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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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Generation of the Mtmr2- allele. (A) Schematic diagrams (from top) show the following: genomic structure of Mtmr2 surrounding exon 4 (wild-type locus); targeting construct where genomic Mtmr2 fragments are indicated with thick lines and vector-derived segments with thin lines (targeting vector); the Mtmr2 locus after homologous recombination in embryonic stem cells (floxed Mtmr2 allele); the floxed Mtmr2 allele after CMV-Cre–mediated excision of exon 4 (exon 4 excised allele). The KpnI (K) and HindIII (H) restriction sites, as well as probes used for Southern blot analysis, are shown. A–D are primers used for genotype analysis. (B) Southern blot analysis of embryonic stem cell clones containing the targeted (floxed, Fl/+) allele after homologous recombination. DNA was digested with KpnI (K) and HindIII (H) and hybridized with probe A. The last two lanes (+/+) contain DNA from wild-type embryonic stem cells. (C) Genotyping of mouse tail DNA using primer pairs C + B and A + D. (+/+) is a wild-type mouse; (Fl/+) is a mouse carrying the targeted floxed allele, before CMV-Cre–mediated excision of exon 4; (+/−) is a heterozygous mouse for the CMV-Cre–mediated excision of exon 4; and (−/−) is a homozygous  mouse where the excision of exon 4 occurred on both Mtmr2 alleles. (D) RT-PCR analysis on sciatic nerve mRNA from wild-type (+/+) and Mtmr2- animals (−/−). When a reverse primer recognizing exon 4 was used, no amplification was detected in Mtmr2 (−/−) mice. cDNA synthesis was performed using both oligo-dT and random hexamers on total RNA from wild-type and mutant nerves. (C and D) White lines indicate that intervening lanes have been spliced out. (E) Western blot analysis of Mtmr2 immunoprecipitated using anti-hMTMR2 antibodies. Brain homogenates from wild-type (+/+) and mutant (−/−) animals were prepared using two different lysis buffers containing either Igepal or Triton X-100 as detergents. Unb, unbound fraction after immunoprecipitation.
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fig1: Generation of the Mtmr2- allele. (A) Schematic diagrams (from top) show the following: genomic structure of Mtmr2 surrounding exon 4 (wild-type locus); targeting construct where genomic Mtmr2 fragments are indicated with thick lines and vector-derived segments with thin lines (targeting vector); the Mtmr2 locus after homologous recombination in embryonic stem cells (floxed Mtmr2 allele); the floxed Mtmr2 allele after CMV-Cre–mediated excision of exon 4 (exon 4 excised allele). The KpnI (K) and HindIII (H) restriction sites, as well as probes used for Southern blot analysis, are shown. A–D are primers used for genotype analysis. (B) Southern blot analysis of embryonic stem cell clones containing the targeted (floxed, Fl/+) allele after homologous recombination. DNA was digested with KpnI (K) and HindIII (H) and hybridized with probe A. The last two lanes (+/+) contain DNA from wild-type embryonic stem cells. (C) Genotyping of mouse tail DNA using primer pairs C + B and A + D. (+/+) is a wild-type mouse; (Fl/+) is a mouse carrying the targeted floxed allele, before CMV-Cre–mediated excision of exon 4; (+/−) is a heterozygous mouse for the CMV-Cre–mediated excision of exon 4; and (−/−) is a homozygous mouse where the excision of exon 4 occurred on both Mtmr2 alleles. (D) RT-PCR analysis on sciatic nerve mRNA from wild-type (+/+) and Mtmr2- animals (−/−). When a reverse primer recognizing exon 4 was used, no amplification was detected in Mtmr2 (−/−) mice. cDNA synthesis was performed using both oligo-dT and random hexamers on total RNA from wild-type and mutant nerves. (C and D) White lines indicate that intervening lanes have been spliced out. (E) Western blot analysis of Mtmr2 immunoprecipitated using anti-hMTMR2 antibodies. Brain homogenates from wild-type (+/+) and mutant (−/−) animals were prepared using two different lysis buffers containing either Igepal or Triton X-100 as detergents. Unb, unbound fraction after immunoprecipitation.

Mentions: To generate a conditional Mtmr2- allele, we flanked exon 4 with lox-P sites, because its excision introduces a frameshift from either ATG start site of translation (exon 1 or 3); virtual translation predicts a short peptide without putative functional domains (Fig. 1 A). In addition, a naturally occurring nonsense mutation in exon 4, thought to produce complete loss of function, was described in an Indian family with typical CMT4B1 (Houlden et al., 2001). The Mtmr2- allele was produced by crossing Mtmr2-floxed mice with cytomegalovirus (CMV) promoter–Cre transgenic mice. The deletion of exon 4 was documented in progeny by PCR analysis of genomic DNA (Fig. 1 C). Heterozygous Mtmr2 exon 4–deleted mice were crossed to generate Mtmr2 homozygous mice. No Mtmr2 mRNAs containing exon 4 or 3′-exons could be detected by RT-PCR analysis of total RNA from the tail, brain, muscle, and sciatic nerve (Fig. 1 D and not depicted). Immunoprecipitation followed by Western blot analysis revealed that Mtmr2 protein was absent from brain lysates in Mtmr2- animals (Fig. 1 E).


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)

Generation of the Mtmr2- allele. (A) Schematic diagrams (from top) show the following: genomic structure of Mtmr2 surrounding exon 4 (wild-type locus); targeting construct where genomic Mtmr2 fragments are indicated with thick lines and vector-derived segments with thin lines (targeting vector); the Mtmr2 locus after homologous recombination in embryonic stem cells (floxed Mtmr2 allele); the floxed Mtmr2 allele after CMV-Cre–mediated excision of exon 4 (exon 4 excised allele). The KpnI (K) and HindIII (H) restriction sites, as well as probes used for Southern blot analysis, are shown. A–D are primers used for genotype analysis. (B) Southern blot analysis of embryonic stem cell clones containing the targeted (floxed, Fl/+) allele after homologous recombination. DNA was digested with KpnI (K) and HindIII (H) and hybridized with probe A. The last two lanes (+/+) contain DNA from wild-type embryonic stem cells. (C) Genotyping of mouse tail DNA using primer pairs C + B and A + D. (+/+) is a wild-type mouse; (Fl/+) is a mouse carrying the targeted floxed allele, before CMV-Cre–mediated excision of exon 4; (+/−) is a heterozygous mouse for the CMV-Cre–mediated excision of exon 4; and (−/−) is a homozygous  mouse where the excision of exon 4 occurred on both Mtmr2 alleles. (D) RT-PCR analysis on sciatic nerve mRNA from wild-type (+/+) and Mtmr2- animals (−/−). When a reverse primer recognizing exon 4 was used, no amplification was detected in Mtmr2 (−/−) mice. cDNA synthesis was performed using both oligo-dT and random hexamers on total RNA from wild-type and mutant nerves. (C and D) White lines indicate that intervening lanes have been spliced out. (E) Western blot analysis of Mtmr2 immunoprecipitated using anti-hMTMR2 antibodies. Brain homogenates from wild-type (+/+) and mutant (−/−) animals were prepared using two different lysis buffers containing either Igepal or Triton X-100 as detergents. Unb, unbound fraction after immunoprecipitation.
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Related In: Results  -  Collection

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fig1: Generation of the Mtmr2- allele. (A) Schematic diagrams (from top) show the following: genomic structure of Mtmr2 surrounding exon 4 (wild-type locus); targeting construct where genomic Mtmr2 fragments are indicated with thick lines and vector-derived segments with thin lines (targeting vector); the Mtmr2 locus after homologous recombination in embryonic stem cells (floxed Mtmr2 allele); the floxed Mtmr2 allele after CMV-Cre–mediated excision of exon 4 (exon 4 excised allele). The KpnI (K) and HindIII (H) restriction sites, as well as probes used for Southern blot analysis, are shown. A–D are primers used for genotype analysis. (B) Southern blot analysis of embryonic stem cell clones containing the targeted (floxed, Fl/+) allele after homologous recombination. DNA was digested with KpnI (K) and HindIII (H) and hybridized with probe A. The last two lanes (+/+) contain DNA from wild-type embryonic stem cells. (C) Genotyping of mouse tail DNA using primer pairs C + B and A + D. (+/+) is a wild-type mouse; (Fl/+) is a mouse carrying the targeted floxed allele, before CMV-Cre–mediated excision of exon 4; (+/−) is a heterozygous mouse for the CMV-Cre–mediated excision of exon 4; and (−/−) is a homozygous mouse where the excision of exon 4 occurred on both Mtmr2 alleles. (D) RT-PCR analysis on sciatic nerve mRNA from wild-type (+/+) and Mtmr2- animals (−/−). When a reverse primer recognizing exon 4 was used, no amplification was detected in Mtmr2 (−/−) mice. cDNA synthesis was performed using both oligo-dT and random hexamers on total RNA from wild-type and mutant nerves. (C and D) White lines indicate that intervening lanes have been spliced out. (E) Western blot analysis of Mtmr2 immunoprecipitated using anti-hMTMR2 antibodies. Brain homogenates from wild-type (+/+) and mutant (−/−) animals were prepared using two different lysis buffers containing either Igepal or Triton X-100 as detergents. Unb, unbound fraction after immunoprecipitation.
Mentions: To generate a conditional Mtmr2- allele, we flanked exon 4 with lox-P sites, because its excision introduces a frameshift from either ATG start site of translation (exon 1 or 3); virtual translation predicts a short peptide without putative functional domains (Fig. 1 A). In addition, a naturally occurring nonsense mutation in exon 4, thought to produce complete loss of function, was described in an Indian family with typical CMT4B1 (Houlden et al., 2001). The Mtmr2- allele was produced by crossing Mtmr2-floxed mice with cytomegalovirus (CMV) promoter–Cre transgenic mice. The deletion of exon 4 was documented in progeny by PCR analysis of genomic DNA (Fig. 1 C). Heterozygous Mtmr2 exon 4–deleted mice were crossed to generate Mtmr2 homozygous mice. No Mtmr2 mRNAs containing exon 4 or 3′-exons could be detected by RT-PCR analysis of total RNA from the tail, brain, muscle, and sciatic nerve (Fig. 1 D and not depicted). Immunoprecipitation followed by Western blot analysis revealed that Mtmr2 protein was absent from brain lysates in Mtmr2- animals (Fig. 1 E).

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