Response to: Pardossi-Piquard et al., “Presenilin-Dependent Transcriptional Control of the Aβ-Degrading Enzyme Neprilysin by Intracellular Domains of βAPP and APLP.” Neuron 46, 541–554

Results Neprilysin Protein Levels Do Not Correlate with Presenilin Genotype and Are Not Rescued by Presenilin Expression To examine whether neprilysin protein levels are regulated by presenilin (PS) expression as reported (Pardossi-Piquard et al., 2005), mouse embryonic stem cells genetically devoid of both PS1 and PS2 (BD8 cells) were analyzed by Western blotting (see Methods in the Supplemental Data available online). Cells were harvested in Tris buffer containing either no detergent, 0.5% Triton X-100, or 1% NP40 and blotted for neprilysin, APP C-terminal fragments (CTFs), and GAPDH. The lack of PS expression resulted in no significant reduction in neprilysin levels in cells harvested in either Tris buffer or Tris-1% NP40 buffer compared to identically prepared wt embryonic stem cells (PBD8) (Figure 1A). In cells harvested in Tris-0.5% Triton buffer, we observed either a modest (Figure 1A) or no (Figure S1A) reduction in neprilysin levels. To determine whether introduction of presenilin could rescue this variable and modest decrease, the BD8 cells were transiently transfected with PS1, PS2, or both. Transfection of PS resulted in the rescue of gamma -secretase complex formation (PS endoproteolysis and enhanced nicastrin maturation) and activity (reduction in the elevated APP CTFs) in the BD8 cells (Figure 1B) ([Chen et al., 2003], [Kimberly et al., 2002] and [Leem et al., 2002]). However, neprilysin levels were unchanged (Figure 1B). When quantified, neprilysin levels (versus control) were 93.7% ± 0.2% (SEM), 105.7% ± 1.1%, and 98.6% ± 18.3% for cells transfected with PS1, PS2, or both, respectively (p > 0.05 in all cases) (Figure S1B).