|M.Sc Student||Perry Luba|
|Subject||The Role of TSPO in Mediating Cell Death Induced by|
|Department||Department of Medicine||Supervisor||Professor Emeritus Moshe Gavish|
|Full Thesis text|
Previous studies have shown that the mitochondrial 18 kDa Translocator Protein (TSPO) as well as nitric oxide (NO) appear to be involved in neurodegeneration. In particular, both TSPO and NO have been associated with processes involved in neurodegenerative diseases. Furthermore, both TSPO and the NO producing enzyme, nitric oxide synthase (NOS) are up - regulated in inflammatory-activated glia that in turn may kill neurons and cause neurodegeneration. A potential physical link connecting between the TSPO and NOS has also been described previously.
In the present study, we wanted to check whether cell death induced by NO involves activation of the TSPO. One possible mechanism of receptor activation by NO previously described is protein S-nitrosylation (i.e. attachment of NO to protein cysteine residue). For our study, we induced cell death by applying nitric oxide donors to two different cell lines, human glioblastoma U118MG cells and rat pheochromocytoma PC12 cells. Furthermore, we studied the effects of the TSPO specific ligands, PK 11195 and Ro5-4864, and also the central benzodiazepine receptor (CBR) specific ligand, clonazepam, at a concentration of 25 ?M. We also applied TSPO knockdown by siRNA and examined its effect on cell death induced by the NO donors.
We found that the TSPO ligand, PK 11195, significantly counteracted induction of cell death otherwise caused by the NO donor, sodium nitroprusside (SNP). This included apoptosis, mitochondrial membrane potential (ΔΨm) collapse, and reactive oxygen species (ROS) generation at mitochondrial levels. Moreover, we found that TSPO knockdown with siRNA significantly protected against mentioned cell death mechanisms. We also found that in siRNA cells, PK 11195 did not exert its effects mentioned. This indicates that the PK 11195 effects observed in this study, regarding mitochondrial activity and cell death, are TSPO specific effects. Displacement assays indicated that application of SNP did not interfere with [3H]PK 11195 binding to the TSPO. In addition, our Western blots and binding analysis showed no significant differences in TSPO protein expression and binding characteristics following SNP application. However, we found that TSPO is S-nitrosylated following SNP application.
In conclusion, our studies show that activation of TSPO by the NO donor SNP, leading to ROS generation, cardiolipin oxidation, collapse of the ΔΨm, and cell death, may involve S-nitrosylation of the TSPO.