Stathmin-Deficient Mice Develop an Age-Dependent Axonopathy of the Central and Peripheral Nervous Systems
2002; Elsevier BV; Volume: 160; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)64866-3
ISSN1525-2191
AutoresWolfgang Liedtke, Elizabeth E. Leman, Robert E.W. Fyffe, Cedric S. Raine, Ulrich K. Schubart,
Tópico(s)Neurogenesis and neuroplasticity mechanisms
ResumoStathmin is a cytosolic protein that binds tubulin and destabilizes cellular microtubules, an activity regulated by phosphorylation. Despite its abundant expression in the developing mammalian nervous system and despite its high degree of evolutionary conservation, stathmin-deficient mice do not exhibit a developmental phenotype.1Schubart UK Yu J Amat JA Wang Z Hoffmann MK Edelmann W Normal development of mice lacking metablastin (P19), a phosphoprotein implicated in cell cycle regulation.J Biol Chem. 1996; 271: 14062-14066Crossref PubMed Scopus (80) Google Scholar Here we report that aging stathmin−/− mice develop an axonopathy of the central and peripheral nervous systems. The pathological hallmark of the early axonal lesions was a highly irregular axoplasm predominantly affecting large, heavily myelinated axons in motor tracts. As the lesions progressed, degeneration of axons, dysmyelination, and an unusual glial reaction were observed. At the functional level, electrophysiology recordings demonstrated a significant reduction of motor nerve conduction velocity in stathmin−/− mice. At the molecular level, increased gene expression of SCG 10-like protein, a stathmin-related gene with microtubule destabilizing activity, was detected in the central nervous system of aging stathmin−/− mice. Together, these findings suggest that stathmin plays an essential role in the maintenance of axonal integrity. Stathmin is a cytosolic protein that binds tubulin and destabilizes cellular microtubules, an activity regulated by phosphorylation. Despite its abundant expression in the developing mammalian nervous system and despite its high degree of evolutionary conservation, stathmin-deficient mice do not exhibit a developmental phenotype.1Schubart UK Yu J Amat JA Wang Z Hoffmann MK Edelmann W Normal development of mice lacking metablastin (P19), a phosphoprotein implicated in cell cycle regulation.J Biol Chem. 1996; 271: 14062-14066Crossref PubMed Scopus (80) Google Scholar Here we report that aging stathmin−/− mice develop an axonopathy of the central and peripheral nervous systems. The pathological hallmark of the early axonal lesions was a highly irregular axoplasm predominantly affecting large, heavily myelinated axons in motor tracts. As the lesions progressed, degeneration of axons, dysmyelination, and an unusual glial reaction were observed. At the functional level, electrophysiology recordings demonstrated a significant reduction of motor nerve conduction velocity in stathmin−/− mice. At the molecular level, increased gene expression of SCG 10-like protein, a stathmin-related gene with microtubule destabilizing activity, was detected in the central nervous system of aging stathmin−/− mice. Together, these findings suggest that stathmin plays an essential role in the maintenance of axonal integrity. Stathmin2Sobel A Boutterin MC Beretta L Chneiweiss H Doye V Peyro-Saint-Paul H Intracellular substrates for extracellular signaling: characterization of a ubiquitous, neuron-enriched phosphoprotein (stathmin).J Biol Chem. 1989; 264: 3765-3772Abstract Full Text PDF PubMed Google Scholar is a 148-amino acid cytosolic phosphoprotein that has also been referred to as p19,3Pasmantier R Danoff A Fleischer N Schubart UK p19, a hormonally regulated phosphoprotein of peptide hormone-producing cells: secretagogue-induced phosphorylation in AtT-20 mouse pituitary tumor cells and in rat and hamster insulinoma cells.Endocrinology. 1986; 119: 1229-1238Crossref PubMed Scopus (66) Google Scholar prosolin,4Cooper HL McDuffie E Braverman R Human peripheral lymphocyte growth regulation and response to phorbol esters is linked to synthesis and phosphorylation of the cytosolic protein, prosolin.J Immunol. 1989; 143: 956-963PubMed Google Scholar Lap18,5Ferrari AC Seuanez HN Hanash SM Atweh GF A gene that encodes for a leukemia-associated phosphoprotein (p18) maps to chromosome bands 1p35–36.1.Genes Chromosom Cancer. 1990; 2: 125-129Crossref PubMed Scopus (29) Google Scholar oncoprotein 18,6Melhem RF Strahler JR Hailat N Zhu XX Hanash SM Involvement of Op18 in cell proliferation.Biochem Biophys Res Comm. 1991; 179: 1649-1655Crossref PubMed Scopus (59) Google Scholar metablastin,7Schubart UK Xu J Fan W Cheng G Goldstein H Alpini G Shafritz DA Amat JA Farooq M Norton WT Owen TA Lian JB Stein GS Widespread, differentiation stage-specific expression of the gene encoding phosphoprotein p19 (Metablastin) in mammalian cells.Differentiation. 1992; 51: 21-32Crossref PubMed Scopus (45) Google Scholar and Op18.8Brattsand G Roos G Marklund U Ueda H Landberg G Nanberg E Sideras P Gullberg M Quantitative analysis of the expression and regulation of an activation-regulated phosphoprotein (oncoprotein 18) in normal and neoplastic cells.Leukemia. 1993; 7: 569-579PubMed Google Scholar This protein undergoes phosphorylation, at four serine residues, in mammalian cells in response to a diverse group of extracellular factors.9Horwitz SB Shen HJ He L Dittmar P Neef R Chen J Schubart UK The microtubule-destabilizing activity of metablastin (p19) is controlled by phosphorylation.J Biol Chem. 1997; 272: 8129-8132Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar Its expression is differentiation stage-specific, occurring in many immature cell types, most abundantly in the developing nervous system,10Schubart UK Expression of phosphoprotein p19 in brain, testis, and neuroendocrine tumor cells. Developmental regulation in rat brain.J Biol Chem. 1988; 263: 12156-12160Abstract Full Text PDF PubMed Google Scholar, 11Koppel J Boutterin MC Doye V Peyro-Saint-Paul H Sobel A Developmental tissue expression and phylogenetic conservation of stathmin, a phosphoprotein associated with cell regulations.J Biol Chem. 1990; 265: 3703-3707Abstract Full Text PDF PubMed Google Scholar, 12Amat JA Fields KL Schubart UK Distribution of phosphoprotein p19 in rat brain during ontogeny: stage-specific expression in neurons and glia.Brain Res Dev Brain Res. 1991; 60: 205-218Crossref PubMed Scopus (47) Google Scholar and is dramatically down-regulated during terminal differentiation.7Schubart UK Xu J Fan W Cheng G Goldstein H Alpini G Shafritz DA Amat JA Farooq M Norton WT Owen TA Lian JB Stein GS Widespread, differentiation stage-specific expression of the gene encoding phosphoprotein p19 (Metablastin) in mammalian cells.Differentiation. 1992; 51: 21-32Crossref PubMed Scopus (45) Google Scholar, 12Amat JA Fields KL Schubart UK Distribution of phosphoprotein p19 in rat brain during ontogeny: stage-specific expression in neurons and glia.Brain Res Dev Brain Res. 1991; 60: 205-218Crossref PubMed Scopus (47) Google Scholar, 13Amat JA Fields KL Schubart UK Stage-specific expression of phosphoprotein p19 during spermatogenesis in the rat.Mol Reprod Dev. 1990; 26: 383-390Crossref PubMed Scopus (30) Google Scholar In cultured cell lines, phosphorylation of stathmin fluctuates during the cell cycle and reaches its maximum during the M phase.14Strahler JR Lamb BJ Ungar DR Fox DA Hanash SM Cell cycle progression is associated with distinct patterns of phosphorylation of Op18.Biochem Biophys Res Commun. 1992; 185: 197-203Crossref PubMed Scopus (37) Google Scholar, 15Luo XN Mookerjee B Ferrari A Mistry S Atweh GF Regulation of phosphoprotein p18 in leukemic cells. Cell cycle regulated phosphorylation by p34cdc2 kinase.J Biol Chem. 1994; 269: 10312-10318Abstract Full Text PDF PubMed Google Scholar, 16Brattsand G Marklund U Nylander K Roos G Gullberg M Cell-cycle-regulated phosphorylation of oncoprotein 18 on Ser16, Ser25 and Ser38.Eur J Biochem. 1994; 220: 359-368Crossref PubMed Scopus (100) Google Scholar Overexpression of phosphorylation site mutants of stathmin results in cell cycle arrest at the G2/M interphase.17Marklund U Osterman O Melander H Bergh A Gullberg M The phenotype of a Cdc2 kinase target site-deficient mutant of oncoprotein 18 reveals a role of this protein in cell cycle control.J Biol Chem. 1994; 269: 30626-30635Abstract Full Text PDF PubMed Google Scholar, 18Larsson N Melander H Marklund U Osterman O Gullberg M G2/M transition requires multisite phosphorylation of oncoprotein 18 by two distinct protein kinase systems.J Biol Chem. 1995; 270: 14175-14183Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar Therefore, the protein has been implicated in cell cycle regulation. However, although stathmin is expressed in a variety of transformed cell lines in culture,10Schubart UK Expression of phosphoprotein p19 in brain, testis, and neuroendocrine tumor cells. Developmental regulation in rat brain.J Biol Chem. 1988; 263: 12156-12160Abstract Full Text PDF PubMed Google Scholar, 11Koppel J Boutterin MC Doye V Peyro-Saint-Paul H Sobel A Developmental tissue expression and phylogenetic conservation of stathmin, a phosphoprotein associated with cell regulations.J Biol Chem. 1990; 265: 3703-3707Abstract Full Text PDF PubMed Google Scholar and in some cancer cells in vivo,19Hanash SM Strahler JR Kuick R Chu EHY Nichols D Identification of a polypeptide associated with the malignant phenotype in acute leukemia.J Biol Chem. 1988; 263: 12813-12815Abstract Full Text PDF PubMed Google Scholar, 20Roos G Brattsand G Landberg G Marklund U Gullberg M Expression of oncoprotein 18 in human leukemias and lymphomas.Leukemia. 1993; 7: 1538-1546PubMed Google Scholar, 21Nylander K Marklund U Brattsand G Gullberg M Roos G Immunohistochemical detection of oncoprotein 18 (Op18) in malignant lymphomas.Histochem J. 1995; 27: 155-160Crossref PubMed Scopus (35) Google Scholar, 22Friedrich B Gronberg H Landstrom M Gullberg M Bergh A Differentiation-stage specific expression of oncoprotein 18 in human and rat prostatic adenocarcinoma.Prostate. 1995; 27: 102-109Crossref PubMed Scopus (90) Google Scholar, 23Bieche I Lachkar S Becette V Cifuentes-Diaz C Sobel A Lidereau R Curmi PA Overexpression of the stathmin gene in a subset of human breast cancer.Br J Cancer. 1998; 78: 701-709Crossref PubMed Scopus (89) Google Scholar expression in normal tissues is primarily restricted to postmitotic cells.7Schubart UK Xu J Fan W Cheng G Goldstein H Alpini G Shafritz DA Amat JA Farooq M Norton WT Owen TA Lian JB Stein GS Widespread, differentiation stage-specific expression of the gene encoding phosphoprotein p19 (Metablastin) in mammalian cells.Differentiation. 1992; 51: 21-32Crossref PubMed Scopus (45) Google Scholar, 12Amat JA Fields KL Schubart UK Distribution of phosphoprotein p19 in rat brain during ontogeny: stage-specific expression in neurons and glia.Brain Res Dev Brain Res. 1991; 60: 205-218Crossref PubMed Scopus (47) Google Scholar, 13Amat JA Fields KL Schubart UK Stage-specific expression of phosphoprotein p19 during spermatogenesis in the rat.Mol Reprod Dev. 1990; 26: 383-390Crossref PubMed Scopus (30) Google Scholar, 24Himi T Okazaki T Wang H McNeill TH Mori N Differential localization of SCG10 and p19/stathmin messenger RNAs in adult rat brain indicates distinct roles for these growth-associated proteins.Neuroscience. 1994; 60: 907-926Crossref PubMed Scopus (65) Google Scholar, 25Sugiura Y Mori N SCG10 expresses growth-associated manner in developing rat brain, but shows a different pattern to p19/stathmin or GAP-43.Brain Res Dev Brain Res. 1995; 90: 73-91Crossref PubMed Scopus (58) Google Scholar Recently, stathmin has been identified as a microtubule (MT)-destabilizing factor. In vitro, the protein binds to tubulin,26Belmont LD Mitchison TJ Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules.Cell. 1996; 84: 623-631Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar, 27Larsson N Marklund U Gradin HM Brattsand G Gullberg M Control of microtubule dynamics by oncoprotein 18: dissection of the regulatory role of multisite phosphorylation during mitosis.Mol Cell Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (168) Google Scholar, 28Curmi PA Andersen SS Lachkar S Gavet O Karsenti E Knossow M Sobel A The stathmin/tubulin interaction in vitro.J Biol Chem. 1997; 272: 25029-25036Crossref PubMed Scopus (192) Google Scholar, 29Jourdain L Curmi P Sobel A Pantaloni D Carlier MF Stathmin: a tubulin-sequestering protein which forms a ternary T2S complex with two tubulin molecules.Biochemistry. 1997; 36: 10817-10821Crossref PubMed Scopus (213) Google Scholar inhibits MT assembly,9Horwitz SB Shen HJ He L Dittmar P Neef R Chen J Schubart UK The microtubule-destabilizing activity of metablastin (p19) is controlled by phosphorylation.J Biol Chem. 1997; 272: 8129-8132Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 27Larsson N Marklund U Gradin HM Brattsand G Gullberg M Control of microtubule dynamics by oncoprotein 18: dissection of the regulatory role of multisite phosphorylation during mitosis.Mol Cell Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (168) Google Scholar, 29Jourdain L Curmi P Sobel A Pantaloni D Carlier MF Stathmin: a tubulin-sequestering protein which forms a ternary T2S complex with two tubulin molecules.Biochemistry. 1997; 36: 10817-10821Crossref PubMed Scopus (213) Google Scholar and promotes MT catastrophes.26Belmont LD Mitchison TJ Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules.Cell. 1996; 84: 623-631Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar, 30Howell B Larsson N Gullberg M Cassimeris L Dissociation of the tubulin-sequestering and microtubule catastrophe-promoting activities of oncoprotein 18/stathmin.Mol Biol Cell. 1999; 10: 105-118Crossref PubMed Scopus (158) Google Scholar Consistent with this activity, overexpression of stathmin in cells leads to disassembly of MTs9Horwitz SB Shen HJ He L Dittmar P Neef R Chen J Schubart UK The microtubule-destabilizing activity of metablastin (p19) is controlled by phosphorylation.J Biol Chem. 1997; 272: 8129-8132Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 27Larsson N Marklund U Gradin HM Brattsand G Gullberg M Control of microtubule dynamics by oncoprotein 18: dissection of the regulatory role of multisite phosphorylation during mitosis.Mol Cell Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (168) Google Scholar, 31Marklund U Larsson N Gradin HM Brattsand G Gullberg M Oncoprotein 18 is a phosphorylation-responsive regulator of microtubule dynamics.EMBO J. 1996; 15: 5290-5298Crossref PubMed Scopus (248) Google Scholar and, hence, inability to enter mitosis.17Marklund U Osterman O Melander H Bergh A Gullberg M The phenotype of a Cdc2 kinase target site-deficient mutant of oncoprotein 18 reveals a role of this protein in cell cycle control.J Biol Chem. 1994; 269: 30626-30635Abstract Full Text PDF PubMed Google Scholar, 18Larsson N Melander H Marklund U Osterman O Gullberg M G2/M transition requires multisite phosphorylation of oncoprotein 18 by two distinct protein kinase systems.J Biol Chem. 1995; 270: 14175-14183Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar Interestingly, the MT-destabilizing activity of stathmin is controlled by phosphorylation.9Horwitz SB Shen HJ He L Dittmar P Neef R Chen J Schubart UK The microtubule-destabilizing activity of metablastin (p19) is controlled by phosphorylation.J Biol Chem. 1997; 272: 8129-8132Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 27Larsson N Marklund U Gradin HM Brattsand G Gullberg M Control of microtubule dynamics by oncoprotein 18: dissection of the regulatory role of multisite phosphorylation during mitosis.Mol Cell Biol. 1997; 17: 5530-5539Crossref PubMed Scopus (168) Google Scholar, 31Marklund U Larsson N Gradin HM Brattsand G Gullberg M Oncoprotein 18 is a phosphorylation-responsive regulator of microtubule dynamics.EMBO J. 1996; 15: 5290-5298Crossref PubMed Scopus (248) Google Scholar, 32Melander Gradin H Marklund U Larsson N Chatila TA Gullberg M Regulation of microtubule dynamics by Ca2+/calmodulin-dependent kinase IV/Gr-dependent phosphorylation of oncoprotein 18.Mol Cell Biol. 1997; 17: 3459-3467Crossref PubMed Scopus (124) Google Scholar Therefore, the biological function of stathmin is possibly related to its MT-destabilizing activity. The high degree of evolutionary conservation of the gene encoding stathmin33Schubart UK Banerjee MD Eng J Homology between the cDNAs encoding phosphoprotein p19 and SCG10 reveals a novel mammalian gene family preferentially expressed in developing brain.DNA. 1989; 8: 389-398Crossref PubMed Scopus (69) Google Scholar, 34Zhu X-X Kozarsky K Strahler JR Eckerskorn C Lottspeich F Melhem R Lowe J Fox DA Hanash SM Atweh GF Molecular cloning of a novel human leukemia-associated gene. Evidence of conservation in animal species.J Biol Chem. 1989; 264: 14556-14560Abstract Full Text PDF PubMed Google Scholar, 35Doye V Soubrier F Bauw G Boutterin M-C Beretta L Koppel J Vanderkerkhove J Sobel A A single cDNA encodes two isoforms of stathmin, a developmentally regulated neuron-enriched phosphoprotein.J Biol Chem. 1989; 264: 12134-12137Abstract Full Text PDF PubMed Google Scholar, 36Melhem RF Zhu XX Hailat N Strahler JR Hanash SM Characterization of the gene for a proliferation-related phosphoprotein (oncoprotein 18) expressed in high amounts in acute leukemia.J Biol Chem. 1991; 266: 17747-17753Abstract Full Text PDF PubMed Google Scholar, 37Maucuer A Moreau J Méchali M Sobel A Stathmin gene family: phylogenetic conservation and developmental regulation in Xenopus.J Biol Chem. 1993; 268: 16420-16429Abstract Full Text PDF PubMed Google Scholar further suggests that it serves an essential function. It was surprising, therefore, that stathmin knockout mice did not exhibit any overt developmental abnormalities.1Schubart UK Yu J Amat JA Wang Z Hoffmann MK Edelmann W Normal development of mice lacking metablastin (P19), a phosphoprotein implicated in cell cycle regulation.J Biol Chem. 1996; 271: 14062-14066Crossref PubMed Scopus (80) Google Scholar This is presumably because of expression of other members of the stathmin gene family,33Schubart UK Banerjee MD Eng J Homology between the cDNAs encoding phosphoprotein p19 and SCG10 reveals a novel mammalian gene family preferentially expressed in developing brain.DNA. 1989; 8: 389-398Crossref PubMed Scopus (69) Google Scholar, 38Okazaki T Yoshida BN Avraham KB Wang H Wuenschell CW Jenkins NA Copeland NG Anderson DJ Mori N Molecular diversity of the SCG10/stathmin gene family in the mouse.Genomics. 1993; 18: 360-373Crossref PubMed Scopus (56) Google Scholar, 39Ozon S Maucuer A Sobel A The stathmin family: molecular and biological characterization of novel mammalian proteins expressed in the nervous system.Eur J Biochem. 1997; 248: 794-806Crossref PubMed Scopus (98) Google Scholar, 40Ozon S Byk T Sobel A SCLIP: a novel SCG10-like protein of the stathmin family expressed in the nervous system.J Neurochem. 1998; 70: 2386-2396Crossref PubMed Scopus (79) Google Scholar which includes the genes encoding SCG10, Rb3 (and related proteins derived by alternative splicing), and SCG10-like protein (SCLIP). With respect to SCG10, we have found no evidence for up-regulation in young stathmin knockout mice (Schubart et al1Schubart UK Yu J Amat JA Wang Z Hoffmann MK Edelmann W Normal development of mice lacking metablastin (P19), a phosphoprotein implicated in cell cycle regulation.J Biol Chem. 1996; 271: 14062-14066Crossref PubMed Scopus (80) Google Scholar and unpublished observations). We have now elucidated a phenotype of stathmin−/− mice by pathological-anatomical, neurophysiological, and molecular studies. Aged stathmin−/− mice were found to develop a progressive axonopathy, suggesting that stathmin is essential for the integrity of the nervous system. The generation of stathmin-deficient mice was described previously.1Schubart UK Yu J Amat JA Wang Z Hoffmann MK Edelmann W Normal development of mice lacking metablastin (P19), a phosphoprotein implicated in cell cycle regulation.J Biol Chem. 1996; 271: 14062-14066Crossref PubMed Scopus (80) Google Scholar The line has been maintained by successively backcrossing heterozygotes (stathmin+/−) into the C57BL/6J strain. Homozygous wild-type (stathmin+/+) and mutant (stathmin−/−) mice were F1 mice obtained by crossing stathmin+/− littermates. Animals used for the initial studies were from the second and third backcrosses (N2 and N3). The neuropathological findings were subsequently recapitulated in mice from the sixth backcross (N6). Animals were subjected to clinical examinations as previously described41Brown A McFarlin DE Raine CS Chronic neuropathology of relapsing experimental allergic encephalomyelitis in the mouse.Lab Invest. 1982; 46: 171-185PubMed Google Scholar for neurological and behavioral assessment of mice with experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. Specifically, we examined cranial nerves, motor function and sensory perception, coordination, balance, and gait. The mice were bred and housed in the barrier facility maintained by an Association for Assessment and Accreditation of Laboratory Animal Care accredited facility at the Albert Einstein College of Medicine. A total of 97 mice were analyzed, 58 for structural studies, 16 for Western and Northern blotting, and 23 for neurophysiology. Investigators were unaware of the genotype of the animals while carrying out the studies. All procedures were in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Animal Institute Committees of the academic institutions involved. Genotyping for the targeted stathmin gene was performed on tail DNA, using the polymerase chain reaction, which was performed in polymerase chain reaction buffer (Boehringer Mannheim Corp., Indianapolis, IN) containing 1.5 mmol/L MgCl2, deoxynucleotide triphosphates (1 mmol/L each), three primers, and Taq polymerase (0.05 U/μl) using 25 temperature cycles (94°C, 30 seconds; 57°C, 30 seconds; 72°C, 60 seconds). The primers used were a wild-type allele-specific forward primer (GAGAATCCATGATTGCCAGCAC, 1 μmol/L), corresponding to a region of intron III deleted in the mutant allele; a mutant allele-specific forward primer (CTAATGGCTATAGTTTCATGTTCC, 1 μmol/L), which anneals in the mPGK-1b 3′-flanking region42Boer PH Potten H Adra CN Jardine K Mullhofer G McBurney MW Polymorphisms in the coding and noncoding regions of murine Pgk-1 alleles.Biochem Genet. 1990; 28: 299-308Crossref PubMed Scopus (83) Google Scholar that is part of the PGKneo cassette used;1Schubart UK Yu J Amat JA Wang Z Hoffmann MK Edelmann W Normal development of mice lacking metablastin (P19), a phosphoprotein implicated in cell cycle regulation.J Biol Chem. 1996; 271: 14062-14066Crossref PubMed Scopus (80) Google Scholar and a reverse primer (AGCAAAACCAAATTAAGGGCCAGC, 2 μmol/L), corresponding to a region of intron III of the stathmin gene that is retained in the mutant allele. The polymerase chain reaction products obtained from the wild-type and mutant alleles were 463 bp and 610 bp in size, respectively. Neuropathological studies were performed as previously described.43Liedtke W Edelmann W Chiu FC Kucherlapati R Raine CS Experimental autoimmune encephalomyelitis in mice lacking glial fibrillary acidic protein is characterized by a more severe clinical course and an infiltrative central nervous system lesion.Am J Pathol. 1998; 152: 251-259PubMed Google Scholar, 44Liedtke W Edelmann W Bieri PL Chiu FC Cowan NJ Kucherlapati R Raine CS GFAP is necessary for the integrity of CNS white matter architecture and long-term maintenance of myelination.Neuron. 1996; 17: 607-615Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar Briefly, mice were anesthetized with ether and perfused transcardially either with fixative [2.5% glutaraldehyde in Millonig's buffer (phosphate-buffered saline [PBS] supplemented with 5.4g dextrose/L) or 10% formalin in PBS, pH 7.4], or PBS, pH 7.4. Fixative-perfused tissue was embedded in paraffin or epoxy (Epon 812; Ladd Research Industries, Williston, VT) and PBS-perfused tissue was used for frozen blocks in OCT/Tissuetek or for tissue homogenization and denaturation before immunoblot analysis. Glutaraldehyde/osmium-tetroxide fixed epoxy sections were sectioned on a Reichert ultramicrotome at 1 μm and stained with toluidine blue. Both longitudinal and cross sections were obtained from the optic nerve, corpus callosum, and brain stem. At least four, in most cases six, different levels of spinal cord were sampled, in addition to spinal roots and sciatic nerve, the latter sampled at mid-thigh level. Either 10-μm frozen or 1-μm epoxy sections were incubated with primary antibody and developed using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). The following primary antibodies were used: rabbit anti-stathmin C-terminal peptide,12Amat JA Fields KL Schubart UK Distribution of phosphoprotein p19 in rat brain during ontogeny: stage-specific expression in neurons and glia.Brain Res Dev Brain Res. 1991; 60: 205-218Crossref PubMed Scopus (47) Google Scholar 1:1000; mouse anti-glial fibrillary acidic protein (GFAP) (Zymed, San Francisco, CA), 1:200; mouse anti-NFH-(strongly phosphorylated) SMI 35 (Sternberger Monoclonals, Baltimore, MD), 1:5000; mouse anti-NFH-(nonphosphorylated) SMI 32 (Sternberger Monoclonals), 1:2000; mouse anti-NFH-(intermediately phosphorylated) SMI 31 (Sternberger Monoclonals), 1:5000; mouse anti-NFL (Sigma Chemical Co., St. Louis, MO), 1:100. Abnormal cellular aggregates in white matter, representing the end-stage of central nervous system (CNS) axonopathy (see Results), were counted in stathmin+/+ and stathmin−/− mice of different ages: 21 months of age, four of five mice each; 14 months of age, five of six mice each, and 6 weeks of age, six of six mice each. Matched cross-sections of cervical, thoracic, and upper lumbar spinal cord were scored at ×100 magnification. Blocks of tissue were selected for electron microscopy after light microscopy of semithin sections. Thin sections (60 to 80 nm) of epon-embedded tissue were mounted on 200-mesh copper grids, stained with lead acetate (1% in H2O) and uranyl acetate (8.5% in ethanol), carbon-coated, and scanned in a Siemens 101 electron microscope. The procedure has been described in more detail elsewhere.45Raine CS Bornstein MB Experimental allergic neuritis. Ultrastructure of serum-induced myelin aberrations in peripheral nervous system cultures.Lab Invest. 1979; 40: 423-432PubMed Google Scholar The basic outline of ultrastructural morphometry has been described elsewhere.43Liedtke W Edelmann W Chiu FC Kucherlapati R Raine CS Experimental autoimmune encephalomyelitis in mice lacking glial fibrillary acidic protein is characterized by a more severe clinical course and an infiltrative central nervous system lesion.Am J Pathol. 1998; 152: 251-259PubMed Google Scholar Briefly, matched cross-sections of spinal cord anterior column and sciatic nerve were scanned at ×7.500 magnification and printed at constant enlargement. Three axons each per spinal cord and per sciatic nerve were selected, n = 2 mice (21 months of age) per genotype. Myelinated axons were randomly selected for wild-type littermates. Only axons with a uniformly hyperdense axoplasm were analyzed for stathmin−/− mice. A square of 2.5-cm side-length was drawn into the micrograph and neurofilaments (NFs) and MTs were counted on the micrograph. Thus a total of six axons per mouse was analyzed. Spinal cord tissue of 17-month-old mice (four stathmin+/+ and four stathmin−/−), which had been perfused with ice-cold PBS in situ, was homogenized immediately in 8 mol/L urea. Protein was measured by the Bradford assay46Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214515) Google Scholar using Bio-Rad reagents (Bio-Rad Laboratories, Hercules, CA). Aliquots (50 μg) were subjected to immunoblot analysis as previously described.10Schubart UK Expression of phosphoprotein p19 in brain, testis, and neuroendocrine tumor cells. Developmental regulation in rat brain.J Biol Chem. 1988; 263: 12156-12160Abstract Full Text PDF PubMed Google Scholar In addition to the antibodies described in the Immunocytochemistry section, the following antibodies were used: mouse anti-tubulin (Sigma Chemical Co.); and mouse anti-GFAP (Boehringer Mannheim Corp.). The blots were developed using ECL reagents (Amersham Life Science Inc., Arlington Heights, IL). Brain and spinal cord tissue was dissected from 19-month-old mice perfused with ice-cold PBS, pH 7.4, pretreated with diethyl pyrocarbonate 0.1%, and total RNA was isolated as described.47Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63088) Google Scholar To average the abundance of expressed mRNAs, RNA from four knockout mice was pooled. Northern blot analysis was performed as previously described.1Schubart UK Yu J Amat JA Wang Z Hoffmann MK Edelmann W Normal development of mice lacking metablastin (P19), a phosphoprotein implicated in cell cycle regulation.J Biol Chem. 1996; 271: 14062-14066Crossref PubMed Scopus (80) Google Scholar, 48Liedtke W Choe Y Marti-Renom MA Bell AM Denis CS Sali A Hudspeth AJ Friedman JM Heller S Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor.Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1057) Google Scholar32P-labeled DNA probes for stathmin-related transcripts were generated by reverse transcriptase-polymerase chain reaction using normal mouse brain RNA as template. The primers used were: forward (stathmin) 5′-TCCCCGACCCCTTCCTAAATATCC; reverse (sta
Referência(s)