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...

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The unlabeled peroxidase-antiperoxidase method has been used with antiserum against "myelin-associated glycoprotein" to establish the presence of the glycoprotein in myelin and myelin-forming cells of the developing rat nervous system. Myelin-associated glycoprotein is found in oligodendroglial cytoplasm before the beginning of myelination. MATERIALS AND METHODS MAG was purified from myelin isolated from the brains of 200-to 250-g Osborne-Mendel rats. The first step in MAG purification was lithium diiodosalicylate extraction of the myelin fraction and subsequent partitioning of the extract with phenol as described (7). The glycoprotein preparation obtained by this procedure was dissolved in a solution containing 2% (wt/vol) sodium dodecyl sulfate, 1.5% (wt/vol) dithiothreitol, and 8% (wt/vol) sucrose for application to a preparative 5% polyacrylamide gel. The procedure for preparative electrophoresis and extraction of MAG from appropriate gel slices was as described (8). The glycoprotein extract was lyophilized, suspended in a small volume of saline, and emulsified with an equal volume of complete Freund's adjuvant for rabbit immunization.The emulsion containing about 0.5 mg of MAG was injected intradermally in multiple sites on the rabbit's back. Similar injections were repeated twice at 3-week intervals, except that the final injection utilized incomplete Freund's adjuvant. Also, a control rabbit was injected on the same time schedule with analogous extracts of preparative polyacrylamide gels to which no glycoprotein samples were applied.A double antibody radioimmunoassay was used for measuring the level of antibodies to the glycoprotein. The MAG antigen used in the assay was purified by the lithium diiodosalicylate/phenol procedure (7) after radiolabeling in vivo by injecting adult rats intracranially with [3H]fucose or 13-day-old rats with [14C]fucose (1). The incubation mixture contained 1 ltg of MAG (1500 dpm 3H or 900 dpm 14C) and 10 ,.d of normal rat serum and was made up to a total volume of 150 ,l with phosphate-buffered saline (pH 7.4). The serum to be tested for MAG antibodies was added as 20 pl of a 1:20 dilution in phosphate-buffered saline, and the assay tubes were incubated for 1 hr at 370C. Then, 100 AI of goat anti-rabbit IgG (Cappel Laboratories, Cochranville, PA) was added, and the tubes were incubated for another 30 min at 370C followed by 1 hr at 4VC. The mixtures were centrifuged at 2000 X g for 30 min, and the precipitates were washed with phosphate-buffered saline. The precipitates were dissolved with 1% sodium dodecyl sulfate and NCS solubilizer (Amersham) for liquid scintillation counting.Newborn to adult Osborne-Mendel rats were anesthetized with chloral hydrate and fixed by intracardiac perfusion for 10 min with a solution containing 76 ml of HgCI2 (saturated at 0'C) and 20 ml of 37% (vol/vol)

Alzheimer tangles, despite their location in neuronal perikarya, react immunocytochemically with monoclonal antibodies to phosphorylated epitopes of neurofilaments. Normal perikarya do not contain phosphorylated neurofilaments. The aberrant phosphorylation in both plaques and tangles seems to be largely restricted to individual phosphorylation sites among the many sites available in neurorilaments. It is suggested that the Alzheimer lesion involves an imbalance within specific kinases responsible for phosphorylation of different sites in neurofilaments. Subsequent studies have shown (3) that the macroheterogeneity was posttranslational and depended on phosphorylation. Thus, antibodies from group II reacted exclusively with phosphorylated neurofilaments; those ofgroup III, with nonphosphorylated epitopes in neurofilaments that are masked by phosphorylation; and antibodies from group IV, apparently with a more accessible, nonphosphorylated neurofilament epitope. In tissue sections, trypsin or phosphatase treatment alone had no effect on the immunocytochemical staining by antibodies from group II. However, trypsin followed by phosphatase reduced the staining. Trypsin treatment abolished the staining by antibodies from group III. However, the staining with these antibodies reappeared by subsequent phosphatase treatment but was converted to axonal staining-i.e., from a group III to a group II pattern. The data permitted the conclusions that neurofilaments in dendrites, perikarya, and proximal axons are nonphosphorylated and that phosphorylation occurs during transport along the axon. Furthermore, it was apparent that phosphorylated neurofilaments were more compact than nonphosphorylated forms.Alzheimer tangles are perikaryonal constituents. They have been shown by Selkoe et al. (4) to differ from normal neurofilaments in their resistance to solubilization by even extensive treatment with sodium dodecyl sulfate and, thus, can be considered highly compacted structures at least with regard to tertiary conformation. In contrast, normal perikaryonal neurofilaments, which are not phosphorylated, seem, according to our data, of noncompact configuration. It appeared, therefore, of interest to study phosphorylation of Alzheimer tangles and plaques and to examine the compactness of these structures with regard to susceptibility to dephosphorylation. MATERIALS AND METHODSThis study includes two cases of Alzheimer disease, one case of Down syndrome, and a case of cerebral infarct. The first three cases exhibited progressive dementia and revealed, on autopsy, severe changes of the Alzheimer type in hippocampus and neocortex. The last case had only few changes of the Alzheimer type.Paraffin sections were stained immunocytochemically (5) by using monoclonal first-layer antibodies, goat anti-mouse 4274 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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