[Frontiers in Bioscience E4, 2365-2377, June 1, 2012]

GSTP1, a novel downstream regulator of LRRK2, G2019S-induced neuronal cell death

Jie Chen1, Anthony Liou2, Lili Zhang2, Zhongfang Weng2, YanqinGao1, Guodong Cao1,2,3 Michael J. Zigmond1, 2, Jun Chen1,2,3

1State Key Laboratory of Medical Neurobiology and Institute of Brain Sciences, Fudan University School of Medicine, Shanghai, China, 200032, 2Department of Neurology and Pittsburgh Institute of Neurodegenerative Diseases, University of Pittsburgh, Pittsburgh, PA 15261, 3Geriatric Research, Educational and Clinical Center, Veterans Affairs Pittsburgh Health Care System, Pittsburgh, PA 15213

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Materials
3.2. Cell culture
3.3. DNA transfection
3.4. Establishment of stable expression cell clones
3.5. Sample preparation and Western blot analysis
3.6. Direct immunofluorescence
3.7. Cell death assessment
3.8. Two dimensional gel electrophoresis and image analysis
3.9. In gel tryptic digest
3.10. MALDI-TOF MS and MS/MS analysis and database search
3.11. Construction of lentivirus encoding LRRK2 wild-type and G2019S mutant
3.12. Real-time PCR
3.13. Methylation-specific PCR
3.14. Statistical analysis
4. Results
4.1. Cellular localization of truncated LRRK2 wild-type and G2019S
4.2. G2019S was more toxic than LRRK2 wild-type when over-expressed in SH-SY5Y cells
4.3. Expression of G2019S mutant decreased endogenous level of GSTP1
4.4. Over-expression of GSTP1 suppressed caspase-3 activation and cell death
4.5. G2019S down-regulate GSTP1 via promoter hyper-methylation
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

The enhanced neurotoxicity of the Parkinson's disease-associated LRRK2 mutant, G2019S, than its wild-type counter-part has recently been reported. Overexpression of LRRK2 (G2019S) in cultured neural cells results in caspase-3-dependent apoptosis via a yet undefined signaling pathway. Elucidation of the mechanism underlying LRRK2 (G2019S) neurotoxicity may offer new insights into the pathogenesis of Parkinson's disease. In this study, we identified glutathione s-transferase P1 (GSTP1) as a selective target whose expression is negatively regulated at the transcriptional levels via promoter hyper-methylation by LRRK2 (G2019S). Overexpression of LRRK2 (G2019S) in the human neuronal cell line SH-SY5Y markedly suppressed the expression of GSTP1 prior to any manifestation of cell death. Moreover, shRNA-mediated knockdown of endogenous GSTP1 expression exacerbated LRRK2 (G2019S) neurotoxicity, whereas overexpression of GSTP1 protected against LRRK2 (G2019S)-induced caspase-3 activation and neuronal apoptosis. In conclusion, the results suggest a previously undefined signaling mechanism underlying the neurotoxic effect of LRRK2 (G2019S), in which LRRK2 (G2019S) triggers oxidative stress in cells and, in turn, results in caspase-dependent apoptosis at least in part by suppressing the expression of GSTP1.