No abstract

To study the metabolism of amyloid ␤ protein (A␤) in Alzheimer's disease, we have developed a new approach for analyzing the profile of soluble A␤ and its variants. In the present method, A␤ and its variants are immunoisolated with A␤-specific monoclonal antibodies. The identities of the A␤ variants are determined by measuring their molecular masses using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The levels of A␤ variants are determined by their relative peak intensities in mass spectrometric measurements by comparison with internal standards of known identities and concentrations. We used this method to examine the A␤ species in conditioned media of mouse neuroblastoma cells transfected with cDNAs encoding wild type or mutant human amyloid precursor protein. In addition to human A␤-(1-40) and A␤-(1-42), more than 40 different human A␤ variants were identified. Endogenous murine A␤ and its variants were also identified by this approach. The present approach is a new and sensitive method to characterize the profile of soluble A␤ in conditioned media and biological fluids. Furthermore, it allows direct measurement of each individual peptide in a peptide mixture and provides comprehensive information on the identity and concentration of A␤ and A␤ variants.Alzheimer's disease (AD) 1 is a progressive neurodegenerative disorder and the most common form of dementia (1). One of the neuropathological features of AD is the presence of amyloid deposits in senile plaques and in blood vessel walls (2, 3). These amyloid deposits are mainly composed of a 4-kDa protein, amyloid ␤ protein (A␤), which contains 39 -43 amino acid residues (4 -6). A␤ is derived from a 695-770-amino acid precursor, amyloid precursor protein (APP), through proteolytic processing (7-9). Since the initial isolation of A␤ from amyloid deposits (4), various forms of A␤ peptides have been reported. Both NH 2 -terminally (5, 10 -13) and COOH-terminally (9, 14) truncated A␤ peptides have been isolated and identified either from plaque cores (11), from neurofibrillary tangles (15), or from cerebrovascular amyloid fibrils (11, 16) from patients with AD or with Down's syndrome (5). The major A␤ peptide in aqueous cerebral cortical extracts from AD brains has been reported as A␤-(1-40) (10). However, recent reports indicate that the insoluble amyloid in senile plaque cores is primarily A␤-(1-42) (11,12) and that diffuse senile plaques are primarily A␤-(17-42) (17). These findings have been verified by measuring A␤ in a 70% formic acid brain extract using sandwich enzyme-linked immunoabsorbent assay (18). In comparison, vascular amyloid is reported to be a mixture of A␤-(1-40) and A␤-(1-42) (11,12).The discovery that soluble A␤ (sA␤) is a constituent of cerebrospinal fluid (CSF) (19 -21) and cultured cell media (20,22) indicates that A␤ is a normal product of cellular metabolism of APP. The major form of A␤ in biological fluids is A␤-(1-40). Both NH 2 -terminal and COOH-terminal truncated sA␤s have been identified in pooled C...

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to generate phosphatidic acid. In mammalian cells this reaction has been implicated in the recruitment of coatomer to Golgi membranes and release of nascent secretory vesicles from the trans-Golgi network. These observations suggest that PLD is associated with the Golgi complex; however, to date, because of its low abundance, the intracellular localization of PLD has been characterized only indirectly through overexpression of chimeric proteins. We have used highly sensitive antibodies to PLD1 together with immunofluorescence and immunogold electron microscopy as well as cell fractionation to identify the intracellular localization of endogenous PLD1 in several cell types. Although PLD1 had a diffuse staining pattern, it was enriched significantly in the Golgi apparatus and was also present in cell nuclei. On fragmentation of the Golgi apparatus by treatment with nocodazole, PLD1 closely associated with membrane fragments, whereas after inhibition of PA synthesis, PLD1 dissociated from the membranes. Overexpression of an hemagglutinin-tagged form of PLD1 resulted in displacement of the endogenous enzyme from its perinuclear localization to large vesicular structures. Surprisingly, when the Golgi apparatus collapsed in response to brefeldin A, the nuclear localization of PLD1 was enhanced significantly. Our data show that the intracellular localization of PLD1 is consistent with a role in vesicle trafficking from the Golgi apparatus and suggest that it also functions in the cell nucleus.