|M.Sc Student||Roim Inbar|
|Subject||In vitro and in vivo Studies of the Potential Involvement|
of TSPO in the Pathological Effects of apoE
|Department||Department of Medicine||Supervisor||Professor Emeritus Moshe Gavish|
|Full Thesis text|
Our invitro experiments focused on the association of TSPO and apoE with apoptosis. High density culture (HDC) was applied to induce apoptosis of the human neuronal cell lines, SK-N-SH and SHSY-5Y. Propidium Iodide (PI) labeling was used to assay cell viability. DNA fragmentation assays with the Cell death detection ELISA PLUS Kit and Pre-G1 measurements were applied to assay apoptosis. With Standard PCR, Western blot, and binding assays potential changes in the expression of TSPO and apoE mRNA and protein were assayed. Furthermore, we studied the effects of the TSPO ligands, PK 11195 and Ro5-4864, including the benzodiazepine clonazepam as a negative control.
HDC led to increases in apoptosis and TSPO expression invitro. At the concentration used (1µM), TSPO ligands did not exert a major influence. Using RT-PCR in SK-N-SH cells, we found enhanced apoE expression under HDC conditions, but not in the SHSY-5Y cell line. In both cell lines, HDC did not affect apoE protein levels. Thus, we did not observe an unequivocal correlation of TSPO and apoptotic levels with apoE expression invitro. Interestingly, HDC, in the SK-N-SH cell line, in combination with the TSPO ligands PK 11195 and Ro5-4864 enhanced apoE levels compared to untreated control.
To determine potential correlations between TSPO and apoE at the level of the organism in vivo, we studied TSPO binding characteristics in various tissues of apoE knockout mice that are involved in the functions mentioned above. TSPO levels appeared to be enhanced in platelets of apoE knockout mice. This may suggest that TSPO and apoE expression are interconnected in relation to some aspect of the host defense response. Liver, testis, brain, heart, aorta, lung, kidney and spleen, did not show a difference in TSPO binding levels between apoE knockout mice and wild type mice. This suggests that enhanced cholesterol levels, which occur in apoE knockout mice, do not play a determining role in TSPO expression in vivo.
Thus, invitro TSPO upregulation can be induced in correlation with enhanced apoptotic levels. In vivo, apoE and TSPO may interact in relation to some aspects of the host defense response.