05-348 Sigma-AldrichAnti-Tau Antibody, clone 5E2

Neuropil threads were quantitated in the neuropil (excluding senile plaques) of the superior frontal gyrus of 6 late stage patients with Alzheimer's disease (AD) and 6 age-matched control subjects using tau immunocytochemistry and computerized morphometric image analysis. The mean percent of the area of the neuropil occupied by neuropil threads was 10.6 for AD and 0.19 for controls (p < 1 x 10(-10)). The mean length of neuropil threads in AD was 21.9 mu compared with 19.7 mu for controls (p < 1 x 10(-10)). The mean area of neuropil threads was 25.3 mu 2 for AD and 21.3 mu 2 for controls (p < 1 x 10(-10)). In AD, the threads were most prominent in mid cortex (lamina 2 and 3) and least prominent in the lower cortex (lamina 5 and 6). Neuropil threads appear to lead to severe disorganization of intracortical and corticocortical connectivity and probably play a role in the cognitive failure in AD.

Cytoskeletal disruption is a key pathological feature of Alzheimer's disease (AD). We used refined immunocytochemical techniques to define the range of abnormalities affecting the microtubule system in AD hippocampus. Minimal tau and tubulin immunoreactivity was granular and accumulated in otherwise normal neuronal perikarya. As tau-reactive neurofibrillary tangles formed, granular tau and tubulin staining diminished, and ubiquitin reactivity developed. In regions of high neurofibrillary tangle density, microtubule-associated protein 2 (MAP2) histochemical features of remaining nontangled neurons included apical dendritic degeneration with proliferation of basal dendrites. In addition to perisomatic dendritic proliferation, there was massive sprouting of tau-immunoreactive distal dystrophic neurites. Sprouting proximal dendrites and dystrophic neurites often demonstrated growth-cone-like lamellipodia and filopodia. Degeneration of the perisomatic proliferating dendrites was characterized by the accumulation of fibrillar tau immunoreactivity. The colocalization of MAP2 and tau in growth structures recapitulated their codistribution in developing neurites. The data suggest that extensive plasticity and growth response occur in tandem with neuronal degeneration in AD, and that reorganization of the cytoskeletal microtubule system may underlie these proliferative changes.

Tau protein has been shown to be an integral component of Alzheimer paired helical filaments (PHF). However, the extent to which tau is incorporated into PHF has not been clear because the antibodies used to label PHF generally do not have precisely defined epitopes. Here we define the antigenic sites for five monoclonal antibodies that react with tau and cross-react with SDS-extracted neurofibrillary tangles. The reactive sites were determined by screening a lambda gt11 sublibrary expressing small fragments of the tau sequence. The mapped epitopes were found to span almost the entire length of tau, suggesting that PHF contains tau in its entirety or nearly in its entirety. One antibody was found to cross-react with microtubule-associated protein 2, implying some degree of homology between the two proteins.

The microtubule-associated protein tau, a major antigenic component of paired helical filaments, has been demonstrated in neurofibrillary tangles and in neurites of senile plaques. With optimal fixation and histochemical methods, we show the normal axonal location of tau protein in human cerebral cortex and the striking alterations of tau distribution that affect the cortical neuropil in Alzheimer's disease. Normally, cortical tau-immunoreactive fiber bundles form a pattern resembling that seen with myelin stains. The prominence of white matter staining suggests that tau may be especially enriched in projection systems. Alzheimer's disease causes massive axonal disruption and the dislocation of tau protein from its usual axonal domain into neuronal cell bodies, dendrites, and presynaptic regions. The normal pattern of axonal staining in cortex is disrupted and white matter staining is reduced. Prominent abnormal tau-immunoreactive neuropil fibers are densely present even in cortical regions without classical neurofibrillary tangle and senile plaque formation. The striking neuropil abnormalities, revealed by the aberrant localization of tau protein, are likely to contribute to neuronal dysfunction in Alzheimer's disease.

The neuronal microtubule-associated phosphoprotein, tau, has been identified as a major antigenic component of paired helical filaments in Alzheimer's disease (AD). The extent and distribution of altered tau antigens in AD brain, other than those found in neurofibrillary tangles (NFT) and senile plaque (SP) neurites, has not been widely discussed. We have examined tau immunoreactivity in AD using the monoclonal antibody (MAb), 5E2, raised against human fetal tau. Four types of abnormalities were recognized by MAb 5E2, each having some counterpart in Bielschowsky silver impregnations: 1) NFT; 2) thickened neurites in SP; 3) diffuse perikaryal staining seen in some neurons apparently lacking NFT; and 4) a dispersed network of randomly oriented thickened neurites not clustered into discrete plaques but found in NFT- and SP-rich cerebral cortex. These four alterations could also be recognized using three different polyclonal antibodies which had strong tau immunoreactivity but were optimally shown by MAb 5E2. Our findings demonstrate the complexity of altered tau-immunoreactive neuronal elements and emphasize the widespread abnormality of microtubule-associated proteins in AD cortex.

We sought to determine whether the strict segregation of MAP2 and tau into somatodendritic and axonal compartments in situ was maintained in dissociated neuronal cultures of the rat cerebrum. Cultures grown under serum-free conditions were immunolabeled with monoclonal antibodies specific for MAP2 and tau. At 14 d after plating, a clear distinction between MAP2- and tau-immunoreactive neurites was apparent. MAP2-immunoreactive neurites were relatively short, thick, tapering, and branched. Tau-immunoreactive neurites formed a crisscrossing meshwork of long, fine-caliber neurites, which, in more densely plated cultures, had a tendency to form thick, ropelike fascicles. Unlike the MAP2 pattern, tau antibodies labeled somata only lightly. Since distinct populations of neurites were labeled with the 2 antibodies, we sought to observe the development of the topographically distinct compartments by double-labeled immunocytochemistry with both polyclonal and monoclonal antibodies to MAP2 and tau. Cells observed within the first 8 hr after plating demonstrated equally intense MAP2 and tau immunoreactivity in a coextensive distribution throughout the cell body and initial neurites. By 16 hr, some neurites began to assume dendritic and axonal features; however, many such processes contained reaction product for both MAP2 and tau. Beginning at this time, neurites that appeared axonal showed a progressively weaker reaction with MAP2 antibodies, and neurites that appeared dendritic showed a progressively weaker reaction with tau antibodies. In most neurites the diminution appeared to occur uniformly over the entire extent of the neurite. During this transformation period there were occasional axon-like neurites that contained MAP2 immunoreactivity proximally, while tau immunoreactivity extended over the entire length of the neurite. We conclude that neurons in culture are able to compartmentalize MAP2 and tau into their appropriate processes and only attain an apparently homogeneous population of one of these MAPs after the neuron has assumed dendritic and axonal features. The analysis also lends indirect support to the hypothesis that microtubule-associated proteins (MAPs) form this association at the distal extent of the growing neurite.