Neuroprotection of rapamycin in lactacystin-induced neurodegeneration via autophagy enhancement

https://doi.org/10.1016/j.nbd.2008.06.003Get rights and content

Abstract

The ubiquitin–proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the two most important cellular mechanisms for protein degradation. To investigate the role of autophagy in reversing neuronal injury, the proteasome inhibitor lactacystin was used to cause UPS dysfunction in differentiated PC12 cells and in C57BL/6 mice and rapamycin was used as an autophagy enhancer. The results showed that rapamycin pre-treatment attenuated lactacystin-induced apoptosis and reduced lactacystin-induced ubiquitinated protein aggregation in differentiated PC12 cells. The observed protection was partially blocked by the autophagy inhibitor 3-methyladenine. Furthermore, post-treatment of mice with rapamycin significantly attenuated lactacystin-induced loss of nigral DA neurons and the reduction of striatal DA levels. The lactacystin-induced high molecular ubiquitinated proteins were also attenuated by rapamycin treatment in vivo. In addition, as a chemical compound, rapamycin caused an increase of bcl2 protein level and blocked the release of cytochrome c from mitochondria to cytosal. We concluded that the neuroprotective effect of rapamycin is partially mediated by autophagy enhancement through enhanced degradation of misfolded proteins and autophagy enhancement may be considered to be a promising strategy to prevent diseases associated with misfolded/aggregated proteins, such as Parkinson's disease.

Introduction

Many neurodegenerative diseases such as Parkinson's disease (PD) are associated with the accumulation and aggregation of misfolded proteins (Lansbury and Lashuel, 2006, McNaught and Olanow, 2006, Rubinsztein, 2006). Preventing aggregation or disaggregating misfolded proteins might provide potential therapeutic benefit by slowing or preventing the progression of PD. The ubiquitin–proteasome system (UPS) is one of the most important degradation mechanisms acting on the aggregated proteins. Proteasomal dysfunction has been recently implicated in the pathogenesis of several neurodegenerative diseases, including PD (Olanow and McNaught, 2006, Sawada et al., 2004). Besides UPS, the autophagy-lysosomal pathway (ALP) is another important protein degradation pathway (Pan et al., 2008, Ravikumar et al., 2002) and its dysfunction has been implicated in a number of neurodegenerative disorders, including PD (Martinez-Vicente and Cuervo, 2007).

Non-specific inhibition of autophagy has been reported to have deleterious effects, including interference with efficient recycling from macromolecules under conditions of starvation and hypoxia (Kuma et al., 2004). This in turn can enhance susceptibility to certain types of apoptosis (Boya et al., 2005, Ravikumar et al., 2006) and eventually lead to the formation of ubiquitinated inclusions (Rideout et al., 2004). Genetic ablation of autophagy in mice (by deleting the ubiquitin- and LC3-binding protein “p62” that normally regulates the formation of protein aggregates), has been recently shown to induce neurodegeneration and accumulation of ubiquitinated proteins (Hara et al., 2006, Komatsu et al., 2006, Komatsu et al., 2007, Kuma et al., 2004, Pandey et al., 2007). Therefore, up-regulating autophagy may have a potential therapeutic value.

Rapamycin, which is a lipophilic, macrolide antibiotic, induces autophagy by inactivating the protein mammalian target of rapamycin (mTOR), and as such serves as an autophagy enhancer (Berger et al., 2006). Several studies have shown that rapamycin, acting through mTOR pathway, is neuroprotective in various neurological diseases (Erlich et al., 2007, Parker et al., 2000, Wu et al., 2008, Zemke et al., 2007). The purpose of our study was to investigate if autophagy enhancement by rapamycin alleviates UPS dysfunction-induced neuron injury. By applying proteasome inhibitor lactacystin to dopaminergic PC12 cell line, we created an in vitro model of proteasomal dysfunction (Keller et al., 2000, McNaught et al., 2002). We also used stereotactic injection with lactacystin into the median forebrain bundle (MFB) of mice to produce an in vivo model of substantia nigra injury, one of the characteristics of PD (Zhang et al., 2005, Zhu et al., 2007). The primary aim of the present study is to demonstrate the neuroprotective effects of rapamycin on lactacystin-induced cell injury and to explore the possible mechanisms involved in the cellular action of rapamycin on protein degrading systems.

Section snippets

Cell culture and treatment

PC12 cells were grown in 5% CO2 at 37 °C. The growth medium consisted of DMEM supplemented with 5% heat-inactivated fetal bovine serum, 5% heat-inactivated horse serum, and penicillin/streptomycin. Cells were plated in poly d-lysine-coated 6-well or 96-well plates and allowed to attach overnight. The next day, differentiated PC12 cells were established by treating PC12 cells with 2.5S NGF (100 ng/ml; Upstate Biotechnology, Lake Placid, NY, USA) in DMEM/1% horse serum for 5 days followed by

Lactacystin-induced injury in differentiated PC12 cells

The differentiated PC12 cells were treated with lactacystin at various concentrations for 24 h. The proteasome activity, as measured using 20S proteasome activity assay kit, was significantly decreased by 40% at 5 μM, 47% at 10 μM and 63% at 20 μM (Fig. 1A). The cell viability was significantly decreased by 20% at 2.5 μM (P < 0.01) and was decreased by 52% at 20 μM (P < 0.01) in cells treated with lactacystin as compared with its vehicle control, which was dose-dependently (Fig. 1B). The

Discussion

Several lines of evidence have converged to suggest that failure of the UPS to degrade misfolded proteins plays an important role in the etiopathogenesis of familial and sporadic PD as well as other neurodegenerative disorders (Betarbet et al., 2005, Olanow and McNaught, 2006; Soto and Estrada, 2008). In this study, we have modeled proteasomal dysfunction through the application of specific pharmacological proteasomal inhibitors to differentiated PC12 cells in vitro or to MFB of mice in vivo.

Acknowledgments

This work was supported by Diana Helis Henry Medical Research Foundation (2007, 2008), and in part by NIH (NS 043567, NS 40370). We also thank the National Parkinson Foundation for supporting the NPF Center of Excellence at Baylor College of Medicine.

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