Astrocytes but not neurons are responsible for the hippocampal increase of amyloid beta peptide induced by neurotoxin quinolinic acid

Background:Mutations in the amyloid precursor protein (APP) cause familial Alzheimer disease (FAD). Our group recently described the aggressive FAD-associated I716FAPPmutation. The aim of our study is to characterise the effects on APP processing of the three known FAD mutations in codon 716 of APP (I716F, I716T and I716V). Methods: APP I716F, I716T and I716V mutations were introduced in human wild-type APP cDNA by sitedirected mutagenesis. Transfected CHO cells were cultured and the conditioned medium was collected 24h later. Human Amyloid-beta40, Amyloid-beta42 and total Amyloid-beta levels were measured by ELISA. APP intracellular domain (AICD) was measured by western-blot using a cellfree assay. For imaging studies, we generated a cDNA construct containing the C-terminal fragment of APP (C99) fused to the fluorescent protein Dendra2. Wild-type C99-Dendra 2 was also mutated to I716F, I716Tand I716V. Fluorescence images were obtained on a Leica SP5 confocal microscope. Nuclear fluorescence intensity was quantified as a measure of AICD generation.Results:Amyloid-beta42/40 ratios were increased in cells transfected with any of the mutations in APP as follows (% of wild-type APP): I716F: 283%, I716T: 276%, I716V: 142% (p< 0.05; one-way ANOVA). Therewas an inversely linear relationship (R2 1⁄4 0.97) between the Amyloid-beta 42/ 40 ratio and the mean age at onset of AD (I716F: 31yo, I716T: 36yo, I716V: 53yo). Total Amyloid-beta was reduced in I716F but not in I716Vor I716T mutations. AICDs generation was decreased in cells transfected with APP I716F measured by a cell-free assay. Cells transfected with I716F mutation showed diminished nuclear fluorescence.Conclusions:Mutations in the codon 716 of APP differentially alter APP processing. All of them increase the amyloid-beta42/40 ratio, which is inversely related with the mean age of onset of AD. However, only the I716Fmutation reduces total gamma-secretase products, namely, AICDs and total Amyloid-beta. Our results indicate that the I716F APP mutation may generate a protein that is poorly processed by gamma-secretase. Taken together, this may indicate a loss-of-function in APP proteolysis as an additional mechanism in FAD.