Preliminary Study on Clusterin Protein (sCLU) Expression in PC-12 Cells Overexpressing Wild-Type and Mutated (Swedish) AβPP genes Affected by Non-Steroid Isoprenoids and Water-Soluble Cholesterol.

Beata Pająk,Elżbieta Kania,Arkadiusz Orzechowski,Anita Gołaszewska

doi:10.3390/ijms20061481

Abstract

In this study we attempted to verify the hypothesis that the mevalonate pathway affects amyloid beta precursor protein (AβPP) processing and regulates clusterin protein levels. AβPP expression was monitored by green fluorescence (FL) and Western blot (WB). WB showed soluble amyloid protein precursor alpha (sAβPPα) presence in AβPP-wt cells and Aβ expression in AβPP-sw cells. Nerve growth factor (NGF)-differentiated rat neuronal pheochromocytoma PC-12 cells were untreated/treated with statins alone or together with non-sterol isoprenoids. Co-treatment with mevalonate, dolichol, ubiquinol, farnesol, geranylgeraniol, or water-soluble cholesterol demonstrated statin-dependent neurotoxicity resulted from the attenuated activity of mevalonate pathway rather than lower cholesterol level. Atorvastatin (50 μM) or simvastatin (50 μM) as well as cholesterol chelator methyl-β-cyclodextrin (0.2 mM) diminished cell viability (p < 0.05) and clusterin levels. Interestingly, co-treatment with mevalonate, dolichol, ubiquinol, farnesol, geranylgeraniol, or water-soluble cholesterol stimulated (p < 0.05) clusterin expression. Effects of non-sterol isoprenoids, but not water soluble cholesterol (Chol-PEG), were the most significant in mock-transfected cells. Geranylgeraniol (GGOH) overcame atorvastatin (ATR)-dependent cytotoxicity. This effect does not seem to be dependent on clusterin, as its level became lower after GGOH. The novelty of these findings is that they show that the mevalonate (MEV) pathway rather than cholesterol itself plays an important role in clusterin expression levels. In mock-transfected, rather than in AβPP-overexpressing cells, GGOH/farnesol (FOH) exerted a protective effect. Thus, protein prenylation with GGOH/FOH might play substantial role in neuronal cell survival.

Highlights

Cholesterol synthesis and non-sterol isoprenoids play a vital role during the development of the central nervous system (CNS) [1,2]
Isoprenoid synthesis starts with five carbon chain (5-C) isoprene formed from decarboxylation of mevalonate pyrophosphate (MEVPP) to form isopentenyl pyrophosphate (IPP)/dimethylallyl diphosphate (DMPP)
Chol-poly(ethylene-glycol 600) (PEG) administration could hardly restore cell viability diminished by ATR, SIM or MβCD (Figure 1, p > 0.05) so we assumed that diminished neuronal cell survival by statins or MβCD could be independent of a reduction in cholesterol level

Summary

Introduction

Cholesterol synthesis and non-sterol isoprenoids play a vital role during the development of the central nervous system (CNS) [1,2]. The bulk of the evidence, suggests that isoprenoids rather than cholesterol itself play a crucial role in the adult brain [4,5]. Isoprenoid synthesis starts with five carbon chain (5-C) isoprene formed from decarboxylation of mevalonate pyrophosphate (MEVPP) to form isopentenyl pyrophosphate (IPP)/dimethylallyl diphosphate (DMPP) This step is followed by several condensations of IPP/DMPP or other isoprenoids resulted in geranyl pyrophosphate (GPP, 10-C), farnesyl pyrophosphate (FPP, 15-C) or geranylgeranyl pyrophosphate (GGPP, C-20). Statins inhibit the MEV pathway, which plays a pivotal role in de novo cholesterol and isoprenoid synthesis. It is a matter of debate which end-product of the MEV pathway and in what circumstances are vital for brain functions in health and disease. With regard to Alzheimer’s disease (AD) overexpressed amyloid beta precursor protein (AβPP), a substrate for amyloid beta (Aβ) synthesis, was reported to suppress HMGCR [8]

Methods

Results

Conclusion