Localization of the O-GlcNAc transferase and O-GlcNAc-modified proteins in rat cerebellar cortex.
O-linked N-acetylglucosamine (O-GlcNAc) is a ubiquitous nucleocytoplasmic protein modification that has a complex interplay with phosphorylation on cytoskeletal proteins, signaling proteins and transcription factors. O-GlcNAc is essential for life at the single cell level, and much indirect evidence suggests it plays an important role in nerve cell biology and neurodegenerative disease. Here we show the localization of O-GlcNAc Transferase (OGTase) mRNA, OGTase protein, and O-GlcNAc-modified proteins in the rat cerebellar cortex. The sites of OGTase mRNA expression were determined by in situ hybridization histochemistry. Intense hybridization signals were present in neurons, especially in the Purkinje cells. Fluorescent-tagged antibody against OGTase stained almost all of the neurons with especially intense reactivity in Purkinje cells, within which the nucleus, perikaryon, and dendrites were most intensely stained. Using immuno-electron microscopic labeling, OGTase was seen to be enriched in euchromatin, in the cytoplasmic matrix, at the nerve terminal, and around microtubules in dendrites. In nerve terminals, immuno-gold labeling was observed around synaptic vesicles, with the enzyme more densely localized in the presynaptic terminals than in the postsynaptic ones. Using an antibody to O-GlcNAc, we found the sugar localizations reflected results seen for OGTase. Collectively, these data support hypothesized roles for O-GlcNAc in key processes of brain cells, including the regulation of transcription, synaptic vesicle secretion, transport, and signal transduction. Thus, by modulating the phosphorylation or protein associations of key regulatory and cytoskeletal proteins, O-GlcNAc is likely important to many functions of the cerebellum.
Akimoto
Yoshihiro
Y
Department of Anatomy, Kyorin University School of Medicine, Mitaka, 181-8611, Tokyo, Japan.
Comer
Frank I
FI
Cole
Robert N
RN
Kudo
Akihiko
A
Kawakami
Hayato
H
Hirano
Hiroshi
H
Hart
Gerald W
GW
D000117:Acetylglucosamine / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002525:Cerebellar Cortex / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D008297:Male; D016253:Microscopy, Immunoelectron; D008954:Models, Biological; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000276:immunology / Q000378:metabolism; D011689:Purkinje Cells / Q000378:metabolism / Q000648:ultrastructure; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure
D021122:Protein Subunits; D012333:RNA, Messenger; D011994:Recombinant Proteins; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; D000117:Acetylglucosamine