טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentYasin Nasra
SubjectNovel TSPO Ligands, Based on a Quinazoline Scaffold, as
Therapy for Traumatic Brain Injury (TBI),
Including the Potential Involvement in
Modulation of Nuclear Gene...
DepartmentDepartment of Medicine
Supervisors Professor Emeritus Moshe Gavish
Professor Rafael M. Nagler
Full Thesis textFull thesis text - English Version


Abstract

Traumatic brain injury (TBI) can lead to death, progressing brain damage and behavioral impairments. It is known that the mitochondrial 18-kDa translocator protein (TSPO) is involved in neurodegenerative diseases and brain injury. TSPO is important for arresting cell death of neurons and glia. We studied the ability of TSPO ligands to regulate gene expression associated with cell differentiation and other processes potentially related to tissue regeneration. We also used the TSPO ligand ( 2-Cl-MGV-1: 2-chlorophenyl quinazolin-4-yl dimethylcarbamate) for treating neurodegeneration in a mouse model for TBI. 2-Cl-MGV-1 modulated brain gene expression, counteracted programmed cell death of astroglia, microglia activation, and induced neuronal differentiation.

Within 15 min the TSPO ligand PK 11195 induced changes in the expression of immediate early genes and transcription factors in U118MG cells. These changes accord real-time, reverse transcriptase (RT)-PCR. The changes in gene expression occurred at the time points of 15, 30, 45, and 60 min, as well as 3 and 24 h after exposure to PK 11195, were relevant to cell viability, proliferation, differentiation, adhesion, migration, tumorigenesis, and angiogenesis. This was corroborated microscopically for cell migration, accumulation, adhesion, and neuronal differentiation. Our triple labeling showed intense TSPO labeling in the mitochondria but without TSPO signal in the cell nuclei. Thus, mitochondrial TSPO appears to be part of the mitochondria-to- nucleus signaling pathway for modulation of nuclear gene expression.

2-Cl-MGV-1 applied at various doses (3.75, 7.5, 15.0 mg / kg) reduced brain injury size by 90%, as observed by MRI. We applied 7.5 mg / kg of 2-Cl-MGV-1 per mouse and found over a period of two months improvement in brain damage as assessed by MRI and behavior as assessed by a treadmill assay. 2-Cl-MGV-1 was started 5.5 hours after induction of TBI and appeared to be a good candidate for treatment of TBI in mice.

Within 18 hours after TBI, 2613 genes displayed changes in expression in the neocortex of mice subjected to TBI (p < 0.01, n = 4) compared to naïve mice.
After treatment with 7.5 mg / kg of 2-Cl-MGV-1, administered 4 hours after TBI, only 231 genes showed changed expression (p < 0.01, n = 4) compared to naïve mice.

We found that 1094 genes have their expression changed in mouse cortex 2 months after TBI. Only 157 genes have their expression changed in TBI suffering, vehicle treated mice. This indicates that 2-Cl-MGV-1 reduces effects caused by both TBI and DMSO.

We applied 7.5 mg / kg of 2-Cl-MGV-1 per mouse and assayed liver and kidney functions and found that this treatment has no toxic effects. DMSO was averse to healing of the brain, while 2-Cl-MGV-1 treatment resulted in recovery in the brain. Our genomics studies in cell culture and in mouse neocortex in situ suggests that the effects of 2-Cl-MGV-1 on regulation of programmed cell death and the mitochondria- to-cell nucleus pathway are important for the therapeutic effects of TSPO ligands in TBI.