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Disruption of Mtmr2 produces CMT4B1-like neuropathy with myelin outfolding and impaired spermatogenesis.

Bolino A, Bolis A, Previtali SC, Dina G, Bussini S, Dati G, Amadio S, Del Carro U, Mruk DD, Feltri ML, Cheng CY, Quattrini A, Wrabetz L - J. Cell Biol. (2004)

Bottom Line: We also identified a novel physical interaction in Schwann cells, between Mtmr2 and discs large 1 (Dlg1)/synapse-associated protein 97, a scaffolding molecule that is enriched at the node/paranode region.Dlg1 homologues have been located in several types of cellular junctions and play roles in cell polarity and membrane addition.We propose that Schwann cell-autonomous loss of Mtmr2-Dlg1 interaction dysregulates membrane homeostasis in the paranodal region, thereby producing outfolding and recurrent loops of myelin.

View Article: PubMed Central - PubMed

Affiliation: Dulbecco Telethon Institute, San Raffaele Scientific Institute, 20132 Milan, Italy. bolino.alessandra@hsr.it

ABSTRACT
Mutations in MTMR2, the myotubularin-related 2 gene, cause autosomal recessive Charcot-Marie-Tooth (CMT) type 4B1, a demyelinating neuropathy with myelin outfolding and azoospermia. MTMR2 encodes a ubiquitously expressed phosphatase whose preferred substrate is phosphatidylinositol (3,5)-biphosphate, a regulator of membrane homeostasis and vesicle transport. We generated Mtmr2- mice, which develop progressive neuropathy characterized by myelin outfolding and recurrent loops, predominantly at paranodal myelin, and depletion of spermatids and spermatocytes from the seminiferous epithelium, which leads to azoospermia. Disruption of Mtmr2 in Schwann cells reproduces the myelin abnormalities. We also identified a novel physical interaction in Schwann cells, between Mtmr2 and discs large 1 (Dlg1)/synapse-associated protein 97, a scaffolding molecule that is enriched at the node/paranode region. Dlg1 homologues have been located in several types of cellular junctions and play roles in cell polarity and membrane addition. We propose that Schwann cell-autonomous loss of Mtmr2-Dlg1 interaction dysregulates membrane homeostasis in the paranodal region, thereby producing outfolding and recurrent loops of myelin.

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Mtmr2 interacts with Dlg1/SAP97. (A) Semithin section analysis of sciatic nerves from mutant mice with conditional ablation of Mtmr2 in Schwann cells (P0-Cre/Mtmr2-) demonstrate myelin outfoldings (arrows). (B) Western blot analysis for Dlg1 in sciatic nerve homogenates from wild-type and Mtmr2- animals. β-Tubulin was used to normalize loading. (C) Coimmunoprecipitation of Mtmr2-Myc and Dlg1. Mtmr2-Myc was detected in immunoprecipitates prepared from lysates of COS-7 cells cotransfected with Dlg1 and Mtmr2-Myc (IP Dlg1 + Mtmr2) but not in untransfected cells (IP COS-7 lysate). Lanes (from left) show the following: untransfected COS-7 cells; cells cotransfected with both Dlg1 and Mtmr2-Myc; unbound fraction of the immunoprecipitation performed with anti-Dlg1 antibody on the lysates of cotransfected cells; immunoprecipitation from cotransfected cells performed with anti-Dlg1 antibody; and immunoprecipitation from untransfected cells. (C′) Converse coimmunoprecipitation in which an anti-Myc antibody was used to immunoprecipitate Mtmr2-Myc bound to Dlg1, as revealed by Western blot using an anti-Dlg1 antibody. (D–I) Immunohistochemistry on rat sciatic nerves using anti-Dlg1 antibody (D and G); anti–NF-H antibody to recognize axons, where Dlg1 is detected in a ring at the ab-axonal surface of a myelin-forming Schwann cell (F); and anti-GFAP to recognize non–myelin-forming Schwann cells (H), showing that Dlg1 is also expressed there (I). (J–L) MTMR2 is enriched at nodal regions of Schwann cells in human fibers stained with anti-hMTMR2 antibody (K) and colocalizes with DLG1 there (J and L). (M–O and P–R) Costaining of DLG1 and CASPR, which identifies paranodal domains, in teased fibers from normal (M) and Mtmr2- (P) mice. Dlg1 is enriched in the nodal–paranodal domain of normal fibers (M). This enrichment was much less obvious on fibers from mutant animals (P). Bar: (D–F and J–R) 12 μm; (G–I) 16 μm.
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fig6: Mtmr2 interacts with Dlg1/SAP97. (A) Semithin section analysis of sciatic nerves from mutant mice with conditional ablation of Mtmr2 in Schwann cells (P0-Cre/Mtmr2-) demonstrate myelin outfoldings (arrows). (B) Western blot analysis for Dlg1 in sciatic nerve homogenates from wild-type and Mtmr2- animals. β-Tubulin was used to normalize loading. (C) Coimmunoprecipitation of Mtmr2-Myc and Dlg1. Mtmr2-Myc was detected in immunoprecipitates prepared from lysates of COS-7 cells cotransfected with Dlg1 and Mtmr2-Myc (IP Dlg1 + Mtmr2) but not in untransfected cells (IP COS-7 lysate). Lanes (from left) show the following: untransfected COS-7 cells; cells cotransfected with both Dlg1 and Mtmr2-Myc; unbound fraction of the immunoprecipitation performed with anti-Dlg1 antibody on the lysates of cotransfected cells; immunoprecipitation from cotransfected cells performed with anti-Dlg1 antibody; and immunoprecipitation from untransfected cells. (C′) Converse coimmunoprecipitation in which an anti-Myc antibody was used to immunoprecipitate Mtmr2-Myc bound to Dlg1, as revealed by Western blot using an anti-Dlg1 antibody. (D–I) Immunohistochemistry on rat sciatic nerves using anti-Dlg1 antibody (D and G); anti–NF-H antibody to recognize axons, where Dlg1 is detected in a ring at the ab-axonal surface of a myelin-forming Schwann cell (F); and anti-GFAP to recognize non–myelin-forming Schwann cells (H), showing that Dlg1 is also expressed there (I). (J–L) MTMR2 is enriched at nodal regions of Schwann cells in human fibers stained with anti-hMTMR2 antibody (K) and colocalizes with DLG1 there (J and L). (M–O and P–R) Costaining of DLG1 and CASPR, which identifies paranodal domains, in teased fibers from normal (M) and Mtmr2- (P) mice. Dlg1 is enriched in the nodal–paranodal domain of normal fibers (M). This enrichment was much less obvious on fibers from mutant animals (P). Bar: (D–F and J–R) 12 μm; (G–I) 16 μm.

Mentions: To determine possible compensation or redundancy in the absence of Mtmr2, we stained nerves for Mtm1 and Mtmr1, which are both highly homologous to Mtmr2. In normal rat sciatic nerve, two different antibodies against Mtm1 revealed expression in the cytoplasms of both myelin-forming and non–myelin-forming Schwann cells, as well as in axons, all of which are locations where Mtmr2 is also expressed (Fig. 5, A–I; Previtali et al., 2003). On the contrary, an antibody against Mtmr1 revealed staining in the cytoplasm of non–myelin-forming Schwann cells and in axons, but not in myelin-forming Schwann cells (Fig. 5, R–W). Each of the Mtm1 or Mtmr1 antibodies produced similar patterns and intensities of staining on Mtmr2 (−/−) and control sciatic nerves (YFig. 5, J, K, X, and Y). These findings suggest that loss of Mtmr2 does not significantly affect the expression of Mtm1 and Mtmr1. It is important to note the difficulty of using antibodies to MTMR family members in mouse nerve. Two different antibodies against human MTMR2 and one against rat Mtmr2 all produced a positive staining in the cytoplasm of both Schwann cells and axons in Mtmr2- nerves, where Mtmr2 was absent in animals as shown by RT-PCR analysis and by immunoprecipitation followed by Western blot analysis (Fig. 1 D). However, these same antibodies produced no staining on frozen sural nerve biopsies of CMT4B1 patients (unpublished data), and produced staining in all cytoplasmic spaces of Schwann cells and axons in control human nerves (Fig. 6, J–L; Previtali et al., 2003). Thus, in immunohistochemistry, it is possible that Mtmr2 antibodies also recognize an MTMR2 homologue in mouse nerves that is distributed similarly to Mtmr2.


Disruption of Mtmr2 produces CMT4B1-like neuropathy with myelin outfolding and impaired spermatogenesis.

Bolino A, Bolis A, Previtali SC, Dina G, Bussini S, Dati G, Amadio S, Del Carro U, Mruk DD, Feltri ML, Cheng CY, Quattrini A, Wrabetz L - J. Cell Biol. (2004)

Mtmr2 interacts with Dlg1/SAP97. (A) Semithin section analysis of sciatic nerves from mutant mice with conditional ablation of Mtmr2 in Schwann cells (P0-Cre/Mtmr2-) demonstrate myelin outfoldings (arrows). (B) Western blot analysis for Dlg1 in sciatic nerve homogenates from wild-type and Mtmr2- animals. β-Tubulin was used to normalize loading. (C) Coimmunoprecipitation of Mtmr2-Myc and Dlg1. Mtmr2-Myc was detected in immunoprecipitates prepared from lysates of COS-7 cells cotransfected with Dlg1 and Mtmr2-Myc (IP Dlg1 + Mtmr2) but not in untransfected cells (IP COS-7 lysate). Lanes (from left) show the following: untransfected COS-7 cells; cells cotransfected with both Dlg1 and Mtmr2-Myc; unbound fraction of the immunoprecipitation performed with anti-Dlg1 antibody on the lysates of cotransfected cells; immunoprecipitation from cotransfected cells performed with anti-Dlg1 antibody; and immunoprecipitation from untransfected cells. (C′) Converse coimmunoprecipitation in which an anti-Myc antibody was used to immunoprecipitate Mtmr2-Myc bound to Dlg1, as revealed by Western blot using an anti-Dlg1 antibody. (D–I) Immunohistochemistry on rat sciatic nerves using anti-Dlg1 antibody (D and G); anti–NF-H antibody to recognize axons, where Dlg1 is detected in a ring at the ab-axonal surface of a myelin-forming Schwann cell (F); and anti-GFAP to recognize non–myelin-forming Schwann cells (H), showing that Dlg1 is also expressed there (I). (J–L) MTMR2 is enriched at nodal regions of Schwann cells in human fibers stained with anti-hMTMR2 antibody (K) and colocalizes with DLG1 there (J and L). (M–O and P–R) Costaining of DLG1 and CASPR, which identifies paranodal domains, in teased fibers from normal (M) and Mtmr2- (P) mice. Dlg1 is enriched in the nodal–paranodal domain of normal fibers (M). This enrichment was much less obvious on fibers from mutant animals (P). Bar: (D–F and J–R) 12 μm; (G–I) 16 μm.
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fig6: Mtmr2 interacts with Dlg1/SAP97. (A) Semithin section analysis of sciatic nerves from mutant mice with conditional ablation of Mtmr2 in Schwann cells (P0-Cre/Mtmr2-) demonstrate myelin outfoldings (arrows). (B) Western blot analysis for Dlg1 in sciatic nerve homogenates from wild-type and Mtmr2- animals. β-Tubulin was used to normalize loading. (C) Coimmunoprecipitation of Mtmr2-Myc and Dlg1. Mtmr2-Myc was detected in immunoprecipitates prepared from lysates of COS-7 cells cotransfected with Dlg1 and Mtmr2-Myc (IP Dlg1 + Mtmr2) but not in untransfected cells (IP COS-7 lysate). Lanes (from left) show the following: untransfected COS-7 cells; cells cotransfected with both Dlg1 and Mtmr2-Myc; unbound fraction of the immunoprecipitation performed with anti-Dlg1 antibody on the lysates of cotransfected cells; immunoprecipitation from cotransfected cells performed with anti-Dlg1 antibody; and immunoprecipitation from untransfected cells. (C′) Converse coimmunoprecipitation in which an anti-Myc antibody was used to immunoprecipitate Mtmr2-Myc bound to Dlg1, as revealed by Western blot using an anti-Dlg1 antibody. (D–I) Immunohistochemistry on rat sciatic nerves using anti-Dlg1 antibody (D and G); anti–NF-H antibody to recognize axons, where Dlg1 is detected in a ring at the ab-axonal surface of a myelin-forming Schwann cell (F); and anti-GFAP to recognize non–myelin-forming Schwann cells (H), showing that Dlg1 is also expressed there (I). (J–L) MTMR2 is enriched at nodal regions of Schwann cells in human fibers stained with anti-hMTMR2 antibody (K) and colocalizes with DLG1 there (J and L). (M–O and P–R) Costaining of DLG1 and CASPR, which identifies paranodal domains, in teased fibers from normal (M) and Mtmr2- (P) mice. Dlg1 is enriched in the nodal–paranodal domain of normal fibers (M). This enrichment was much less obvious on fibers from mutant animals (P). Bar: (D–F and J–R) 12 μm; (G–I) 16 μm.
Mentions: To determine possible compensation or redundancy in the absence of Mtmr2, we stained nerves for Mtm1 and Mtmr1, which are both highly homologous to Mtmr2. In normal rat sciatic nerve, two different antibodies against Mtm1 revealed expression in the cytoplasms of both myelin-forming and non–myelin-forming Schwann cells, as well as in axons, all of which are locations where Mtmr2 is also expressed (Fig. 5, A–I; Previtali et al., 2003). On the contrary, an antibody against Mtmr1 revealed staining in the cytoplasm of non–myelin-forming Schwann cells and in axons, but not in myelin-forming Schwann cells (Fig. 5, R–W). Each of the Mtm1 or Mtmr1 antibodies produced similar patterns and intensities of staining on Mtmr2 (−/−) and control sciatic nerves (YFig. 5, J, K, X, and Y). These findings suggest that loss of Mtmr2 does not significantly affect the expression of Mtm1 and Mtmr1. It is important to note the difficulty of using antibodies to MTMR family members in mouse nerve. Two different antibodies against human MTMR2 and one against rat Mtmr2 all produced a positive staining in the cytoplasm of both Schwann cells and axons in Mtmr2- nerves, where Mtmr2 was absent in animals as shown by RT-PCR analysis and by immunoprecipitation followed by Western blot analysis (Fig. 1 D). However, these same antibodies produced no staining on frozen sural nerve biopsies of CMT4B1 patients (unpublished data), and produced staining in all cytoplasmic spaces of Schwann cells and axons in control human nerves (Fig. 6, J–L; Previtali et al., 2003). Thus, in immunohistochemistry, it is possible that Mtmr2 antibodies also recognize an MTMR2 homologue in mouse nerves that is distributed similarly to Mtmr2.

Bottom Line: We also identified a novel physical interaction in Schwann cells, between Mtmr2 and discs large 1 (Dlg1)/synapse-associated protein 97, a scaffolding molecule that is enriched at the node/paranode region.Dlg1 homologues have been located in several types of cellular junctions and play roles in cell polarity and membrane addition.We propose that Schwann cell-autonomous loss of Mtmr2-Dlg1 interaction dysregulates membrane homeostasis in the paranodal region, thereby producing outfolding and recurrent loops of myelin.

View Article: PubMed Central - PubMed

Affiliation: Dulbecco Telethon Institute, San Raffaele Scientific Institute, 20132 Milan, Italy. bolino.alessandra@hsr.it

ABSTRACT
Mutations in MTMR2, the myotubularin-related 2 gene, cause autosomal recessive Charcot-Marie-Tooth (CMT) type 4B1, a demyelinating neuropathy with myelin outfolding and azoospermia. MTMR2 encodes a ubiquitously expressed phosphatase whose preferred substrate is phosphatidylinositol (3,5)-biphosphate, a regulator of membrane homeostasis and vesicle transport. We generated Mtmr2- mice, which develop progressive neuropathy characterized by myelin outfolding and recurrent loops, predominantly at paranodal myelin, and depletion of spermatids and spermatocytes from the seminiferous epithelium, which leads to azoospermia. Disruption of Mtmr2 in Schwann cells reproduces the myelin abnormalities. We also identified a novel physical interaction in Schwann cells, between Mtmr2 and discs large 1 (Dlg1)/synapse-associated protein 97, a scaffolding molecule that is enriched at the node/paranode region. Dlg1 homologues have been located in several types of cellular junctions and play roles in cell polarity and membrane addition. We propose that Schwann cell-autonomous loss of Mtmr2-Dlg1 interaction dysregulates membrane homeostasis in the paranodal region, thereby producing outfolding and recurrent loops of myelin.

Show MeSH
Related in: MedlinePlus