Alzheimer's disease (AD) is characterized by overproduction of A beta derived from APP cleavage via beta- and gamma-secretase pathway. Recent evidence has linked altered cholesterol metabolism to AD pathogenesis. In this study, we show that AD brain had significant cholesterol retention and high beta- and gamma-secretase activities as compared to age-matched non-demented controls (ND). Over one-half of AD patients had an apoE4 allele but none of the ND. beta- and gamma-secretase activities were significantly stimulated in vitro by 40 and 80 microM cholesterol in AD and ND brains, respectively. Both secretase activities in AD brain were more sensitive to cholesterol (40 microM) than those of ND (80 microM). Filipin-stained cholesterol overlapped with BACE and A beta in AD brain sections. Cholesterol (10-80 microM) added to N2a cultures significantly increased cellular cholesterol, beta- and gamma-secretase activities and A beta secretion. Similarly, addition of cholesterol (20-80 microM) to cell lysates stimulated both in vitro secretase activities. Ergosterol slightly decreased beta-secretase activity at 20-80 microM, but strongly inhibited gamma-secretase activity at 40 microM. Cholesterol depletion reduced cellular cholesterol, beta-secretase activity and A beta secretion. Transcription factor profiling shows that several key nuclear receptors involving cholesterol metabolism were significantly altered in AD brain, including decreased LXR-beta, PPAR and TR, and increased RXR. Treatment of N2a cells with LXR, RXR or PPAR agonists strongly stimulated cellular cholesterol efflux to HDL and reduced cellular cholesterol and beta-/gamma-secretase activities. This study provides direct evidence that cholesterol homeostasis is impaired in AD brain and suggests that altered levels or activities of nuclear receptors may contribute to cholesterol retention which likely enhances beta- and gamma-secretase activities and A beta production in human brain.

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The billions of specimens housed in natural science collections provide a tremendous source of under–utilized data that are useful for scientific research, conservation, commerce, and education. Digitization and mobilization of specimen data and images promises to greatly accelerate their utilization. While digitization of natural science collection specimens has been occurring for decades, the vast majority of specimens remain un–digitized. If the digitization task is to be completed in the near future, innovative, high–throughput approaches are needed. To create a dataset for the study of global change in New England, we designed and implemented an industrial–scale, conveyor–based digitization workflow for herbarium specimen sheets. The workflow is a variation of an object–to–image–to–data workflow that prioritizes imaging and the capture of storage container–level data. The workflow utilizes a novel conveyor system developed specifically for the task of imaging flattened herbarium specimens. Using our workflow, we imaged and transcribed specimen–level data for almost 350,000 specimens over a 131–week period; an additional 56 weeks was required for storage container–level data capture. Our project has demonstrated that it is possible to capture both an image of a specimen and a core database record in 35 seconds per herbarium sheet (for intervals between images of 30 minutes or less) plus some additional overhead for container–level data capture. This rate was in line with the pre–project expectations for our approach. Our throughput rates are comparable with some other similar, high–throughput approaches focused on digitizing herbarium sheets and is as much as three times faster than rates achieved with more conventional non–automated approaches used during the project. We report on challenges encountered during development and use of our system and discuss ways in which our workflow could be improved. The conveyor apparatus software, database schema, configuration files, hardware list, and conveyor schematics are available for download on GitHub.

This expands the number of families reported with mutations in the coding region of the amyloid precursor protein gene. Cerebral hemorrhage appears to be less frequent in this family than in the previously reported Flemish pedigree with the same mutation.