Pathogenic Aβ production by heterozygous PSEN1 mutations is intrinsic to the mutant protein and not mediated by conformational hindrance of wild-type PSEN1

Presenilin-1 (PSEN1) is the catalytic subunit of the intramembrane protease y-secretase and undergoes endoproteolysis during its maturation. Heterozygous mutations in the PSEN1 gene cause early-onset familial Alzheimer's disease (eFAD) and increase the proportion of longer aggregation-prone amyloid-P peptides (AP42 and/or AP43). Previous studies had suggested that PSEN1 mutants might act in a dominant-negative fashion by functional impediment of wild-type PSEN1, but the exact mechanism by which PSEN1 mutants promote pathogenic AP production remains controversial. Using dual recombinasemediated cassette exchange (dRMCE), here we generated a panel of isogenic embryonic and neural stem cell lines with heterozygous, endogenous expression of PSEN1 mutations. When catalytically inactive PSEN1 was expressed alongside the wild-type protein, we found the mutant accumulated as a fulllength protein, indicating that endoproteolytic cleavage occurred strictly as an intramolecular event. Heterozygous expression of eFAD-causing PSEN1 mutants increased the AP42/AP40 ratio. In contrast, catalytically inactive PSEN1 mutants were still incorporated into the y-secretase complex but failed to change the AP42/AP40 ratio. Finally, interaction and enzyme activity assays demonstrated the binding of mutant PSEN1 to other y-secretase subunits, but no interaction between mutant and wild-type PSEN1 was observed. These reproperty of PSEN1 mutants and strongly argue against a dominant-negative effect in which PSEN1 mutants would compromise the catalytic activity of wild-type PSEN1 through conformational effects.