Adaptive changes in autophagy after UPS impairment in Parkinson's disease
Abstract
Yu-fei SHEN1, #, Yu TANG2, #, Xiao-jie ZHANG2, Kai-xing Huang2, Wei-dong LE1, 2, *
1Institute of Neurology, Ruijin Hospital, Jiao Tong University School of Medicine, Shanghai 200025, China; 2The Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
Aim: Ubiquitin-proteasome system (UPS) and autophagosome-lysosome pathway (ALP) are the most important machineries responsible for protein degradation in Parkinson’s disease (PD). The aim of this study is to investigate the adaptive alterations in autophagy upon proteasome inhibition in dopaminergic neurons in vitro and in vivo.
Methods: Human dopaminergic neuroblastoma SH-SY5Y cells were treated with the proteasome inhibitor lactacystin (5 μmol/L) for 5, 12, or 24 h. The expression of autophagy-related proteins in the cells was detected with immunoblotting. UPS-impaired mouse model of PD was established by microinjection of lactacystin (2 μg) into the left hemisphere of C57BL/6 mice that were sacrificed 2 or 4 weeks later. The midbrain tissues were dissected to assess alterations in autophagy using immunofluorescence, immunoblotting and electron microscopy assays.
Results: Both in SH-SY5Y cells and in the midbrain of UPS-impaired mouse model of PD, treatment with lactacystin significantly increased the expression levels of LC3-I/II and Beclin 1, and reduced the levels of p-mTOR, mTOR and p62/SQSTM1. Furthermore, lactacystin treatment in UPS-impaired mouse model of PD caused significant loss of TH-positive neurons in the substantia nigra, and dramatically increased the number of autophagosomes in the left TH-positive neurons.
Conclusion: Inhibition of UPS by lactacystin in dopaminergic neurons activates another protein degradation system, the ALP, which includes both the mTOR signaling pathway and Beclin 1-associated pathway.
Keywords: proteasome inhibitor; lactacystin; ubiquitin-proteasome system; dopaminergic neuron; Parkinson’s disease; autophagy-lysosomal pathway; autophagosome; mTOR; Beclin 1
This study was supported by funding from the National Natural Sciences Foundation of China (No 81171201) and the National Basic Research Program of China (No 2011CB510003).
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail wdle@sibs.ac.cn
Received 2012-11-17 Accepted 2012-12-30
Keywords:
1Institute of Neurology, Ruijin Hospital, Jiao Tong University School of Medicine, Shanghai 200025, China; 2The Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
Aim: Ubiquitin-proteasome system (UPS) and autophagosome-lysosome pathway (ALP) are the most important machineries responsible for protein degradation in Parkinson’s disease (PD). The aim of this study is to investigate the adaptive alterations in autophagy upon proteasome inhibition in dopaminergic neurons in vitro and in vivo.
Methods: Human dopaminergic neuroblastoma SH-SY5Y cells were treated with the proteasome inhibitor lactacystin (5 μmol/L) for 5, 12, or 24 h. The expression of autophagy-related proteins in the cells was detected with immunoblotting. UPS-impaired mouse model of PD was established by microinjection of lactacystin (2 μg) into the left hemisphere of C57BL/6 mice that were sacrificed 2 or 4 weeks later. The midbrain tissues were dissected to assess alterations in autophagy using immunofluorescence, immunoblotting and electron microscopy assays.
Results: Both in SH-SY5Y cells and in the midbrain of UPS-impaired mouse model of PD, treatment with lactacystin significantly increased the expression levels of LC3-I/II and Beclin 1, and reduced the levels of p-mTOR, mTOR and p62/SQSTM1. Furthermore, lactacystin treatment in UPS-impaired mouse model of PD caused significant loss of TH-positive neurons in the substantia nigra, and dramatically increased the number of autophagosomes in the left TH-positive neurons.
Conclusion: Inhibition of UPS by lactacystin in dopaminergic neurons activates another protein degradation system, the ALP, which includes both the mTOR signaling pathway and Beclin 1-associated pathway.
Keywords: proteasome inhibitor; lactacystin; ubiquitin-proteasome system; dopaminergic neuron; Parkinson’s disease; autophagy-lysosomal pathway; autophagosome; mTOR; Beclin 1
This study was supported by funding from the National Natural Sciences Foundation of China (No 81171201) and the National Basic Research Program of China (No 2011CB510003).
# These authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail wdle@sibs.ac.cn
Received 2012-11-17 Accepted 2012-12-30