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O-Glycosylation of the Tail Domain of Neurofilament Protein M in Human Neurons and in Spinal Cord Tissue of a Rat Model of Amyotrophic Lateral Sclerosis (ALS) *

O-Glycosylation of the Tail Domain of Neurofilament Protein M in Human Neurons and in Spinal Cord... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 36, Issue of September 9, pp. 31648–31658, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. O-Glycosylation of the Tail Domain of Neurofilament Protein M in Human Neurons and in Spinal Cord Tissue of a Rat Model of Amyotrophic Lateral Sclerosis (ALS)* Received for publication, April 21, 2005, and in revised form, July 8, 2005 Published, JBC Papers in Press, July 8, 2005, DOI 10.1074/jbc.M504395200 ¶  ¶ Nina Lu¨ demann‡§, Albrecht Clement , Volkmar H. Hans , Julia Leschik‡, Christian Behl , and Roland Brandt‡** From the ‡Department of Neurobiology, University of Osnabru¨ck, Barbarastrasse 11, D-49076 Osnabru¨ck, the §Department of Neurobiology, IZN, University of Heidelberg, INF 345, 69120 Heidelberg, the Institute for Physiological Chemistry and Pathobiochemistry, Johannes-Gutenberg-University, Duesbergweg 6, 55099 Mainz, and the Institute of Neuropathology, University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany components in large-diameter myelinated axons. Mammalian Mammalian neurofilaments (NFs) are modified by post-translational modifications that are thought to reg- NFs consist of three major subunits of 200, 160, and 70 kDa, ulate NF assembly and organization. Whereas phospho- termed NF-H, NF-M, and NF-L, respectively (1). Structurally, rylation has been intensely studied, the role of another the NFs contain a N-terminal head and a helical rod domain common modification, the attachment of O-linked N- that are required for filament assembly (2), and a C-terminal acetylglucosamine (GlcNAc) to individual serine and tail domain. NFs are the intrinsic determinant of radial growth threonine residues, is hardly understood. We generated of axons, which is most probably mediated by the tail domains a novel monoclonal antibody that specifically recog- of NF-M and NF-H. Whereas genetic deletion of NF-H or its tail nizes an O-glycosylated epitope in the tail domain of domain has relative little effect on radial growth (3), the tail of NF-M and allows determination of the glycosylation NF-M has an important role in the formation of cross-links state at this residue. The antibody displays strong spe- between NFs and modulation of axonal caliber (4). Misaccumu- cies preference for human NF-M, shows some reactivity lation of NFs is a frequent hallmark of several neurodegenera- with rat but not with mouse or bovine NF-M. By immu- tive diseases including amyotrophic lateral sclerosis (ALS), nohistochemistry and Western blot analysis of biopsy- spinal muscular atrophy, and Alzheimer disease (5). derived human temporal lobe tissue we show that im- NFs are among the most highly phosphorylated neuronal munoreactivity is highly enriched in axons parallel to proteins. Second messenger-dependent protein kinases such as hyperphosphorylated NFs. Treatment of cultured neu- protein kinase C and cyclic AMP-dependent protein kinase rons with the GlcNAcase inhibitor PUGNAc causes a appear to be the principal kinases targeting the head domain. 40% increase in immunoreactivity within 1 h, which is The tail domain is a preferred substrate for second messenger- completely reversible and parallels the total increase in independent kinases (6), e.g. casein kinase I and II (7), cdk5 (8), cellular O-GlcNAc modification. Treatment with the mito- and mitogen-activated protein kinases (ERK1/2) (9). Phospho- gen-activated protein kinase kinase inhibitor PD-98059 leads to a similar increase in immunoreactivity. In spinal rylation of the head domain appears to regulate the assembly of cord tissue of a transgenic rat model for amyotrophic NFs (10, 11), whereas tail domain phosphorylation may affect lateral sclerosis, immunoreactivity is strongly de- their ability to control the axon caliber (12). Normally, tail creased compared with wild-type animals while phos- domain phosphorylation appears to be restricted to the axon, phorylation is increased. The data suggest that hyper- but in ALS hyperphosphorylation of this domain occurs also in phosphorylation and tail domain O-glycosylation of NFs perikarya (13). are synchronously regulated in axons of human neurons Attachment of O-linked N-acetylglucosamine (GlcNAc) to in- in situ and that O-glycosylation of NF-M is highly dy- dividual serine and threonine residues is a common post-trans- namic and closely interweaved with phosphorylation lational modification of many nuclear and cytoskeletal proteins cascades and may have a pathophysiological role. (14–16). The enzyme catalyzing O-GlcNAc modification (O- GlcNAc transferase) is essential for cell viability in mammals (17). O-Glycosylation by GlcNAc appears to be as dynamic as Neurofilaments (NFs) are the most abundant structural protein phosphorylation and is often reciprocal to phosphoryl- ation at the same or adjacent sites (18). Also NFs are modified by O-linked GlcNAc with several identified sites being located * This work was supported by Deutsche Forschungsgemeinschaft in the head region of NF-L and NF-M (19). It is not known how Grant SFB 488 project B1 and the Ministry for Science and Culture of Lower Saxony. The costs of publication of this article were defrayed in O-glycosylation of NFs is regulated, how it relates to the phos- part by the payment of page charges. This article must therefore be phorylation of NFs, and where O-glycosylated NFs are hereby marked “advertisement” in accordance with 18 U.S.C. Section distributed. 1734 solely to indicate this fact. Here we describe a novel monoclonal antibody (NL6) that ** To whom correspondence should be addressed. Tel.: 49-541- 9692338; Fax: 49-541-9692354; E-mail: [email protected]. specifically recognizes an O-glycosylated epitope in the projec- de. tion domain of NF-M. The antibody displayed a strong species The abbreviations used are: NF, neurofilament; ALS, amyotrophic preference for human NF-M. NL6-positive NF-M was enriched lateral sclerosis; ERK, extracellular signal-regulated kinase; MEK, mi- togen-activated protein kinase kinase; PUGNAc, O-(2-acetamido-2-de- oxy-D-glucopyranosylidene)amino-N-phenylcarbamate; GFP, green flu- orescent protein; PBS, phosphate-buffered saline; FCS, fetal calf serum; ride; PIPES, 1,4-piperazinediethanesulfonic acid; MES, 4-morpho- MEM, minimal essential medium; PMSF, phenylmethylsulfonyl fluo- lineethanesulfonic acid. 31648 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. O-Glycosylation of NF-M 31649 was prepared by changing A at position 2248 against G. Mutated in the axons of human neurons in situ. Pharmacological ma- constructs were verified by dideoxy sequencing using T7 Sequenase nipulations demonstrated that O-glycosylation as detected (Amersham Biosciences). with the NL6 antibody is highly dynamic, reversible, and in- Cell Culture and Transfection—NT2 cells were grown in serum- versely regulated to the activity of mitogen-activated protein DMEM (Dulbecco’s modified Eagle’s medium supplemented with 10% kinase kinase (MEK). In a rat model for ALS, NL6 immunore- FCS, 5% horse serum (HS), 292 g/ml glutamine, 50 units/ml penicillin, activity was strongly decreased compared with wild-type and 50 g/ml streptomycin) and differentiated essentially as described previously (24). NT2-N neurons were obtained after 5 weeks differen- animals. tiation with retinoic acid and 2 weeks treatment with mitotic inhibitors EXPERIMENTAL PROCEDURES 2 as described previously (20), plated at 4000 cells/cm onto collagen- Materials—Chemicals were purchased from Sigma, cell culture me- coated coverslips and cultured for 3 days in serum/Dulbecco’s modified dia and supplements from Life Technologies, culture flasks and dishes Eagle’s medium. After this time, almost all neurons had established from Nunc (Roskilde, Denmark) unless otherwise stated. Collagen was polarity as judged from axon-specific tau staining. SK-N-BE(2) cells (25) prepared from rat tails by acetic acid extraction (20). O-(2-Acetamido- were grown in serum/Opti-MEM (Opti-MEM supplemented with 5% 2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) iron enriched FCS, 292 g/ml glutamine, 50 units/ml penicillin, and 50 was purchased from Carbogen (Aarau, Switzerland), the MEK inhibitor g/ml streptomycin) and differentiated with 10 M retinoic acid for 2 PD-98059 was from New England Biolabs (Beverly, MA). Mouse mono- weeks. 3T3 cells (26) were cultured in serum/Dulbecco’s modified Ea- clonal M20 antibody against a phosphorylation-independent site on NF-M gle’s medium supplemented with 10% FCS, 584 g/ml glutamine, 50 (21) was a kind gift from Dr. B. M. Riederer (Lausanne, Switzerland). units/ml penicillin, and 50 g/ml streptomycin, HeLa cells (27) in se- Antibody SMI31 against phosphorylated NF-M and NF-H was pur- rum/minimal essential Earle’s medium (MEM Earle’s Medium supple- chased from Sternberger Monoclonals Inc. (Baltimore, MD), mouse mented with 10% FCS, 292 g/ml glutamine, 50 units/ml penicillin, and monoclonal antibody against O-glycosylated GlcNAc (CTD110.6), which 50 g/ml streptomycin) and Neuro2A cells (28) in serum/MEM (mini- detects GlcNAc independent from the protein backbone (35), was a kind mal essential medium with Earle’s salts supplemented with 10% FCS, gift of Covance (Cumberland, VA), rabbit polyclonal anti-GFP was 292 g/ml glutamine and nonessential amino acids for MEM). In some obtained from Molecular Probes (Leiden, Netherlands), mouse mono- experiments cells were treated with PUGNAc or the MEK inhibitor clonal antibody against the rod domain of NF-M (RMO44) from Stress- PD-98059 for1horthe times indicated prior to analysis. Gene (Victoria, BC, Canada), mouse monoclonal antibody against One day before transfections, cells were plated at a density of 3  10 MAP2 from Chemicon (Temecula, CA), mouse monoclonal 2F11 react- cells/coverslip on collagen-coated coverslips and transfected with Fu- ing with the phosphorylated form of NF-L from Dako (Glostrup, GENE 6 (Roche Molecular Biochemicals) at a ratio of 1 g of DNA to 4 Denmark), rabbit polyclonal antibodies against phospho-p44/42 and l of FuGENE per coverslip. Cells were processed for immunocytochem- total p44/42 MAP kinase from Cell Signaling (Beverly, MA), and mouse istry 48 h after transfection. monoclonal antibody against actin from Oncogene (San Diego, CA). Preparation of Cellular and Tissue Lysates and Cell Fractionation— Cy3-conjugated donkey anti-mouse antibody, fluorescein isothiocya- For preparation of lysates, cells were washed twice with PBS, scraped nate-conjugated goat anti-rabbit and horseradish peroxidase-conju- off into ice-cold PBS, and collected for 5 min at 300  g. Tissue was gated donkey anti-mouse and goat anti-rabbit antibody were obtained shock frozen in liquid nitrogen, cut with a cryostat in sections to allow from Dianova (Hamburg, Germany), Alexa 488-conjugated goat anti- a better penetration of the lysis buffer, and collected in a test tube. mouse IgG1 and Alexa 543-conjugated goat anti-mouse IgG2A from Pellet and tissue slices were resuspended in RIPA buffer (50 mM Tris/ Molecular Probes (Leiden, Netherlands). HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% Preparation of the NL6 Antibody—Mice were immunized with a deoxycholate, 0.1% SDS) containing protease and phosphatase inhibi- fraction enriched for components of the human neuronal cytoskeleton. tors (1 mM PMSF, 10 g/ml each of leupeptin and pepstatin, 1 mM The fraction was prepared from NT2-N neurons and SK-N-BE(2) neu- sodium orthovanadate, 20 mM NaF, 1 mM sodium pyrophosphate), roblastoma cells as described previously (22). The immunization was incubated for 30 min at 4 °C, centrifuged for 10 min at 13,000  g, and performed with the PBS-insoluble material that was solubilized with 8 the supernatant (lysate) collected. M urea and diluted to a final concentration of 0.85 M urea in PBS. For detergent extraction, SK-N-BE(2) cells from one 15-cm TC dish BALB/c mice were injected 4 times intraperitonally with 50 gofthe were washed twice with PBS, scraped off into extraction buffer (10 mM protein fraction at intervals of 3 to 4 weeks. Four days after the final PIPES/KOH, pH 6.8, 50 mM KCl, 3 mM MgCl ,10mM EGTA), and injection, spleens were removed and the spleenocytes were fused with collected for 5 min at 300  g with all steps at 4 °C. Cells were the myeloma line X63Ag8.653 as described previously (23). The result- resuspended in 1 ml of extraction buffer containing 1%(v/v) Triton ing hybridoma clones were screened for immunoreaction against cy- X-100 and protease inhibitors and incubated for 10 min. 250 lofthe toskeletal proteins. Positive clones were recloned twice to obtain stable solution were transferred on a 0.85 M sucrose cushion and centrifuged antibody producing hybridoma lines and cultured in RPMI 1640, 10% for 30 min at 72,000  g. The pellet was resuspended in 100 lof fetal calf serum (FCS), 292 g/ml glutamine. Antibodies were concen- extraction buffer. trated by ammonium sulfate precipitation of the hybridoma superna- For reassembly of NFs, spinal cords from several adult rats were tant, resuspended in PBS, dialyzed against PBS, and stored in the prepared and frozen in liquid nitrogen. An equal amount (w/v) of dis- presence of 50% (v/v) glycerol at 20 °C. assembly buffer (50 mM MES, pH 6.8, 0.5 mM MgCl ,1mM EGTA, 1 mM For isotype determination, the “isostrip mouse monoclonal antibody PMSF) was added, the mixture was homogenized on ice using an isotyping kit” from Roche Diagnostics was used according to the man- electrical blade homogenizer and centrifuged for 30 min at 72,000  g. ufacturer’s protocol. NL6 is a IgG2a antibody with a -chain. Because The supernatant was collected, cleared by centrifugation, and an equal most of the commercially available mouse antibodies belong to the volume of assembly buffer (50 mM MES, pH 6.8, 0.5 mM MgCl ,1mM subtype G1, double fluorescence analysis using isotype-specific second- EGTA, 8 M glycerol) was added. The mixture was incubated for 15 min ary antibodies becomes possible. Characterization of the antibody by at 4 °C and centrifuged for 45 min at 75,000  g. The pellet was immunocytochemistry revealed that it was sensitive to glutaraldehyde resuspended in 100 l of assembly buffer. fixation and that the signal was completely lost after fixation for 10 min Tissue extracts from different species (human, bovine, rat, and with 0.3%(v/v) glutaraldehyde. mouse) were prepared as previously described (29). Tissues were ho- Construction of Expression Vectors—Eukaryotic expression plasmids mogenized on ice in 50 mM Tris, pH 7.5, 0.5 mM EDTA, 1 mM of the for hNF-M, mNF-M, and deletion constructs of hNF-M with the C- protease inhibitors PMSF, aprotinin, and chymostatin. An equal vol- terminal fused GFP epitope were constructed using the CT-GFP fusion ume of extraction buffer was added (50 mM Tris, pH 7.5, 150 mM NaCl, TOPO®TA expression kit (Invitrogen, Karlsruhe, Germany). To con- 1% Nonidet P-40, 1% sodium deoxycholate, 2% SDS), the homogenates struct mutated hNF-M, respective codons were changed using the were sonicated for 20 s, boiled for 10 min, centrifuged for 5 min at QuikChange® site-directed mutagenesis kit (Stratagene, Amsterdam, 16,000  g, and the supernatants saved. Netherlands). HNF-M(385–472) was prepared by deleting the respec- Immunohistochemistry, Immunocytochemistry, and Immunoelectron tive fragment with the restriction enzyme Eco0109I. Constructs with Microscopy—Human temporal lobe tissue was obtained neurosurgically N-terminal-fused GFP were prepared in EGFP C3 Vector (Clontech, from five patients suffering from focal epilepsy. Material was kept on ice, Palo Alto, CA). HNF-M(891) was produced using BCUI385, hNF- and gray and white matter were dissected apart. Tissue samples were M(552), with BamHI. To prepare hNF-M(603), an Eco32I restriction transferred to liquid nitrogen and kept at 80 °C. For immunohistochem- site was inserted by changing GT at positions 1813/1814 to AT followed istry, samples were immediately fixed in 4% formalin for 24 h after by the respective digest. For hNF-M(789), a Eco32I restriction site was surgical removal and routinely paraffin-embedded. Deparaffinized 4-m inserted by changing AA at positions 2369/2370 to TG. HNF-M(T747A) thick sections were blocked and incubated with primary antibodies over- 31650 O-Glycosylation of NF-M night at 4 °C. Delay of formalin fixation for a few hours, or incubation for to4mM. Samples were centrifuged for 20 min at 4 °C and 12,800  g 1 h at room temperature resulted in substantial loss of staining intensity and equal amounts of pellet and supernatant were analyzed by immu- in 14 samples of surgically resected hippocampi (data not shown). Biotin- noblot. Comparison between experimental groups was based on paired labeled secondary antibodies were visualized using peroxidase-conjugated Student’s t test (*, p  0.05; **, p  0.01; ***, p  0.001). streptavidin with diaminobenzidine as the substrate. All solutions for the visualization of the antigens were contained in a commercially available RESULTS TM detection kit (DAKO ChemMate , DakoCytomation GmbH, Hamburg, NL6, A Novel Monoclonal Antibody against a Human Neu- Germany). Staining procedures were performed on an autostainer accord- ronal Cytoskeleton Fraction, Recognizes an O-Glycosylated ing to routine protocols provided by the manufacturer (DAKO TechMate TM 500 ). As negative controls, primary antibodies were omitted. The study Epitope of Human NF-M—In an immunological approach to was approved by the local ethics committee, and informed consent was obtain novel antibodies against components of the human neu- received from all tissue donors. ronal cytoskeleton, a PBS-insoluble cytoskeletal fraction of hu- Cultured cells were fixed for 20 min with 4% paraformaldehyde in man model neurons was prepared and solubilized with urea PBS (10 mM phosphate buffer, pH 7.4, 2.7 mM KCl, and 137 mM NaCl) (see “Experimental Procedures”). The material was used to containing 4% (w/v) sucrose at room temperature, washed with PBS, immunize mice and produce hybridoma lines. One of the anti- incubated for 20 min with 0.1 M glycine in PBS, and permeabilized with 0.2% (v/v) Triton X-100 for 5 min. Staining was performed as described bodies (NL6) detected a protein with an apparent molecular earlier (30) in PBS containing 1% (w/v) bovine serum albumin using mass of 160 kDa. The antigen was highly enriched in the appropriate antibody combinations. 5 g/ml 4,6-Diamidino-2-phenylin- Triton X-100-insoluble fraction of homogenates from differen- dole was included in the mixture of the secondary antibodies to detect tiated human neuroblastoma (SK-N-BE(2)) cells (Fig. 1A), in- the nuclei. Cells were analyzed using a Leica DMIRB fluorescence dicating that NL6 recognizes an intermediate filament protein. microscope or a Leica TCS SP2 True confocal scanner (Leica, Solms, The NL6 antigen was up-regulated during differentiation of Germany). For immunoelectron microscopy, reassembled rat NFs were trans- human neuroblastoma cell lines parallel to NF-M (SK-N-BE(2), ferred on pioloform-coated and glow-discharged copper grids (400 SH-SY5Y cells; Fig. 1B, left panel). To determine whether the mesh), fixed for 20 min with 4% paraformaldehyde in PBS containing antigen of NL6 is also expressed in neuronal precursor cells 4% (w/v) sucrose, incubated for 20 min with 0.1 M glycine in PBS, and and when it is produced during differentiation, the NT2/NT2-N labeled with primary antibodies. After five washes with PBS containing system was employed. NT2 cells are a human teratocarcinoma 1% (w/v) bovine serum albumin and 0.1% (v/v) Tween 20, grids were cell line with characteristics of central nervous system neuro- incubated with polyclonal rabbit anti-mouse antibody (ICN Biomedi- cals, Aurora, OH), washed five times with PBS containing 1% (w/v) nal progenitor cells that can be differentiated in vitro to yield bovine serum albumin and 0.1% (v/v) Tween 20, and incubated with terminally differentiated, polar, and postmitotic neurons (24). protein A coupled to 15 nm gold. Grids were washed with PBS, postfixed The NL6 antigen was present in lysates of NT2-N cells. In for 30 min with 2% (v/v) glutaraldehyde in PBS, washed 3  10 min contrast, no signal was observed in NT2 cell lysates (Fig. 1B, with water, and contrasted with uranyl acetate. Electron microscopy right panel), indicating that the NL6 antigen is expressed only was performed on a Zeiss 10CR electron microscope (Zeiss, Oberkochen, Germany). in differentiated neurons. The expression pattern was similar Other Methods—Protein concentrations were determined using the albeit not identical to total NF-M as detected using a previously BCA microplate assay (Pierce). SDS-polyacrylamide gel electrophoresis described antibody recognizing NF-M independent of post- and transfer onto polyvinylidene difluoride were performed as de- translational modifications (M20 (21, 33)). Taken together the scribed previously (31) using the benchmark protein molecular weight data strongly suggest that NL6 detects a subpopulation of marker (Life Technologies). For dot-blot analysis, samples were diluted NF-M. with 2 volumes of dot-blot buffer (30% methanol, 0.5% deoxycholate, 9 g/liter NaCl, 1.21 g/liter Tris, pH 7.4) and immobilized on Immobilon It is known that in neurons NF-M is highly post-translation- NC pure (Millipore Corp., Bedford, MA) using a dot-blot hybridization ally modified (for a review, see Ref. 1). To test whether NL6 chamber (Loxo, Dossenheim, Germany). In some cases, blots were detects a phosphorylated epitope, cellular homogenates were stained for total protein using Ponceau S or Coomassie Brilliant Blue separated by SDS-PAGE, blotted onto polyvinylidene difluo- according to standard procedures (32). Immunodetection used en- ride, and treated with alkaline phosphatase. Phosphatase hanced chemiluminescence (ECL) (Amersham Biosciences) and was treatment resulted in a strong reduction of the immunoreac- performed according to the manufacturer’s protocol. Image acquisition employed a chemo CAM-System (Intas Science Imaging Instruments, tivity with an antibody that detects phosphorylated epitopes in Go¨ttingen, Germany) or Fuji LAS3000-System (Raytest, Straubenhart, NF-H and NF-M (SMI31) but caused no reduction in M20 or Germany). Quantification of the blots was carried out with NIH Image NL6 immunoreactivity (Fig. 1C, arrowheads) indicating that 1.61/ppc (rsb.info.nih.gov/nih-image/index.html). binding of NL6 does not depend on phosphorylation of the Phosphatase treatment was performed by incubating the membrane epitope. To test whether NL6 detects an O-glycosylated for 24 h at 37 °C with 20 units/ml alkaline phosphatase (from calf epitope, blots from cellular lysates were treated with 55 mM intestinal mucosa) in 100 mM Tris/HCl, pH 8.0, 1 mM PMSF. Phos- phatase-treated blots were subjected for immunodetection in parallel NaOH, which is known to remove -glycosidically linked Glc- with control blots that have been washed with buffer only. NAc from serine or threonine residues by a reaction called For chemical cleavage of -O-glycosidic linkages, blots were washed -elimination (34). The treatment resulted in a complete loss of for 3  10 min with water, incubated for 16 h with 55 mM NaOH at immunoreactivity with an antibody that recognizes O-linked 40 °C, and washed again for 3  10 min with water. Blots were sub- GlcNAc independent of protein determinants (CTD110.6 (35); jected for immunodetection in parallel with control blots that have been Fig. 1D, top left panel). Blots from fractions enriched for NFs incubated with water only. Enzymatic digestion of -glycosidic linkages was performed with Triton-extracted fractions of BE2 cells. 20 gof were treated similarly and probed with NL6 and M20 antibody. protein were incubated with 0.5 unit of -N-acetylglucosaminidase Treatment with NaOH resulted in an almost complete loss of (from bovine kidney) in a total volume of 50 lin10mM sodium NL6 immunoreactivity, whereas M20 staining remained un- cacodylate, pH 5.0, 1 mM PMSF, and 10 g/ml each of leupeptin and changed (Fig. 1D, top right panel). To check whether GlcNAc at pepstatin for 16 h at 37 °C. The reaction was stopped by addition of 12.5 the NL6 epitope can also be removed enzymatically, a fraction lof5 Laemmli buffer. Control samples were treated identical, but in the absence of -N-acetylglucosaminidase. 50% of the samples were enriched for neurofilaments was incubated with -N-acetylglu- subjected for immunodetection and processed in parallel for NL6 and cosaminidase from bovine kidney. The treatment resulted in a M20 staining. strong reduction in NL6 immunoreactivity compared with a Partial chymotryptic digest of intact NFs was performed with Triton- control sample, whereas M20 staining remained unchanged extracted fractions of BE2 cells. 10 g of protein were incubated with 50 (Fig. 1D, bottom left panel). Furthermore, inclusion of 1 M ng of chymotrypsin in a total volume of 18 l in digestion buffer (100 mM GlcNAc in the primary antibody reaction abrogated binding of Tris/HCl, pH 7.8, 10 mM CaCl ,10 g/ml each of leupeptin and pepsta- tin). The reaction was stopped after different times by addition of PMSF the NL6 antibody (Fig. 1D, bottom right panel). Taken together O-Glycosylation of NF-M 31651 the data indicate that NL6 recognizes an O-glycosylated epitope of NF-M. To test whether NF-M remains O-glycosylated at the NL6 epitope during disassembly and reassembly, NFs were recon- stituted from high-speed supernatants of rat spinal cord homo- genates and analyzed by immunoelectron microscopy. NL6 im- munoreactivity was evenly distributed on reconstituted NFs similar to M20 reactivity (Fig. 1E). The data indicate that O-glycosylated NF-M is assembly-competent and uniformly in- corporates into NFs. The NL6 Epitope Is Localized in the Tail Domain of NF-M— NF-M consists of a central -helical rod domain that forms the filament backbone, a small globular head domain, and an ex- tended carboxyl-terminal tail domain that protrudes from the backbone (1, 36). To determine in which of these regions the NL6 epitope is localized, a partial chymotryptic digest of intact NFs was performed. It has previously been shown that, under these conditions, at first the tail domain is cleaved off from the remainder of the protein close to the filament backbone, which can then be separated by centrifugation (37). After short treat- ment with chymotrypsin (2 min), NL6 reactivity was exclu- sively present in the supernatant at molecular masses of about 100 and 110 kDa (Fig. 2A), which closely corresponds to the apparent size of the tail domain as reported earlier (37). After longer incubation periods (8 min) NL6 immunoreactivity dis- appeared because of further digestion of the protein (data not shown). The data indicates that the NL6 epitope is localized in the carboxyl-terminal tail domain of NF-M. As a tool to determine the position of the NL6 epitope, a fusion protein between human NF-M and GFP was constructed and transfected into the human neuronal precursor cell line NT2. As shown above (Fig. 1B) and confirmed by immunocyto- chemistry (data not shown), these cells do not express detect- FIG.1. Generation and identification of a novel monoclonal able amounts of endogenous NF-M. Individual transfected cells antibody (NL6) against an O-glycosylated epitope of NF-M. A, showed a filamentous pattern of GFP fluorescence (Fig. 3B, top immunoblot of subcellular fractions of differentiated SK-N-BE(2) cells left) indicative of the incorporation of the exogenous construct after detergent extraction with Triton X-100 (TX-100). The antigen has an apparent molecular mass of 160 kDa and is highly enriched in the into the cellular intermediate filaments. NL6 staining colocal- Triton-insoluble fraction (pellet, P), indicating that the NL6 antibody ized with the GFP fluorescence (Fig. 3B, top right) indicating recognizes an intermediate filament protein. S, supernatant. B, expres- that exogenously expressed NF-M becomes O-glycosylated at sion of the NL6 epitope during in vitro differentiation of human neuro- the NL6 epitope. To map the region of NL6 immunoreactivity, nal cells. The antigen is up-regulated in differentiated SK-N-BE(2) cells a panel of deletion mutants and mutated constructs were pre- (BE2), SH-SY5Y cells, and NT2-N cells similar to NF-M as detected using an antibody (M20) that recognizes NF-M independent of post- pared and tested in similar transfection assays. The only translational modifications. C, effect of phosphatase treatment on im- known glycosylation site in the tail domain of NF-M is localized munoreactivity against the NL6 epitope. Phosphatase treatment at Thr (19). However, NL6 immunoreactivity persisted after strongly reduces immunoreactivity against the antibody SMI31 that deletion of a part of NF-M containing Thr (hNH-M(385– reacts with a phosphorylated epitope in NF-H (arrow) and NF-M (ar- rowhead) but does not affect immunoreactivity against M20 or NL6 472)) (Fig. 2B, middle), indicating that NL6 recognizes a so far (arrowheads), indicating that the NL6 epitope is phosphorylation inde- unknown glycosylation site on NF-M. The deletion resulted in pendently. D, effect of -elimination, glucosaminidase treatment, and a loss of the filamentous pattern and the formation of immu- competition with GlcNAc on NL6 immunoreactivity. Treatment with noreactive aggregates because of the deletion of part of the core NaOH strongly reduces reactivity of cellular lysates against an anti- body that detects -glycosidically linked N-acetylglucosamine and NL6 domain that is known to be required for filament assembly (2). immunoreactivity (top). Glucosaminidase treatment resulted in a A construct truncated at amino acid 789 was immunoreactive strong reduction of NL6 immunoreactivity (bottom left) and inclusion of against NL6 (Fig. 2B, top), whereas immunoreactivity was lost GlcNAc in the antibody reaction abrogated NL6 binding (bottom right), with a construct truncated at amino acids 603 (Fig. 2B, bottom) indicating that NL6 recognizes an O-glycosylated epitope of NF-M. E, immunoelectron micrograph of reconstituted NFs. NL6 immunoreactiv- or 552 (Fig. 2C). This indicates that the epitope is located ity is evenly distributed on NFs indicating assembly competence of between 604 and 789. This region encompasses the KSP repeat O-glycosylated NF-M. Cell culture, fractionations, immunoblot analy- region (amino acids 613–691), which consists of 6 repeats con- sis, treatment with phosphatase, -elimination, and electron micros- taining 2 KSP motifs each in human NF-M and a region car- copy were performed as described under “Experimental Procedures.” For A, 2% (v/v) of each fraction was separated by SDS-PAGE on 10% boxyl-terminal flanking the KSP repeats (amino acids 692– acrylamide and stained with Coomassie (left) or NL6 (right). For B,20 789). Within this region 3 O-glycosylation sites are predicted by (BE2/SH-SY5Y; immunoblot with NL6, M20), 10 (NT2/NT2-N; immu- the program YinOYang (www.cbs.dtu.dk/services/YinOYang/) noblot with NL6 and M20), and 1 g (immunoblot with actin) of protein based on 40 experimentally determined O--GlcNAc acceptor lysates were separated by SDS-PAGE on 7.5 (NL6/M20) and 10% (actin) acrylamide. For C,10or20 g of protein lysates from BE2 cells were separated by SDS-PAGE on 7.5% acrylamide. For -elimination in D,1 (GlcNAc) and 10 g(NL6/M20) of Triton extracts from BE2 cells were arated per lane and processed in parallel for NL6 and M20 immunode- separated by SDS-PAGE on 7.5% acrylamide, blotted, and incubated tection. GlcNAc competition was performed with or without 1 M GlcNAc with or without 55 mM NaOH and immunostained. Following immuno- in the primary antibody reaction. For E, rat NFs from spinal cord were detection, the blot was stained with Ponceau Red for total protein reconstituted and processed for immunoelectron microscopy. Scale bar, detection. After glucosaminidase treatment, 10 g of protein were sep- 500 nm. 31652 O-Glycosylation of NF-M FIG.2. Localization of the NL6 epitope on NF-M. A, immunoblot after chymotryptic cleavage of intact NFs. Af- ter cleavage, NL6 detects protein frag- ments at 100 and 110 kDa in the super- natant (sup.), indicating the localization of the NL6 epitope in the tail domain as represented in the schematic drawing. B, double fluorescence staining for GFP and the NL6 epitope of NT2 cells transfected with GFP-tagged NF-M deletion con- structs. NL6 reactivity is detected in NF-M truncated at amino acid 789 (hNF- M(789)). No NL6 reactivity is detected in the fragment truncated after amino acid 603 (hNF-M(603)), indicating that the NL6 epitope is located between amino acids 604 and 789. Note that the construct hNF-M(385–472), where part of the core domain is deleted, is deficient to assemble into filaments but still forms immunore- active aggregates. C, schematic represen- tation of the constructs, position of known and predicted O-GlcNAc sites, and fluo- rescence staining results after transfec- tion. The data suggest that the O-GlcNAc site that is detected by the NL6 epitope is located in or close to the KSP region. Chy- motryptic cleavage, immunoblot analysis, construction of expression plasmids, and immunofluorescence staining were per- formed as described under “Experimental Procedures.” For A, equal amounts of pel- let and supernatant from samples that have been treated with or without chymo- trypsin were separated by SDS-PAGE on 10% acrylamide. Scale bar,10 m. sites, to recognize the sequence context and surface accessibil- tive immunoreactive epitope. As expected, staining was also ity (38). One of the predicted O-glycosylation sites was located absent in transfected neuronal and non-neuronal mouse lines outside the KSP repeat region at position Thr . To test (Neuro 2A and 3T3) (Fig. 3C). In contrast, expression of human whether this site is part of the epitope we mutated Thr to NF-M (hNF-M) in any of these cell lines resulted in NL6 im- alanine. This construct was still immunoreactive with NL6 munoreactivity, indicating that formation of the NL6 epitope after transfection, suggesting that Thr is not involved but depends on the NF-M sequence but not on the cell type where that the glycosylated NL6 epitope is localized within the KSP it is expressed. repeat region. It was not possible to mutate the candidate sites The number and distribution of the repeats strongly differs within the KSP region directly by site-directed mutagenesis among vertebrates. In human NF-M, 12 KSP repeats are lo- because of the repetitive nature of this region. Taken together, cated between residues 611 and 688, rat and mouse NF-M the data indicate that the NL6 epitope is localized in or close to contain only 2 KSP repeats in the respective region (Fig. 3D). the KSP region of human NF-M. The repeats of the human sequence are very similar to each Immunoreactivity of the NL6 Epitope Shows a Strong Pref- other and in five of six repeats the sequence PVPK, which erence for Human Species—NFs are highly conserved across reflects, except for the missing Gln, the glycosylation motif of species. The region where most changes occur represents the O-GlcNAc transferase is located in front of the second KSP KSP repeat region that displays a major deletion of about 60 sequence. The rat KSP region is identical to human NF-M, amino acids in mouse and rat compared with the human se- whereas the mouse sequence exhibits a Val to Met exchange. quence. Thus, also NL6 immunoreactivity may differ across The NL6 epitope may encompass the PVPKS sequence with Ser species. In fact, NFs from mouse and bovine spinal cord were being O-glycosylated. Because this sequence is present five not reactive with NL6 in immunoblot analysis (Fig. 3A). In times in human NF-M but only once in rat NF-M, this may contrast, some signal, albeit less than with human material, explain the stronger immunoreactivity of human versus rat material. The PVPKS motif is missing in the bovine NF-M was observed with rat NFs. In agreement with the immunoblot results, NL6 immunoreactive neurons were observed in orga- sequence, which is consistent with the lack of immunoreactiv- ity in this material. notypic cultures of rat but not of mouse brain (data not shown). To test whether glycosylation of the NL6 epitope depends on O-Glycosylated NF-M as Recognized by NL6 Is Enriched in the Axons of Human Neurons in Situ—It is known that phos- the sequence or is cell type-specific, GFP-tagged NF-M from mouse (mNF-M) was transfected into human neuronal (NT2) phorylated subpopulations of NF-M are enriched in the axon in situ as detected by phosphorylation-sensitive antibodies (39). and non-neuronal (HeLa) cells. The construct was expressed as evidenced by the filamentous GFP fluorescence and reactivity However, it is not known whether differentially glycosylated NFs also show a compartment-specific distribution. Because with M20 (Fig. 3B, bottom panel). No staining with NL6 was observed in both cell lines, indicating the absence of the respec- O-glycosylation may be very labile during post-mortem proces- O-Glycosylation of NF-M 31653 FIG.3. Species specificity of the NL6 epitope. A, immunoblot of NFs from human (H), bovine (B), rat (R), and mouse (M) spinal cord. NL6 shows strong immunoreactivity against human NF-M, some reactivity against rat NF-M, but no reactivity against bovine or mouse NF-M. B, double fluorescence staining for GFP, NL6, and M20 of NT2 cells transfected with human (top; hNF-M) or mouse (bottom; mNF-M) GFP-tagged NF-M sequence. C, summary of experiments where human (hNF-M) or mouse (mNF-M) NF-M sequence are expressed in neural (NT2, Neuro 2A) or non-neural cell lines (HeLa, 3T3) of human or mouse origin. In all cell lines, NL6 reactivity is detected only with the human NF-M sequence indicating that the formation of the NL6 epitope depends on the NF-M sequence. D, sequence comparison of the KSP regions of human, rat, mouse, and bovine NF-M. The PVPKS motif, which is present 5 times in human and once in rat NF-M, but absent from mouse or bovine NF-M, is indicated by a box. Preparation of tissue extracts, immunoblot analysis, and immunofluorescence staining were performed as described under “Experimental Procedures.” For A,20 g of protein were separated by SDS-PAGE on 7.5% acrylamide. Scale bar,10 m. sion of human material, we aimed to perform NL6 staining on in situ in parallel to phosphorylated NF-L subpopulations. as fresh material as possible. In tissue from human temporal O-Glycosylation of NF-M Is Dynamically Influenced by In- lobe obtained at surgery of epilepsy patients, NL6 immunore- hibitors of GlcNAcase and Mitogen-activated Protein Kinase activity was highly enriched in the subcortical white matter Kinase—O-Glycosylation of cellular proteins appears to be similar to staining with an antibody against phosphorylated highly dynamic similar to phosphorylation (40–42). In some NF-L that is known to be enriched in the axon (2F11; Fig. 4A). instances a direct reciprocity of phosphorylation and O-glyco- Both antibodies labeled radial and horizontal fibers as well as sylation on the same or neighboring serines or threonines was recurrent collaterals in the infragranular layers (layers IV–VI). observed (43, 44). To test the dynamicity of O-glycosylation at In high power magnifications it was evident that NL6 immu- the NL6 epitope, the human neuroblastoma cell line SK-N- noreactivity was exclusively found in axons, whereas 2F11 also BE(2) was treated with the drug PUGNAc, a membrane-per- labeled the perinuclear soma of some neurons (Fig. 4B, aster- meable derivative of N-acetylglucosamine that binds and com- isks). We also performed immunoblot analysis with NL6 and petitively inhibits the cytoplasmic GlcNAcase (45). Immunoblot M20 antibody of temporal lobe tissue lysates from white and analysis of cellular lysates revealed that PUGNAc increased gray matter obtained from 5 different patients (Fig. 4C). Quan- NL6 immunoreactivity relative to total NF-M in a concentration- titation revealed that the ratio of immunoreactivity of white dependent manner with a maximum of 140–150% compared versus gray matter was significantly higher (p  0.001; n  5) with the control (set at 100%) (Fig. 5A). PUGNAc caused a fast with NL6 compared with M20 that recognizes total NF-M (Fig. increase in NL6 immunoreactivity because already after 10 4D), which was well in accordance with the findings from min a significant rise was observed (Fig. 5B). The changes in immunohistochemistry. When autopsy-derived human brain immunoreactivity were fully reversible indicating fast O-glyco- material with considerable postmortem intervals (12–72 h) was sylation and -deglycosylation at the NL6 epitope. Total O- used for immunoblot analysis, no significant enrichment of glycosylation as detected using an antibody that recognizes NL6 immunoreactivity in white matter was observed (data not O-linked GlcNAc independent of protein determinants showed a very similar increase after PUGNAc treatment (Fig. 5C), shown), indicating that O-glycosylation of NF-M in neuronal axons is very sensitive to degradation. The data suggest that suggesting that the dynamic glycosylation/deglycosylation at the NL6 epitope closely reflects changes in the general level of O-glycosylated NF-M as recognized by NL6 is a dynamic mod- ification that is highly enriched in the axons of human neurons O-glycosylation within the cell. 31654 O-Glycosylation of NF-M FIG.4. Distribution of NL6 immuno- reactivity in brain tissue. A, staining of human temporal lobe tissue with NL6 and 2F11. NL6 immunoreactivity is highly enriched in the subcortical white matter similar to 2F11 staining, which detects an axonally enriched phosphoryl- ated NF-H isoform. B, temporal lobe tis- sue stained for NL6 and 2F11 epitopes at higher magnification. NL6 reactivity is restricted to radial and horizontal fibers, whereas staining of perinuclear soma of some neurons is observed with 2F11 (as- terisks), as also shown at high power mag- nifications in the insets. C and D, immu- noblot (C) of gray and white matter of temporal lobe tissue lysates from five dif- ferent patients, and quantitation of the relative ratio (D) for NL6 and M20 immu- noreactivity. The ratio of white versus gray matter is significantly increased for NL6 reactivity compared with M20 (***, p  0.001, n  5; paired Student’s t test), indicating a strong axonal enrichment of the NL6 epitope in situ. Tissue prepara- tion, stainings, and immunoblot analysis were performed as described under “Ex- perimental Procedures.” For C,10or30 g of protein lysates were separated by SDS-PAGE on 7.5% acrylamide. Nuclei in sections A and B were counterstained with hematoxylin. Scale bars,20 m(in- sets of B),1mm(A), and 100 m(B). 2 2 It is known that the KSP repeat region of NF-M is phospho- model where Cu /Zn -superoxide dismutase 1 harboring an G93A rylated by the proline-directed protein kinase ERK1/2 (9), ALS-linked familial genetic mutation (SOD1 ) is overex- which is the effector kinase of the mitogen-activated protein pressed and that develops ALS-like pathology was used (48). (MAP) kinase pathway. Thus, to test for the effect of phospho- NL6 immunoreactivity was strongly reduced in spinal cord rylation in the KSP region, cells were treated with PD-98059, a extracts from mutant animals compared with wild-type rats specific inhibitor of activation of MEK (46). PD-98059 caused (Fig. 6A). The reduction was evident at postnatal day 0 as well 202 204 an about 35% decrease of activated (Thr /Tyr phosphoryl- as after disease onset in end-stage animals that expressed full ated) ERK1/2 relative to total ERK1/2, indicating inhibition of pathology. Also total NF-M reactivity decreased in these ani- MEK and an increase in relative NL6 immunoreactivity (Fig. mals indicating neurofilament loss in axons. To determine the 5D). This suggests that phosphorylation of NF-M in the tail decrease in NL6 immunoreactivity compared with changes in domain dynamically affects O-glycosylation in the same region total NF-M, blots were quantitated and relative ratios of NL6 and is reciprocally related to O-glycosylation at the NL6 and RMO44 reactivity to tubulin from transgenic animals were epitope. Treatment of the cells with the protein phosphatase 2A determined and compared with the respective ratios in wild- inhibitor cyclosporin A (5 nM, 2 h) resulted in an about 25% type animals. At all time points, the reduction in NL6 immu- decrease in relative NL6 immunoreactivity, which is also con- noreactivity was higher than in total neurofilament (Fig. 6B). sistent with a reciprocal O-glycosylation and phosphorylation In particular, in spinal cord material from end-stage animals, (data not shown). Considering these findings and the fact that NL6 immunoreactivity was barely detectable. In contrast, re- neurofilament shows an ongoing process of phosphorylation/ activity for SMI31 (phosphorylated NF-M) was increased com- dephosphorylation in human brain tissue ex vivo, the rapid loss pared with total NF-M at every age analyzed. The data suggest of NL6-reactive epitopes as described above might well be that O-glycosylation at the NL6 epitope is selectively decreased explained by a co-regulated deglycosylation (47). However, it during ALS, whereas phosphorylation is increased. cannot be excluded from these experiments that MEK inhibi- DISCUSSION tion and cyclosporin A treatment exert a systemic effect on NF-M glycosylation that is independent of the phosphorylation Using a cytoskeletal fraction prepared from human neurons of the KSP repeats. as immunogenic material we generated a novel monoclonal O-Glycosylation of NF-M at the NL6 Epitope Is Down-regu- antibody that specifically detects an O-glycosylated epitope in lated in a Transgenic Rat Model of ALS—To determine a po- NF-M. To our knowledge, this is the first antibody that specif- tential pathophysiological role of disturbed O-glycosylation in ically detects this modification in NF-M and one of very few neurodegenerative diseases, an animal model for ALS was antibodies that recognizes O-linked GlcNAc in cellular pro- employed. Because NFs from mouse tissue were not reactive teins. An example for the latter is an antibody that detects with NL6 (Fig. 3A), a previously established transgenic rat O-GlcNAc at threonine 58 in c-Myc protein and that was used O-Glycosylation of NF-M 31655 FIG.5. Regulation of O-glycosylation at the NL6 epitope. A and B, effect of the GlcNAcase inhibitor PUGNAc on the relative ratio of NL6 to M20 reactivity. Incubations were at different concentrations of PUGNAc for1h(A), or at different times at 20 M PUGNAc (B). C, effect of 20 M PUGNAc on total O-glycosylation as determined from the relative ratio of immunoreactivity with an antibody that detects -glycosidically linked GlcNAc and actin. D, effect of the MEK inhibitor PD-98059 on ERK1/2 phosphorylation and the relative ratio of NL6 to M20 reactivity. 202 205 Incubations were for 1 h. Activated (Thr /Tyr phosphorylated) ERK1/2 is decreased relative to total ERK1/2, and reactivity against the NL6 epitope relative to total NF-M is increased after inhibition of MEK. Cell culture of SK-N-BE(2) cells and immunoblot analysis were performed as described under “Experimental Procedures.” For quantitations by Western blot (A, B, and D), 20 g of protein lysates were separated by SDS-PAGE on 7.5% acrylamide. For dot-blot analysis (C), 15 g of protein lysates were immobilized on nitrocellulose. Mean  S.E. are shown. * indicates significant differences to carrier control experiments (p  0.05; n  5; paired Student’s t test). to analyze the interplay between phosphorylation and glycosy- GlcNAc no strict consensus sequence can be discerned yet. lation at this position (49). Several antibodies that more gen- Thus, it cannot be completely excluded that the NL6 antibody erally detect O-GlcNAc-modified proteins have been developed. detects an O-glycosylated epitope outside the KSP repeats. However, most of the antibodies are limited in their use be- We have shown that O-glycosylation of NF-M can quickly cause they only detect part of all modified proteins and require change, is fully reversible, and is affected by changes in protein additional epitopes (50–52). An exception is an antibody that phosphorylation. It is also very sensitive to postmortem/ex vivo has been developed by Comer et al. (35) that detects O-GlcNAc delay in the processing of neuronal tissue. Thus, O-glycosyla- independent from the protein backbone and is very useful to tion of NF-M in neurons appears to be highly dynamic similar determine overall O-GlcNAc modifications (see Figs. 1D and to the rapid turnover of O-GlcNAc-modified proteins after lym- 5C). phocyte activation (40) or the dynamic O-GlcNAcylation of the Many antibodies have been described that detect phospho- small heat shock protein B-crystallin (42). rylated epitopes in NF proteins, mostly in NF-H, which is The level of glycosylation can be very different in various cell heavily phosphorylated with a stoichiometry of about 20 mol of types (45). Thus, also during neuronal development, the cycling phosphate/mol of protein (53). NF-M is much less phosphoryl- enzymes that are responsible for GlcNAcylation may be differ- ated with about 4–9 mol of phosphate/mol of protein. The entially regulated. This would result in a change of the level of epitopes that are detected by phosphorylation-dependent anti- NL6 reactivity relative to total NF-M. We did not observe bodies are located predominantly in the tail region of NF-M and obvious changes during the differentiation of NT2 cells, SK-N- NF-H that contain multiple repeats of lysine-serine-proline BE(2) cells, or SH-SY5Y cells, indicating that the enzymes that (KSP). Especially the serines in KSP repeats appear to be are responsible for the GlcNAc modification are constitutively heavily phosphorylated in axons (54–56). The number and active in neuronal precursor cells and neurons. In agreement, distribution of the repeats strongly differ among vertebrates. strong glycosylation was observed also in non-neuronal cells NF-H contains 42–51 of these repeats. In human NF-M, 12 both of human and mouse origin after transfection of human KSP repeats are located between residues 614 and 689, rat and NF-M sequence. O-Glycosylation could be increased by phar- mouse NF-M contain only 2 KSP repeats in the respective macological inhibition of the deglycosylating enzyme with region. We have mapped the position of the O-glycosylated PUGNAc by 40–50%. Thus, the amount of glycosylation is in a epitope that is detected by the NL6 antibody in or close to the medial range that would allow the cell to react to stimuli with KSP repeat region between residues 604 and 789. This region a change in the glycosylation level. A similar increase after contains 3 predicted sites for O-linked GlcNAc modifications PUGNAc treatment was observed for the O-glycosylation of all with two of them being located in the KSP region and one proteins, indicating that NF-M glycosylation closely reflects 747 747 (Thr ) carboxyl-terminal flanking it. Mutating Thr to ala- the general activity of the cycling enzymes. nine did not affect NL6 immunoreactivity, suggesting that the O-Glycosylation of NF proteins may affect their functional site that is recognized by the antibody is located within the properties. We did not observe a differential stability of NL6- KSP repeats. It should, however, be noted that because of the positive NF-M versus total NF-M to urea-induced disassembly low number of identified sites that are modified by O-linked of NFs (data not shown) and O-glycosylated NF-M incorporated 31656 O-Glycosylation of NF-M FIG.6. Expression of the NL6 epitope in spinal cords of SOD1 transgenic rats. A, immunoblots of spinal cord extracts from wild-type and G93A mutant SOD1 expressing rats at postnatal day 0 (top) and at the end-stage of the disease (bottom). NL6 immunoreactivity is strongly reduced in NF-M from mutant animals compared with wild-type rats. B, relative intensities of the immunoreactivity against total NF-M (RMO44), the NL6 G93A epitope, and phosphorylated NF-M (SMI31) of spinal cord extracts from wild-type and mutant SOD1 expressing rats of different ages. NF-M from mutant animals shows a stronger reduction of immunoreactivity against the NL6 epitope compared with total NF-M at every age analyzed, whereas phosphorylated NF-M is increased. Preparation of spinal cord extracts and immunoblot analysis were performed as described under “Experimental Procedures.” Immunoblots of spinal cord extract from animals with different ages were quantified for NL6, SMI31, and RMO44 signals and normalized to tubulin (monoclonal antibody DM1A). Three animals of the same genotype were analyzed for every time point, and the data for wild-type animals were set as 1.0. Blots were performed at least twice. Mean  S.E. are shown. into reassembled NFs similar to total NF-M. It is known that properties of NFs different from a strongly negatively the head and rod of NF proteins, but not the tail, are essential charged phosphate group that may repel the filaments and for assembly and that phosphorylation of the head region of increase radial growth (54, 60). intermediate filaments affects assembly or disassembly (10, It is known that the tail regions of NF-M and NF-H are 11, 57, 58). Because the NL6 epitope is located in the tail heavily phosphorylated in axons (54–56). In some cases, a region, it is conceivable that O-glycosylation in this region reciprocal relation between phosphorylation and O-glycosyla- does not affect assembly properties but other features that tion (“yin-yang” relationship) has been reported (15, 61). How- are thought to be mediated by the tail region. These could ever, in contrast to what might be expected from these results, include modulation of the radial growth of axons that are also the GlcNAc modification in the tail region shows a strong critically affected by the tail domain of NF-M (4), interactions enrichment in the white matter, which appears to be even more between filaments and other cellular components, and slow pronounced than the distribution of the phosphoepitope in that axonal transport rates that are also thought to be affected by it is completely absent from neuronal cell bodies in situ. Thus, phosphorylation in the tail region (1, 6). Very recent data in white matter, phosphorylation and O-glycosylation of NF-M have implicated a role for NF-M for myelin-directed “outside- are increased in parallel. Dong et al. (62) suggested that O- in” signaling cascades (59). It will be interesting to determine glycosylation of the KSP region in NF-H occurs directly after the effect of O-glycosylation on these features. Because Glc- synthesis before the subunits are incorporated into NFs and NAc is neutral, it is possible that it modulates the surface are transported into the axon. In the axon, GlcNAc would be O-Glycosylation of NF-M 31657 10. Sihag, R. K., and Nixon, R. A. (1990) J. Biol. Chem. 265, 4166–4171 removed and substituted by phosphate groups. At least with 11. Sihag, R. K., and Nixon, R. A. (1991) J. Biol. Chem. 266, 18861–18867 respect to NF-M our data are not consistent with such an 12. de Waegh, S. M., Lee, V. M., and Brady, S. T. (1992) Cell 68, 451–463 13. Julien, J. P. (1997) Trends Cell Biol. 7, 243–249 hypothesis. In contrast, the data argue for a synchronous phos- 14. Hart, G. W. (1997) Annu. Rev. Biochem. 66, 315–335 phorylation and O-glycosylation of the tail region of NF-M 15. Comer, F. I., and Hart, G. W. (2000) J. Biol. Chem. 275, 29179–29182 within the axon. Despite this synchronicity of phosphorylation 16. Vosseller, K., Sakabe, K., Wells, L., and Hart, G. W. (2002) Curr. Opin. Chem. Biol. 6, 851–857 and O-glycosylation, inhibition of MEK with PD-98059 in- 17. Shafi, R., Iyer, S. P., Ellies, L. G., O’Donnell, N., Marek, K. W., Chui, D. H., creased O-GlcNAcylation at the NL6 epitope, suggesting a pos- Hart, G. W., and Marth, J. D. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5735–5739 sible reciprocal regulation between phosphorylation and O- 18. Slawson, C., and Hart, G. W. (2003) Curr. Opin. Struct. Biol. 13, 631–636 glycosylation in the KSP region. Whether such a reciprocal 19. Dong, D. L., Xu, Z. S., Chevrier, M. R., Cotter, R. J., Cleveland, D. W., and regulation occurs also in vivo remains to be shown. Hart, G. W. (1993) J. Biol. Chem. 268, 16679–16687 20. Piontek, J., Chen, C. C., Kempf, M., and Brandt, R. (1999) J. Neurochem. 73, The development of a polar cytoarchitecture alone was not 139–146 sufficient to cause a compartment-specific enrichment of O- 21. Riederer, B. M., Porchet, R., and Marugg, R. (1996) J. Comp. 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(1987) scribed in patients with ALS (65), and in mice and rats trans- J. Neurosci. 7, 3474–3488 40. Kearse, K. P., and Hart, G. W. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, genic for SOD1 with familial ALS-linked mutations (48, 66, 1701–1705 67), which is in agreement with our observation. In ALS, also 41. Chou, C. F., Smith, A. J., and Omary, M. B. (1992) J. Biol. Chem. 267, an increased nonenzymatic glycation (leading to an irrevers- 3901–3906 42. Roquemore, E. P., Chevrier, M. R., Cotter, R. J., and Hart, G. W. (1996) ible formation of advanced glycation end products) has been Biochemistry 35, 3578–3586 reported (reviewed in Ref. 68). However, this reaction is 43. Kelly, W. G., Dahmus, M. E., and Hart, G. W. (1993) J. Biol. Chem. 268, 10416–10424 mechanistically completely different from the dynamic enzy- 44. Chou, T. Y., Hart, G. W., and Dang, C. V. (1995) J. Biol. 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A. 99, role of disturbed O-glycosylation in common neurodegenera- 1604–1609 tive diseases. 49. Kamemura, K., Hayes, B. K., Comer, F. I., and Hart, G. W. (2002) J. Biol. Chem. 277, 19229–19235 Acknowledgments—We thank Drs. Don Cleveland and Christian 50. Holt, G. D., Snow, C. M., Senior, A., Haltiwanger, R. S., Gerace, L., and Hart, Lobsiger for providing rat spinal cord tissues, Dr. Beat Riederer for G. W. (1987) J. Cell Biol. 104, 1157–1164 51. Snow, C. M., Senior, A., and Gerace, L. (1987) J. Cell Biol. 104, 1143–1156 antibody M20, Dr. Eva-Maria Mandelkow for Neuro2A cells, Dr. Jochen 52. Turner, J. R., Tartakoff, A. M., and Greenspan, N. S. (1990) Proc. Natl. Acad. Walter for HeLa cells, Andrea Hellwig for electron microsocopy, Sci. U. S. A. 87, 5608–5612 Angelika Hilderink, Claudia Mandl, Silvia Nekum, and Brigitte Welsch 53. Jones, S. M., and Williams, R. C., Jr. (1982) J. Biol. Chem. 257, 10467–10470 for expert technical assistance, and Dr. Neelam Shahani for critically 54. 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Rev. 41, 306–323 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

O-Glycosylation of the Tail Domain of Neurofilament Protein M in Human Neurons and in Spinal Cord Tissue of a Rat Model of Amyotrophic Lateral Sclerosis (ALS) *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2005 Elsevier Inc.
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0021-9258
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10.1074/jbc.m504395200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 36, Issue of September 9, pp. 31648–31658, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. O-Glycosylation of the Tail Domain of Neurofilament Protein M in Human Neurons and in Spinal Cord Tissue of a Rat Model of Amyotrophic Lateral Sclerosis (ALS)* Received for publication, April 21, 2005, and in revised form, July 8, 2005 Published, JBC Papers in Press, July 8, 2005, DOI 10.1074/jbc.M504395200 ¶  ¶ Nina Lu¨ demann‡§, Albrecht Clement , Volkmar H. Hans , Julia Leschik‡, Christian Behl , and Roland Brandt‡** From the ‡Department of Neurobiology, University of Osnabru¨ck, Barbarastrasse 11, D-49076 Osnabru¨ck, the §Department of Neurobiology, IZN, University of Heidelberg, INF 345, 69120 Heidelberg, the Institute for Physiological Chemistry and Pathobiochemistry, Johannes-Gutenberg-University, Duesbergweg 6, 55099 Mainz, and the Institute of Neuropathology, University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany components in large-diameter myelinated axons. Mammalian Mammalian neurofilaments (NFs) are modified by post-translational modifications that are thought to reg- NFs consist of three major subunits of 200, 160, and 70 kDa, ulate NF assembly and organization. Whereas phospho- termed NF-H, NF-M, and NF-L, respectively (1). Structurally, rylation has been intensely studied, the role of another the NFs contain a N-terminal head and a helical rod domain common modification, the attachment of O-linked N- that are required for filament assembly (2), and a C-terminal acetylglucosamine (GlcNAc) to individual serine and tail domain. NFs are the intrinsic determinant of radial growth threonine residues, is hardly understood. We generated of axons, which is most probably mediated by the tail domains a novel monoclonal antibody that specifically recog- of NF-M and NF-H. Whereas genetic deletion of NF-H or its tail nizes an O-glycosylated epitope in the tail domain of domain has relative little effect on radial growth (3), the tail of NF-M and allows determination of the glycosylation NF-M has an important role in the formation of cross-links state at this residue. The antibody displays strong spe- between NFs and modulation of axonal caliber (4). Misaccumu- cies preference for human NF-M, shows some reactivity lation of NFs is a frequent hallmark of several neurodegenera- with rat but not with mouse or bovine NF-M. By immu- tive diseases including amyotrophic lateral sclerosis (ALS), nohistochemistry and Western blot analysis of biopsy- spinal muscular atrophy, and Alzheimer disease (5). derived human temporal lobe tissue we show that im- NFs are among the most highly phosphorylated neuronal munoreactivity is highly enriched in axons parallel to proteins. Second messenger-dependent protein kinases such as hyperphosphorylated NFs. Treatment of cultured neu- protein kinase C and cyclic AMP-dependent protein kinase rons with the GlcNAcase inhibitor PUGNAc causes a appear to be the principal kinases targeting the head domain. 40% increase in immunoreactivity within 1 h, which is The tail domain is a preferred substrate for second messenger- completely reversible and parallels the total increase in independent kinases (6), e.g. casein kinase I and II (7), cdk5 (8), cellular O-GlcNAc modification. Treatment with the mito- and mitogen-activated protein kinases (ERK1/2) (9). Phospho- gen-activated protein kinase kinase inhibitor PD-98059 leads to a similar increase in immunoreactivity. In spinal rylation of the head domain appears to regulate the assembly of cord tissue of a transgenic rat model for amyotrophic NFs (10, 11), whereas tail domain phosphorylation may affect lateral sclerosis, immunoreactivity is strongly de- their ability to control the axon caliber (12). Normally, tail creased compared with wild-type animals while phos- domain phosphorylation appears to be restricted to the axon, phorylation is increased. The data suggest that hyper- but in ALS hyperphosphorylation of this domain occurs also in phosphorylation and tail domain O-glycosylation of NFs perikarya (13). are synchronously regulated in axons of human neurons Attachment of O-linked N-acetylglucosamine (GlcNAc) to in- in situ and that O-glycosylation of NF-M is highly dy- dividual serine and threonine residues is a common post-trans- namic and closely interweaved with phosphorylation lational modification of many nuclear and cytoskeletal proteins cascades and may have a pathophysiological role. (14–16). The enzyme catalyzing O-GlcNAc modification (O- GlcNAc transferase) is essential for cell viability in mammals (17). O-Glycosylation by GlcNAc appears to be as dynamic as Neurofilaments (NFs) are the most abundant structural protein phosphorylation and is often reciprocal to phosphoryl- ation at the same or adjacent sites (18). Also NFs are modified by O-linked GlcNAc with several identified sites being located * This work was supported by Deutsche Forschungsgemeinschaft in the head region of NF-L and NF-M (19). It is not known how Grant SFB 488 project B1 and the Ministry for Science and Culture of Lower Saxony. The costs of publication of this article were defrayed in O-glycosylation of NFs is regulated, how it relates to the phos- part by the payment of page charges. This article must therefore be phorylation of NFs, and where O-glycosylated NFs are hereby marked “advertisement” in accordance with 18 U.S.C. Section distributed. 1734 solely to indicate this fact. Here we describe a novel monoclonal antibody (NL6) that ** To whom correspondence should be addressed. Tel.: 49-541- 9692338; Fax: 49-541-9692354; E-mail: [email protected]. specifically recognizes an O-glycosylated epitope in the projec- de. tion domain of NF-M. The antibody displayed a strong species The abbreviations used are: NF, neurofilament; ALS, amyotrophic preference for human NF-M. NL6-positive NF-M was enriched lateral sclerosis; ERK, extracellular signal-regulated kinase; MEK, mi- togen-activated protein kinase kinase; PUGNAc, O-(2-acetamido-2-de- oxy-D-glucopyranosylidene)amino-N-phenylcarbamate; GFP, green flu- orescent protein; PBS, phosphate-buffered saline; FCS, fetal calf serum; ride; PIPES, 1,4-piperazinediethanesulfonic acid; MES, 4-morpho- MEM, minimal essential medium; PMSF, phenylmethylsulfonyl fluo- lineethanesulfonic acid. 31648 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. O-Glycosylation of NF-M 31649 was prepared by changing A at position 2248 against G. Mutated in the axons of human neurons in situ. Pharmacological ma- constructs were verified by dideoxy sequencing using T7 Sequenase nipulations demonstrated that O-glycosylation as detected (Amersham Biosciences). with the NL6 antibody is highly dynamic, reversible, and in- Cell Culture and Transfection—NT2 cells were grown in serum- versely regulated to the activity of mitogen-activated protein DMEM (Dulbecco’s modified Eagle’s medium supplemented with 10% kinase kinase (MEK). In a rat model for ALS, NL6 immunore- FCS, 5% horse serum (HS), 292 g/ml glutamine, 50 units/ml penicillin, activity was strongly decreased compared with wild-type and 50 g/ml streptomycin) and differentiated essentially as described previously (24). NT2-N neurons were obtained after 5 weeks differen- animals. tiation with retinoic acid and 2 weeks treatment with mitotic inhibitors EXPERIMENTAL PROCEDURES 2 as described previously (20), plated at 4000 cells/cm onto collagen- Materials—Chemicals were purchased from Sigma, cell culture me- coated coverslips and cultured for 3 days in serum/Dulbecco’s modified dia and supplements from Life Technologies, culture flasks and dishes Eagle’s medium. After this time, almost all neurons had established from Nunc (Roskilde, Denmark) unless otherwise stated. Collagen was polarity as judged from axon-specific tau staining. SK-N-BE(2) cells (25) prepared from rat tails by acetic acid extraction (20). O-(2-Acetamido- were grown in serum/Opti-MEM (Opti-MEM supplemented with 5% 2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) iron enriched FCS, 292 g/ml glutamine, 50 units/ml penicillin, and 50 was purchased from Carbogen (Aarau, Switzerland), the MEK inhibitor g/ml streptomycin) and differentiated with 10 M retinoic acid for 2 PD-98059 was from New England Biolabs (Beverly, MA). Mouse mono- weeks. 3T3 cells (26) were cultured in serum/Dulbecco’s modified Ea- clonal M20 antibody against a phosphorylation-independent site on NF-M gle’s medium supplemented with 10% FCS, 584 g/ml glutamine, 50 (21) was a kind gift from Dr. B. M. Riederer (Lausanne, Switzerland). units/ml penicillin, and 50 g/ml streptomycin, HeLa cells (27) in se- Antibody SMI31 against phosphorylated NF-M and NF-H was pur- rum/minimal essential Earle’s medium (MEM Earle’s Medium supple- chased from Sternberger Monoclonals Inc. (Baltimore, MD), mouse mented with 10% FCS, 292 g/ml glutamine, 50 units/ml penicillin, and monoclonal antibody against O-glycosylated GlcNAc (CTD110.6), which 50 g/ml streptomycin) and Neuro2A cells (28) in serum/MEM (mini- detects GlcNAc independent from the protein backbone (35), was a kind mal essential medium with Earle’s salts supplemented with 10% FCS, gift of Covance (Cumberland, VA), rabbit polyclonal anti-GFP was 292 g/ml glutamine and nonessential amino acids for MEM). In some obtained from Molecular Probes (Leiden, Netherlands), mouse mono- experiments cells were treated with PUGNAc or the MEK inhibitor clonal antibody against the rod domain of NF-M (RMO44) from Stress- PD-98059 for1horthe times indicated prior to analysis. Gene (Victoria, BC, Canada), mouse monoclonal antibody against One day before transfections, cells were plated at a density of 3  10 MAP2 from Chemicon (Temecula, CA), mouse monoclonal 2F11 react- cells/coverslip on collagen-coated coverslips and transfected with Fu- ing with the phosphorylated form of NF-L from Dako (Glostrup, GENE 6 (Roche Molecular Biochemicals) at a ratio of 1 g of DNA to 4 Denmark), rabbit polyclonal antibodies against phospho-p44/42 and l of FuGENE per coverslip. Cells were processed for immunocytochem- total p44/42 MAP kinase from Cell Signaling (Beverly, MA), and mouse istry 48 h after transfection. monoclonal antibody against actin from Oncogene (San Diego, CA). Preparation of Cellular and Tissue Lysates and Cell Fractionation— Cy3-conjugated donkey anti-mouse antibody, fluorescein isothiocya- For preparation of lysates, cells were washed twice with PBS, scraped nate-conjugated goat anti-rabbit and horseradish peroxidase-conju- off into ice-cold PBS, and collected for 5 min at 300  g. Tissue was gated donkey anti-mouse and goat anti-rabbit antibody were obtained shock frozen in liquid nitrogen, cut with a cryostat in sections to allow from Dianova (Hamburg, Germany), Alexa 488-conjugated goat anti- a better penetration of the lysis buffer, and collected in a test tube. mouse IgG1 and Alexa 543-conjugated goat anti-mouse IgG2A from Pellet and tissue slices were resuspended in RIPA buffer (50 mM Tris/ Molecular Probes (Leiden, Netherlands). HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% Preparation of the NL6 Antibody—Mice were immunized with a deoxycholate, 0.1% SDS) containing protease and phosphatase inhibi- fraction enriched for components of the human neuronal cytoskeleton. tors (1 mM PMSF, 10 g/ml each of leupeptin and pepstatin, 1 mM The fraction was prepared from NT2-N neurons and SK-N-BE(2) neu- sodium orthovanadate, 20 mM NaF, 1 mM sodium pyrophosphate), roblastoma cells as described previously (22). The immunization was incubated for 30 min at 4 °C, centrifuged for 10 min at 13,000  g, and performed with the PBS-insoluble material that was solubilized with 8 the supernatant (lysate) collected. M urea and diluted to a final concentration of 0.85 M urea in PBS. For detergent extraction, SK-N-BE(2) cells from one 15-cm TC dish BALB/c mice were injected 4 times intraperitonally with 50 gofthe were washed twice with PBS, scraped off into extraction buffer (10 mM protein fraction at intervals of 3 to 4 weeks. Four days after the final PIPES/KOH, pH 6.8, 50 mM KCl, 3 mM MgCl ,10mM EGTA), and injection, spleens were removed and the spleenocytes were fused with collected for 5 min at 300  g with all steps at 4 °C. Cells were the myeloma line X63Ag8.653 as described previously (23). The result- resuspended in 1 ml of extraction buffer containing 1%(v/v) Triton ing hybridoma clones were screened for immunoreaction against cy- X-100 and protease inhibitors and incubated for 10 min. 250 lofthe toskeletal proteins. Positive clones were recloned twice to obtain stable solution were transferred on a 0.85 M sucrose cushion and centrifuged antibody producing hybridoma lines and cultured in RPMI 1640, 10% for 30 min at 72,000  g. The pellet was resuspended in 100 lof fetal calf serum (FCS), 292 g/ml glutamine. Antibodies were concen- extraction buffer. trated by ammonium sulfate precipitation of the hybridoma superna- For reassembly of NFs, spinal cords from several adult rats were tant, resuspended in PBS, dialyzed against PBS, and stored in the prepared and frozen in liquid nitrogen. An equal amount (w/v) of dis- presence of 50% (v/v) glycerol at 20 °C. assembly buffer (50 mM MES, pH 6.8, 0.5 mM MgCl ,1mM EGTA, 1 mM For isotype determination, the “isostrip mouse monoclonal antibody PMSF) was added, the mixture was homogenized on ice using an isotyping kit” from Roche Diagnostics was used according to the man- electrical blade homogenizer and centrifuged for 30 min at 72,000  g. ufacturer’s protocol. NL6 is a IgG2a antibody with a -chain. Because The supernatant was collected, cleared by centrifugation, and an equal most of the commercially available mouse antibodies belong to the volume of assembly buffer (50 mM MES, pH 6.8, 0.5 mM MgCl ,1mM subtype G1, double fluorescence analysis using isotype-specific second- EGTA, 8 M glycerol) was added. The mixture was incubated for 15 min ary antibodies becomes possible. Characterization of the antibody by at 4 °C and centrifuged for 45 min at 75,000  g. The pellet was immunocytochemistry revealed that it was sensitive to glutaraldehyde resuspended in 100 l of assembly buffer. fixation and that the signal was completely lost after fixation for 10 min Tissue extracts from different species (human, bovine, rat, and with 0.3%(v/v) glutaraldehyde. mouse) were prepared as previously described (29). Tissues were ho- Construction of Expression Vectors—Eukaryotic expression plasmids mogenized on ice in 50 mM Tris, pH 7.5, 0.5 mM EDTA, 1 mM of the for hNF-M, mNF-M, and deletion constructs of hNF-M with the C- protease inhibitors PMSF, aprotinin, and chymostatin. An equal vol- terminal fused GFP epitope were constructed using the CT-GFP fusion ume of extraction buffer was added (50 mM Tris, pH 7.5, 150 mM NaCl, TOPO®TA expression kit (Invitrogen, Karlsruhe, Germany). To con- 1% Nonidet P-40, 1% sodium deoxycholate, 2% SDS), the homogenates struct mutated hNF-M, respective codons were changed using the were sonicated for 20 s, boiled for 10 min, centrifuged for 5 min at QuikChange® site-directed mutagenesis kit (Stratagene, Amsterdam, 16,000  g, and the supernatants saved. Netherlands). HNF-M(385–472) was prepared by deleting the respec- Immunohistochemistry, Immunocytochemistry, and Immunoelectron tive fragment with the restriction enzyme Eco0109I. Constructs with Microscopy—Human temporal lobe tissue was obtained neurosurgically N-terminal-fused GFP were prepared in EGFP C3 Vector (Clontech, from five patients suffering from focal epilepsy. Material was kept on ice, Palo Alto, CA). HNF-M(891) was produced using BCUI385, hNF- and gray and white matter were dissected apart. Tissue samples were M(552), with BamHI. To prepare hNF-M(603), an Eco32I restriction transferred to liquid nitrogen and kept at 80 °C. For immunohistochem- site was inserted by changing GT at positions 1813/1814 to AT followed istry, samples were immediately fixed in 4% formalin for 24 h after by the respective digest. For hNF-M(789), a Eco32I restriction site was surgical removal and routinely paraffin-embedded. Deparaffinized 4-m inserted by changing AA at positions 2369/2370 to TG. HNF-M(T747A) thick sections were blocked and incubated with primary antibodies over- 31650 O-Glycosylation of NF-M night at 4 °C. Delay of formalin fixation for a few hours, or incubation for to4mM. Samples were centrifuged for 20 min at 4 °C and 12,800  g 1 h at room temperature resulted in substantial loss of staining intensity and equal amounts of pellet and supernatant were analyzed by immu- in 14 samples of surgically resected hippocampi (data not shown). Biotin- noblot. Comparison between experimental groups was based on paired labeled secondary antibodies were visualized using peroxidase-conjugated Student’s t test (*, p  0.05; **, p  0.01; ***, p  0.001). streptavidin with diaminobenzidine as the substrate. All solutions for the visualization of the antigens were contained in a commercially available RESULTS TM detection kit (DAKO ChemMate , DakoCytomation GmbH, Hamburg, NL6, A Novel Monoclonal Antibody against a Human Neu- Germany). Staining procedures were performed on an autostainer accord- ronal Cytoskeleton Fraction, Recognizes an O-Glycosylated ing to routine protocols provided by the manufacturer (DAKO TechMate TM 500 ). As negative controls, primary antibodies were omitted. The study Epitope of Human NF-M—In an immunological approach to was approved by the local ethics committee, and informed consent was obtain novel antibodies against components of the human neu- received from all tissue donors. ronal cytoskeleton, a PBS-insoluble cytoskeletal fraction of hu- Cultured cells were fixed for 20 min with 4% paraformaldehyde in man model neurons was prepared and solubilized with urea PBS (10 mM phosphate buffer, pH 7.4, 2.7 mM KCl, and 137 mM NaCl) (see “Experimental Procedures”). The material was used to containing 4% (w/v) sucrose at room temperature, washed with PBS, immunize mice and produce hybridoma lines. One of the anti- incubated for 20 min with 0.1 M glycine in PBS, and permeabilized with 0.2% (v/v) Triton X-100 for 5 min. Staining was performed as described bodies (NL6) detected a protein with an apparent molecular earlier (30) in PBS containing 1% (w/v) bovine serum albumin using mass of 160 kDa. The antigen was highly enriched in the appropriate antibody combinations. 5 g/ml 4,6-Diamidino-2-phenylin- Triton X-100-insoluble fraction of homogenates from differen- dole was included in the mixture of the secondary antibodies to detect tiated human neuroblastoma (SK-N-BE(2)) cells (Fig. 1A), in- the nuclei. Cells were analyzed using a Leica DMIRB fluorescence dicating that NL6 recognizes an intermediate filament protein. microscope or a Leica TCS SP2 True confocal scanner (Leica, Solms, The NL6 antigen was up-regulated during differentiation of Germany). For immunoelectron microscopy, reassembled rat NFs were trans- human neuroblastoma cell lines parallel to NF-M (SK-N-BE(2), ferred on pioloform-coated and glow-discharged copper grids (400 SH-SY5Y cells; Fig. 1B, left panel). To determine whether the mesh), fixed for 20 min with 4% paraformaldehyde in PBS containing antigen of NL6 is also expressed in neuronal precursor cells 4% (w/v) sucrose, incubated for 20 min with 0.1 M glycine in PBS, and and when it is produced during differentiation, the NT2/NT2-N labeled with primary antibodies. After five washes with PBS containing system was employed. NT2 cells are a human teratocarcinoma 1% (w/v) bovine serum albumin and 0.1% (v/v) Tween 20, grids were cell line with characteristics of central nervous system neuro- incubated with polyclonal rabbit anti-mouse antibody (ICN Biomedi- cals, Aurora, OH), washed five times with PBS containing 1% (w/v) nal progenitor cells that can be differentiated in vitro to yield bovine serum albumin and 0.1% (v/v) Tween 20, and incubated with terminally differentiated, polar, and postmitotic neurons (24). protein A coupled to 15 nm gold. Grids were washed with PBS, postfixed The NL6 antigen was present in lysates of NT2-N cells. In for 30 min with 2% (v/v) glutaraldehyde in PBS, washed 3  10 min contrast, no signal was observed in NT2 cell lysates (Fig. 1B, with water, and contrasted with uranyl acetate. Electron microscopy right panel), indicating that the NL6 antigen is expressed only was performed on a Zeiss 10CR electron microscope (Zeiss, Oberkochen, Germany). in differentiated neurons. The expression pattern was similar Other Methods—Protein concentrations were determined using the albeit not identical to total NF-M as detected using a previously BCA microplate assay (Pierce). SDS-polyacrylamide gel electrophoresis described antibody recognizing NF-M independent of post- and transfer onto polyvinylidene difluoride were performed as de- translational modifications (M20 (21, 33)). Taken together the scribed previously (31) using the benchmark protein molecular weight data strongly suggest that NL6 detects a subpopulation of marker (Life Technologies). For dot-blot analysis, samples were diluted NF-M. with 2 volumes of dot-blot buffer (30% methanol, 0.5% deoxycholate, 9 g/liter NaCl, 1.21 g/liter Tris, pH 7.4) and immobilized on Immobilon It is known that in neurons NF-M is highly post-translation- NC pure (Millipore Corp., Bedford, MA) using a dot-blot hybridization ally modified (for a review, see Ref. 1). To test whether NL6 chamber (Loxo, Dossenheim, Germany). In some cases, blots were detects a phosphorylated epitope, cellular homogenates were stained for total protein using Ponceau S or Coomassie Brilliant Blue separated by SDS-PAGE, blotted onto polyvinylidene difluo- according to standard procedures (32). Immunodetection used en- ride, and treated with alkaline phosphatase. Phosphatase hanced chemiluminescence (ECL) (Amersham Biosciences) and was treatment resulted in a strong reduction of the immunoreac- performed according to the manufacturer’s protocol. Image acquisition employed a chemo CAM-System (Intas Science Imaging Instruments, tivity with an antibody that detects phosphorylated epitopes in Go¨ttingen, Germany) or Fuji LAS3000-System (Raytest, Straubenhart, NF-H and NF-M (SMI31) but caused no reduction in M20 or Germany). Quantification of the blots was carried out with NIH Image NL6 immunoreactivity (Fig. 1C, arrowheads) indicating that 1.61/ppc (rsb.info.nih.gov/nih-image/index.html). binding of NL6 does not depend on phosphorylation of the Phosphatase treatment was performed by incubating the membrane epitope. To test whether NL6 detects an O-glycosylated for 24 h at 37 °C with 20 units/ml alkaline phosphatase (from calf epitope, blots from cellular lysates were treated with 55 mM intestinal mucosa) in 100 mM Tris/HCl, pH 8.0, 1 mM PMSF. Phos- phatase-treated blots were subjected for immunodetection in parallel NaOH, which is known to remove -glycosidically linked Glc- with control blots that have been washed with buffer only. NAc from serine or threonine residues by a reaction called For chemical cleavage of -O-glycosidic linkages, blots were washed -elimination (34). The treatment resulted in a complete loss of for 3  10 min with water, incubated for 16 h with 55 mM NaOH at immunoreactivity with an antibody that recognizes O-linked 40 °C, and washed again for 3  10 min with water. Blots were sub- GlcNAc independent of protein determinants (CTD110.6 (35); jected for immunodetection in parallel with control blots that have been Fig. 1D, top left panel). Blots from fractions enriched for NFs incubated with water only. Enzymatic digestion of -glycosidic linkages was performed with Triton-extracted fractions of BE2 cells. 20 gof were treated similarly and probed with NL6 and M20 antibody. protein were incubated with 0.5 unit of -N-acetylglucosaminidase Treatment with NaOH resulted in an almost complete loss of (from bovine kidney) in a total volume of 50 lin10mM sodium NL6 immunoreactivity, whereas M20 staining remained un- cacodylate, pH 5.0, 1 mM PMSF, and 10 g/ml each of leupeptin and changed (Fig. 1D, top right panel). To check whether GlcNAc at pepstatin for 16 h at 37 °C. The reaction was stopped by addition of 12.5 the NL6 epitope can also be removed enzymatically, a fraction lof5 Laemmli buffer. Control samples were treated identical, but in the absence of -N-acetylglucosaminidase. 50% of the samples were enriched for neurofilaments was incubated with -N-acetylglu- subjected for immunodetection and processed in parallel for NL6 and cosaminidase from bovine kidney. The treatment resulted in a M20 staining. strong reduction in NL6 immunoreactivity compared with a Partial chymotryptic digest of intact NFs was performed with Triton- control sample, whereas M20 staining remained unchanged extracted fractions of BE2 cells. 10 g of protein were incubated with 50 (Fig. 1D, bottom left panel). Furthermore, inclusion of 1 M ng of chymotrypsin in a total volume of 18 l in digestion buffer (100 mM GlcNAc in the primary antibody reaction abrogated binding of Tris/HCl, pH 7.8, 10 mM CaCl ,10 g/ml each of leupeptin and pepsta- tin). The reaction was stopped after different times by addition of PMSF the NL6 antibody (Fig. 1D, bottom right panel). Taken together O-Glycosylation of NF-M 31651 the data indicate that NL6 recognizes an O-glycosylated epitope of NF-M. To test whether NF-M remains O-glycosylated at the NL6 epitope during disassembly and reassembly, NFs were recon- stituted from high-speed supernatants of rat spinal cord homo- genates and analyzed by immunoelectron microscopy. NL6 im- munoreactivity was evenly distributed on reconstituted NFs similar to M20 reactivity (Fig. 1E). The data indicate that O-glycosylated NF-M is assembly-competent and uniformly in- corporates into NFs. The NL6 Epitope Is Localized in the Tail Domain of NF-M— NF-M consists of a central -helical rod domain that forms the filament backbone, a small globular head domain, and an ex- tended carboxyl-terminal tail domain that protrudes from the backbone (1, 36). To determine in which of these regions the NL6 epitope is localized, a partial chymotryptic digest of intact NFs was performed. It has previously been shown that, under these conditions, at first the tail domain is cleaved off from the remainder of the protein close to the filament backbone, which can then be separated by centrifugation (37). After short treat- ment with chymotrypsin (2 min), NL6 reactivity was exclu- sively present in the supernatant at molecular masses of about 100 and 110 kDa (Fig. 2A), which closely corresponds to the apparent size of the tail domain as reported earlier (37). After longer incubation periods (8 min) NL6 immunoreactivity dis- appeared because of further digestion of the protein (data not shown). The data indicates that the NL6 epitope is localized in the carboxyl-terminal tail domain of NF-M. As a tool to determine the position of the NL6 epitope, a fusion protein between human NF-M and GFP was constructed and transfected into the human neuronal precursor cell line NT2. As shown above (Fig. 1B) and confirmed by immunocyto- chemistry (data not shown), these cells do not express detect- FIG.1. Generation and identification of a novel monoclonal able amounts of endogenous NF-M. Individual transfected cells antibody (NL6) against an O-glycosylated epitope of NF-M. A, showed a filamentous pattern of GFP fluorescence (Fig. 3B, top immunoblot of subcellular fractions of differentiated SK-N-BE(2) cells left) indicative of the incorporation of the exogenous construct after detergent extraction with Triton X-100 (TX-100). The antigen has an apparent molecular mass of 160 kDa and is highly enriched in the into the cellular intermediate filaments. NL6 staining colocal- Triton-insoluble fraction (pellet, P), indicating that the NL6 antibody ized with the GFP fluorescence (Fig. 3B, top right) indicating recognizes an intermediate filament protein. S, supernatant. B, expres- that exogenously expressed NF-M becomes O-glycosylated at sion of the NL6 epitope during in vitro differentiation of human neuro- the NL6 epitope. To map the region of NL6 immunoreactivity, nal cells. The antigen is up-regulated in differentiated SK-N-BE(2) cells a panel of deletion mutants and mutated constructs were pre- (BE2), SH-SY5Y cells, and NT2-N cells similar to NF-M as detected using an antibody (M20) that recognizes NF-M independent of post- pared and tested in similar transfection assays. The only translational modifications. C, effect of phosphatase treatment on im- known glycosylation site in the tail domain of NF-M is localized munoreactivity against the NL6 epitope. Phosphatase treatment at Thr (19). However, NL6 immunoreactivity persisted after strongly reduces immunoreactivity against the antibody SMI31 that deletion of a part of NF-M containing Thr (hNH-M(385– reacts with a phosphorylated epitope in NF-H (arrow) and NF-M (ar- rowhead) but does not affect immunoreactivity against M20 or NL6 472)) (Fig. 2B, middle), indicating that NL6 recognizes a so far (arrowheads), indicating that the NL6 epitope is phosphorylation inde- unknown glycosylation site on NF-M. The deletion resulted in pendently. D, effect of -elimination, glucosaminidase treatment, and a loss of the filamentous pattern and the formation of immu- competition with GlcNAc on NL6 immunoreactivity. Treatment with noreactive aggregates because of the deletion of part of the core NaOH strongly reduces reactivity of cellular lysates against an anti- body that detects -glycosidically linked N-acetylglucosamine and NL6 domain that is known to be required for filament assembly (2). immunoreactivity (top). Glucosaminidase treatment resulted in a A construct truncated at amino acid 789 was immunoreactive strong reduction of NL6 immunoreactivity (bottom left) and inclusion of against NL6 (Fig. 2B, top), whereas immunoreactivity was lost GlcNAc in the antibody reaction abrogated NL6 binding (bottom right), with a construct truncated at amino acids 603 (Fig. 2B, bottom) indicating that NL6 recognizes an O-glycosylated epitope of NF-M. E, immunoelectron micrograph of reconstituted NFs. NL6 immunoreactiv- or 552 (Fig. 2C). This indicates that the epitope is located ity is evenly distributed on NFs indicating assembly competence of between 604 and 789. This region encompasses the KSP repeat O-glycosylated NF-M. Cell culture, fractionations, immunoblot analy- region (amino acids 613–691), which consists of 6 repeats con- sis, treatment with phosphatase, -elimination, and electron micros- taining 2 KSP motifs each in human NF-M and a region car- copy were performed as described under “Experimental Procedures.” For A, 2% (v/v) of each fraction was separated by SDS-PAGE on 10% boxyl-terminal flanking the KSP repeats (amino acids 692– acrylamide and stained with Coomassie (left) or NL6 (right). For B,20 789). Within this region 3 O-glycosylation sites are predicted by (BE2/SH-SY5Y; immunoblot with NL6, M20), 10 (NT2/NT2-N; immu- the program YinOYang (www.cbs.dtu.dk/services/YinOYang/) noblot with NL6 and M20), and 1 g (immunoblot with actin) of protein based on 40 experimentally determined O--GlcNAc acceptor lysates were separated by SDS-PAGE on 7.5 (NL6/M20) and 10% (actin) acrylamide. For C,10or20 g of protein lysates from BE2 cells were separated by SDS-PAGE on 7.5% acrylamide. For -elimination in D,1 (GlcNAc) and 10 g(NL6/M20) of Triton extracts from BE2 cells were arated per lane and processed in parallel for NL6 and M20 immunode- separated by SDS-PAGE on 7.5% acrylamide, blotted, and incubated tection. GlcNAc competition was performed with or without 1 M GlcNAc with or without 55 mM NaOH and immunostained. Following immuno- in the primary antibody reaction. For E, rat NFs from spinal cord were detection, the blot was stained with Ponceau Red for total protein reconstituted and processed for immunoelectron microscopy. Scale bar, detection. After glucosaminidase treatment, 10 g of protein were sep- 500 nm. 31652 O-Glycosylation of NF-M FIG.2. Localization of the NL6 epitope on NF-M. A, immunoblot after chymotryptic cleavage of intact NFs. Af- ter cleavage, NL6 detects protein frag- ments at 100 and 110 kDa in the super- natant (sup.), indicating the localization of the NL6 epitope in the tail domain as represented in the schematic drawing. B, double fluorescence staining for GFP and the NL6 epitope of NT2 cells transfected with GFP-tagged NF-M deletion con- structs. NL6 reactivity is detected in NF-M truncated at amino acid 789 (hNF- M(789)). No NL6 reactivity is detected in the fragment truncated after amino acid 603 (hNF-M(603)), indicating that the NL6 epitope is located between amino acids 604 and 789. Note that the construct hNF-M(385–472), where part of the core domain is deleted, is deficient to assemble into filaments but still forms immunore- active aggregates. C, schematic represen- tation of the constructs, position of known and predicted O-GlcNAc sites, and fluo- rescence staining results after transfec- tion. The data suggest that the O-GlcNAc site that is detected by the NL6 epitope is located in or close to the KSP region. Chy- motryptic cleavage, immunoblot analysis, construction of expression plasmids, and immunofluorescence staining were per- formed as described under “Experimental Procedures.” For A, equal amounts of pel- let and supernatant from samples that have been treated with or without chymo- trypsin were separated by SDS-PAGE on 10% acrylamide. Scale bar,10 m. sites, to recognize the sequence context and surface accessibil- tive immunoreactive epitope. As expected, staining was also ity (38). One of the predicted O-glycosylation sites was located absent in transfected neuronal and non-neuronal mouse lines outside the KSP repeat region at position Thr . To test (Neuro 2A and 3T3) (Fig. 3C). In contrast, expression of human whether this site is part of the epitope we mutated Thr to NF-M (hNF-M) in any of these cell lines resulted in NL6 im- alanine. This construct was still immunoreactive with NL6 munoreactivity, indicating that formation of the NL6 epitope after transfection, suggesting that Thr is not involved but depends on the NF-M sequence but not on the cell type where that the glycosylated NL6 epitope is localized within the KSP it is expressed. repeat region. It was not possible to mutate the candidate sites The number and distribution of the repeats strongly differs within the KSP region directly by site-directed mutagenesis among vertebrates. In human NF-M, 12 KSP repeats are lo- because of the repetitive nature of this region. Taken together, cated between residues 611 and 688, rat and mouse NF-M the data indicate that the NL6 epitope is localized in or close to contain only 2 KSP repeats in the respective region (Fig. 3D). the KSP region of human NF-M. The repeats of the human sequence are very similar to each Immunoreactivity of the NL6 Epitope Shows a Strong Pref- other and in five of six repeats the sequence PVPK, which erence for Human Species—NFs are highly conserved across reflects, except for the missing Gln, the glycosylation motif of species. The region where most changes occur represents the O-GlcNAc transferase is located in front of the second KSP KSP repeat region that displays a major deletion of about 60 sequence. The rat KSP region is identical to human NF-M, amino acids in mouse and rat compared with the human se- whereas the mouse sequence exhibits a Val to Met exchange. quence. Thus, also NL6 immunoreactivity may differ across The NL6 epitope may encompass the PVPKS sequence with Ser species. In fact, NFs from mouse and bovine spinal cord were being O-glycosylated. Because this sequence is present five not reactive with NL6 in immunoblot analysis (Fig. 3A). In times in human NF-M but only once in rat NF-M, this may contrast, some signal, albeit less than with human material, explain the stronger immunoreactivity of human versus rat material. The PVPKS motif is missing in the bovine NF-M was observed with rat NFs. In agreement with the immunoblot results, NL6 immunoreactive neurons were observed in orga- sequence, which is consistent with the lack of immunoreactiv- ity in this material. notypic cultures of rat but not of mouse brain (data not shown). To test whether glycosylation of the NL6 epitope depends on O-Glycosylated NF-M as Recognized by NL6 Is Enriched in the Axons of Human Neurons in Situ—It is known that phos- the sequence or is cell type-specific, GFP-tagged NF-M from mouse (mNF-M) was transfected into human neuronal (NT2) phorylated subpopulations of NF-M are enriched in the axon in situ as detected by phosphorylation-sensitive antibodies (39). and non-neuronal (HeLa) cells. The construct was expressed as evidenced by the filamentous GFP fluorescence and reactivity However, it is not known whether differentially glycosylated NFs also show a compartment-specific distribution. Because with M20 (Fig. 3B, bottom panel). No staining with NL6 was observed in both cell lines, indicating the absence of the respec- O-glycosylation may be very labile during post-mortem proces- O-Glycosylation of NF-M 31653 FIG.3. Species specificity of the NL6 epitope. A, immunoblot of NFs from human (H), bovine (B), rat (R), and mouse (M) spinal cord. NL6 shows strong immunoreactivity against human NF-M, some reactivity against rat NF-M, but no reactivity against bovine or mouse NF-M. B, double fluorescence staining for GFP, NL6, and M20 of NT2 cells transfected with human (top; hNF-M) or mouse (bottom; mNF-M) GFP-tagged NF-M sequence. C, summary of experiments where human (hNF-M) or mouse (mNF-M) NF-M sequence are expressed in neural (NT2, Neuro 2A) or non-neural cell lines (HeLa, 3T3) of human or mouse origin. In all cell lines, NL6 reactivity is detected only with the human NF-M sequence indicating that the formation of the NL6 epitope depends on the NF-M sequence. D, sequence comparison of the KSP regions of human, rat, mouse, and bovine NF-M. The PVPKS motif, which is present 5 times in human and once in rat NF-M, but absent from mouse or bovine NF-M, is indicated by a box. Preparation of tissue extracts, immunoblot analysis, and immunofluorescence staining were performed as described under “Experimental Procedures.” For A,20 g of protein were separated by SDS-PAGE on 7.5% acrylamide. Scale bar,10 m. sion of human material, we aimed to perform NL6 staining on in situ in parallel to phosphorylated NF-L subpopulations. as fresh material as possible. In tissue from human temporal O-Glycosylation of NF-M Is Dynamically Influenced by In- lobe obtained at surgery of epilepsy patients, NL6 immunore- hibitors of GlcNAcase and Mitogen-activated Protein Kinase activity was highly enriched in the subcortical white matter Kinase—O-Glycosylation of cellular proteins appears to be similar to staining with an antibody against phosphorylated highly dynamic similar to phosphorylation (40–42). In some NF-L that is known to be enriched in the axon (2F11; Fig. 4A). instances a direct reciprocity of phosphorylation and O-glyco- Both antibodies labeled radial and horizontal fibers as well as sylation on the same or neighboring serines or threonines was recurrent collaterals in the infragranular layers (layers IV–VI). observed (43, 44). To test the dynamicity of O-glycosylation at In high power magnifications it was evident that NL6 immu- the NL6 epitope, the human neuroblastoma cell line SK-N- noreactivity was exclusively found in axons, whereas 2F11 also BE(2) was treated with the drug PUGNAc, a membrane-per- labeled the perinuclear soma of some neurons (Fig. 4B, aster- meable derivative of N-acetylglucosamine that binds and com- isks). We also performed immunoblot analysis with NL6 and petitively inhibits the cytoplasmic GlcNAcase (45). Immunoblot M20 antibody of temporal lobe tissue lysates from white and analysis of cellular lysates revealed that PUGNAc increased gray matter obtained from 5 different patients (Fig. 4C). Quan- NL6 immunoreactivity relative to total NF-M in a concentration- titation revealed that the ratio of immunoreactivity of white dependent manner with a maximum of 140–150% compared versus gray matter was significantly higher (p  0.001; n  5) with the control (set at 100%) (Fig. 5A). PUGNAc caused a fast with NL6 compared with M20 that recognizes total NF-M (Fig. increase in NL6 immunoreactivity because already after 10 4D), which was well in accordance with the findings from min a significant rise was observed (Fig. 5B). The changes in immunohistochemistry. When autopsy-derived human brain immunoreactivity were fully reversible indicating fast O-glyco- material with considerable postmortem intervals (12–72 h) was sylation and -deglycosylation at the NL6 epitope. Total O- used for immunoblot analysis, no significant enrichment of glycosylation as detected using an antibody that recognizes NL6 immunoreactivity in white matter was observed (data not O-linked GlcNAc independent of protein determinants showed a very similar increase after PUGNAc treatment (Fig. 5C), shown), indicating that O-glycosylation of NF-M in neuronal axons is very sensitive to degradation. The data suggest that suggesting that the dynamic glycosylation/deglycosylation at the NL6 epitope closely reflects changes in the general level of O-glycosylated NF-M as recognized by NL6 is a dynamic mod- ification that is highly enriched in the axons of human neurons O-glycosylation within the cell. 31654 O-Glycosylation of NF-M FIG.4. Distribution of NL6 immuno- reactivity in brain tissue. A, staining of human temporal lobe tissue with NL6 and 2F11. NL6 immunoreactivity is highly enriched in the subcortical white matter similar to 2F11 staining, which detects an axonally enriched phosphoryl- ated NF-H isoform. B, temporal lobe tis- sue stained for NL6 and 2F11 epitopes at higher magnification. NL6 reactivity is restricted to radial and horizontal fibers, whereas staining of perinuclear soma of some neurons is observed with 2F11 (as- terisks), as also shown at high power mag- nifications in the insets. C and D, immu- noblot (C) of gray and white matter of temporal lobe tissue lysates from five dif- ferent patients, and quantitation of the relative ratio (D) for NL6 and M20 immu- noreactivity. The ratio of white versus gray matter is significantly increased for NL6 reactivity compared with M20 (***, p  0.001, n  5; paired Student’s t test), indicating a strong axonal enrichment of the NL6 epitope in situ. Tissue prepara- tion, stainings, and immunoblot analysis were performed as described under “Ex- perimental Procedures.” For C,10or30 g of protein lysates were separated by SDS-PAGE on 7.5% acrylamide. Nuclei in sections A and B were counterstained with hematoxylin. Scale bars,20 m(in- sets of B),1mm(A), and 100 m(B). 2 2 It is known that the KSP repeat region of NF-M is phospho- model where Cu /Zn -superoxide dismutase 1 harboring an G93A rylated by the proline-directed protein kinase ERK1/2 (9), ALS-linked familial genetic mutation (SOD1 ) is overex- which is the effector kinase of the mitogen-activated protein pressed and that develops ALS-like pathology was used (48). (MAP) kinase pathway. Thus, to test for the effect of phospho- NL6 immunoreactivity was strongly reduced in spinal cord rylation in the KSP region, cells were treated with PD-98059, a extracts from mutant animals compared with wild-type rats specific inhibitor of activation of MEK (46). PD-98059 caused (Fig. 6A). The reduction was evident at postnatal day 0 as well 202 204 an about 35% decrease of activated (Thr /Tyr phosphoryl- as after disease onset in end-stage animals that expressed full ated) ERK1/2 relative to total ERK1/2, indicating inhibition of pathology. Also total NF-M reactivity decreased in these ani- MEK and an increase in relative NL6 immunoreactivity (Fig. mals indicating neurofilament loss in axons. To determine the 5D). This suggests that phosphorylation of NF-M in the tail decrease in NL6 immunoreactivity compared with changes in domain dynamically affects O-glycosylation in the same region total NF-M, blots were quantitated and relative ratios of NL6 and is reciprocally related to O-glycosylation at the NL6 and RMO44 reactivity to tubulin from transgenic animals were epitope. Treatment of the cells with the protein phosphatase 2A determined and compared with the respective ratios in wild- inhibitor cyclosporin A (5 nM, 2 h) resulted in an about 25% type animals. At all time points, the reduction in NL6 immu- decrease in relative NL6 immunoreactivity, which is also con- noreactivity was higher than in total neurofilament (Fig. 6B). sistent with a reciprocal O-glycosylation and phosphorylation In particular, in spinal cord material from end-stage animals, (data not shown). Considering these findings and the fact that NL6 immunoreactivity was barely detectable. In contrast, re- neurofilament shows an ongoing process of phosphorylation/ activity for SMI31 (phosphorylated NF-M) was increased com- dephosphorylation in human brain tissue ex vivo, the rapid loss pared with total NF-M at every age analyzed. The data suggest of NL6-reactive epitopes as described above might well be that O-glycosylation at the NL6 epitope is selectively decreased explained by a co-regulated deglycosylation (47). However, it during ALS, whereas phosphorylation is increased. cannot be excluded from these experiments that MEK inhibi- DISCUSSION tion and cyclosporin A treatment exert a systemic effect on NF-M glycosylation that is independent of the phosphorylation Using a cytoskeletal fraction prepared from human neurons of the KSP repeats. as immunogenic material we generated a novel monoclonal O-Glycosylation of NF-M at the NL6 Epitope Is Down-regu- antibody that specifically detects an O-glycosylated epitope in lated in a Transgenic Rat Model of ALS—To determine a po- NF-M. To our knowledge, this is the first antibody that specif- tential pathophysiological role of disturbed O-glycosylation in ically detects this modification in NF-M and one of very few neurodegenerative diseases, an animal model for ALS was antibodies that recognizes O-linked GlcNAc in cellular pro- employed. Because NFs from mouse tissue were not reactive teins. An example for the latter is an antibody that detects with NL6 (Fig. 3A), a previously established transgenic rat O-GlcNAc at threonine 58 in c-Myc protein and that was used O-Glycosylation of NF-M 31655 FIG.5. Regulation of O-glycosylation at the NL6 epitope. A and B, effect of the GlcNAcase inhibitor PUGNAc on the relative ratio of NL6 to M20 reactivity. Incubations were at different concentrations of PUGNAc for1h(A), or at different times at 20 M PUGNAc (B). C, effect of 20 M PUGNAc on total O-glycosylation as determined from the relative ratio of immunoreactivity with an antibody that detects -glycosidically linked GlcNAc and actin. D, effect of the MEK inhibitor PD-98059 on ERK1/2 phosphorylation and the relative ratio of NL6 to M20 reactivity. 202 205 Incubations were for 1 h. Activated (Thr /Tyr phosphorylated) ERK1/2 is decreased relative to total ERK1/2, and reactivity against the NL6 epitope relative to total NF-M is increased after inhibition of MEK. Cell culture of SK-N-BE(2) cells and immunoblot analysis were performed as described under “Experimental Procedures.” For quantitations by Western blot (A, B, and D), 20 g of protein lysates were separated by SDS-PAGE on 7.5% acrylamide. For dot-blot analysis (C), 15 g of protein lysates were immobilized on nitrocellulose. Mean  S.E. are shown. * indicates significant differences to carrier control experiments (p  0.05; n  5; paired Student’s t test). to analyze the interplay between phosphorylation and glycosy- GlcNAc no strict consensus sequence can be discerned yet. lation at this position (49). Several antibodies that more gen- Thus, it cannot be completely excluded that the NL6 antibody erally detect O-GlcNAc-modified proteins have been developed. detects an O-glycosylated epitope outside the KSP repeats. However, most of the antibodies are limited in their use be- We have shown that O-glycosylation of NF-M can quickly cause they only detect part of all modified proteins and require change, is fully reversible, and is affected by changes in protein additional epitopes (50–52). An exception is an antibody that phosphorylation. It is also very sensitive to postmortem/ex vivo has been developed by Comer et al. (35) that detects O-GlcNAc delay in the processing of neuronal tissue. Thus, O-glycosyla- independent from the protein backbone and is very useful to tion of NF-M in neurons appears to be highly dynamic similar determine overall O-GlcNAc modifications (see Figs. 1D and to the rapid turnover of O-GlcNAc-modified proteins after lym- 5C). phocyte activation (40) or the dynamic O-GlcNAcylation of the Many antibodies have been described that detect phospho- small heat shock protein B-crystallin (42). rylated epitopes in NF proteins, mostly in NF-H, which is The level of glycosylation can be very different in various cell heavily phosphorylated with a stoichiometry of about 20 mol of types (45). Thus, also during neuronal development, the cycling phosphate/mol of protein (53). NF-M is much less phosphoryl- enzymes that are responsible for GlcNAcylation may be differ- ated with about 4–9 mol of phosphate/mol of protein. The entially regulated. This would result in a change of the level of epitopes that are detected by phosphorylation-dependent anti- NL6 reactivity relative to total NF-M. We did not observe bodies are located predominantly in the tail region of NF-M and obvious changes during the differentiation of NT2 cells, SK-N- NF-H that contain multiple repeats of lysine-serine-proline BE(2) cells, or SH-SY5Y cells, indicating that the enzymes that (KSP). Especially the serines in KSP repeats appear to be are responsible for the GlcNAc modification are constitutively heavily phosphorylated in axons (54–56). The number and active in neuronal precursor cells and neurons. In agreement, distribution of the repeats strongly differ among vertebrates. strong glycosylation was observed also in non-neuronal cells NF-H contains 42–51 of these repeats. In human NF-M, 12 both of human and mouse origin after transfection of human KSP repeats are located between residues 614 and 689, rat and NF-M sequence. O-Glycosylation could be increased by phar- mouse NF-M contain only 2 KSP repeats in the respective macological inhibition of the deglycosylating enzyme with region. We have mapped the position of the O-glycosylated PUGNAc by 40–50%. Thus, the amount of glycosylation is in a epitope that is detected by the NL6 antibody in or close to the medial range that would allow the cell to react to stimuli with KSP repeat region between residues 604 and 789. This region a change in the glycosylation level. A similar increase after contains 3 predicted sites for O-linked GlcNAc modifications PUGNAc treatment was observed for the O-glycosylation of all with two of them being located in the KSP region and one proteins, indicating that NF-M glycosylation closely reflects 747 747 (Thr ) carboxyl-terminal flanking it. Mutating Thr to ala- the general activity of the cycling enzymes. nine did not affect NL6 immunoreactivity, suggesting that the O-Glycosylation of NF proteins may affect their functional site that is recognized by the antibody is located within the properties. We did not observe a differential stability of NL6- KSP repeats. It should, however, be noted that because of the positive NF-M versus total NF-M to urea-induced disassembly low number of identified sites that are modified by O-linked of NFs (data not shown) and O-glycosylated NF-M incorporated 31656 O-Glycosylation of NF-M FIG.6. Expression of the NL6 epitope in spinal cords of SOD1 transgenic rats. A, immunoblots of spinal cord extracts from wild-type and G93A mutant SOD1 expressing rats at postnatal day 0 (top) and at the end-stage of the disease (bottom). NL6 immunoreactivity is strongly reduced in NF-M from mutant animals compared with wild-type rats. B, relative intensities of the immunoreactivity against total NF-M (RMO44), the NL6 G93A epitope, and phosphorylated NF-M (SMI31) of spinal cord extracts from wild-type and mutant SOD1 expressing rats of different ages. NF-M from mutant animals shows a stronger reduction of immunoreactivity against the NL6 epitope compared with total NF-M at every age analyzed, whereas phosphorylated NF-M is increased. Preparation of spinal cord extracts and immunoblot analysis were performed as described under “Experimental Procedures.” Immunoblots of spinal cord extract from animals with different ages were quantified for NL6, SMI31, and RMO44 signals and normalized to tubulin (monoclonal antibody DM1A). Three animals of the same genotype were analyzed for every time point, and the data for wild-type animals were set as 1.0. Blots were performed at least twice. Mean  S.E. are shown. into reassembled NFs similar to total NF-M. It is known that properties of NFs different from a strongly negatively the head and rod of NF proteins, but not the tail, are essential charged phosphate group that may repel the filaments and for assembly and that phosphorylation of the head region of increase radial growth (54, 60). intermediate filaments affects assembly or disassembly (10, It is known that the tail regions of NF-M and NF-H are 11, 57, 58). Because the NL6 epitope is located in the tail heavily phosphorylated in axons (54–56). In some cases, a region, it is conceivable that O-glycosylation in this region reciprocal relation between phosphorylation and O-glycosyla- does not affect assembly properties but other features that tion (“yin-yang” relationship) has been reported (15, 61). How- are thought to be mediated by the tail region. These could ever, in contrast to what might be expected from these results, include modulation of the radial growth of axons that are also the GlcNAc modification in the tail region shows a strong critically affected by the tail domain of NF-M (4), interactions enrichment in the white matter, which appears to be even more between filaments and other cellular components, and slow pronounced than the distribution of the phosphoepitope in that axonal transport rates that are also thought to be affected by it is completely absent from neuronal cell bodies in situ. Thus, phosphorylation in the tail region (1, 6). Very recent data in white matter, phosphorylation and O-glycosylation of NF-M have implicated a role for NF-M for myelin-directed “outside- are increased in parallel. Dong et al. (62) suggested that O- in” signaling cascades (59). It will be interesting to determine glycosylation of the KSP region in NF-H occurs directly after the effect of O-glycosylation on these features. Because Glc- synthesis before the subunits are incorporated into NFs and NAc is neutral, it is possible that it modulates the surface are transported into the axon. In the axon, GlcNAc would be O-Glycosylation of NF-M 31657 10. Sihag, R. K., and Nixon, R. A. (1990) J. Biol. Chem. 265, 4166–4171 removed and substituted by phosphate groups. At least with 11. Sihag, R. K., and Nixon, R. A. (1991) J. Biol. Chem. 266, 18861–18867 respect to NF-M our data are not consistent with such an 12. de Waegh, S. M., Lee, V. M., and Brady, S. T. (1992) Cell 68, 451–463 13. Julien, J. P. (1997) Trends Cell Biol. 7, 243–249 hypothesis. In contrast, the data argue for a synchronous phos- 14. Hart, G. W. (1997) Annu. Rev. Biochem. 66, 315–335 phorylation and O-glycosylation of the tail region of NF-M 15. Comer, F. I., and Hart, G. W. (2000) J. Biol. Chem. 275, 29179–29182 within the axon. Despite this synchronicity of phosphorylation 16. Vosseller, K., Sakabe, K., Wells, L., and Hart, G. W. (2002) Curr. Opin. Chem. Biol. 6, 851–857 and O-glycosylation, inhibition of MEK with PD-98059 in- 17. Shafi, R., Iyer, S. P., Ellies, L. G., O’Donnell, N., Marek, K. W., Chui, D. H., creased O-GlcNAcylation at the NL6 epitope, suggesting a pos- Hart, G. W., and Marth, J. D. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5735–5739 sible reciprocal regulation between phosphorylation and O- 18. Slawson, C., and Hart, G. W. (2003) Curr. Opin. Struct. Biol. 13, 631–636 glycosylation in the KSP region. Whether such a reciprocal 19. Dong, D. L., Xu, Z. S., Chevrier, M. R., Cotter, R. J., Cleveland, D. W., and regulation occurs also in vivo remains to be shown. Hart, G. W. (1993) J. Biol. Chem. 268, 16679–16687 20. Piontek, J., Chen, C. C., Kempf, M., and Brandt, R. (1999) J. Neurochem. 73, The development of a polar cytoarchitecture alone was not 139–146 sufficient to cause a compartment-specific enrichment of O- 21. Riederer, B. M., Porchet, R., and Marugg, R. (1996) J. Comp. Neurol. 364, glycosylation because NL6 immunoreactivity was present in all 704–717 22. Piontek, J., and Brandt, R. (2003) J. Biol. Chem. 278, 38970–38979 processes and in the cell body of dissociated human model 23. Faissner, A., and Kruse, J. (1990) Neuron 5, 627–637 neurons (data not shown). It is possible that O-glycosylation in 24. Pleasure, S. J., Page, C., and Lee, V. M. (1992) J. Neurosci. 12, 1802–1815 25. Biedler, J. L., Roffler-Tarlov, S., Schachner, M., and Freedman, L. S. (1978) the axon is ultimately linked to, and may be regulated by, Cancer Res. 38, 3751–3757 myelination as has been suggested for phosphorylation (1). It 26. Todaro, G. J., Habel, K., and Green, H. (1965) Virology 27, 179–185 will be very interesting to determine whether O-glycosylation 27. Scherer, W. F., Syverton, J. T., and Gey, G. O. (1953) J. Exp. Med. 97, 695–709 28. Olmsted, J. B., Carlson, K., Klebe, R., Ruddle, F., and Rosenbaum, J. 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Histo- chem. Cytochem. 41, 609–616 brains (64), NL6 might also become a valuable tool to further 34. Duk, M., Ugorski, M., and Lisowska, E. (1997) Anal. Biochem. 253, 98–102 investigate the pathophysiological role of disturbed O-glycosy- 35. Comer, F. I., Vosseller, K., Wells, L., Accavitti, M. A., and Hart, G. W. (2001) lation in common neurodegenerative diseases. Anal. Biochem. 293, 169–177 36. Hirokawa, N., Glicksman, M. A., and Willard, M. B. (1984) J. Cell Biol. 98, We have shown that in an animal model for ALS, O-glyco- 1523–1536 sylation of NF-M as detected using the NL6 antibody is de- 37. Julien, J. P., and Mushynski, W. E. (1983) J. Biol. Chem. 258, 4019–4025 creased, whereas phosphorylation is increased relative to total 38. Blom, N., Sicheritz-Ponten, T., Gupta, R., Gammeltoft, S., and Brunak, S. (2004) Proteomics 4, 1633–1649 NF-M. Hyperphosphorylation of NF proteins has been de- 39. Lee, V. M., Carden, M. J., Schlaepfer, W. W., and Trojanowski, J. Q. 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Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Sep 9, 2005

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