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To test the hypothesis that tau-immunoreactive curly fibers represent a regenerative attempt of cortical neurons in the brain affected with Alzheimer's disease (AD), we established 2 monoclonal antibodies to isolated neurofibrillary tangles. Both antibodies extensively stained curly fibers and specifically labeled a novel protein of Mr 70,000 (p70), which was abundantly expressed in fetal brain, but undetectable in adult brain. Furthermore, the antibodies visualized curly fibers in newborn rat brain, which were very similar to those in the AD brain, and also intensely stained extending neurites of NGF-primed PC 12 cells. These observations suggest that P70 is a fetal antigen associated with growing neurites, and thus support the sprouting hypothesis.

Hirano bodies are intraneuronal inclusions whose frequency increases with age and Alzheimer's disease. These paracrystalline inclusions have been shown previously using immunocytochemistry to share epitopes with actin, tropomyosin, alpha-actinin and vinculin. Hirano bodies have not previously been demonstrated to share components with neurofibrillary tangles, another intraneuronal inclusion characteristic of Alzheimer's disease. In this study, we show that Hirano bodies bind antibodies to the microtubule-associated protein tau, a component of Alzheimer neurofibrillary tangles.

We have utilized monoclonal antibodies to investigate the antigenic diversity of MAP2-immunoreactive proteins in the nervous system of vertebrates. We found that domains defined by the monoclonal antibodies differed in their conservation across vertebrate evolution, ranging from wide cross-reactivity with almost all vertebrates (mammals, birds, reptiles and amphibians) to a very limited cross-reactivity with only few mammalian species. However, we did not find MAP2-immunoreactive proteins in fish species with either of the monoclonal or polyclonal antibodies. There was also a significant divergence in the apparent molecular weight of MAP2, even in closely related species. For example, different species of wild mice and strains of laboratory mice showed variations of up to 30 kDa in their apparent molecular mass. Using alkaline phosphatase, under conditions that dephosphorylate neurofilaments, we showed that the observed heterogeneity was not the result of variations in the phosphate content. The heterogeneity in molecular weight of MAP2 may, therefore, be the result of changes in primary structure, transcriptional variations or different post-translational modifications. The heterogeneity of MAP2, as well as its specific distribution and implicated interactions with other molecules, underscore the complexity of MAP2 and its potential for structural and functional diversity. The phylogenic analysis of such a complex molecule also provides a method to establish the uniqueness of monoclonal antibodies and the degree of their conservation for their corresponding epitopes.

We used a monoclonal antibody to study the immunocytochemical distribution of microtubule-associated protein 2 (MAP 2) in human fetal neocortex between the ages of 16 and 22 weeks gestation. The staining pattern was lamina-specific. Neuronal somata and dendrites in all cortical layers and in the intermediate zone were labelled. Cajal-Retzius cells of layer I, large pyramidal neurons in the inner cortical plate and neurons in the subplate were most strongly immunoreactive. Separate from the underlying cortical plate a thin sheet of small neurons in the inner marginal zone was highlighted by MAP 2 immunoreactivity. The morphologic diversity, density and regional distribution of the interstitial neurons in the subplate was emphasized by MAP 2 staining. In general, the intensity of MAP 2 immunoreactivity in cell somata and dendrites correlated with the degree of neuronal differentiation but the pattern of intracellular staining also varied as a function of laminar position, and presumably cell type.

The developing neocortex in mice from embryonic day 13 (E13) until birth (E19) was immunoreacted with a monoclonal antibody for microtubule-associated protein 2 (MAP2) that is highly specific for neuronal somata and dendrites. In E13 neocortex there was no detectable MAP2 immunoreactivity on tissue sections or on gel blots. From E14 to birth the MAP2 immunoreactivity was present in both tissue sections and immunoblots of homogenized cortex. In the neocortex the staining pattern was lamina-specific. The molecular layer and the cortical subplate contained the most dense staining of dendrites and cell somata. The cortical plate showed weak to moderate staining at these ages while the intermediate and ventricular zones were not stained above background control levels. Gel blots correspondingly did not show detectable levels of MAP2 until E14. Ultrastructural data suggest that MAP2 is present in dendrites in each of the laminae. The laminar pattern of MAP2 immunoreactivity may be due to either the higher density of differentiating dendrites in the molecular and subplate layers or to compartmentalization of MAP2 within individual cortical neurons.

Huntington's disease is an autosomal dominant neuronal degeneration characterized by age-related neuronal loss principally affecting caudate and putamen and, to a lesser extent, cerebral cortex. In order to identify selective polypeptide alterations in HD brain, we analyzed unfractionated homogenates and purified neuronal perikarya from striatum and cortex of 12 control and 14 HD brains by gel electrophoresis and immunochemical techniques. SDS polyacrylamide gel electrophoresis (SDS-PAGE) revealed a 3- to 8-fold increase in a 50,000 MW (50K) protein in HD striatal homogenates. Neuronal fractions isolated from the same tissue almost never showed this change. In cortex, 50 K protein was either normal or minimally increased. The increase at 50 K in striatal homogenates was often associated with variable increases of proteins at 40 K and 43 K. No other consistent polypeptide changes in HD brain tissue were found by one-dimensional SDS-PAGE. The increased 40 K protein in HD striatum extracts showed a strong immunoprecipitant line with an antiserum to GFA. This antiserum also produced greater immunofluorescent staining of HD than control striatum. Direct immunostaining of polypeptides in gels demonstrated selective staining of the 50 K, 43 K and 40 K proteins in HD striatum. The pattern was highly similar to that reported by Dahl et al. 6 for glial filament preparations that underwent postmortem proteolysis. We conclude that these polypeptide changes are related to increased glial filaments in affected HD tissue, and that similar protein changes reported in other human neuronal degenerations also reflect secondary astrocytic gliosis rather than the primary gene product.

Microtubule associated protein 2 (MAP2) forms a long outward projection from the microtubule wall and is localized to the microtubule population of neuronal cell bodies and dendrites. In Alzheimer's disease, MAP2 epitopes are shared with the neurofibrillary tangle (NFT). Direct information about the molecular composition of the NFT can be obtained by cloning the genes for protein epitopes shared with NFT. Using two monoclonal antibodies to distinct MAP2 epitopes, one of which recognizes NFT, we screened a complex human fetal brain cDNA expression library constructed in lambda gt11. A 2.4 kilobase (kb) cDNA reacted with both antibodies independently. Northern blot analysis showed hybridization of the clone to a 9 kb RNA specific not only to brain but also to neurons. The DNA sequence of the cDNA will include the information for the MAP2 epitope contained in NFT.

We examined the expression and distribution of microtubule-associated protein 2 (MAP2) during the differentiation in culture of both mouse NB2a neuroblastoma and primary embryonic rat neurons. The differentiation of NB2a cells was induced with retinoic acid (RA) which stimulated the extension of a highly branched neuritic network and dibutyryl cAMP which stimulated the outgrowth of long bipolar or monopolar processes. We found that although monoclonal antibodies to MAP2 stained the cell bodies of control and differentiated cells, only the RA-induced neurites were positive for this antigen. These data support our ultrastructural studies indicating that the RA-induced neurites were dendrite-like and that the dibutyryl cAMP-induced processes were axon-like. Studies on the biosynthesis of MAP2 indicated that RA induced a 2–3-fold increase in MAP2 synthesis in 24 h; however, this effect was transient, with the synthesis of MAP2 in RA-treated cells returning to control level by 72 h. Although biosynthetic studies suggested the synthesis of species at 250–300 kdalton, the major molecular weight form in the neuroblastoma cells was 230 kdalton. Immunocytochemical analysis of primary neurons showed staining of neuronal cell bodies and of short processes, but virtually no staining of the long axon-like processes. The of neuronal cell bodies and processes was evident at all stages of cell differentiation. This finding was corroborated by immunoblots which showed significant amounts of MAP2 throughout cell development. The molecular weight of the immunoreactive material was ca. 300 kdalton in both primary neurons and rat brain. Immunoblots also revealed that embryonic neurons expressed only MAP2B as they differentiated in culture for 14 days. Biosynthesis studies suggested that early in culture there was a modest increase in MAP2 synthesis, but no detectable change was observed thereafter. We concluded therefore that both neuroblastoma cells and primary neurons can differentiate neuritic processes, which show dendritic properties in terms of morphology and preferential distribution of MAP2.