Tobun T. Cheung

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.

The 4-kilodalton (39 to 43 amino acids) amyloid beta protein (beta AP), which is deposited as amyloid in the brains of patients with Alzheimer's diseases, is derived from a large protein, the amyloid beta protein precursor (beta APP). Human mononuclear leukemic (K562) cells expressing a beta AP-bearing, carboxyl-terminal beta APP derivative released significant amounts of a soluble 4-kilodalton beta APP derivative essentially identical to the beta AP deposited in Alzheimer's disease. Human neuroblastoma (M17) cells transfected with constructs expressing full-length beta APP and M17 cells expressing only endogenous beta APP also released soluble 4-kilodalton beta AP, and a similar, if not identical, fragment was readily detected in cerebrospinal fluid from individuals with Alzheimer's disease and normal individuals. Thus cells normally produce and release soluble 4-kilodalton beta AP that is essentially identical to the 4-kilodalton beta AP deposited as insoluble amyloid fibrils in Alzheimer's disease.

Salmonella typhimurium contains three distinct transport systems (CorA, MgtA, and MgtB) that move Mg2+ across the cytoplasmic membrane. Mutant strains containing only one of these three systems have been constructed and used to study each system in isolation. Characterization of these systems has been hampered, however, by the need to use 28Mg2+, a relatively unavailable, extremely expensive, and short lived radioisotope. This paper reports that 63Ni2+ is transported into the cell by all three of the S typhimurium Mg2+ transport systems. In a strain deficient in all three systems, uptake of 63Ni2+ was undetectable under the conditions used. Comparison of 63Ni2+ uptake kinetics and inhibition of 63Ni2+ transport by other divalent cations suggest that Ni2+ can be used as an analog of Mg2+ in the study of these three transport systems. Using 63Ni2+ to measure uptake, the effect of Mg2+ levels in the growth medium on transport by each system was tested. Transport by the CorA system was unaffected by changes in the amount of Mg2+ in the growth medium. In contrast, uptake via MgtA and MgtB was significantly increased in cells grown in 10 microM extracellular Mg2+ compared to cells grown in 10 mM Mg2+. The increases in uptake were the result of increases in Vmax without change in Km. This result suggests that, in low Mg2+ medium, cells contained higher levels of the transporters. Production of beta-galactosidase from mgtA::lacZ and mgtB::lacZ but not corA::lacZ fusions was also increased when cells were grown in low extracellular concentrations of Mg2+ indicating that the regulation occurs at the level of transcription. Expression of beta-galactosidase was also inhibited by the addition of other divalent cations including Ca2+ and Mn2+. Regulation of transcription from the mgtA and mgtB promoters was similar over the range of extracellular Mg2+ concentrations from 10 microM to 10 mM. At 1 microM, however, transcription from the mgtB promoter, as measured by beta-galactosidase levels in a mgtB::lacZ transcriptional fusion strain, was increased over 800-fold, and Ca2+ could no longer inhibit transcription effectively. In contrast, growth at 1 microM extracellular Mg2+ increased transcription from the mgtA promoter only about 30-fold and Ca2+ could still inhibit this increase. These results suggest that at least two distinct mechanisms are responsible for regulation of the mgtA and mgtB transcription in response to extracellular cation concentration.

The 4-kd amyloid beta protein (A beta) deposited as amyloid in Alzheimer's disease (AD) is produced and released by normal proteolytic processing of the amyloid beta protein precursor (beta APP) and is readily detected in cerebrospinal fluid (CSF). Here, we present the levels of A beta in CSF from a total of 95 subjects, including 38 patients with AD, 14 with early-onset AD and 24 with late-onset AD, 25 normal control subjects, and 32 patients with other neurological diseases. The level of A beta decreased with normal aging, and there was a significant elevation in the level of A beta in the CSF of early-onset AD patients (4.14 +/- 1.37 pmol/ml, p < 0.01). Neither Mini-Mental State nor Functional Assessment Staging were correlated with the amount of A beta in the CSF. The A beta/secreted form of beta APP ratio was elevated, but the level of alpha 1-antichymotrypsin in the CSF did not correlate with the level of CSF A beta in early-onset AD patients. Thus, the level of A beta in the CSF is elevated in early-onset AD patients and is suggested to be correlated with the pathology in the brain that characterizes AD.

The 39-43 amino acid beta amyloid protein (A beta) that deposits as amyloid in the brains of patients with Alzheimer's disease (AD) is encoded as an internal sequence within a larger membrane-associated protein known as the amyloid protein precursor (APP). In cultured cells, the APP is normally cleaved within the A beta to generate a large secreted derivative and a small membrane-associated fragment. Neither of these derivatives can produce amyloid because neither contains the entire A beta. Our study was designed to determine whether the soluble APP derivatives in human brain end within the A beta as described in cell culture or whether AD brain produces potentially amyloidogenic soluble derivatives that contain the entire A beta. We find that both AD and control brain contain nonamyloidogenic soluble derivatives that end at position 15 of the A beta. We have been unable to detect any soluble derivatives that contain the entire A beta in either the AD or control brain.