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Essential role of oligodendrocytes in the formation and maintenance of central nervous system nodal regions

Carole Mathis1, Natalia Denisenko-Nehrbass2, Jean-Antoine Girault2 and Emiliana Borrelli1,*

1 Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM/CNRS/ULP, BP 163, 67404 Illkirch Cedex, C.U. de Strasbourg, France
2 INSERM U536, Institut du Fer à Moulin et Collège de France, 75005 Paris, France



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  		Fig. 1. Distribution of axonal proteins is dramatically altered at nodes of Ranvier in the absence of oligodendrocytes. Brain sections from wild-type (A,C,E) and MBP-TK (B,D,F) animals treated from postnatal day 1 to 20 with FIAU were double-labeled with anti-paranodin (PND) (red) and either anti-ankyrin G (green; A,B), anti-Na+ channel (PAN) (green; C,D) or anti-Kv1.1 (green; E,F) antibodies. Wild-type treated mice show a normal localization of paranodin in the paranodal regions (A,C,E) with typical ankyrin G (A) and Na+ channels clusters (C) distribution in the node of Ranvier. In treated MBP-TK mice, paranodin was not detectable in the paranodes, even if the red was enhanced to reveal nonspecific background (B,D,F). Localization of ankyrin G and Na+ channels was also markedly altered and immunoreactivity was mainly found along the axons (arrows in B,D) of treated MBP-TK animals. Double immunostaining for ankyrin G and Na+ channels showed that these proteins are colocalized (Fig. 1D inset). In contrast to the specific distribution of Kv1.1 clusters in the juxtaparanodes in the wild type (E), K+ channel clustering was completely absent after FIAU treatment in MBP-TK mice (F). Pictures represent single optical sections of the corpus callosum region using confocal scanning microscopy. Scale bars: in F, 10 µm in A-F; in inset, 4 µm.

 


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  		Fig. 2. Protein levels of paranodin, Na+ and K+ channel {alpha} subunits in the brain of wild-type and MBP-TK mice treated from day 1 to 20. Forebrain tissue from chronically treated (1-20d) wild-type and MBP-TK mice were homogenized as described in the Methods, and equal amounts of proteins were analyzed by immunoblotting (10 µg for paranodin or 30 µg for K+ and Na+ channels). No statistical differences in the levels of paranodin (180 kDa), Na+ channel {alpha} subunit (260 kDa) and K+ channels (86 kDa) were observed after quantification of the western blots (less than 10% variation between samples). The results are representative of three different experiments.

 


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  		Fig. 3. Immunofluorescence studies of MBP and PND/Caspr in the corpus callosum and the cortex of wild-type and treated MBP-TK mice. Myelinated fibers in the cortex and the corpus callosum are strongly labeled by the MBP antibody in wild-type animals (A). Absence of cortical MBP-positive fibers and a significant reduction of MBP labeling is observed in the corpus callosum of MBP-TK mice (1-20d) (B). Arrowhead in B indicates residual myelinated axons in the first cortical layer of MBP-TK mice brain. MBP-positive myelinated fibers are less abundant in MBP-TK mice corpus callosum after 1-6d treatment (C) than after 6-20d treatment (D). (E,F) Double immunostaining using anti-MBP (red) and anti-paranodin (green) antibodies. The presence of myelin sheaths correlates with the focal localization of paranodin at the paranodal zone in a wild-type animal (E). Paranodin staining is concentrated at the paranode only in myelinated fibers (arrow, F). Scale bars: in D, 100 µm for A-D; in F 10 µm for E,F.

 


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  		Fig. 4. Localization of ankyrin G and paranodin in jimpy mice. Sections from corpus callosum of wild-type (A,C) or jimpy (B,D) littermates were examined by double immunostaining with antibodies against paranodin (green) and ankyrin G (red) at postnatal day 15 (A,B) or 21 (C,D). In wild-type mice, paranodin immunoreactivity was concentrated in paranodes, in most instances flanking the nodal aggregates of ankyrin G on either side (A,C). In jimpy mice, paranodin immunoreactivity was diffuse, along the axonal membranes (arrows), with no identifiable paranodal cluster. Although ankyrin G clusters were similar in both genotypes at P15, their limits appeared less well delineated in jimpy mice at P21. Scale bar: 10 µm.

 


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  		Fig. 5. Localization of Na+ and K+ channels in jimpy mice. Sections from corpus callosum of wild-type (A,C) or jimpy (B,D) littermates were examined by double immunostaining with antibodies against the Na+ channels (green) and the K+ channels Kv1.1 (red). Postnatal day 15 (A,B). In wild-type mice, Kv1.1 immunoreactivity was clearly separated from Na+ channels clusters by an empty space corresponding to paranodes (A). In jimpy mice, although the shape of Na+ and K+ channel clusters was roughly comparable with that in wild-type littermates, the localization of Kv1.1 immunoreactivity was abnormal as it was not separated from the nodal Na+ channels (arrowhead, B). Postnatal day 21 (C,D). The alteration of K+ channels distribution was much more severe than at P15. Immunoreactivity appeared more diffuse, and Kv1.1 aggregates were irregular and fragmented. The shape of Na+ channels clusters was also altered with fuzzy and irregular limits. Scale bar: 10 µm.

 


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  		Fig. 6. Early aspect of nodal and paranodal regions in wild-type and jimpy mice. Sections from corpus callosum of wild-type (A) or jimpy (B) littermates, were examined by double immunostaining with antibodies against paranodin (green) and Na+ channels (red) at postnatal day 8. In wild-type mice, paranodin immunoreactivity was concentrated in binary or single paranodes, flanking the nodal cluster of Na+ channels. In many instances, Na+ channels clusters were observed independently from paranodes (arrows in A). In jimpy, paranodin was distributed irregularly, without bona fide identifiable paranodal labeling, whereas the clusters of Na+ channels appeared essentially normal and comparable with those in wild-type littermates. Scale bar: 20 µm.

 


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  		Fig. 7. Localization of Na+ channel clusters in the initial axonal segment of cortical neurons. Comparable brain sections from wild-type (A) and treated MBP-TK (B) 1-20d mice were immunolabeled with anti-Na+ channel (PAN) antibodies. Nuclei of cortical neurons are visualized with DAPI staining. In both wild-type and treated MBP-TK animals, we observed a high concentration of Na+ channels (A,B) in the initial axonal segment of the cortical pyramidal neurons (arrowheads). Scale bar: 30 µm.

 

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