Novel monoclonal antibodies provide evidence for the in situ existence of a nonphosphorylated form of the largest neurofilament subunit.
We have obtained five monoclonal antibodies to the Mr 200,000 neurofilament component (NF200) after immunization with polypeptides purified from enzymatically dephosphorylated bovine neurofilaments. In immunoblots of untreated neurofilament protein and protein from filaments exposed to phosphatase, these antibodies recognize nonphosphorylated or dephosphorylated, but not phosphorylated, forms of NF200. The epitopes recognized by these new monoclonal antibodies reside in the carboxyterminal domain of the NF200 polypeptide as defined by immunoreaction with limited chymotryptic fragments. Immunohistochemical studies of bovine cerebellum, spinal cord, trigeminal ganglion, and trigeminal nerve with these new monoclonal antibodies demonstrate immunoreactivity primarily in neuronal perikarya; axons and dendrites are weakly or infrequently immunostained. After enzymatic dephosphorylation of these tissues, a more extensive distribution of immunoreactivity is seen, especially in axons and dendrites. Immunostaining of cultured rat sympathetic neurons is restricted to cell bodies. These data provide evidence for the in situ existence of NF200 epitopes that are not phosphorylated in some classes of neurons or regions of a neuron, but are modified by phosphorylation in other neurons or neuronal domains. These new monoclonal antibodies are distinctly different from those in a large library (over 100) raised to, and specific for, phosphorylated neurofilament proteins. They are novel tools for probing neurofilament distribution, metabolism, structure, and possibly function.
V M
VM
Lee
M J
MJ
Carden
J Q
JQ
Trojanowski
D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002531:Cerebellum / Q000166:cytology; D002918:Chymotrypsin / Q000378:metabolism; D003599:Cytoskeleton / Q000032:analysis*; D000939:Epitopes / Q000032:analysis; D005455:Fluorescent Antibody Technique; D005724:Ganglia / Q000032:analysis / Q000166:cytology; D006651:Histocytochemistry; D007163:Immunosorbent Techniques; D007382:Intermediate Filaments / Q000032:analysis*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010766:Phosphorylation; D013116:Spinal Cord / Q000166:cytology; D014276:Trigeminal Nerve / Q000032:analysis / Q000166:cytology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D002918:Chymotrypsin