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).
Mammalian neurofilaments (NFs) are modified by post-translational modifications that are thought to regulate NF assembly and organization. Whereas phosphorylation has been intensely studied, the role of another common modification, the attachment of O-linked N-acetylglucosamine (GlcNAc) to individual serine and threonine residues, is hardly understood. We generated a novel monoclonal antibody that specifically recognizes an O-glycosylated epitope in the tail domain of NF-M and allows determination of the glycosylation state at this residue. The antibody displays strong species preference for human NF-M, shows some reactivity with rat but not with mouse or bovine NF-M. By immunohistochemistry and Western blot analysis of biopsy-derived human temporal lobe tissue we show that immunoreactivity is highly enriched in axons parallel to hyperphosphorylated NFs. Treatment of cultured neurons with the GlcNAcase inhibitor PUGNAc causes a 40% increase in immunoreactivity within 1 h, which is completely reversible and parallels the total increase in cellular O-GlcNAc modification. Treatment with the mitogen-activated protein kinase kinase inhibitor PD-98059 leads to a similar increase in immunoreactivity. In spinal cord tissue of a transgenic rat model for amyotrophic lateral sclerosis, immunoreactivity is strongly decreased compared with wild-type animals while phosphorylation is increased. The data suggest that hyperphosphorylation and tail domain O-glycosylation of NFs are synchronously regulated in axons of human neurons in situ and that O-glycosylation of NF-M is highly dynamic and closely interweaved with phosphorylation cascades and may have a pathophysiological role.
Lüdemann
Nina
N
Department of Neurobiology, University of Osnabrück, Barbarastrasse 11, D-49076 Osnabrück.
Clement
Albrecht
A
Hans
Volkmar H
VH
Leschik
Julia
J
Behl
Christian
C
Brandt
Roland
R
D000118:Acetylglucosaminidase / Q000037:antagonists & inhibitors; D000595:Amino Acid Sequence; D000690:Amyotrophic Lateral Sclerosis / Q000378:metabolism*; D000818:Animals; D030801:Animals, Genetically Modified; D000911:Antibodies, Monoclonal; D001369:Axons / Q000378:metabolism; D002417:Cattle; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D000939:Epitopes / Q000276:immunology; D006031:Glycosylation; D006801:Humans; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D016900:Neurofilament Proteins / Q000276:immunology / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D013116:Spinal Cord / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D016900:Neurofilament Proteins; C061195:neurofilament protein M; D020929:Mitogen-Activated Protein Kinase Kinases; D000118:Acetylglucosaminidase