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HDAC1/2-Dependent P0 Expression Maintains Paranodal and Nodal Integrity Independently of Myelin Stability through Interactions with Neurofascins.

Brügger V, Engler S, Pereira JA, Ruff S, Horn M, Welzl H, Münger E, Vaquié A, Sidiropoulos PN, Egger B, Yotovski P, Filgueira L, Somandin C, Lühmann TC, D'Antonio M, Yamaguchi T, Matthias P, Suter U, Jacob C - PLoS Biol. (2015)

Bottom Line: We identify P0 as a novel binding partner of NFasc155 and NFasc186, both in vivo and by in vitro adhesion assay.Furthermore, we demonstrate that HDAC1/2-dependent P0 expression is crucial for the maintenance of paranodal/nodal integrity and axonal function through interaction of P0 with neurofascins.In addition, we show that the latter mechanism is impaired by some P0 mutations that lead to late onset Charcot-Marie-Tooth disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Fribourg, Fribourg, Switzerland.

ABSTRACT
The pathogenesis of peripheral neuropathies in adults is linked to maintenance mechanisms that are not well understood. Here, we elucidate a novel critical maintenance mechanism for Schwann cell (SC)-axon interaction. Using mouse genetics, ablation of the transcriptional regulators histone deacetylases 1 and 2 (HDAC1/2) in adult SCs severely affected paranodal and nodal integrity and led to demyelination/remyelination. Expression levels of the HDAC1/2 target gene myelin protein zero (P0) were reduced by half, accompanied by altered localization and stability of neurofascin (NFasc)155, NFasc186, and loss of Caspr and septate-like junctions. We identify P0 as a novel binding partner of NFasc155 and NFasc186, both in vivo and by in vitro adhesion assay. Furthermore, we demonstrate that HDAC1/2-dependent P0 expression is crucial for the maintenance of paranodal/nodal integrity and axonal function through interaction of P0 with neurofascins. In addition, we show that the latter mechanism is impaired by some P0 mutations that lead to late onset Charcot-Marie-Tooth disease.

No MeSH data available.


Related in: MedlinePlus

P0 interacts with neurofascins.(A–E) Adhesion assay in HEK293T cells showing homophilic adhesion of P0 extracellular domain, as well as binding to the extracellular domain of NFasc155 and NFasc186. Confocal images of P0-Fc or control-Fc (Neg-Fc) particles (green, or false-colored green for Contactin-GFP) and Myc (A, red), neurofascins (B and C, red), Caspr (D, red), or Contactin (E, red) coimmunofluorescence and GFP fluorescence (E, false-colored turquoise; right side of the panel) in HEK293T cells expressing P0-Myc (A), NFasc155 (B), NFasc186 (C), Caspr (D) or Contactin-GFP (E), indicated by arrows. Overlays appear yellow. Nuclei are labeled in blue with DAPI. Single optical sections are shown. Each experiment was done at least three times and representative pictures are shown. (F) Immunoprecipitation (IP) IgY (negative control with chicken IgY) and IP P0 (with chicken anti-P0 antibody) from adult dKO and control littermate sciatic nerve lysates and detection with pan-NFasc antibody, and western blot for total NFasc on a 10% acrylamide SDS-Page gel, and reblot of immunoprecipitations (IPs) and western blot with P0 antibody. GAPDH western blot on lysates used for IgY and P0 IPs shows input. The band marked by an asterisk is most likely nonspecific. The graphs showing the quantification of coimmunoprecipitated (IPed) NFasc186 or NFasc155 with P0 (normalized to GAPDH input and calculated as fold change compared to IgY IP which was set to 1) in sciatic nerves of three dKO and three control littermate mice at 8 wk post-tamoxifen indicate that binding of P0 to neurofascins occurs in vivo in sciatic nerves of wild type mice, but is largely lost in dKO mice. Both sciatic nerves of each animal were pooled and split into two equal volumes for each IP IgY and P0. P-values (unpaired two-tailed Student's t test): * = p < 0.05, ** = p < 0.01, error bars = SEM.
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pbio.1002258.g007: P0 interacts with neurofascins.(A–E) Adhesion assay in HEK293T cells showing homophilic adhesion of P0 extracellular domain, as well as binding to the extracellular domain of NFasc155 and NFasc186. Confocal images of P0-Fc or control-Fc (Neg-Fc) particles (green, or false-colored green for Contactin-GFP) and Myc (A, red), neurofascins (B and C, red), Caspr (D, red), or Contactin (E, red) coimmunofluorescence and GFP fluorescence (E, false-colored turquoise; right side of the panel) in HEK293T cells expressing P0-Myc (A), NFasc155 (B), NFasc186 (C), Caspr (D) or Contactin-GFP (E), indicated by arrows. Overlays appear yellow. Nuclei are labeled in blue with DAPI. Single optical sections are shown. Each experiment was done at least three times and representative pictures are shown. (F) Immunoprecipitation (IP) IgY (negative control with chicken IgY) and IP P0 (with chicken anti-P0 antibody) from adult dKO and control littermate sciatic nerve lysates and detection with pan-NFasc antibody, and western blot for total NFasc on a 10% acrylamide SDS-Page gel, and reblot of immunoprecipitations (IPs) and western blot with P0 antibody. GAPDH western blot on lysates used for IgY and P0 IPs shows input. The band marked by an asterisk is most likely nonspecific. The graphs showing the quantification of coimmunoprecipitated (IPed) NFasc186 or NFasc155 with P0 (normalized to GAPDH input and calculated as fold change compared to IgY IP which was set to 1) in sciatic nerves of three dKO and three control littermate mice at 8 wk post-tamoxifen indicate that binding of P0 to neurofascins occurs in vivo in sciatic nerves of wild type mice, but is largely lost in dKO mice. Both sciatic nerves of each animal were pooled and split into two equal volumes for each IP IgY and P0. P-values (unpaired two-tailed Student's t test): * = p < 0.05, ** = p < 0.01, error bars = SEM.

Mentions: NFasc155 is necessary for the maintenance of paranodes and is the only known transmembrane component of the paranodal complex that is expressed on the SC side [27,36]. Because P0 was necessary for the maintenance of Caspr and neurofascins, and was localized in paranodes and microvilli, we hypothesized that P0 is another binding partner within the paranodal complex and of the nodal complex. To test this hypothesis, we generated Fc particles fused to the extracellular domain of P0 (P0-Fc, S12A and S12B Fig) and control Fc particles (neg-Fc, S12A Fig) and carried out adhesion assays. The extracellular domain of P0 forms homotetramers connecting two myelin wraps [18], and as expected, P0-Fc bound to HEK293T cells expressing P0-myc at their cell surface, whereas neg-Fc particles did not (Fig 7A and S13A Fig). We found that P0-Fc also decorated the surface of HEK293T cells expressing NFasc155 or NFasc186 at their plasma membrane, whereas neg-Fc particles did not (Fig 7B and 7C and S13B and S13C Fig). P0-Fc did not bind to HEK293T cells expressing Caspr or Contactin (Fig 7D and 7E and S13D and S13E Fig). These data indicate that the extracellular domain of P0 is capable of interacting with the extracellular domain of NFasc155 and of NFasc186. We then tested by coimmunoprecipitation whether these interactions also occur in vivo. Indeed, NFasc155 and NFasc186 coimmunoprecipitated with P0 (Fig 7F) in adult control sciatic nerves, showing that P0 interacts with these two neurofascins in vivo, whereas coimmunoprecipitated NFasc levels were strongly reduced in dKO sciatic nerves (Fig 7F). These data demonstrate that P0 belongs to the paranodal and nodal complexes in the adult PNS.


HDAC1/2-Dependent P0 Expression Maintains Paranodal and Nodal Integrity Independently of Myelin Stability through Interactions with Neurofascins.

Brügger V, Engler S, Pereira JA, Ruff S, Horn M, Welzl H, Münger E, Vaquié A, Sidiropoulos PN, Egger B, Yotovski P, Filgueira L, Somandin C, Lühmann TC, D'Antonio M, Yamaguchi T, Matthias P, Suter U, Jacob C - PLoS Biol. (2015)

P0 interacts with neurofascins.(A–E) Adhesion assay in HEK293T cells showing homophilic adhesion of P0 extracellular domain, as well as binding to the extracellular domain of NFasc155 and NFasc186. Confocal images of P0-Fc or control-Fc (Neg-Fc) particles (green, or false-colored green for Contactin-GFP) and Myc (A, red), neurofascins (B and C, red), Caspr (D, red), or Contactin (E, red) coimmunofluorescence and GFP fluorescence (E, false-colored turquoise; right side of the panel) in HEK293T cells expressing P0-Myc (A), NFasc155 (B), NFasc186 (C), Caspr (D) or Contactin-GFP (E), indicated by arrows. Overlays appear yellow. Nuclei are labeled in blue with DAPI. Single optical sections are shown. Each experiment was done at least three times and representative pictures are shown. (F) Immunoprecipitation (IP) IgY (negative control with chicken IgY) and IP P0 (with chicken anti-P0 antibody) from adult dKO and control littermate sciatic nerve lysates and detection with pan-NFasc antibody, and western blot for total NFasc on a 10% acrylamide SDS-Page gel, and reblot of immunoprecipitations (IPs) and western blot with P0 antibody. GAPDH western blot on lysates used for IgY and P0 IPs shows input. The band marked by an asterisk is most likely nonspecific. The graphs showing the quantification of coimmunoprecipitated (IPed) NFasc186 or NFasc155 with P0 (normalized to GAPDH input and calculated as fold change compared to IgY IP which was set to 1) in sciatic nerves of three dKO and three control littermate mice at 8 wk post-tamoxifen indicate that binding of P0 to neurofascins occurs in vivo in sciatic nerves of wild type mice, but is largely lost in dKO mice. Both sciatic nerves of each animal were pooled and split into two equal volumes for each IP IgY and P0. P-values (unpaired two-tailed Student's t test): * = p < 0.05, ** = p < 0.01, error bars = SEM.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4583457&req=5

pbio.1002258.g007: P0 interacts with neurofascins.(A–E) Adhesion assay in HEK293T cells showing homophilic adhesion of P0 extracellular domain, as well as binding to the extracellular domain of NFasc155 and NFasc186. Confocal images of P0-Fc or control-Fc (Neg-Fc) particles (green, or false-colored green for Contactin-GFP) and Myc (A, red), neurofascins (B and C, red), Caspr (D, red), or Contactin (E, red) coimmunofluorescence and GFP fluorescence (E, false-colored turquoise; right side of the panel) in HEK293T cells expressing P0-Myc (A), NFasc155 (B), NFasc186 (C), Caspr (D) or Contactin-GFP (E), indicated by arrows. Overlays appear yellow. Nuclei are labeled in blue with DAPI. Single optical sections are shown. Each experiment was done at least three times and representative pictures are shown. (F) Immunoprecipitation (IP) IgY (negative control with chicken IgY) and IP P0 (with chicken anti-P0 antibody) from adult dKO and control littermate sciatic nerve lysates and detection with pan-NFasc antibody, and western blot for total NFasc on a 10% acrylamide SDS-Page gel, and reblot of immunoprecipitations (IPs) and western blot with P0 antibody. GAPDH western blot on lysates used for IgY and P0 IPs shows input. The band marked by an asterisk is most likely nonspecific. The graphs showing the quantification of coimmunoprecipitated (IPed) NFasc186 or NFasc155 with P0 (normalized to GAPDH input and calculated as fold change compared to IgY IP which was set to 1) in sciatic nerves of three dKO and three control littermate mice at 8 wk post-tamoxifen indicate that binding of P0 to neurofascins occurs in vivo in sciatic nerves of wild type mice, but is largely lost in dKO mice. Both sciatic nerves of each animal were pooled and split into two equal volumes for each IP IgY and P0. P-values (unpaired two-tailed Student's t test): * = p < 0.05, ** = p < 0.01, error bars = SEM.
Mentions: NFasc155 is necessary for the maintenance of paranodes and is the only known transmembrane component of the paranodal complex that is expressed on the SC side [27,36]. Because P0 was necessary for the maintenance of Caspr and neurofascins, and was localized in paranodes and microvilli, we hypothesized that P0 is another binding partner within the paranodal complex and of the nodal complex. To test this hypothesis, we generated Fc particles fused to the extracellular domain of P0 (P0-Fc, S12A and S12B Fig) and control Fc particles (neg-Fc, S12A Fig) and carried out adhesion assays. The extracellular domain of P0 forms homotetramers connecting two myelin wraps [18], and as expected, P0-Fc bound to HEK293T cells expressing P0-myc at their cell surface, whereas neg-Fc particles did not (Fig 7A and S13A Fig). We found that P0-Fc also decorated the surface of HEK293T cells expressing NFasc155 or NFasc186 at their plasma membrane, whereas neg-Fc particles did not (Fig 7B and 7C and S13B and S13C Fig). P0-Fc did not bind to HEK293T cells expressing Caspr or Contactin (Fig 7D and 7E and S13D and S13E Fig). These data indicate that the extracellular domain of P0 is capable of interacting with the extracellular domain of NFasc155 and of NFasc186. We then tested by coimmunoprecipitation whether these interactions also occur in vivo. Indeed, NFasc155 and NFasc186 coimmunoprecipitated with P0 (Fig 7F) in adult control sciatic nerves, showing that P0 interacts with these two neurofascins in vivo, whereas coimmunoprecipitated NFasc levels were strongly reduced in dKO sciatic nerves (Fig 7F). These data demonstrate that P0 belongs to the paranodal and nodal complexes in the adult PNS.

Bottom Line: We identify P0 as a novel binding partner of NFasc155 and NFasc186, both in vivo and by in vitro adhesion assay.Furthermore, we demonstrate that HDAC1/2-dependent P0 expression is crucial for the maintenance of paranodal/nodal integrity and axonal function through interaction of P0 with neurofascins.In addition, we show that the latter mechanism is impaired by some P0 mutations that lead to late onset Charcot-Marie-Tooth disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Fribourg, Fribourg, Switzerland.

ABSTRACT
The pathogenesis of peripheral neuropathies in adults is linked to maintenance mechanisms that are not well understood. Here, we elucidate a novel critical maintenance mechanism for Schwann cell (SC)-axon interaction. Using mouse genetics, ablation of the transcriptional regulators histone deacetylases 1 and 2 (HDAC1/2) in adult SCs severely affected paranodal and nodal integrity and led to demyelination/remyelination. Expression levels of the HDAC1/2 target gene myelin protein zero (P0) were reduced by half, accompanied by altered localization and stability of neurofascin (NFasc)155, NFasc186, and loss of Caspr and septate-like junctions. We identify P0 as a novel binding partner of NFasc155 and NFasc186, both in vivo and by in vitro adhesion assay. Furthermore, we demonstrate that HDAC1/2-dependent P0 expression is crucial for the maintenance of paranodal/nodal integrity and axonal function through interaction of P0 with neurofascins. In addition, we show that the latter mechanism is impaired by some P0 mutations that lead to late onset Charcot-Marie-Tooth disease.

No MeSH data available.


Related in: MedlinePlus