g 1. The APPNLI responder transgene construct. The 695 amino acid-long amyloid precursor protein cDNA harboring the Swedish mutation was inserted into the XhoI site of MoPrP.Xho fragment, which was further excised at two XbaI sites. The resulting fragment of prnp.APPNL was cloned into the unique XbaI site in the inducible expression vector pTRE. The London mutation was further introduced into the pTRE.prnp.APPNL plasmid using site-directed mutagenesis.Beta-secretase-mediated APP processing Beta-secretase-mediated digestion of APP to release C-terminal fragments is the first step in amyloidogenic A production. This 99 amino acid-long APP fragment is associated with multiple neurological ill-effects, including neuroinflammation and neurodegeneration, disruption of neuronal ionic homeostasis, and learning and memory impairments. We measured the levels of CTF at different ages in rTg9191 mice and found an age-dependent increase in the level of CTF, despite the fact that the level of APPNLI remained constant with age. We also compared the levels of CTF in rTg9191 mice to the level found in Tg2576 mice. At 21 months of age, rTg9191 mice generate a level of CTF equivalent to that of age-matched Tg2576 mice, as might be expected, since both lines harbor the Swedish mutation. Age-dependent progression of A plaques We tracked the onset and accumulation of A plaques in cerebral cortex and hippocampus of rTg9191 mice from 2 to 26 months of age. Plaques were visualized using four antibodies: 6E10, 4G8, 1395 Bigenic activator-repsonder system. rTg9191 mice employ a bigenic system in which a calcium-calmodulin kinase II protomer drives constitutive expression of the tetracycline-controlled transactivator gene, and a responder transgene for human APP695 containing the Swedish and London mutations is under control of the tetracycline response element. Regulatable expression of the APP transgene in the rTg9191 line is under the control of doxycycline. In the absence of DOX, tTA binds the tetO promoter and APPNLI is expressed; in the presence of DOX, the tTA-tetO interaction is blocked, and expression of APPNLI is suppressed. Expression of APPNLI. Representative immunoblot probed with monoclonal antibody 22C11, which recognizes both mouse and human APP; numbers above the blot show amounts of protein Crenolanib manufacturer loaded in each lane. Quantification. Thirty-five g of protein from brains of of 2-month-old non-transgenic mice is required to produce the same APP signal as 7 g of protein from age-matched rTg9191 littermates, indicating that transgenic mice have 5 times more APP than non-transgenic mice. Therefore, rTg9191 mice express 4 times PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19783938 more APPNLI relative to mouse APP. DLU, densitometric light unit. 4 / 26 Characterizing a Model of -Amyloid Toxicity Suppression of APPNLI expression. Representative immunoblot using monoclonal antibody 6E10, which recognizes human A116; 10 g of protein was loaded in each lane. Alpha-tubulin served as the loading control. 8Mon and 10Mon: 8- and 10-month-old rTg9191 mice without DOX treatment; 8-10Moff: 10-month-old rTg9191 mice, treated with DOX from 8 to 10 months of age. Quantification. Administration of DOX to rTg9191 mice decreased levels of 
APPNLI by 87%. p < 0.0001, one-way ANOVA followed by Fisher's post PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19786154 hoc analysis. doi:10.1371/journal.pone.0126317.g002 end-specific antibody), and 1-11-3. For all four antibodies, we found that plaques emerged first in the cerebral cortex, as early as 8 months of age, and then appeared in the h
