Missense mutations in the presenilin 1 (PS1) gene cause the most common form of dominant early-onset familial Alzheimer's disease (FAD) and are associated with increased levels of amyloid beta-peptides (A beta) ending at residue 42 (A beta 42) in plasma and skin fibroblast media of gene carriers. A beta 42 aggregates readily and appears to provide a nidus for the subsequent aggregation of A beta 40 (ref. 4), resulting in the formation of innumerable neuritic plaques. To obtain in vivo information about how PS1 mutations cause AD pathology at such early ages, we characterized the neuropathological phenotype of four PS1-FAD patients from a large Colombian kindred bearing the codon 280 Glu to Ala substitution (Glu280Ala) PS1 mutation. Using antibodies specific to the alternative carboxy-termini of A beta, we detected massive deposition of A beta 42, the earliest and predominant form of plaque A beta to occur in AD (ref. 6-8), in many brain regions. Computer-assisted quantification revealed a significant increase in A beta 42, but not A beta 40, burden in the brains from 4 PS1-FAD patients compared with those from 12 sporadic AD patients. Severe cerebellar pathology included numerous A beta 42-reactive plaques, many bearing dystrophic neurites and reactive glia. Our results in brain tissue are consistent with recent biochemical evidence of increased A beta 42 levels in PS1-FAD patients and strongly suggest that mutant PS1 proteins alter the proteolytic processing of the beta-amyloid precursor protein at the C-terminus of A beta to favor deposition of A beta 42.

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.

The C-terminus Hsp70 interacting protein (CHIP) has dual function as both co-chaperone and ubiquitin ligase. CHIP is increasingly implicated in the biology of polyglutamine expansion disorders, Parkinson's disease and tau protein in Alzheimer's disease. We investigated the involvement of CHIP in the metabolism of the beta-amyloid precursor protein and its derivative beta-amyloid (Abeta). Using immunoprecipitation, fluorescence localization and crosslinking methods, endogenous CHIP and betaAPP interact in brain and cultured skeletal myotubes as well as when they are expressed in stable HEK cell lines. Their interaction is confined to Golgi and ER compartments. In the presence of the proteasome inhibitor with MG132, endogenous and expressed betaAPP levels are significantly increased and accordingly, the interaction with CHIP enhanced. Concurrently, levels of Hsp70 were most consistently induced by proteasome inhibition among the various heat shock proteins (HSPs) tested. Thus, complexes of CHIP, Hsp70 and holo-betaAPP (as well as C-terminal fragments) were stabilized by the action of MG132. Moreover, CHIP itself is shown to both increase cellular holo-betaAPP levels and protect it from oxidative stress and degradation. Interestingly, CHIP also promotes the association of ubiquitin with betaAPP, implying that a smaller pool of betaAPP is destined for proteasomal processing. In neuronal cultures, CHIP and Hsp70/90 expression reduce steady-state cellular Abeta levels and hasten its degradation in pulse-chase experiments. The functional significance of CHIP and HSP interactions, especially with Hsp70, was tested using siRNA and in neuronal cells where protection from Abeta-induced toxicity is shown. We conclude that CHIP, as a bimolecular switch, interacts with HSP to stabilize normal holo-betaAPP on the one hand while also assisting in the ubiquitination of a subpopulation of betaAPP molecules that are destined for proteasome degradation. CHIP also hastens the clearance of Abeta in a manner consistent with its known neuroprotective properties.

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