| Summary: | Alzheimer's disease (AD) is the most common neurodegenerative disorder in the developed world. While the more common late-onset AD (LOAD) is associated with genetic and environmental risk factors, the early-onset (EOAD) form is most commonly caused by mutations in the Presenilin 1 (PSi) gene. Studying EOAD can be useful as the neuropathological hallmarks of the disease are the same as in LOAD. We have used human genomic constructs of PSi in order to investigate the role of this gene in the aetiology of AD. This method offers several advantages over cDNA over-expression studies as it allows gene expression from the entire genomic locus. The construct containing the human PSi gene was subcloned to include considerable upstream (~10 kb) and downstream (~15 kb) flanking genomic sequence. Expression levels were found to be physiologically relevant and the PSi protein was shown to undergo correct post-translational processing into PSi N-terminal and C- terminal fragments (NTF and CTF, respectively). We used this construct as the genetic background for several genetic manipulations including generation of separate mutant constructs each containing one of the following FAD mutations: M146V, Delta E9 and C410Y or the artificial proteolytically-dead D257A mutation. Clonal cell lines generated from these genomic constructs were shown to express partially or completely endoproteolytically impaired PSi proteins. In addition, the mutations were found to cause a partial to complete loss of y-secretase function with respect to Notch processing highlighting the loss of function mechanism of FAD mutations. Having engineered these mutations onto a WT-PSl-2A-Luciferase genomic construct, we expressed these in neuronal-like and PSdKO cell lines and found repression of expression with the FAD mutations tested (M146V, Delta E9 and C4i0Y). Interestingly, M146V showed a neuron-specific repression of expression highlighting an as yet unknown pathological mechanism for PSi FAD mutations. Together, these findings show that a physiologically relevant cell culture model system can be used to detect pathological mechanisms that are not detectable in an over-expressed cDNA transgenic system, which lacks the full genetic complexity of the human PSi locus.
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