Efficacy of NPl in the rescue of baseline 𝛾-secretase activity following knockdown with RNAi treatment
Alzheimer's disease, a neurodegenerative disorder that afflicts one in ten people over the age of 65, is the most common cause of memory loss and dementia in the elderly. Histologically characterized by amyloid plaques localized in the brain, Alzheimer's results in a slow degeneration in cognitive ability due to neuronal death and subsequent brain dysfunction. The amyloid plaques associated with this cell death result from aggregation of protein fragments produced by enzymatic cleavage of the amyloid precursor protein (APP) by 𝛾-secretase and 𝛽-secretase. These cleavage events release a 4 kD peptide known as A𝛽. NP1, a type I trafficking protein previously described elsewhere but never before implicated in Alzheimer's disease, has been shown to bind with all the components of the 𝛾-secretase enzymatic complex. RNAi knockdown of NPl expression in human embryonic kidney cells increases AP secretion, suggesting a possible link between normal NP1 expression and basal levels of A𝛽 expression. In order to explore this phenomenon, recombinant NP1 was expressed in E. coli and collected. Two versions of the recombinant NPI were produced: N-terminally-tagged NPI and C-terminally tagged NPI. In addition, the N-terminally-tagged NPI were purified using two different methods. It was expected that addition of exogenously produced recombinant NPI could restore A𝛽 levels to normal after they had been elevated by RNAi knockdown. This study indicates that producing recombinant NP1 in E. coli may be ineffective in recovery of baseline A𝛽 secretion. However, prior results suggest that NPI produced in eukaryotic cells can be effective in doing so, and therefore more research into the role of NP1 in Alzheimer's disease is merited.