Barney E. Miller

Phosphorylated tau epitope of Alzheimer's disease is coupled to axon development in the avian central nervous system

The monoclonal antibody PHF-1 recognizes phosphorylated tau isoforms present in paired helical filaments of Alzheimers disease. We have found that PHF-1 immunoreactivity is present in chick brain, which expresses three major PHF-1-reactive proteins at the same molecular weights seen in humans. The developmental pattern of expression suggests a functional role in differentiation, rather than in programmed nerve cell death. Expression of PHF-1 immunoreactivity in developing retina was highly cell selective, showing robust staining of ganglion cells, the only long-axon neuron of the seven major retina cell types. The majority of ganglion cells were PHF-1 positive. The developmental window of expression extended at least from E6 through P0, well outside the period of embryonic ganglion cell death. Mature cells did not show PHF-1 immunoreactivity. In the embryo, staining was particularly robust in ganglion cell axons (optic fiber layer), and association of PHF-1 reactivity with axonal tracts also was seen in developing forebrain. PHF-1 polarization occurred at ages when staining with polyclonal anti-tau did not show axonal selectivity. Similarly, in cell cultures, PHF-1 immunoreactivity became localized to single neurites, but polyclonal anti-tau did not. These results indicate that, rather than being associated with cell degeneration, PHF-1 immunoreactivity in the developing nervous system is associated with early stages of axon formation, both in vivo and in vitro. Therefore, expression of PHF-1 immunoreactive proteins in Alzheimers disease suggests that paired helical filament formation might be triggered by mechanisms related to axon regeneration.

Detection of amyloid beta protein precursor immunoreactivity in normal and Alzheimer's disease cerebrospinal fluid

Abstract The amyloid A4 (or beta protein), a 4.2 kD polypeptide, is a major component of amyloid deposits in the brains of patients with Alzheimers Disease (AD). The self-aggregating amyloid A4 protein of AD is encoded as part of three larger proteins by the amyloid A4 precursor gene. The corresponding proteins have 695, 751 and 770 amino acid residues. To investigate the utility of amyloid beta protein precursor (AβPP) as a diagnostic marker for AD an antiserum against a synthetic peptide (175–186), predicted from cDNA sequence for AβPP, was used. The immunoreactivity of AβPP in normal and AD cerebrospinal fluid (CSF) was measured by Western blot and detected with radiolabeled protein A. A total of fifty-seven CSF samples (AD=27 and normal=30) were analyzed for AβPP immunoreactivity. A polyclonal antibody detected two major protein bands with apparent molecular weights of 105kD and 90kD both in normal and AD CSF. The difference between normal and AD CSF was not significant. These results indicate that immunoreactivity of AβPP is present both in normal and AD CSF, and that the difference is too small to be used as a diagnostic marker.

Cathepsin D from Alzheimer's-Diseased and Normal Brains

An acid protease activity from human brain was found to cleave a fluorogenic peptide substrate encompassing the amino terminus of Alzheimers amyloid-beta peptide (A beta). The protease was isolated and determined to be cathepsin D based on chromatographic, immunological, and enzymatic data. Analysis of the cleavage sites indicated that cathepsin D hydrolyzed the methionine--aspartate bond generating the in vivo amino terminus of A beta. These data suggested that cathepsin D could be involved in amyloidogenic processing of the amyloid precursor protein. Consequently, cathepsin D from both Alzheimers-diseased and control brains was compared to determine whether there were any differences which could account for an increase in A beta production in Alzheimers disease. No differences were detected in isoform composition or tissue content of cathepsin D as measured by 2-D IEF-SDS-PAGE. Enzymological characterization of brain cathepsin D demonstrated that it could undergo a previously undescribed pH-dependent reversible activation. However, that activation appeared identical for both AD and normal brain enzymes. These data demonstrate that concentration, isoform distribution, and several enzymological characteristics of cathepsin D are not distinguishable between AD and normal brain. The pH dependence of cathepsin D activity suggests, however, that its intracellular localization may be important in considering the potential role of cathepsin D in Alzheimers disease.

Recent advances in the neurochemical pathology of Alzheimer's disease. Studies of neuropeptides, cholinergic function and Alzheimer's disease-associated protein.

Our findings from three postmortem tissue studies in Alzheimers disease (AD) are presented. We investigated (1) alterations in somatostatin (SRIF) and corticotropin-releasing factor (CRF) in AD; (2) regulatory changes in presynaptic cholinergic function in AD; and (3) use of Alzheimers disease-associated protein (ADAP) as a diagnostic test for AD in postmortem tissue. Taken together, these findings reveal marked reductions in SRIF and CRF concentrations in many cerebrocortical areas in AD, a marked up-regulation of cholinergic neuronal activity in surviving cholinergic neurons in AD, and excellent specificity and sensitivity for the use of the ADAP assay as a diagnostic test for AD in postmortem tissue.

Immunochemical characterization of a monoclonal antibody specific for Alzheimer's disease associated protein

In this study, the monoclonal antibody PHF-1 which recognizes epitopes unique to Alzheimers disease associated proteins (ADAP) has been characterized. Crossed affinity immunoelectrophoresis was used to estimate the binding constant for the interaction of PHF-1 with ADAP and to estimate the fraction of PHF-1 reactive protein. The binding constant of PHF-1 was determined to be 1.3 x 10(-8) M. Furthermore, the effect of dephosphorylation on the electrophoretic pattern of the PHF-1 reactive protein and the ensuing changes in its immunoreactivity were demonstrated.