| Abstract No.: | B-C2101 |
| Country: | Canada |
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| Title: | REGULATION OF JNK SIGNALLING BY SNAM11 AND HSP70 IN DOPAMINERGIC CELL DEATH |
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| Authors/Affiliations: | 1 Lorraine V. Kalia*; 1 Hien Chau; 1 Suneil K. Kalia; 1 Andres M. Lozano;
1 Toronto Western Research Institute, University Health Network, Division of Brain Imaging and Behaviour Systems - Neuroscience, ON, Canada
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| Content: | Objectives: Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the loss of selected but heterogeneous populations of neurons, including dopaminergic neurons in the substantia nigra pars compacta. Despite the identification of genes linked to PD, the molecular mechanisms underlying the pathogenesis of PD remain poorly understood. SNam11 and c-Jun N-terminal kinase (JNK) are two molecules which have recently been identified as mediators of dopaminergic neurodegeneration. SNam11 is a member of the bcl-2 associated athanogene (BAG) family of co-chaperone molecules and JNK is a component of a mitogen-activated protein (MAP) kinase signalling pathway. We have previously shown SNam11 to be a negative modulator of the molecular chaperone Hsp70. Emerging evidence supports a role for Hsp70 as an inhibitor of JNK-mediated cell death. The objective of this study was to investigate whether SNam11, through its effects on Hsp70, regulates JNK signalling associated with dopaminergic cell death.
Materials and Methods: Protein-protein interactions were tested using in vitro pull down assays with purified recombinant proteins and co-immunoprecipitation experiments with lysates from transfected HEK293 cells. Interacting proteins were separated by SDS-PAGE and identified by Western blot. JNK signalling was studied in a cell culture model system in which cell death was induced in the human dopaminergic SH-SY5Y cell line using the proteasome inhibitor MG132. We investigated for activation of the JNK signalling pathway with and without overexpression of SNam11 by first separating cell lysate proteins by SDS-PAGE and then probing for the phosphorylation states of components of the JNK pathway. c-Jun activity, the final step of the JNK pathway, was measured in Hsp70-expressing SH-SY5Y cells with and without SNam11 using a luciferase-based reporting system.
Results: We found that SNam11 interacted with Hsp70 and JNK in both pull down assays and co-immunoprecipitation experiments. We also found that phosphorylation of JNK and its substrate c-Jun was increased in MG132-treated SH-SY5Y cells versus vehicle-treated cells. Over-expression of SNam11 resulted in a further increase in JNK phosphorylation. When the JNK pathway was activated in SH-SY5Y cells by expression of MEKK1, c-Jun activity measured by the luciferase reporter was increased relative to control. Co-expression of Hsp70 resulted in a decrease in c-Jun activity which was occluded by the addition of SNam11.
Conclusion: From these results, we conclude that JNK forms a protein complex in vitro with SNam11 and Hsp70. Furthermore, we suggest that SNam11, by inhibiting Hsp70, may upregulate a JNK-mediated cell death pathway in dopaminergic cells. Future studies will investigate whether SNam11-mediated signalling through the JNK pathway promotes death of dopaminergic neurons in vivo.
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