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• W2034336725 abstract "The cell adhesion molecule L1 is a Lewisx-carrying glycoprotein that plays important roles in the developing and adult nervous system. Here we show that myelin basic protein (MBP) binds to L1 in a Lewisx-dependent manner. Furthermore, we demonstrate that MBP is released by murine cerebellar neurons as a sumoylated dynamin-containing protein upon L1 stimulation and that this MBP cleaves L1 as a serine protease in the L1 extracellular domain at Arg687 yielding a transmembrane fragment that promotes neurite outgrowth and neuronal survival in cell culture. L1-induced neurite outgrowth and neuronal survival are reduced in MBP-deficient cerebellar neurons and in wild-type cerebellar neurons in the presence of an MBP antibody or L1 peptide containing the MBP cleavage site. Genetic ablation of MBP in shiverer mice and mutagenesis of the proteolytically active site in MBP or of the MBP cleavage site within L1 as well as serine protease inhibitors and an L1 peptide containing the MBP cleavage site abolish generation of the L1 fragment. Our findings provide evidence for novel functions of MBP in the nervous system. The cell adhesion molecule L1 is a Lewisx-carrying glycoprotein that plays important roles in the developing and adult nervous system. Here we show that myelin basic protein (MBP) binds to L1 in a Lewisx-dependent manner. Furthermore, we demonstrate that MBP is released by murine cerebellar neurons as a sumoylated dynamin-containing protein upon L1 stimulation and that this MBP cleaves L1 as a serine protease in the L1 extracellular domain at Arg687 yielding a transmembrane fragment that promotes neurite outgrowth and neuronal survival in cell culture. L1-induced neurite outgrowth and neuronal survival are reduced in MBP-deficient cerebellar neurons and in wild-type cerebellar neurons in the presence of an MBP antibody or L1 peptide containing the MBP cleavage site. Genetic ablation of MBP in shiverer mice and mutagenesis of the proteolytically active site in MBP or of the MBP cleavage site within L1 as well as serine protease inhibitors and an L1 peptide containing the MBP cleavage site abolish generation of the L1 fragment. Our findings provide evidence for novel functions of MBP in the nervous system. Development of the nervous system and its correct functioning in the adult depend on many cellular features, such as cell proliferation and migration, neuronal survival and programmed cell death, neuritogenesis, synapse formation, and synaptic plasticity. The protein backbones and distinct carbohydrate structures of cell adhesion molecules participate in these important cellular events (1Kleene R. Schachner M. Glycans and neural cell interactions.Nat. Rev. Neurosci. 2004; 5: 195-208Crossref PubMed Scopus (433) Google Scholar, 2Maness P.F. Schachner M. Neural recognition molecules of the immunoglobulin superfamily: signaling transducers of axon guidance and neuronal migration.Nat. Neurosci. 2007; 10: 19-26Crossref PubMed Scopus (667) Google Scholar, 3Schmid R.S. Maness P.F. L1 and NCAM adhesion molecules as signaling coreceptors in neuronal migration and process outgrowth.Curr. Opin. Neurobiol. 2008; 18: 245-250Crossref PubMed Scopus (170) Google Scholar). In particular, recognition molecules at the cell surface and in the extracellular matrix contribute to these events, and among these the immunoglobulin superfamily member L1 has been implicated in all these functions. L1 is a transmembrane cell surface glycoprotein and is not only a glycan-carrying but also a glycan binding cell adhesion molecule (4Streit A. Yuen C.T. Loveless R.W. Lawson A.M. Finne J. Schmitz B. Feizi T. Stern C.D. The Le(x) carbohydrate sequence is recognized by antibody to L5, a functional antigen in early neural development.J. Neurochem. 1996; 66: 834-844Crossref PubMed Scopus (78) Google Scholar5Wing D.R. Rademacher T.W. Schmitz B. Schachner M. Dwek R.A. Comparative glycosylation in neural adhesion molecules.Biochem. Soc. Trans. 1992; 20: 386-390Crossref PubMed Scopus (26) Google Scholar, 6Lieberoth A. Splittstoesser F. Katagihallimath N. Jakovcevski I. Loers G. Ranscht B. Karagogeos D. Schachner M. Kleene R. Lewis(x) and α2,3-sialyl glycans and their receptors TAG-1, Contactin, and L1 mediate CD24-dependent neurite outgrowth.J. Neurosci. 2009; 29: 6677-6690Crossref PubMed Scopus (56) Google Scholar, 7Kleene R. Yang H. Kutsche M. Schachner M. The neural recognition molecule L1 is a sialic acid-binding lectin for CD24, which induces promotion and inhibition of neurite outgrowth.J. Biol. Chem. 2001; 276: 21656-21663Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) that plays a crucial role in neuronal cell migration and survival, neurite outgrowth, axon guidance and fasciculation, myelination, synaptic plasticity, and regeneration after injury (1Kleene R. Schachner M. Glycans and neural cell interactions.Nat. Rev. Neurosci. 2004; 5: 195-208Crossref PubMed Scopus (433) Google Scholar, 2Maness P.F. Schachner M. Neural recognition molecules of the immunoglobulin superfamily: signaling transducers of axon guidance and neuronal migration.Nat. Neurosci. 2007; 10: 19-26Crossref PubMed Scopus (667) Google Scholar, 8Guseva D. Zerwas M. Xiao M.F. Jakovcevski I. Irintchev A. Schachner M. Adhesion molecule L1 overexpressed under the control of the neuronal Thy-1 promoter improves myelination after peripheral nerve injury in adult mice.Exp. Neurol. 2011; 229: 339-352Crossref PubMed Scopus (28) Google Scholar, 9Lutz D. Wolters-Eisfeld G. Joshi G. Djogo N. Jakovcevski I. Schachner M. Kleene R. Generation and nuclear translocation of a sumoylated transmembrane fragment of the cell adhesion molecule L1.J. Biol. Chem. 2012; 287: 17161-17175Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 10Lutz D. Wolters-Eisfeld G. Schachner M. Kleene R. Cathepsin E generates a sumoylated intracellular fragment of the cell adhesion molecule L1 to promote neuronal and Schwann cell migration as well as myelination.J. Neurochem. 2014; 128: 713-724Crossref PubMed Scopus (28) Google Scholar). In addition, it is associated with neural disorders, such as the L1 syndrome, fetal alcohol syndrome, and schizophrenia as well as Alzheimer, Parkinson, Huntington, and Hirschsprung diseases (2Maness P.F. Schachner M. Neural recognition molecules of the immunoglobulin superfamily: signaling transducers of axon guidance and neuronal migration.Nat. Neurosci. 2007; 10: 19-26Crossref PubMed Scopus (667) Google Scholar, 9Lutz D. Wolters-Eisfeld G. Joshi G. Djogo N. Jakovcevski I. Schachner M. Kleene R. Generation and nuclear translocation of a sumoylated transmembrane fragment of the cell adhesion molecule L1.J. Biol. Chem. 2012; 287: 17161-17175Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar10Lutz D. Wolters-Eisfeld G. Schachner M. Kleene R. Cathepsin E generates a sumoylated intracellular fragment of the cell adhesion molecule L1 to promote neuronal and Schwann cell migration as well as myelination.J. Neurochem. 2014; 128: 713-724Crossref PubMed Scopus (28) Google Scholar, 11Strekalova H. Buhmann C. Kleene R. Eggers C. Saffell J. Hemperly J. Weiller C. Müller-Thomsen T. Schachner M. Elevated levels of neural recognition molecule L1 in the cerebrospinal fluid of patients with Alzheimer disease and other dementia syndromes.Neurobiol. Aging. 2006; 27: 1-9Crossref PubMed Scopus (73) Google Scholar, 12Wakabayashi Y. Uchida S. Funato H. Matsubara T. Watanuki T. Otsuki K. Fujimoto M. Nishida A. Watanabe Y. State-dependent changes in the expression levels of NCAM-140 and L1 in the peripheral blood cells of bipolar disorders, but not in the major depressive disorders.Prog. Neuropsychopharmacol. Biol. Psychiatry. 2008; 32: 1199-1205Crossref PubMed Scopus (28) Google Scholar, 13Schäfer M.K. Altevogt P. L1CAM malfunction in the nervous system and human carcinomas.Cell. Mol. Life Sci. 2010; 67: 2425-2437Crossref PubMed Scopus (103) Google Scholar, 14Poltorak M. Khoja I. Hemperly J.J. Williams J.R. el-Mallakh R. Freed W.J. Disturbances in cell recognition molecules (N-CAM and L1 antigen) in the CSF of patients with schizophrenia.Exp. Neurol. 1995; 131: 266-272Crossref PubMed Scopus (88) Google Scholar, 15Kurumaji A. Nomoto H. Okano T. Toru M. An association study between polymorphism of L1CAM gene and schizophrenia in a Japanese sample.Am. J. Med. Genet. 2001; 105: 99-104Crossref PubMed Scopus (33) Google Scholar). Besides other functionally important glycan residues, L1 carries the Lewisx glycan epitope (Lex), 3The abbreviations used are:LexLewisxLeaLewisaMBPmyelin basic proteinPNGase Fpeptide N-glycosidase FRFPred fluorescent proteinFnIIIfibronectin type IIIAAV1adeno-associated virus 1. which has been implicated in cell adhesion and migration as well as neurite outgrowth (6Lieberoth A. Splittstoesser F. Katagihallimath N. Jakovcevski I. Loers G. Ranscht B. Karagogeos D. Schachner M. Kleene R. Lewis(x) and α2,3-sialyl glycans and their receptors TAG-1, Contactin, and L1 mediate CD24-dependent neurite outgrowth.J. Neurosci. 2009; 29: 6677-6690Crossref PubMed Scopus (56) Google Scholar, 16Katagihallimath N. Mehanna A. Guseva D. Kleene R. Schachner M. Identification and validation of a Lewis x glycomimetic peptide.Eur. J. Cell Biol. 2010; 89: 77-86Crossref PubMed Scopus (12) Google Scholar, 17Sajdel-Sulkowska E.M. Immunofluorescent detection of CD15-fucosylated glycoconjugates in primary cerebellar cultures and their function in glial-neuronal adhesion in the central nervous system.Acta Biochim. Pol. 1998; 45: 781-790Crossref PubMed Scopus (17) Google Scholar, 18Brito C. Escrevente C. Reis C.A. Lee V.M. Trojanowski J.Q. Costa J. Increased levels of fucosyltransferase IX and carbohydrate Lewis(x) adhesion determinant in human NT2N neurons.J. Neurosci. Res. 2007; 85: 1260-1270Crossref PubMed Scopus (20) Google Scholar). Lewisx Lewisa myelin basic protein peptide N-glycosidase F red fluorescent protein fibronectin type III adeno-associated virus 1. In search of binding partners for the Lex glycan, we identified myelin basic protein (MBP) as a Lex-binding protein and show that it interacts with L1 in a Lex-dependent manner. MBP is a major structural component of mature myelin and is implicated in formation, compaction, and maintenance of myelin (19Boggs J.M. Myelin basic protein: a multifunctional protein.Cell. Mol. Life Sci. 2006; 63: 1945-1961Crossref PubMed Scopus (417) Google Scholar, 20Readhead C. Takasashi N. Shine H.D. Saavedra R. Sidman R. Hood L. Role of myelin basic protein in the formation of central nervous system myelin.Ann. N.Y. Acad. Sci. 1990; 605: 280-285Crossref PubMed Scopus (47) Google Scholar). Different MBP isoforms originate from a gene complex called golli (gene in the oligodendrocyte lineage), which in mice comprises 11 exons and includes the 7 exons coding for the so-called classic MBP proteins (19Boggs J.M. Myelin basic protein: a multifunctional protein.Cell. Mol. Life Sci. 2006; 63: 1945-1961Crossref PubMed Scopus (417) Google Scholar, 21Feng J.M. Minireview: expression and function of golli protein in immune system.Neurochem. Res. 2007; 32: 273-278Crossref PubMed Scopus (20) Google Scholar, 22Harauz G. Boggs J.M. Myelin management by the 18.5-kDa and 21.5-kDa classic myelin basic protein isoforms.J. Neurochem. 2013; 125: 334-361Crossref PubMed Scopus (95) Google Scholar). Golli and MBP proteins are generated from different transcription start sites and are encoded by different exons (19Boggs J.M. Myelin basic protein: a multifunctional protein.Cell. Mol. Life Sci. 2006; 63: 1945-1961Crossref PubMed Scopus (417) Google Scholar, 21Feng J.M. Minireview: expression and function of golli protein in immune system.Neurochem. Res. 2007; 32: 273-278Crossref PubMed Scopus (20) Google Scholar, 22Harauz G. Boggs J.M. Myelin management by the 18.5-kDa and 21.5-kDa classic myelin basic protein isoforms.J. Neurochem. 2013; 125: 334-361Crossref PubMed Scopus (95) Google Scholar). The different MBP proteins are expressed in the central and peripheral nervous system, whereas the golli proteins are found in both the immune and nervous system but not in compact myelin. The importance of MBP proteins for myelination is well exemplified by the severe phenotype of the naturally occurring MBP-deficient shiverer mutant mouse, which lacks most of the compact myelin structures in the central nervous system (23Wolf M.K. Billings-Gagliardi S. CNS hypomyelinated mutant mice (jimpyshivererquaking): in vitro evidence for primary oligodendrocyte defects.Adv. Exp. Med. Biol. 1984; 181: 115-133Crossref PubMed Scopus (11) Google Scholar, 24Readhead C. Hood L. The dysmyelinating mouse mutations shiverer (shi) and myelin deficient (shimld).Behav. Genet. 1990; 20: 213-234Crossref PubMed Scopus (100) Google Scholar). In trying to identify the molecular binding partners of L1 on myelin-forming cells, we found abnormalities in L1 proteolytic processing in the shiverer mouse mutant. We, therefore, undertook a more detailed investigation of the relationship between neuronal L1 and myelin forming glial cells, with a particular view on MBP. Using a phage display approach, we found that MBP binds to Lex and to Lex carrying L1 from mouse brain but not to recombinantly expressed L1 devoid of Lex. Binding of MBP to L1 leads to proteolytic cleavage of L1 at Arg687. This proteolytic cleavage induces L1-mediated functions in vitro, such as L1-dependent neurite outgrowth and neuronal survival. Our findings describing these novel functions of MBP point to more direct roles of this protein in the pathogenesis of demyelinating and neurodegenerative diseases. L1-deficient mice (25Dahme M. Bartsch U. Martini R. Anliker B. Schachner M. Mantei N. Disruption of the mouse L1 gene leads to malformations of the nervous system.Nat. Genet. 1997; 17: 346-349Crossref PubMed Scopus (414) Google Scholar) and MBP-deficient shiverer mice (23Wolf M.K. Billings-Gagliardi S. CNS hypomyelinated mutant mice (jimpyshivererquaking): in vitro evidence for primary oligodendrocyte defects.Adv. Exp. Med. Biol. 1984; 181: 115-133Crossref PubMed Scopus (11) Google Scholar, 26Mikoshiba K. Takamatsu K. Tsukada Y. Peripheral nervous system of shiverer mutant mice: developmental change of myelin components and immunohistochemical demonstration of the absence of MBP and presence of P2 protein.Brain Res. 1983; 283: 71-79Crossref PubMed Scopus (16) Google Scholar) were kept as heterozygous breeding pairs. L1-deficient mice were maintained on a mixed genetic background (129SVJ × C57BL/6 × Black Swiss), and MBP-deficient shiverer mice were maintained on an inbred C57BL/6J background. In experiments using L1- or MBP-deficient mice, wild-type littermates were used. In all other experiments C57BL/6J mice were used. All experiments were conducted in accordance with the German and European Community laws on protection of experimental animals and were approved by the responsible committee of The State of Hamburg. L1 antibody 172-R was from HISS Diagnostics. Pan-MBP antibody (sc-13914) and dynamin I antibody (sc-12724) were purchased from Santa Cruz Biotechnology, and βIII-tubulin antibody (PRB-435P) was from Covance. The MBP antibody against the exon II-encoded domain (27Pedraza L. Fidler L. Staugaitis S.M. Colman D.R. The active transport of myelin basic protein into the nucleus suggests a regulatory role in myelination.Neuron. 1997; 18: 579-589Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) was kindly provided by David Colman (Montreal Neurological Institute and Hospital of McGill University, Montreal, Canada). Polyclonal L1 antibody, L1 antibodies 555 and 557, antibody L3 against oligomannoses, antibody L5 against Lex, and antibody HNK-1 against the HNK-1 glycan have been described (4Streit A. Yuen C.T. Loveless R.W. Lawson A.M. Finne J. Schmitz B. Feizi T. Stern C.D. The Le(x) carbohydrate sequence is recognized by antibody to L5, a functional antigen in early neural development.J. Neurochem. 1996; 66: 834-844Crossref PubMed Scopus (78) Google Scholar, 7Kleene R. Yang H. Kutsche M. Schachner M. The neural recognition molecule L1 is a sialic acid-binding lectin for CD24, which induces promotion and inhibition of neurite outgrowth.J. Biol. Chem. 2001; 276: 21656-21663Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 28Appel F. Holm J. Conscience J.F. von Bohlen und Halbach F. Faissner A. James P. Schachner M. Identification of the border between fibronectin type III homologous repeats 2 and 3 of the neural cell adhesion molecule L1 as a neurite outgrowth promoting and signal transducing domain.J. Neurobiol. 1995; 28: 297-312Crossref PubMed Scopus (70) Google Scholar, 29Heller M. von der Ohe M. Kleene R. Mohajeri M.H. Schachner M. The immunoglobulin-superfamily molecule basigin is a binding protein for oligomannosidic carbohydrates: an anti-idiotypic approach.J. Neurochem. 2003; 84: 557-565Crossref PubMed Scopus (40) Google Scholar). Carbohydrates and glycoconjugates were from Dextra Laboratories, PNGase F was from Roche Diagnostics, and α-(1–3,4)-fucosidase from QA-Bio. Synthetic peptides were from Schafer-N, and MBP purified from bovine brain was from AbD Serotec (#6420-0100). Secondary antibodies were from Dianova, and streptavidin coupled to horseradish peroxidase (HRP) was obtained from Thermo Fisher Scientific. Phage display screening of a Pre-Made T7 Select Library of normal human brain tissue (Novagen; catalogue number 70637-7) was performed as described in the manufacturer's protocol using BSA-Lex or BSA-Lea in PBS (100 μg/ml) as substrate-coated baits and 100 μg/ml antibody L5 in PBS for elution. After four rounds of panning, DNA was isolated from individual clones and sequenced. Western blot analysis, cell surface biotinylation, immunopurification of L1 from early postnatal mouse brains, and isolation of crude myelin from adult mouse brains were described (7Kleene R. Yang H. Kutsche M. Schachner M. The neural recognition molecule L1 is a sialic acid-binding lectin for CD24, which induces promotion and inhibition of neurite outgrowth.J. Biol. Chem. 2001; 276: 21656-21663Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 30Makhina T. Loers G. Schulze C. Ueberle B. Schachner M. Kleene R. Extracellular GAPDH binds to L1 and enhances neurite outgrowth.Mol. Cell. Neurosci. 2009; 41: 206-218Crossref PubMed Scopus (41) Google Scholar, 31Norton W.T. Poduslo S.E. Myelination in rat brain: method of myelin isolation.J. Neurochem. 1973; 21: 749-757Crossref PubMed Scopus (1267) Google Scholar). L1 was immunopurified from early postnatal mouse brains (7Kleene R. Yang H. Kutsche M. Schachner M. The neural recognition molecule L1 is a sialic acid-binding lectin for CD24, which induces promotion and inhibition of neurite outgrowth.J. Biol. Chem. 2001; 276: 21656-21663Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). For N-deglycosylation, brain L1 was denatured at 95 °C in 1% SDS and 20 mm sodium phosphate (pH 7.2), diluted 1:10, and incubated overnight with 0.1 units of PNGase F and 0.5% CHAPS at 37 °C. For defucosylation, it was resuspended in 250 mm sodium phosphate (pH 5.0) and incubated with 2 milliunits of α-(1–3,4)-fucosidase overnight at 37 °C. For the protease assay, 1 μg of brain L1 and 2 μg of bovine MBP were incubated overnight at 37 °C in 30 μl of PBS containing 1 mm CaCl2 and 1 mm MgCl2 without or with 15 μg of aprotinin (5.4 trypsin inhibitory units/mg). ELISA using 5 μg/ml brain L1, L1-Fc, or MBP in TBS as substrate coats, different concentrations of MBP, brain L1, or L1-Fc in PBS as soluble binding partners, and L1 antibody 555 or pan-MBP antibody, HRP-coupled secondary antibodies (Dianova), and o-phenylenediamine dihydrochloride (Thermo Fisher Scientific) for detection of binding was described (7Kleene R. Yang H. Kutsche M. Schachner M. The neural recognition molecule L1 is a sialic acid-binding lectin for CD24, which induces promotion and inhibition of neurite outgrowth.J. Biol. Chem. 2001; 276: 21656-21663Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Peak wavelength shift of reflected light was measured in a label-free binding assay using BIND Technology (SRU Biosystems), a 384-well plate with a TiO2 biosensor surface for substrate-coating, and soluble binding partners (32Loers G. Makhina T. Bork U. Dörner A. Schachner M. Kleene R. The interaction between cell adhesion molecule L1, matrix metalloproteinase 14, and adenine nucleotide translocator at the plasma membrane regulates L1-mediated neurite outgrowth of murine cerebellar neurons.J. Neurosci. 2012; 32: 3917-3930Crossref PubMed Scopus (34) Google Scholar). Dissociated murine cerebellar neurons were prepared from 6–8-day-old mice and maintained on poly-l-lysine for 24 h in serum-containing medium. For MBP surface immunostaining, pan-MBP antibody was added to live cells and incubated for 30 min at 4 °C. For double immunostaining of MBP and L1, cells were incubated with serum-free medium for 4 h, with L1 antibody 557 or 555 for 30 min, and with pan-MBP antibody for 20 min at 37 °C. After fixation with 4% paraformaldehyde for 30 min and blocking with 2% BSA in PBS for 1 h at room temperature, cells were incubated with Cy2- or Cy3-conjugated secondary antibodies in PBS for 2 h at room temperature. For double immunostaining of MBP and βIII-tubulin, fixed cells were incubated in 2% BSA, 0.3% Triton X-100 in PBS for 1 h at room temperature followed by incubation with pan-MBP and βIII-tubulin antibodies in PBS for 2 h at room temperature and with Cy2- or Cy3-conjugated secondary antibodies in PBS for 1 h at room temperature. Coverslips were embedded in Aqua-Poly/Mount (Polysciences) or Roti®-Mount FluorCare DAPI (Carl Roth), and confocal images were taken with an Olympus Fluoview FV1000 confocal laser scanning microscope. Preparation and analysis of cerebellar neurons from 6–8-day-old mice were described, and cells were cultured in chemically defined serum-free medium (33Loers G. Chen S. Grumet M. Schachner M. Signal transduction pathways implicated in neural recognition molecule L1 triggered neuroprotection and neuritogenesis.J. Neurochem. 2005; 92: 1463-1476Crossref PubMed Scopus (89) Google Scholar). Neurite outgrowth was quantified 24 h after seeding by measuring total neurite length of at least 100 neurons per condition using an AxioVision system 4.6 (Carl Zeiss). Glycans (10 μm), Lex peptide (16Katagihallimath N. Mehanna A. Guseva D. Kleene R. Schachner M. Identification and validation of a Lewis x glycomimetic peptide.Eur. J. Cell Biol. 2010; 89: 77-86Crossref PubMed Scopus (12) Google Scholar) and scrambled peptide (100 μg/ml), glycan-specific antibodies (10 μg/ml), and aprotinin (0.03 trypsin inhibitory units/ml) were added 1 h (neurite outgrowth) or 15 h (cell survival) after cell seeding. For induction of cell death, hydrogen peroxide (10 μm) or sodium glutamate (100 μm) was added 16 h after plating. After an additional 24 h, cells were treated with 1 μg/ml calcein AM (Molecular Probes) and 1 μg/ml propidium iodide (Sigma) for 1 h at 37 °C. Viability of cells was assessed by counting the numbers of calcein versus propidium iodide-positive cells. Twelve randomly chosen areas of a microscopic field (20× magnification) from three wells per treatment and experiment were counted. Site-directed mutagenesis and transfection of HEK293 cells were described (9Lutz D. Wolters-Eisfeld G. Joshi G. Djogo N. Jakovcevski I. Schachner M. Kleene R. Generation and nuclear translocation of a sumoylated transmembrane fragment of the cell adhesion molecule L1.J. Biol. Chem. 2012; 287: 17161-17175Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). For mutation of R687A (L1R/A) or of F686L/R687A (L1FR/LA), the following primers were used: fwR/A (5′-CCC TAT GTC CAC TAC ACC TT T GCG GTC ACT GCC ATT AAC AAA TAT-3′) and revR/A (5′-GTT AAT GGC AGT GAC CGC AAA GGT GTA GTG GAC ATA GGG GGA CAG-3′); fwFR/LA (5′-CCC TAT GTC CAC TAC ACC CTT GCG GTC ACT GCC ATT AAC AAA TAT-3′) and revFR/LA (5′-GTT AAT GGC AGT GAC CGC AAG GGT GTA GTG GAC ATA GGG GGA CAG-3′). For site-directed mutagenesis of Ser139 and Ser176 in MBP to Ala139 and Ala176, the following primers and a vector coding for RFP N-terminally fused to the murine 21.5-kDa MBP (34Smith G.S. Paez P.M. Spreuer V. Campagnoni C.W. Boggs J.M. Campagnoni A.T. Harauz G. Classical 18.5-and 21.5-kDa isoforms of myelin basic protein inhibit calcium influx into oligodendroglial cells, in contrast to golli isoforms.J. Neurosci. Res. 2011; 89: 467-480Crossref PubMed Scopus (30) Google Scholar) were used: fw139 (5′-GGC CTG TCC CTC AGC AGA TTT GCC TGG GGG GCC GAG GGG CAG AAG CCA G-3′) and rev139 (5′-CCC CTC GGC CCC CCA GGC AAA TCT GCT GAG GGA CAG GCC TCT CCC-3′); fw179 (5′-TAC GAC GCC CAG GCC ACG CTT GCC AAA ATC TTT AAG CTG GGA GGA-3′) and rev179 (5′-CAG CTT AAA GAT TTT GGC AAG CGT GCC CTG GGC GTC GTA GGC CCC-3′). For subcloning of RFP-tagged wild-type and mutated MBP into pAAV-MCS vector (Cellbiolabs; CMV promoter) via an SalI restriction site using the InFusion Cloning kit (Clontech), PCR amplification with Phusion Polymerase (New England Biolabs) and the following primers was performed: fw (5′-ATCCTCTAGAGTCGAC ATG GTG AGC AAG GGC GAG GAG-3′) and rev (5′-GCTTCTGCAGGTCGAC TCA GCG TCT CGC CAT GGG-3). AAV1 pseudotyped vectors were generated by co-transfection of HEK293-AAV cells (Cellbiolabs) with the particular pAAV transfer plasmid (Cellbiolabs) and the AAV packaging plasmid pDP1rs (a kind gift from Jürgen Kleinschmidt, DKFZ Heidelberg, Germany), which provides the AAV2 rep and AAV1 cap genes and adenoviral helper functions (35Grimm D. Kay M.A. Kleinschmidt J.A. Helper virus-free, optically controllable, and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6.Mol. Ther. 2003; 7: 839-850Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Generation of recombinant AAV1 particles was carried out as described previously (36Grieger J.C. Choi V.W. Samulski R.J. Production and characterization of adeno-associated viral vectors.Nat. Protoc. 2006; 1: 1412-1428Crossref PubMed Scopus (398) Google Scholar) with some modifications. Briefly, 1 × 107 HEK293-AAV cells were transfected with 17.5 μg of pDP1rs and 4.5 μg of pAAV plasmid per plate complexed with Max-polyethyleneimine (PEI, Polysciences) at a PEI:DNA ratio of 3:1 (37Fukumoto Y. Obata Y. Ishibashi K. Tamura N. Kikuchi I. Aoyama K. Hattori Y. Tsuda K. Nakayama Y. Yamaguchi N. Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine.Cytotechnology. 2010; 62: 73-82Crossref PubMed Scopus (88) Google Scholar). After 72 h cells were harvested, washed 3 times with PBS, and resuspended in 5 ml of lysis buffer (50 mm Tris base, 150 m NaCl, 5 mm MgCl2 (pH 8.5)). After three freeze-thaw cycles, lysates were incubated with 250 units/ml Benzonase (Merck) for 1 h at 37 °C. Cell debris was pelleted, and vector containing lysates were purified using iodixanol step gradients and ultrafiltration (Amicon: Ultra Cartridges, 50 Mr cutoff). The genomic titers of DNase-resistant recombinant AAV particles were determined after alkaline treatment and subsequent neutralization by quantitative PCR (50 °C for 2 min and 95 °C for 10 min followed by 35 cycles of 95 °C for 15 s and 60 °C for 60 s) using the SYBR Green qPCR Master MIX 2 (ThermoScientific), CMV promoter sequences (5′-GGCGGAGTTGTTACGACAT-3′ and 5′-GGGACTTTCCTACTTGGCA-3′), and an ABI PRISM® 7900HT cycler (Applied Biosystems). pAAV-MCS plasmid was used as a copy number standard, and calculations were done using the SDS 2.4 software (Applied Biosystems). For viral transduction, AAV1 carrying RFP-tagged wild-type and mutant MBP were incubated at a 1000-fold multiplicity of infection with MBP-deficient cerebellar neurons for 24 h in culture medium (Neurobasal A supplemented with 10% fetal calf serum, 1 mm sodium pyruvate, 2 mm l-glutamine, 4 nm l-tyrosine, 1× B27 supplement, 10 μg/ml BSA, 100 μg/ml transferrin, 10 μg/ml insulin, 10 ng/ml selenium, 50 units/ml penicillin, and 50 units/ml streptomycin). The templates for the synthesis of cRNA hybridization probes were amplified by PCR using the vector coding for murine 21.5-kDa MBP (34Smith G.S. Paez P.M. Spreuer V. Campagnoni C.W. Boggs J.M. Campagnoni A.T. Harauz G. Classical 18.5-and 21.5-kDa isoforms of myelin basic protein inhibit calcium influx into oligodendroglial cells, in contrast to golli isoforms.J. Neurosci. Res. 2011; 89: 467-480Crossref PubMed Scopus (30) Google Scholar), the forward primers 5′-TAATACGACTCACTATAGGGAGA atg gca tca cag aag aga ccc tc-3′ or 5′-TAATACGACTCACTATAGGGAGA gtg aca cct cga aca cca cct cc-3′, both containing the T7 promoter (capital letters), and the reverse primers 5′-tca gcg tct cgc cat ggg aga tc-3′ or 5′-cag ctt aaa gat ttt gga aag cgt gcc-3′. For the preparation of the fluorescently labeled probes the FISH TagTM RNA kit (Invitrogen) and Alexa Fluor® 488 dye were used, and hybridization was done as suggested in the manufacturer's manual. After exposure of the uterine horns, AAV1 carrying wild-type MBP or mutated MBP (1 μl; 4.5 × 105 virus particles) were in utero-injected into the brain ventricle system of 13.5-day-old shiverer embryos. On postnatal day 5.5, cerebellar explant cultures were prepared and analyzed as described (10Lutz D. Wolters-Eisfeld G. Schachner M. Kleene R. Cathepsin E generates a sumoylated intracellular fragment of the cell adhesion molecule L1 to promote neuronal and Schwann cell migration as well as myelination.J. Neurochem. 2014; 128: 713-724Crossref PubMed Scopus (28) Google Scholar). Briefly, explants were maintained on Matrigel (BD Biosciences) diluted 1:3 in serum-free medium for 1 h followed by incubation in serum-free medium, fixation, and staining. Total neurite lengths per explant and the number of migrating cells for 10 explants per gro" @default.
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• W2034336725 date "2014-05-01" @default.
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• W2034336725 title "Myelin Basic Protein Cleaves Cell Adhesion Molecule L1 and Promotes Neuritogenesis and Cell Survival" @default.
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• W2034336725 doi "https://doi.org/10.1074/jbc.m113.530238" @default.
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