JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Expression and processing of fluorescent fusion proteins of amyloid precursor protein (APP).
Biochim. Biophys. Acta
PUBLISHED: 01-04-2013
Show Abstract
Hide Abstract
Processing of ?-amyloid precursor protein (APP) by ?- and ?-secretases in neurons produces amyloid-? (A?), whose excess accumulation leads to Alzheimers disease (AD). Knowledge on subcellular trafficking pathways of APP and its fragments is important for the understanding of AD pathogenesis. We designed fusion proteins comprising a C-terminal fragment of APP (app) and fluorescent proteins GFP (G) and DsRed (D) to permit the tracking of the fusion proteins and fragments in cells. CAD cells expressing these proteins emitted colocalized green and red fluorescence and produce ectodomains, sGapp and sRapp, and A?, whose level was reduced by inhibitors of ?- and ?-secretases. The presence of GappR in endosomes was observed via colocalization with Rab5. These observations indicated that the fusion proteins were membrane inserted, transported in vesicles and proteolytically processed by the same mechanism for APP. By attenuating fusion protein synthesis with cycloheximide, individual fluorescent colors from the C-terminus of the fusion proteins appeared in the cytosol which was strongly suppressed by ?-secretase inhibitor, suggesting that the ectodomains exit the cell rapidly (t1/2 about 20min) while the C-terminal fragments were retained longer in cells. In live cells, we observed the fluorescence of the ectodomains located between parental fusion proteins and plasma membrane, suggesting that these ectodomain positions are part of their secretion pathway. Our results indicate that the native ectodomain does not play a decisive role for the key features of APP trafficking and processing and the new fusion proteins may lead to novel insights in intracellular activities of APP.
Related JoVE Video
Beta-secretase inhibitor GRL-8234 rescues age-related cognitive decline in APP transgenic mice.
FASEB J.
PUBLISHED: 11-08-2010
Show Abstract
Hide Abstract
Alzheimer disease is intimately linked to an excess amount of amyloid-? (A?) in the brain. Thus, therapeutic inhibition of A? production is an attractive clinical approach to treat this disease. Here we provide the first direct experimental evidence that the treatment of Tg2576 transgenic mice with an inhibitor of ?-secretase, GRL-8234, rescues the age-related cognitive decline. We demonstrated that the injected GRL-8234 effectively enters the brain and rapidly decreases soluble A? in the brain of Tg2576 mice. The rescue of cognition, which was observed only after long-term inhibitor treatment ranging from 5 to 7.5 mo, was associated with a decrease of brain amyloid-? plaque load. We also found no accumulation of amyloid-? precursor protein after several months of inhibitor treatment. These observations substantiate the idea that A? accumulation plays a major role in the cognitive decline of Tg2576 mice and support the concept of A? reduction therapy as a treatment of AD.
Related JoVE Video
Structure-based design, synthesis, and biological evaluation of dihydroquinazoline-derived potent ?-secretase inhibitors.
Bioorg. Med. Chem. Lett.
Show Abstract
Hide Abstract
Structure-based design, synthesis, and biological evaluation of a series of dihydroquinazoline-derived ?-secretase inhibitors incorporating thiazole and pyrazole-derived P2-ligands are described. We have identified inhibitor 4f which has shown potent enzyme inhibitory (K(i)=13 nM) and cellular (IC(50)=21 nM in neuroblastoma cells) assays. A model of 4f was created based upon the X-ray structure of 3a-bound ?-secretase. The model suggested possible interactions in the active site.
Related JoVE Video
Structure-based design of highly selective ?-secretase inhibitors: synthesis, biological evaluation, and protein-ligand X-ray crystal structure.
J. Med. Chem.
Show Abstract
Hide Abstract
The structure-based design, synthesis, and X-ray structure of protein-ligand complexes of exceptionally potent and selective ?-secretase inhibitors are described. The inhibitors are designed specifically to interact with S(1) active site residues to provide selectivity over memapsin 1 and cathepsin D. Inhibitor 5 has exhibited exceedingly potent inhibitory activity (K(i) = 17 pM) and high selectivity over BACE 2 (>7000-fold) and cathepsin D (>250000-fold). A protein-ligand crystal structure revealed important molecular insight into these selectivities. These interactions may serve as an important guide to design selectivity over the physiologically important aspartic acid proteases.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.