טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentPe'er Yelena
SubjectThe Role of TSPO and TSPO Ligands in Glutamate Induced
Astrocyte Cell Death
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Moshe Gavish
Full Thesis textFull thesis text - English Version


Abstract

Astrocyte dysfunction occurs in many neuropathological conditions and astrocytes themselves are susceptible to toxic glutamate overload. Noteworthy, astrocytes express 18 kDa Translocator proteins (TSPO), a mitochondrial protein that regulates mitochondrial apoptosis and other cellular processes, including generation of reactive oxygen species at mitochondrial levels, which typically are also affected by glutamate. Therefore we studied the role of the TSPO in lethal effects of glutamate on cultures of the U118MG human glioblastoma cell line and primary rat astrocytes. Also regarding rat brain and behavior we studied the effectiveness of TSPO ligands to counteract damaging effects of kainic acid (KA), which acts on kainate receptors for glutamate.

We found that in U118MG cells glutamate induced cell death included glutathione depletion, nitric oxide synthase activation, calpain activation, mitochondrial membrane potential (DYm) collapse, and cardiolipin oxidation. Interestingly, U118MG cells detached due to glutamate exposure showed increases in TSPO protein expression as well as maximal binding density (Bmax). TSPO knockdown and TSPO classical ligands as well as our novel TSPO ligands BD-287 and BD-42 prevented these lethal effects. Pre-treatment (24 hrs) with TSPO ligands was needed to achieve these protective effects, suggesting the involvement of changes in protein expression. Subsequently, we found that glutamate induced increases in expression of Hexokinase2, voltage dependent anion channel (VDAC), and WNT1 induced signal pathway protein 1 (WISP1), while it induced a decrease in phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) expression. The TSPO ligands PK 11195, BD-287, and BD-42 attenuated the changes in VDAC and HK2 expression.

In contrast to U118MG cells, lethal effects of glutamate on primary rat astrocytes was not accompanied with the appearance of detached cells, and decreased TSPO protein expression and Bmax was observed in the remaining attached cells. Furthermore regarding primary astrocytes, classical TSPO ligands provided very little protection, but the novel TSPO ligands BD-287 and BD-42 did show dose dependent protective effects very similar to those found for U118MG cells.

We also tested the novel TSPO ligand MGV-1, in rats. Systemic KA injections in rats are known to cause seizures associated with neural damage in hippocampus, amygdala, and piriform cortex, which are associated with subsequent behavioral abberations. MGV-1 pretreatment for 1.5 hours had no effect on seizure activity and TSPO levels in hippocampal and cortical extracts in the present study. However, neuron depletion and astrocytes activation in cornuammonis (CA) 1 area of hippocampus associated with seizure induction was attenuated by one and two week-long post treatments with MGV-1. This post treatment also provided maintenance of spontaneous alternation in a Y-maze, and attenuated TSPO increase in CA1. In contrast to hippocampus, pre- and post treatment with MGV-1 did not fully prevent neuron depletion, astrocyte and glial activation, astrocytic cell death and TSPO increase in amygdala and piriform cortex.

Thus, TSPO may be an essential element for processes underlying glutamate induced changes in normal astrocytes as well as glioblastoma cells (astrocytic activation and astrocytic cell death) and may serve as a venue for treatments of brain disease and brain injury.