Rapid and direct transport of cell surface APP to the lysosome defines a novel selective pathway.

BACKGROUND: A central feature of Alzheimer's disease is the cleavage of the amyloid precursor protein (APP) to form beta-amyloid peptide (Abeta) by the beta-secretase and gamma-secretase enzymes. Although this has been shown to occur after endocytosis of APP from the cell surface, the exact compartments of APP processing are not well defined. We have previously demonstrated that APP and gamma-secretase proteins and activity are highly enriched in purified rat liver lysosomes. In order to examine the lysosomal distribution and trafficking of APP in cultured cells, we generated constructs containing APP fused to a C-terminal fluorescent protein tag and N-terminal HA-epitope tag. These were co-transfected with a panel of fluorescent-protein tagged compartment markers. RESULTS: Here we demonstrate using laser-scanning confocal microscopy that although APP is present throughout the endosomal/lysosomal system in transfected Cos7 and neuronal SN56 cell lines as well as in immunostained cultured mouse neurons, it is enriched in the lysosome. We also show that the Swedish and London mutations reduce the amount of APP in the lysosome. Surprisingly, in addition to its expected trafficking from the cell surface to the early and then late endosomes, we find that cell-surface labelled APP is transported rapidly and directly from the cell surface to lysosomes in both Cos7 and SN56 cells. This rapid transit to the lysosome is blocked by the presence of either the London or Swedish mutations. CONCLUSIONS: These results demonstrate the presence of a novel, rapid and specific transport pathway from the cell surface to the lysosomes. This suggests that regulation of lysosomal traffic could regulate APP processing and that the lysosome could play a central role in the pathophysiology of Alzheimer's disease.

Persistence of testosterone and 17beta-estradiol in soils receiving swine manure or municipal biosolids.

Natural and synthetic steroidal hormones can be carried to agricultural soil through fertilization with municipal biosolids, livestock manure, or poultry manure. The persistence and pathways of dissipation of [4-(14)C]-testosterone and of [4-(14)C]-17beta-estradiol in organic-amended soils were investigated using laboratory microcosms. Testosterone dissipation was investigated over a range of amendment concentrations, temperatures, and soil types. Under all conditions the parent compound and transformation products were dissipated within a few days. Addition of swine manure slurry to soil hastened the transformation of testosterone and 17beta-estradiol to the corresponding less hormonally active ketones, 4-androstene-3,17-dione and estrone. Two other testosterone transformation products, 5alpha-androstan-3,17-dione and 1,4-androstadiene-3,17-dione, were also detected. Experiments with sterilized soil and sterilized swine manure slurry suggested that the transformation of (14)C-labeled hormonal parent compounds was mainly caused by microorganisms in manure slurry, while mineralization of the hormones to (14)CO(2) required viable soil microorganisms. Organic amendments transiently inhibited the mineralization of [4-(14)C]-testosterone, perhaps by inhibiting soil microorganisms, or by enhancing sorption and reducing the bioavailability of testosterone or transformation products. Overall, organic amendments influenced the pathways and kinetics of testosterone and estradiol dissipation, but did not increase their persistence.

Persistence and pathways of testosterone dissipation in agricultural soil.

The persistence and pathways of dissipation of testosterone in three agricultural soils were examined in laboratory microcosm incubations at different soil moistures (1.7-39%) and temperatures (4-30 degrees C) using (14)C- and (3)H-labeled and unlabeled testosterone. Sterilized loam was also examined to assess possible abiotic pathways. Extractable (14)C decreased rapidly for all three soils at 30 degrees C with times to dissipate 50% of material (DT(50)) ranging from 8.5 to 21 h. Respired (14)CO(2) accounted for approximately 50% of the applied (14)C after 120 h. Androgenic activity of soil extracts declined faster than the extractable (14)C levels demonstrating that testosterone was not being converted to compounds with greater activity. Dissipation rates of nonvolatile, extractable (3)H in loam at 7, 15, and 39% moisture were similar, but the rate in air-dried loam (1.7% moisture) was significantly reduced. High performance liquid chromatography (HPLC) analysis of extracts of (14)C-testosterone-treated loam incubated at 30 degrees C for 6 h revealed that the (14)C was distributed among the remaining testosterone and three major metabolites (4-androstene-3,17-dione, 5alpha-androstane-3,17-dione, and 1,4-androstadiene-3,17-dione), which accounted for 48.7, 23.7, and 9.6% of the remaining (14)C, respectively. Periodic analysis of soil incubated at 23, 12, and 4 degrees C showed that the rates of testosterone dissipation and metabolite appearance and subsequent dissipation were temperature dependent with rates decreasing with decreasing temperature. In sterilized loam, 4-androstene-3,17-dione was the only metabolite detected. We conclude that testosterone is rapidly and thoroughly biodegraded in agricultural soils under a range of conditions typical of a temperate growing season and thus is unlikely to pose a long-term risk to adjacent aquatic environments.

Survey of hormone activities in municipal biosolids and animal manures.

The potential exists for natural or synthetic hormonal chemicals present in agricultural fertilizers to be transferred to adjacent aquatic environments in order to alter endocrine function in exposed wildlife. Recombinant yeast and mammalian cell line (BG1Luc4E2) assays were used to screen crude organic extracts of municipal biosolids and animal manures for estrogen-, androgen-, and progesterone receptor gene transcription activities. Of the biosolid extracts, those samples that had undergone aerobic digestion had no or minimal estrogen- and no androgen receptor gene transcription activities. In contrast, those biosolid samples that had undergone anaerobic digestion had much higher estrogen- and, for all but one site, androgen receptor gene transcription activities. Extracts prepared from animal manure samples had variable levels of androgen- and estrogen receptor gene transcription activities, which may be related to the type, sex, age, and reproductive status of the animals. The diet and treatment of animals with hormone implants also appeared to be factors influencing hormone activity in animal manure. Progesterone receptor gene transcription activity was observed for only one chicken litter sample. Overall, results of this study suggest that in vitro bioassays can be used to survey and detect hormone activity in municipal biosolids and animal manures. Furthermore, results of these assays can be used to develop practices that will minimize the potential environmental endocrine-disrupting effects of these substances.

Determination of the estrogenic and antiestrogenic effects of environmental contaminants in chicken embryo hepatocyte cultures by quantitative-polymerase chain reaction.

A method was developed to measure the estrogenic and antiestrogenic effects of various chemicals and organochlorine extracts in chicken embryo primary hepatocyte cultures. Messenger RNAs (mRNAs) for the estrogen-inducible egg yolk proteins, vitellogenin II (VTGII), and very low-density lipoprotein apoprotein II (apoII), were measured by multiplex quantitative reverse transcription-polymerase chain reaction (Q-PCR). After 48 h of exposure, both VTGII and apoII mRNA levels were induced by moxestrol (1-1,000 nM), 17 beta-estradiol (10-1,000 nM), o,p'-DDT (apoII: 1,000 and 10,000 nM, VTGII: 10,000 nM), 4-tertoctylphenol ([OP]; apoII: 20 and 50 microM, VTGII: 10-50 microM), and methoxychlor ([MXCL]; apoII: 5-50 microM, VTGII: 20 and 50 microM). Tamoxifen (100 and 1,000 nM) induced apoII mRNA only, and bisphenol A (BPA) was not estrogenic. Inhibition of moxestrol-mediated VTGII or apoII mRNA induction by MXCL, o,p'-DDT and tamoxifen indicated that these chemicals were also antiestrogenic at concentrations similar to those which caused estrogenic responses. Organochlorine extracts prepared from herring gull embryo yolk sacs obtained from three Great Lakes sites and one Atlantic coast site (reference site) did not show any estrogenic activities. However, the same extracts from all three Great Lakes sites had antiestrogenic activities. These results indicate that wild birds may be susceptible to the estrogenic or antiestrogenic effects of environmental contaminants.