APP Processing and Tyrosine682 Figure 7. The YG mutation does not alter synaptic proteins. A, Schematic representation of synaptosomal preparation. B, Synaptic proteins distribution is similar in APPwt/wt and APPYG/YG brains. doi:10.1371/journal.pone.0015503.g007 Figure 8. Loss of APP/Fe65 interaction in APPYG/YG mouse brain. A, IP of APP from a membrane enriched brain fraction using anti-APP antibody. WB analysis for Fe65 shows an absence of detectable signal in both APP2/2 and APPYG/YG compared to APPwt/wt. B, IP of Fe65 from a membrane enriched brain fraction using anti-Fe65 antibody (I12). WB analysis for APP shows an absence of detectable signal in APPYG/YG mutant mice compared to APPwt/wt. doi:10.1371/journal.pone.0015503.g008 proteasomal pathways, it would explain why detection of pY682- APP and pY682-APP-CTFs proteins would be very difficult in a normal brain. Given the functional redundancy provided by APLP1 and APLP2 much of the phenotype in APPYG/YG mice is potentially masked. Therefore a very important goal is to cross APPYG/YG mice with APLP22/2 mice. If significant APP functionality is facilitated by Tyr682 then we may see aspects of the severe APP2/2/APLP22/2 mice phenotype reproduced in a context that allows a much more detailed picture of APP function to be dissected. Physiological characterization of this model in addition to APPYG/YG mice is therefore of great interest. In summary, we have successfully generated two APP KI mouse lines, APP Y682G & T668A, and carried out an initial characterization focusing on APP Y682G. Both APP processing and the APP/Fe65 interaction are significantly altered as a result of this mutation, in agreement with previous in vitro studies. In addition these findings have a therapeutic implication, demon- strating that manipulation of this amino acid could increase production of the sAPPa, considered a protective protein, and decrease the generation of toxic fragments such as Ab40 & 42. without negative physiological consequences. Overall, the data suggests a very important in vivo role for Tyr682 in the brain. Author Contributions Conceived and designed the experiments: LD. Performed the experiments: AB RW RT DZ LG OF LD. Analyzed the data: AB RW DZ OF LD. Wrote the paper: RW LD. References 1. Hardy JA, Higgins GA (1992) Alzheimer’s disease: the amyloid cascade hypothesis. Science 256: 184–185. 2. Selkoe DJ (2000) The genetics and molecular pathology of Alzheimer’s disease: roles of amyloid and the presenilins. Neurol Clin 18: 903–922. PLoS ONE | www.plosone.org 9 November 2010 | Volume 5 | Issue 11 | e15503