## 3 Discussion  
Previously, we reported that Swedish mutation \(A\beta\)PP-sw under control of \(GFP\) gene promoter reduced viability in NGF-induced neuronal PC-12 cells compared to empty-vector transfected cells with symptoms of aberrant autophagy and increased clusterin protein expression [40]. We showed the incomplete autophagy-like process induced by human \(A\beta\)PP-sw gene inserted in neuronal PC-12 cells. The evidence of excess autophagosomes and multivesicular bodies found in \(A\beta\)PP-sw- but not in \(A\beta\)PP-wt- or empty vector-bearing neuronal cells was accompanied by elevated sCLU expression levels. In this work we tried to figure out how the MEV pathway alters \(A\beta\)PP-sw gene dependent sCLU protein expression levels. We report here that, compared to non-treated cells, the viability of \(A\beta\)PP-sw- and \(A\beta\)PP-wt-transfected neuronal PC-12 cells exemplified on respective photographs (Supplementary data 1) was similarly reduced by statins and M\(\beta\)CD (Supplementary data 2, Figure 1). Chol-PEG administration could hardly restore cell viability diminished by ATR, SIM or M\(\beta\)CD (Figure 1, \(p\) > 0.05) so we assumed that diminished neuronal cell survival by statins or M\(\beta\)CD could be independent of a reduction in cholesterol level. We also tested the effects of metabolic inhibitors of MEV pathway on cell viability. As shown in respective plots (Supplementary data 3), the inhibitors of squalene synthase (SQS) - zaragozic acid [10-100 \(\upmu\)M], famesyl transferase - L-744,832 [10-100 \(\upmu\)M], and geranylgeranyl transferase I - GGTI-286 [1-10 \(\upmu\)M] decreased significantly the viability of neuronal PC-12 cells expressing the Alzheimer pertinent protein A\(\beta\)PP695 NL (\(A\beta\)PP-sw-transfected cells). These data point to the important role played by the MEV pathway in neuroprotection other than cholesterol itself. Recently, some authors have highlighted the role of non-sterol isoprenoids in neurons, suggesting that there is a shift of the MEV pathway towards the non-sterol isoprenoids which are essential for synaptic activity [41]. In a process known as prenylation, isoprenoids are covalently bound to small guanosine triphosphate binding proteins (sGTPases) of primary importance for neurons [42]. Among them, Rab3 and RhoA are involved in neurotransmitter release, and synaptic plasticity, respectively [41]. statins, including simvastatin, cause marked drop in RhoA prenylation  
Figure 4: Effect of mevalonate (MEV, 100 \(\upmu\)M), farnesol (FOH, 10 \(\upmu\)M), geranylgeraniol (GGOH, 10 \(\upmu\)M) or water soluble cholesterol (Chol-PEG, 1 mM) on selected protein expressions in cells affected by MEV pathway modulators (**A** for atorvastatin - ATR, **B** for simvastatin - SIM). Western blot analysis of proteins: amyloid precursor protein (A\(\beta\)PP, 110 kDa), soluble amyloid protein precursor alpha (sA\(\beta\)PP\(\alpha\), 100 kDa), clusterin (CLU, 60 and 40 kDa), amyloid-B 1-16 (A\(\beta\), 4 kDa), actin (42 kDa) as house-keeping protein in empty vector (G), \(A\beta\)PP wild type gene (\(A\beta\)PP-wt, W), and \(A\beta\)PP Swedish mutations gene (\(A\beta\)PP-sw, S) transfected cells. The results are indicative of three independent experiments with similar results. which is associated with Akt kinase phosphorylation (activation) followed by cAMP responsive element binding protein 1 (CREB 1) activation. In consequence, this transcription factor upregulates cognition and memory by genomic regulation of _c-Fos_ and _Egr-1_ gene transcription [43; 44]. Thus, depending on type and dose of statin, it may play either a beneficial or harmful role in brain function. As a matter of fact, we decided to test if MEV pathway intermediates could prevent ATR-, SIM- or M\(\beta\)CD-retarded cell viability. Cell viability was used as an index to evaluate cytoprotective/cytotoxic attributes of particular substance.