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J. Biol. Chem., Vol. 280, Issue 36, 31648-31658, September 9, 2005

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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)^*

Nina Lüdemann, Albrecht Clement¶, Volkmar H. Hans||, Julia Leschik, Christian Behl¶, and Roland Brandt**

From the Department of Neurobiology, University of Osnabrück, Barbarastrasse 11, D-49076 Osnabrück, 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

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.

Received for publication, April 21, 2005 , and in revised form, July 8, 2005.

^* This work was supported by Deutsche Forschungsgemeinschaft 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 part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed. Tel.: 49-541-9692338; Fax: 49-541-9692354; E-mail: Brandt{at}biologie.uni-osnabrueck.de.

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