In summary, in vitro studies have provided conclusive evidence that many PSEN mutations cause a sub-stantial loss of ??-secretase activity. However, the results from knock-in mice either with heterozygous expression of PSEN mutants indicate that these frequently used cellular assays and, in particular, kinetic in vitro assays of PSEN mutants might underestimate the enzymatic activity of ??-secretase in a cellular context where both WT and mutant PSEN alleles contribute to the expressed ??-secretase complexes [66-69]. Importantly, in humans, validated loss-of-function mutations in the genes encoding NOTCH or the ??- secretase subunits PEN-2, PSEN1 and Nicastrin cause skin phenotypes ranging from acne inversa to cutaneous squamous cell carcinomas, as well as chronic myelomonocytic leukaemia [87-89].
In addition, genetic deletion of ??-secretase complex components in mice has demonstrated that a 30% reduction in ??-secretase activity is sufficient to induce a myeloproliferative disease resembling chronic myelomonocytic leukaemia . However, these phenotypes, likely provoked by reduced NOTCH processing and signaling, have never been associated with FAD, further arguing that heterozygous expression of PSEN mutations does not result in a substantial loss of ??-secretase activity . In addition, it has been proposed that FAD PSEN1 mutations impair ??-secretase-independent functions of PSEN proteins in signal transduction, autophagy and calcium homeostasis. The anti-apoptotic phosphatidylinositol 3-kinase-AKT signaling pathway seems to be positively regulated by PSEN proteins.
PSEN deficiency or expression of PSEN1 FAD mutants reduced AKT phosphorylation and activity, and increased activity of its downstream target glycogen synthase kinase-3 (GSK-3) [91,92]. In knock-in mice for the PSEN1-I213T mutation, activation of GSK-3?? was observed and correlated with increased phosphorylation of its substrate Tau and the formation of intracellular Tau inclusions . Absence of PSEN or expression of PSEN1 FAD mutants has further been demonstrated to impair intracellular protein degradation, caused by a reduced turnover of autophagic vacuoles that fail to become acidified and fuse with lysosomes . Another consistent observation has been that the induced release of calcium from endoplasmic reticulum stores is strongly increased by PSEN FAD mutants, which might result in deregulation of synaptic transmission Carfilzomib and plasticity .
A few studies using primary cells from FAD carriers have confirmed that PSEN inhibitor bulk mutants negatively affect the role of PSEN in autophagic protein degradation and calcium homeostasis [60,61]. With respect to all of these proposed ??-secretase-independent functions, FAD PSEN mutants mimic the phenotype of PSEN deficiency, indicating that the mutants behave as true loss-of-function alleles.