might functionally interact to induce apoptosis mediated neurodegeneration when misregulated. To test this possibility, we first performed TUNEL assays in larval brains co-expressing either Tip60E431Q and APP or Tip60E431Q and APP dCT under the control of the pan-neuronal 179 y-GAL4 Transgenic Fly Line b dTip60E431Q Buffy ALiX CalpA TRAF4 1, 1, 1 1 1, Gene Namea dTip60WT 1.6 2.1 3.5 3.9 21.5 21.7 23.2 2 14.8139954 APP; dTip60E431Q 21.5 1.5 22.1 21.5 22.1 21.9 22.5 1 824.094897 APP; dTip60WT 3.5 2.3 21.8 21.7 21.5 21.5 22.3 1.8 2472.348951 1.4 1.4 1.5 1.7 2.3 2.4 3.8 1 Frizzled 1 Wingless dMyc PDCD5 24.7 1193.4 Dmel\CG9418 a Quantitative RT-PCR analysis was performed for the indicated target genes. Staged second instar larvae ubiquitously expressing the indicated transgene were used for cDNA preparation. Quantitative RT-PCR reactions were carried out in triplicate and the relative fold change was calculated using the 22DDCT method using RP49 as control. 1 Genes that were differentially regulated between flies expressing the Tip60 HAT mutant dTip60E431Q alone and in conjunction with APP. Gene that were differentially regulated between flies overexpressing dTip60WT alone or together with APP. doi:10.1371/journal.pone.0041776.t004 b 9 Tip60 Mediates APP Induced Cell Death PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22205030 in the CNS driver. For these studies, we used our lower expressing APP; Tip60E431Q line A and APP dCT; Tip60E431Q line A fly lines, as co-expression of higher expressing Tip60E431Q line B and APP induced lethality at the second instar larvae stage which was too early to assess by TUNEL stain. Indeed, as shown in interaction between Tip60 and APP in neuronal apoptotic induction. Importantly, and as we predicted, this interaction was dependent upon the C-terminus of APP that interacts with Tip60 as co-expression of Tip60E431Q and APP dCT resulted in only a moderate level of neuronal apoptosis induction that was approximately equivalent to that observed for Tip60E431Q alone. To determine whether additional Tip60 817204-33-4 levels would suppress the APP induced neuronal 10 Tip60 Mediates APP Induced Cell Death in the CNS apoptotic phenotype as well as to confirm the specificity of the interaction, we performed TUNEL assays in larval brains coexpressing Tip60WT with APP using APP; Tip60WT line C. This line was selected because line Tip60WT C expressed the highest levels of wild type dTip60 for all of our dTip60WT lines and also displayed the highest level of rescue for APP induced lethality. Remarkably, we found that additional levels of Tip60 
partially rescued APP induced apoptotic cell death as evidenced by a visible reduction of the presence of TUNELpositive cells in these brains when compared to APP alone. Co-expression of dTip60WT and APP also appeared to suppress neuronal apoptosis induced by Tip60 overexpression alone, as we observed less TUNEL-positive cells in brains co-expressing dTip60WT and APP when compared with brains expressing equivalent levels of Tip60WT alone. Interestingly, rescue of cell death appeared more prominent in the proximal central brain of APP; dTip60WT flies, as we consistently observed virtually no apoptotic cell death in this area, where vital structures like the Drosophila learning and memory center mushroom body are located. Importantly, and as we predicted, partial rescue of APP induced neuronal apoptosis by Tip60 was dependent upon the Tip60 interacting C-terminus of APP, as brains co-expressing both Tip60WT and APP dCT showed no rescue as shown by th