We have isolated cDNA clones encoding a 383‐amino acid isoform of the human microtubule‐associated protein tau. It differs from previously determined tau sequences by the presence of an additional repeat of 31 amino acids, giving four, rather than three, tandem repeats in its carboxy‐terminal half. The extra repeat is encoded by a separate exon. Probes derived from cDNA clones encoding the three (type I) and four repeat (type II) tau protein isoforms detected mRNAs for both forms in all adult human brain areas examined. However, in foetal brain only type I mRNA was found. Type I and type II mRNAs were present in pyramidal cells in cerebral cortex. In the hippocampal formation, type I mRNA was found in pyramidal and granule cells; type II mRNA was detected in most, though not all, pyramidal cells but not in granule cells. These observations indicate that tau protein mRNAs are expressed in a stage‐ and cell‐specific manner. Tau protein is found in the protease‐resistant core of the paired helical filament, the major constituent of the neurofibrillary tangle in Alzheimer's disease. Taken in conjunction with previous findings, the present results indicate that both the three and four repeat‐containing tau protein isoforms are present in the core of the paired helical filament.

Aging of the brain involves not only appreciable shrinkage of the cortex and other gray matter structures but above all loss of white matter. This could be due to a decline in the number of myelinated fibers or to a loss of water. To assess the role played by each of these factors we studied brains from 33 neurologically intact subjects at autopsy representing three different age groups: 15-50, 51-70, and 71-93 years. The precentral gyrus, gyrus rectus, and corpus callosum were selected for investigation, with staining for alkaline phosphatase on native cryostat sections to visualize the capillary network, and staining for myelin on semithin sections for nerve fiber visualization. Atrophy was objectified by measuring the number of capillaries, the intercapillary distance, and capillary length, since the capillary network remains constant throughout normal life. A mean difference of 16-20% was found, representing white matter atrophy, between the oldest and youngest age-groups. The cortex of the corresponding gyri, on the other hand, showed a difference of less than 6%. Morphometric investigation of sections stained for myelin showed that the brains with a mean age of 78.7 +/- 6.6 years had 10-15% fewer myelinated fibers. This was only partly offset by an increase in the volume of extracellular space. Our findings show that the age-related decline in brain volume is much more a question of white matter atrophy than of brain cortex atrophy. White matter atrophy could be an indirect indicator of nerve cell loss, since the volume of a nerve cell is much smaller than its myelinated fiber.

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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