potentials along nerve fibers. Moreover, myelin ensheathment is thought to be necessary for normal axonal transport, functional integrity and long-term survival of axons. Demyelination results in characteristic impairment of motor and sensory functions and lead to severe neurological disorders, e.g. leukodystrophies, neuropathies, and multiple sclerosis. Also subtle changes in myelin structure may contribute to psychiatric disorders including schizophrenia (Nave, 2010).
Being a highly specialized structure, multilayered myelin sheath requires coordinated integration of large quantities of specific proteins and lipids (Bosse et al., 2003). The most abundant proteins of CNS myelin are proteolipid protein (PLP) and its smaller isoform DM20 (Yool et al., 2001). According to the latest data PLP constitutes 15, 43% of the total myelin protein (Werner et al., in press). Despite of its abundance, the biological role of PLP is not completely understood. It was shown that PLP"M// mice synthesize fully functional myelin with only minor ultrastructural changes of the intraperiod line and lower stability (Klugmann et al., 1997). Also in the absence PLP/DM20 small diameter axons are prone to axonal swellings and degenerations, which indicates role of mentioned proteins in the maintenance of axon-oligodendrocyte interactions. Some proportion of small diameter fibres doesn’t form compacted myelin or myelinate with delay (Yool et al., 2001). On the other hand, overexpression of PLP induces severe neuropathology, including dysmyelination, progressive tremors, ataxia and premature death (Möbius et al., 2009).
Myelin membranes are unusually enriched in lipids (>70% of dry weight), and especially in cholesterol (>25% of total lipid content). Dynamics of lipid ‘rafts’ or cholesterol-rich membrane- microdomains modulate biological function of associated proteins and might have a crucial role in myelin biogenesis (Werner et al., in press; Gielen et al., 2006). PLP induces cholesterol accumulation by molecular association and co-transport, which, accordingly, facilitates myelination. However, the ability of PLP""H mice to produce fully functional myelin indicates existence of other proteins which compensate for PLP deficiency. A homolog of PLP, M6B, was shown to contribute to myelination and cholesterol enrichment in absence of proteolipid protein (Werner et al., in press). Moreover, mice lacking both PLP and M6B have limited myelination suggesting that other proteins may also have compensatory qualities. Among possible candidates are other transmembrane tetraspans, e.g. plasmolipin, CD9, CD81, CD82, tetraspanin 2 (Tspan2) (Werner et al., in press). Differential myelin proteome analysis revealed over 160 proteins in myelin, but only quantity of Tspan2 is greatly increased in myelin of PLP"M//animals (Werner et al., 2007). This indicates that Tspan2 may be involved in myelination and neuroprotection in the absence of PLP.
The tetraspanin family includes proteins which have four hydrophobic transmembrane domains and two extracellular domains (Birling et al., 1999). They are found in all cells except erythrocytes and involved in cell differentiation, proliferation and motility, mediation of signal transduction, and, possibly, organization of membrane topology. Tetraspan proteins interact with each other forming so called tetraspanin webs at the cell surface. Precipitation with digitonin showed that these supramolecular complexes are also tightly associated with cholesterol (Charrin et al., 2003). Tspan2 is a member of tetraspanin family which is restricted to the nervous system and predominantly expressed in oligodendrocytes. It is detectable in the rats’ brain from the postnatal day 3 and strongly increases in concentration to P22. High level of expression of Tspan2 at early stages of development and involvement in integrin signalling may indicate its role in differentiation of oligodendrocytes and myelin formation. Persistence of Tspan2 at later stages suggests that it may contribute to the stabilization of mature myelin, trophic support of axons and neuroprotection (Birling et al., 1999).
In current project we employed morphometric analysis of optic nerves cross sections of P14 and 40 weeks old mice in order to find out whether Tspan2 can reduce delay of myelination or axonal degeneration in mice with PLP-deficient myelin.
MATERIALS AND METHODS
Animals
Wild type, Tspan2"M//, ррр""й and Tspan2"M//PLP"M// mice were included in the experiments. Constitutive mutants were bred into the C75BL/6 background using the mice from the breeding colony of the Max Plank Institute of Experimental Medicine. Only male mice of ages P14 and 40 weeks were used. Animals were sacrificed by cervical dislocation. All experiments were carried out in accordance with the animal protection law and approved by the German Federal State of Niedersachsen.
Electron and light microscopy
Optic nerves of 40 weeks old mice were fixed with 4% formaldehyde and 2% glutaraldehyde in PBS, and then postfixed with 2% osmium tetroxide. Samples were dehydrated in series of ethanol solutions of increasing concentrations. Ethanol was substituted with propylenoxid, and tissues were processed into epoxy resin according to standard procedures. Ultrathin sections (-50 nm) were cut on Leica Ultracut S ultramicrotome, stained with 4% aqueous uranyl acetate and lead citrate, and examined in Zeiss EM 900 electron microscope. 10 pictures per animal were collected at nominal magnification of7, 000 with an on-axis 2048x2048 CCD camera. Pictures ofP14 mice optic nerves were provided by Dr. Wiebke Möbius (5 picture per animal; LEO EM 912AB electron microscope). Embedded in epoxy resin optic nerves were also used to prepare semi thin (-500 nm) sections, which were stained with methylenblue-azur2 solutions and observed in bright field microscope.
Morphometric analysis of electron micrographs
Electron microphotographs were analyzed with the ImageJ (Fiji) software. In order to randomize axons we placed a grid on the picture and counted only axons crossed by the grating. For validation of axon pathologies all axons on the microphotographs were calculated.
Quantification of myelinated vs. nonmyelinated axons
To reveal a delay in myelination, we compared the proportion of myelinated and nonmyelinated axons inP14 mice. An axon was counted as myelinated if it was encircled at least by one compacted myelin layer. Axons were distinguished from other cells by their shape and characteristic appearance of cytoplasm (Figure 1). 700-1200 axons were validated per animal (3-4 animals in each group). The quantity of nonmyelinated axons was calculated as percentage of all validated axons.
illustration not visible in this excerpt
Figure 1. Transmission electron microscopy of optic nerve. Morphology of myelinated and nonmyelinated fibers. Axons appear as nearly circular profile in cross section. Microtubules, mitochondria, and elements of endoplasmic reticulum could be observed in axoplasm of myelinated (M) and nonmyelinated (N) axons. Processes of oligodendrocytes typically look darker than nonmyelinated axons. Only fibers crossed by the grid (green line) were calculated.
Validation of axonpathologies
We calculated the percentage of pathological axons in 40 weeks old mice in order to reveal if absence of Tspan-2 has effect on axon degeneration. Fibers were validated in 6 categories: healthy myelinated axons, healthy nonmyelinated axons, axons with increased adaxonal space, axonal swellings, unhealthy looking axons, and degenerating axons (Figure 2). Groups of axons with increased adaxonal space included fibers which had multivesicular bodies and other inclusions in adaxonal (periaxonal) space and/or between innermost myelin layers. Degenerating axons were identified by tubovesicular structures and amorphous cytoplasm. Axons which were not included in any other category (e.g. invaginations of the axons by the inner tongue of the myelin sheath) and those with mild signs of pathologies made up the group of unhealthy looking axons. Each fiber was assigned only to one category.
g-ratio and axon diameter
The g-ratio is defined as the ratio of axon Feret diameter to the myelinated fiber Feret diameter. Measurements were made from electron microphotographs of randomly selected fields of optic nerves of wild type and Tspan2WM// mice of ages P14 and 40 weeks. A minimum 150 axons was assessed for each animal. Measurements were stratified by axon diameter in ranges of 0,4-0,7; 0,71,0; 1,0-1,3; 1,3-1,6; 1,6-1,9; 1,9-2,2; >2,2 pm.
[...]
-
-
-
-
Upload your own papers! Earn money and win an iPhone X. -
-
Upload your own papers! Earn money and win an iPhone X. -
-
Upload your own papers! Earn money and win an iPhone X. -
-
Upload your own papers! Earn money and win an iPhone X. -
-
Upload your own papers! Earn money and win an iPhone X. -
-
Upload your own papers! Earn money and win an iPhone X.