Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.

Abstract

The immunocytochemical staining patterns of 37 neuron-specific monoclonal antibodies previously described fell into four groups: (i) anti-synapse-associated, (ii) anti-neurofibrillar, (iii) anti-perikaryonal-neurofibrillar, and (iv).a single antibody reactive with a widely distributed epitope that covered the patterns of groups ii and iii. Antibodies of groups ii, iii, and iv were shown to be specific to neurofilament triplet subunits, even though there was little overlap in staining patterns between groups ii and iii. We examined nine of these antibodies as to their ability to distinguish functional states of neurofilaments dependent upon phosphorylation. Upon digestion with phosphatase, electroblot staining of neurofilament components was abolished with the five antibodies from group ii, enhanced with the three antibodies from group iii, and unaffected with antibody iv. Immunocytochemical staining of Bouin-fixed paraffin sections of rat brain was unaffected by phosphatase pretreatment. With antibodies of group ii, digestion with trypsin also left staining unaffected, but when followed by digestion with phosphatase, staining was diminished with three out of five antibodies. In contrast, digestion with trypsin abolished all staining with each antibody from group iii. If followed by digestion with phosphatase, staining reappeared, but the group iii pattern was replaced by a group ii pattern. Staining of this pattern was again abolished upon a second treatment with trypsin. The antibody from group iv lost most of its groups ii and iii staining patterns when sections were digested with trypsin. The group ii pattern reappeared and, indeed, was enhanced upon a subsequent phosphatase treatment and was reduced again upon a second trypsin treatment. Staining by four out of five antibodies from group ii was inhibited by inorganic phosphate. The data indicate that certain nerve cell bodies, their dendrites, and at least proximal axons possess nonphosphorylated neurofilaments and that long fibers, including terminal axons, possess phosphorylated neurofilaments. We propose that phosphorylation may be a factor in stabilizing compacted forms of neurofilaments and that heterogeneity of the compacted structures may play a role in a possible multiplicity of function within individual nerve cells.Out of 135 monoclonal antibodies previously obtained upon immunization with hypothalamus, 37 were neuron-specific and delineated heterogeneous antigens (1, 2). Their general staining distribution permitted classification into four groups: (i) an anti-synapse-associated group that stained gray matter to the exclusion of the interior of cell bodies or discernible nerve fibers and reacted with isolated synaptosomes; (ii) an anti-neurofibrillar group that generally stained white and gray matter fibers, basket cell fibers in the cerebellum, and transverse fibers in the cerebral cortex, but never any cell bodies or their proximal axons or dendrites; (iii) an anti-perikaryonal-neurofibrillar group that stained selected perikarya, their...

Localisation of Microtubule‐Associated Protein 1B Phosphorylation Sites Recognised by Monoclonal Antibody SMI‐31

“…Our data also indicate a proline-directed serine kinase is responsible for generating the mAb SMI-31 epitope on MAP lB. mAb SMI-31 also recognises phosphorylation epitopes on tau and the high and intermediate weight proteins of neurofilaments (Sternberger and Sternberger, 1983;Lichtenberg-Kraag et al, 1992;Ulba et al, 1993) and these epitopes contain serine! proline sites (Lichtenberg-Kraag et al, 1992;Doroudchi and Durham, 1996).…”

Regional modulation of neurofilament organization by myelination in normal axons

“…Myelination correlates with neurojilament phosphorylation and spacing Previous studies have found low levels of phosphorylated neurofilament immunoreactivity in the perikarya, dendrites, and short initial segments, and usually have found that phosphorylation is high within the axons (Stemberger and Stemberger, 1983;Lee et al, 1986Lee et al, , 1987Oblinger, 1987). Because the initial segment is very short in the neurons, it was not clear whether increased phosphorylation is an intrinsic feature of axon per se, or whether it is influenced by the presence of the myelination.…”

Tau gene transfer, but not alpha-synuclein, induces both progressive dopamine neuron degeneration and rotational behavior in the rat

“…They did not colocalize with tau filaments (not shown), but neurons with NFTs did show abnormal compartmentalization of neurofilament epitopes. Specifically, there was aberrant phosphorylated neurofilament in neuronal perikarya and non-phosphorylated neurofilaments (Sternberger and Sternberger, 1983). …”

“…Specifically, there was aberrant phosphorylated neurofilament in neuronal perikarya and non-phosphorylated neurofilaments in myelinated axons (not shown). Non-phosphorylated neurofilaments are usually compartmentalized to neuronal cell bodies and phosphorylated neurofilaments to axons (Sternberger and Sternberger, 1983). …”