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 γ-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-β peptides (Aβ42 and/or Aβ43). 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 Aβ production remains controversial. Using dual recombinase-mediated 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 full-length protein, indicating that endoproteolytic cleavage occurred strictly as an intramolecular event. Heterozygous expression of eFAD-causing PSEN1 mutants increased the Aβ42/Aβ40 ratio. In contrast, catalytically inactive PSEN1 mutants were still incorporated into the γ-secretase complex, but failed to change the Aβ42/Aβ40 ratio. Finally, interaction and enzyme activity assays demonstrated binding of mutant PSEN1 to other γ-secretase subunits, but no interaction between mutant and wild type PSEN1 was observed. These results establish that pathogenic Aβ production is an intrinsic property 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.