Simvastatin Induced Neurite Outgrowth Unveils Role of Cell Surface Cholesterol and Acetyl CoA Carboxylase in SH-SY5Y Cells

Abstract

Statins are known to modulate cell surface cholesterol (CSC) and AMP-activated protein kinase (AMPK) in non-neural cells; however no study demonstrates whether CSC and AMPK may regulate simvastatin induced neuritogenesis (SIN). We found that simvastatin (SIM) maintains CSC as shown by Fillipin III staining, Flotillin-2 protein expression / localization and phosphorylation of various receptor tyrosine kinases (RTKs) in the plasma membrane. Modulation of CSC revealed that SIN is critically dependent on this CSC. Simultaneously, phospho array for mitogen activated protein kinases (MAPKs) revealed PI3K / Akt as intracellular pathway which modulates lipid pathway by inhibiting AMPK activation. Though, SIM led to a transient increase in AMPK phosphorylation followed by a sudden decline; the effect was independent of PI3K. Strikingly, AMPK phosphorylation was regulated by protein phosphatase 2A (PP2A) activity which was enhanced upon SIM treatment as evidenced by increase in threonine phosphorylation. Moreover, it was observed that addition of AMP analogue and PP2A inhibitor inhibited SIN. Bio-composition of neurites shows that lipids form a major part of neurites and AMPK is known to regulate lipid metabolism majorly through acetyl CoA carboxylase (ACC). AMPK activity is negative regulator of ACC activity and we found that phosphorylation of ACC started to decrease after 6 hrs which becomes more pronounced at 12 hrs. Addition of ACC inhibitor showed that SIN is dependent on ACC activity. Simultaneously, addition of Fatty acid synthase (FAS) inhibitor confirmed that endogenous lipid pathway is important for SIN. We further investigated SREBP-1 pathway activation which controls ACC and FAS at transcriptional level. However, SIM did not affect SREBP-1 processing and transcription of its target genes likes ACC1 and FAS. In conclusion, this study highlights a distinct role of CSC and ACC in SIN which might have implication in process of neuronal differentiation induced by other agents.

Modulation of the Isoprenoid/Cholesterol Biosynthetic Pathway During Neuronal Differentiation In Vitro

“…Our results show that the pharmacological inhibition of MVA pathway increases the rate of neurite outgrowth. These results are in good agreement with those obtained by other research groups [Maltese and Sheridan, 1985;Sato-Suzuki and Murota, 1996;Holmberg et al, 2006;Pooler et al, 2006;Raina et al, 2013]. These effects are completely prevented by MVA administration, indicating that simvastatin-induced neurite elongation depends on the specific HMGR inhibition and not by an effect exerted by simvastatin per se.…”

“…The effects of statins on the developing brain are less clear. Previous studies investigated how statins affect neuronal differentiation in vitro [Maltese and Sheridan, 1985;Fan et al, 2002;Schulz et al, 2004;Kim et al, 2009;Raina et al, 2013;Samuel et al, 2014], but the results vary depending on the experimental protocols and the statins used. Moreover, it is unclear, whether neuronal differentiation modulates the mevalonate pathway per se.…”

Harnessing the trophic and modulatory potential of statins in a dopaminergic cell line

“…Trophic activity of statins on total neurite growth has been previously reported (Evangelopoulos et al, ; Jin et al, ; Raina et al, ; Samuel et al, ). However, their potential impact on the complexity of neuritic structures and the specificity versus the other neurotrophic compounds remains unclear.…”

“…These pathways regulate the polymerization of actin filaments involved in the neurite sprouting and have been proposed to contribute to statins trophic properties by inhibition of PI3K and Rho‐associated protein kinase (ROCK, a downstream effector of RhoA) (Fig. I) (Evangelopoulos et al, ; Jin et al, ; Racchetti et al, ; Raina et al, ; Schulz et al, ). In this context, we examined the effects of ROCK and PI3K inhibitors on statin‐induced neurite growth.…”

“…In addition to the neuroprotective potential, statins might also stimulate neurorestoration by promoting neurogenesis in rat dentate gyrus (Lu et al, ; Robin et al, ) and neurite growth in rat cortical and hippocampal primary neuronal cultures (Jin et al, ; Pooler et al, ) and also in different neuronal cell lines such as PC12 (Sato‐Suzuki and Murota, ; Schulz et al, ), SH‐SY5Y (Raina et al, ), and neuro‐2A (Evangelopoulos et al, ; Watanabe et al, ). The precise molecular mechanisms for these statin‐induced actions remain unclear but it has been suggested that Phosphoinositide 3‐kinase/Akt (PI3K/Akt) and ras homolog gene family, member A (RhoA) signaling pathways might play a role in statin‐induced neurite growth (Evangelopoulos et al, ; Jin et al, ; Raina et al, ; Samuel et al, ; Schulz et al, ). Inhibition of HMG‐CoA reductase by statins leads to inhibition of the mevalonate pathway and cholesterol synthesis.…”