A System for Studying the Effect(s) of Familial Alzheimer Disease Mutations on the Processing of the β-amyloid Peptide Precursor

Abstract

Three different point mutations have been observed in some familial Alzheimer′s disease pedigrees at a unique valine, Val717, near the carboxyl end of the β Amyloid Peptide Precursor (βAPP). The effects of these mutations on the processing and cellular functions of βAPP can best be determined in the absence of the normal form(s) of the protein. We have used targeted mRNA degradation by a trans-acting hammerhead ribozyme to cleave and inactivate βAPP expression in vitro. The consensus ribozyme cleavage site, 5′GUC↓X3, matches the Val717 nucleotide sequence in βAPP mRNA. Introduction of FAD point mutations which change Val717 decrease the rate of ribozyme cleavage by more than three orders of magnitude. Thus, ribozyme targeting of this site should allow the study of protein processing in vivo. Furthermore, a ribozyme targeted to mutant βAPP mRNA (Va717⇒Ile) cleaved the mutant sequence 300-fold faster than the normal sequence. This suggests that ribozymes might lower mutant βAPP mRNA levels in FAD cells.