|Ph.D Student||Azrad Maya|
|Subject||TSPO- A Regulator of Apoptosis and Inflammation in|
Cells of Glial Origin
|Department||Department of Medicine||Supervisor||Professor Emeritus Moshe Gavish|
|Full Thesis text|
The 18 kDa Translocator Protein (TSPO) is believed to protect brain from injury and diseases. We were interested in how TSPO regulates cell death and microglial activation in brain damage.
Regarding cell death, TSPO was shown to be involved in the mitochondrial apoptosis pathway including reactive oxygen species (ROS) generation, cardiolipin oxidation, and collapse of mitochondrial membrane potential (Δψm) leading to apoptosis. As one possible mechanism that may explain TSPO pro-apoptotic affects, TSPO may down-regulate anti-apoptotic proteins, such as Hexokinase II (HK II).
HK II and TSPO have opposite effects on cell death. The cell death inducer CoCl2 caused ROS generation, cardiolipin oxidation, ΔΨm collapse, and cell death in wild type U118MG cells which express TSPO. However, CoCl2 was not able to induce these events in TSPO knockdown U118MG cells, indicating that TSPO is required for cell death induction by CoCl2. We assumed that HK II protein levels in CoCl2-treated cells will be decreased, as part of the TSPO pro-apoptotic pathway. We believed that TSPO knockdown inhibiting TSPO function will prevent this decrease. Additionally, as HK II fulfills its anti-apoptotic role primarily when it is connected to the mitochondria, TSPO may regulate HK II intracellular localization.
To address these questions we treated wild-type U118MG glioblastoma cells and TSPO knockdown U118MG cells with CoCl2 and determined HK II protein expression and intracellular localization by western blot and mitochondrial/cytosolic fractionation, respectively.
In the current study we have found that HK II protein expression is elevated in TSPO knockdown cells indicating that TSPO regulates HK II protein expression. As opposed to our hypothesis, HK II expression increased in response to cell death induced by CoCl2 via TSPO activation and so we believe that this is a mechanism to counteract TSPO pro-apoptotic function.
Regarding TSPO regulation of microglial activation, it is known that TSPO expression is enhanced after CNS injury, in activated microglia and astrocytes. Manipulations of TSPO function either by TSPO ligands or by TSPO over-expression, can decrease neuroinflammation and neurotoxicity.
In the present study we tested the efficacy of two new TSPO ligands, designed and synthesized by us, MGV-1 and 2-Cl-MGV-1, in mitigating neuroinflammatory processes in comparison to the classic ligand, PK 11195.
To address this question, we assayed the capability of MGV-1 and 2- Cl- MGV-1 to reduce the levels of inflammatory markers, including COX-2, iNOS (western blot) and NO (Griess reaction), in LPS-induced microglia cells (BV-2). In the current study we have found that both MGV-1 and 2- Cl- MGV-1 reduced the levels of COX-2, iNOS and NO in LPS- induced BV-2 cells more efficiently than PK 11195. Additionally, MGV-1 and 2- Cl- MGV-1 were able to diminish the inflammatory response, even when added one hour after LPS administration.
In conclusion, brain homeostatic control by TSPO includes regulation of cell death potentially in interaction with HK II and regulation of microglial activation; therefore TSPO presents a promising target for treatment of brain injury and diseases.