Normal processing of the amyloid beta protein precursor (beta APP) results in secretion of a soluble 4-kilodalton protein essentially identical to the amyloid beta protein (A beta) that forms insoluble fibrillar deposits in Alzheimer's disease. Human neuroblastoma (M17) cells transfected with constructs expressing wild-type beta APP or the beta APP717 mutants linked to familial Alzheimer's disease were compared by (i) isolation of metabolically labeled 4-kilodalton A beta from conditioned medium, digestion with cyanogen bromide, and analysis of the carboxyl-terminal peptides released, or (ii) analysis of the A beta in conditioned medium with sandwich enzyme-linked immunosorbent assays that discriminate A beta 1-40 from the longer A beta 1-42. Both methods demonstrated that the 4-kilodalton A beta released from wild-type beta APP is primarily but not exclusively A beta 1-40. The beta APP717 mutations, which are located three residues carboxyl to A beta 43, consistently caused a 1.5- to 1.9-fold increase in the percentage of longer A beta generated. Long A beta (for example, A beta 1-42) forms insoluble amyloid fibrils more rapidly than A beta 1-40. Thus, the beta APP717 mutants may cause Alzheimer's disease because they secrete increased amounts of long A beta, thereby fostering amyloid deposition.

Alzheimer's disease is the most common cause of progressive intellectual failure. The lesions that develop, called senile plaques, are extracellular deposits principally composed of insoluble aggregates of beta-amyloid protein (A beta), infiltrated by reactive microglia and astrocytes. Although A beta, and a portion of it, the fragment 25-35 (A beta (25-35)), have been shown to exert a direct toxic effect on neurons, the role of microglia in such neuronal injury remains unclear. Here we report a synergistic effect between A beta and interferon-gamma (IFN-gamma) in triggering the production of reactive nitrogen intermediates and tumour-necrosis factor-alpha (TNF-alpha) from microglia. Furthermore, using co-culture experiments, we show that activation of microglia with IFN-gamma and A beta leads to neuronal cell injury in vitro. These findings suggest that A beta and IFN-gamma activate microglia to produce reactive nitrogen intermediates and TNF-alpha, and this may have a role in the pathogenesis of neuronal degeneration observed in ageing and Alzheimer's disease.

Putative Alzheimer disease (AD)-specific proteins (A68) were purified to homogeneity and shown to be major subunits of one form of paired helical filaments (PHFs). The amino acid sequence and immunological data indicate that the backbone of A68 is indistinguishable from that of the protein tau (tau), but A68 could be distinguished from normal human tau by the degree to which A68 was phosphorylated and by the specific residues in A68 that served as phosphate acceptors. The larger apparent relative molecular mass (Mr) of A68, compared to normal human tau, was attributed to abnormal phosphorylation of A68 because enzymatic dephosphorylation of A68 reduced its Mr to close to that of normal tau. Moreover, the LysSerProVal motif in normal human tau appeared to be an abnormal phosphorylation site in A68 because the Ser in this motif was a phosphate acceptor site in A68, but not in normal human tau. Thus, the major subunits of a class of PHFs are A68 proteins and the excessive or inappropriate phosphorylation of normal tau may change its apparent Mr, thus transforming tau into A68.

No abstract

The microtubule-associated protein tau is hyperphosphorylated in the paired helical filaments (PHFs) of Alzheimer's disease. Immunological and direct chemical studies have identified Ser396 and Ser404 as two of the phosphorylated sites. Previously, we have demonstrated, using synthetic tau peptides containing phosphorylated Ser396, that this site is recognized by the monoclonal antibody PHF-1. The present study extends this observation by showing that PHF-1 recognizes tau peptides containing either individually phosphorylated Ser396 or Ser404, but that there is a > 10-fold increase in the sensitivity of detection of tau peptides by PHF-1 when both serines are phosphorylated. The recognition of singly or doubly phosphorylated Ser396 and Ser404 in tau by PHF-1 can also be demonstrated in Chinese hamster ovary cells transfected with full-length wild-type tau constructs or mutant constructs with Ala substituted for Ser396 or Ser404. We conclude that the PHF-1 epitope contains both phosphorylated Ser396 and Ser404.