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. 2003 Nov 3;22(21):5666-78.
doi: 10.1093/emboj/cdg570.

Nogo-A at CNS paranodes is a ligand of Caspr: possible regulation of K(+) channel localization

Du-Yu Nie  1 , Zhi-Hong Zhou, Beng-Ti Ang, Felicia Y H Teng, Gang Xu, Tao Xiang, Chao-Yang Wang, Li Zeng, Yasuo Takeda, Tian-Le Xu, Yee-Kong Ng, Catherine Faivre-Sarrailh, Brian Popko, Eng-Ang Ling, Melitta Schachner, Kazutada Watanabe, Catherine J Pallen, Bor Luen Tang, Zhi-Cheng Xiao
Affiliations

Nogo-A at CNS paranodes is a ligand of Caspr: possible regulation of K(+) channel localization

Du-Yu Nie et al. EMBO J. .

Abstract

We report Nogo-A as an oligodendroglial component congregating and interacting with the Caspr-F3 complex at paranodes. However, its receptor Nogo-66 receptor (NgR) does not segregate to specific axonal domains. CHO cells cotransfected with Caspr and F3, but not with F3 alone, bound specifically to substrates coated with Nogo-66 peptide and GST-Nogo-66. Binding persisted even after phosphatidylinositol- specific phospholipase C (PI-PLC) removal of GPI-linked F3 from the cell surface, suggesting a direct interaction between Nogo-66 and Caspr. Both Nogo-A and Caspr co-immunoprecipitated with Kv1.1 and Kv1.2, and the developmental expression pattern of both paralleled compared with Kv1.1, implicating a transient interaction between Nogo-A-Caspr and K(+) channels at early stages of myelination. In pathological models that display paranodal junctional defects (EAE rats, and Shiverer and CGT(-/-) mice), distances between the paired labeling of K(+) channels were shortened significantly and their localization shifted toward paranodes, while paranodal Nogo-A congregation was markedly reduced. Our results demonstrate that Nogo-A interacts in trans with axonal Caspr at CNS paranodes, an interaction that may have a role in modulating axon-glial junction architecture and possibly K(+)-channel localization during development.

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Figures

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Fig. 1. Nogo-A clusters at the paranodes in the CNS. (A) Adult rat brainstem sections were double immunolabeled for Nogo-A (green: a, c, d, f, g and i) and the Kv1.1 K+ channel α-subunit (red: b, c, h and i) or PAN Na+ channel (red: e and f). For negative control, Nogo-A antiserum (1:200) was premixed with antigen before staining an adult brainstem section (j). Nogo-A antibodies used were those developed in our laboratory (a–f) or from Dr Strittmatter’s laboratory (g–i). Images c, f and i are merged images of a and b, d and e, and g and h, respectively. (B) Ultrastructural localization of Nogo-A at the paranodes in rat spinal cord: (a) immunogold labeling of cross-sections of myelinated axons revealed that the gold particles were detected at the inner and outer myelin sheaths; (b and c) immunogold particles of Nogo-A are found within glial loops and the compacted myelin; (d and e) in longitudinal sections of paranodes, immunogold particles of Nogo-A located at the tips of glial loops in the axoglial junction and some within the axon in (d). The boxed areas in (d) and (e) are shown at higher magnification in (d′) and (e′).OL, oligodendrocyte; ax, axon. Gold particles are indicated with arrows. Scale bars: 5 µm for (A, a–i), 10 µm for (A, j), 200 nm for (B, a, d and e) and 100 nm for (B, b, c, d′ and e′).
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Fig. 2. Expression and localization of Nogo-A at the nodal areas in EAE rats and CGT–/– mice. (A) Expression and localization of Nogo-A and Caspr in EAE afflicted spinal cord. (a and b) Double immunostaining for Nogo-A (green, a), Caspr (green, b) and Kv1.1 (red, a and b). The star marks a Nogo-A-positive cell body and the arrowhead indicates an undisrupted paranodal Nogo-A labeling. (c) Numbers of Nogo-A and Caspr clusters in several microscope fields of both normal and EAE rat spinal cord sections were counted. Values are given as mean ± SEM from at least three independent experiments (** P < 0.01). (d) Western blotting results show that Nogo-A was significantly downregulated in the spinal cord of EAE animals, but Caspr expression was only affected slightly (* P < 0.05). Nor. SC, spinal cord of normal rats; EAE SC, spinal cord of EAE rats. Scale bars: 10 µm for (a) and (b). (B) Distribution of Nogo-A in spinal cord sections from wild-type and CGT–/– mice. (a and b) double labeling for Nogo-A (green) and Na+ channel in wild-type and CGT–/– spinal cord (P16). In CGT–/– mice (b), the correlation between the congregations of both Nogo-A and Na+ channels were barely detected compared with the wild-type sections (a), in which Nogo-A clustered at paranodes and Na+ channels congregated at the nodes of Ranvier. (c and d) Spinal cord sections from wild-type and CGT–/– mice were double labeled for Nogo-A (green) and 200 kDa neurofilament (NF-200, red). Nogo-A labeling appears to be loosely spiraled around the axon in CGT–/– mice (P21; d), but clusters specifically into the paranodal region in wild-type mice (P21) (c). Arrows in (d) indicate the Nogo-A-labeled spirals. (e and f) Immunogold labeling of Nogo-A in longitudinal sections of paranodes from P16 CGT–/– mouse spinal cord demonstrated that gold particles were visible in the abnormally reversed loops. The boxed areas in (e) and (f) are shown at higher magnification in (e′) and (f′), respectively. Arrows indicate the 10 nm gold particles. Stars indicate the reversed papanodal loops. OL, oligodendrocyte; ax, axon. Scale bars: 10 µm for (a)–(d), 200 nm for (e) and (f), and 50 nm for (e′) and (f′).
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Fig. 3. NgR is not localized to the paranode. (A and B) Tissue lysates from various regions of the CNS of adult rats and brain lysates at various developmental stages were subjected to western blot using antibodies against NgR and γ-tubulin. (C–E) Adult hippocampus sections were double stained for NgR (green, C) and MAP2 (red, D). (E) is a merged image of (C) and (D). Scale bar: 10 µm. (F–H) Brainstem sections were double stained for NgR (red, F) and Kv1.2 (green, G). (H) is a merged image of (F) and (G). Scale bar: 50 µm.
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Fig. 4. Caspr associates with Nogo-66 in an F3-independent manner. (A) Nogo-A associates in vivo with Caspr–F3. (a) Detergent lysates of brain membrane fractions from adult mice were immunoprecipitated with Caspr, Nogo-A and NB3 antibodies as well as non-immune IgG. The immunoprecipitates and detergent extracts from brain (Brain) together with the protein-A beads (Beads) were subjected to western blot using antibodies against Caspr, Nogo-A, F3 and NB3. (b) Membrane fractions of Nogo-A–Caspr–F3-, Nogo-A–F3-, Nogo-A- and Caspr–F3-transfected CHO cells were immunoprecipitated with antibodies to Caspr and Nogo-A as well as non-immune IgG. The immunoprecipitates and brain extracts (Brain) were subjected to western blot analysis using antibodies against Nogo-A, Caspr and F3. (B) Caspr-expressing cells adhere to Nogo-66. (a–l) Caspr–F3-CHO cells (a, e and i), F3-CHO cells (b, f and j) and mock CHO cells (c, g and k), as well as PI-PLC-treated Caspr–F3- CHO cells (d, h and l) were plated onto substrates coated with Nogo-66 peptide, recombinant GST–Nogo-66 protein and GST, respectively. Scale bar: 8 µm. (m and n) Quantification of cells adherent to various substrates. Caspr–F3-CHO cells (in the presence or absence of PI-PLC treatment), but neither F3-CHO nor wild-type CHO cells, bound to Nogo-66 peptide and GST–Nogo-66. Bars represent the number of adherent cells (expressed as mean ± SEM) from at least three independent experiments (m). (o) At the end of cell adhesion assay after PI-PLC treatment, Caspr–F3-CHO and F3-CHO cells and their culture supernatant were collected and subjected to western blotting analysis after normalizing for total protein to detect F3/contactin: 1, mouse brain; 2, F3-CHO cell lysate; 3, Caspr–F3-CHO cell lysate after PI-PLC treatment; 4, Caspr–F3-CHO cell medium after PI-PLC treatment. *P < 0.05. Caspr–F3- CHO cells adhered to Nogo-66 and GST–Nogo-66 with equal efficiency after treatment with increasing concentrations of PI-PLC (0.02, 0.04 and 0.06 U/ml) (n).
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Fig. 5. The Nogo-A–Caspr complex interacts with K+ channels. (A) (a) P7 mouse brain membrane extracts were immunoprecipitated with Caspr, Nogo-A, Kv1.1, Kv1.2 and NB3 antibodies as well as non-immune IgG. The indicated immunoprecipitates and brain extracts (Brain) were subjected to western blot analysis using antibodies against Kv1.1, Kv1.2, Nogo-A and Caspr. (b) GST pulldown assay was performed using recombinant GST–Nogo-66, GST–Nogo-N and GST from adult mouse brain extracts. The indicated precipitates and brain extracts (Brain) were probed with Caspr and Kv1.1 antibodies, following SDS–PAGE separation. (B) (a) Membrane fraction of CHO, F3-CHO and Caspr–F3 CHO cells, as well as brain extracts, were immunoblotted using Caspr antibodies following SDS–PAGE separation. (b) After transient transfection with the Kv1.1 expression construct RBG4/Kv1.1, membrane extracts of CHO, F3-CHO and Caspr–F3 CHO cells were incubated with GST–Nogo-66 or GST, respectively. The eluted proteins were separated by SDS–PAGE and probed with Caspr and Kv1.1 antibodies. IP, immunoprecipitation; WB, western blot.
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Fig. 6. Immunohistochemical labeling of Nogo-A, Caspr and Kv1.1 at different postnatal days in rat brainstem. (A) Brainstem sections of P5 to adult rats were double labeled for Caspr (green) and Kv1.1 (red). Scale bar: 5 µm. (B) Sections from P1–P30 rats were double labeled for Nogo-A (green) and Kv1.1 (red). Scale bar (o): 5 µm. (C) (a) The lengths of Nogo-A and Kv1.1 immunostaining and their overlap were measured from micrographs (µm, mean ± SEM). (b) The number of overlapping Nogo-A–Kv1.1 or Caspr–Kv1.1 clusters at paranodes was counted.
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Fig. 7. Distribution of Nogo-A, Caspr and Kv1.1 in EAE rats and Shiverer mice. (A and B) Double immunofluorescence labeling for Caspr (green) and Kv1.1 (red) in brainstem sections of (A) EAE and (B) control rats. The insets represent magnified views of the Kv1.1 labeling. Scale bars: 10 µm for (A) and (B), and 5 µm for insets to (A) and (B). (C, D, E, F and G) Double labeling for Nogo-A (green) and Kv1.1 (red) in spinal cord sections of (C) wild-type and (D–G) Shiverer mice. Image (F) is a merged image of (D) and (E). Arrows in (C), (F) and (G) indicate Nogo-A-positive cell bodies. Scale bars: 10 µm. (H and I) Double immunofluorescence staining of Caspr (green) and Kv1.1 (red) in brainstem sections of (H) wild-type and (I) Shiverer mice. Scale bar: 10 µm. (J) The distances between paired Kv1.1 staining in EAE with control rats and Shiverer (Shi) with wild-type (WT) mice were measured from micrographs (µm, mean ± SEM), respectively. The values in EAE rats or Shiverer mice were significantly reduced when compared with their controls, respectively (**P < 0.01).
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Fig. 8. The interaction between Nogo-A and Caspr at the paranodes may play a role during myelination. Paranodal Nogo-A trans interacts with axonal Caspr and may play a role in K+-channel localization during the early stages of myelination (from P5). With the firm establishment of axoglial junctions in the adults, K+ channels were excluded from paranodes where Nogo-A–Caspr interaction is maintained. The mechanisms for this separation remain to be further explored. N, node of Ranvier; PN, paranode; JPN, juxtaparanode.
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