2018 Theses Doctoral
Translocator Protein 18 kDa: from Biomarker to Function
Translocator Protein 18 kDa (TSPO) is a protein that is expressed at low levels in the brain, but upon brain injury or inflammation, increases its expression in the areas of the brain specific to injury. In this way, TSPO can be used as a biomarker of brain inflammation and injury. TSPO is primarily expressed in two cell types, microglia and astrocytes, and is used as a marker of reactive gliosis in various brain pathologies. Currently, there is a paucity of knowledge on the function(s) of TSPO in glial cells. Recent studies using conditional and global TSPO knockout mice have questioned the role of TSPO in translocating cholesterol across the outer mitochondrial membrane as the first step in steroidogenesis.
In the brain, microglia and astrocytes exhibit distinct spatial and temporal patterns of TSPO upregulation. These differential patterns are not well characterized across disease models and in particular, are poorly characterized in the early stages of disease, prior to behavioral and clinical disease manifestations. Importantly, these distinct patterns of TSPO upregulation may indicate different functions of TSPO in microglia and astrocytes.
We examined TSPO levels in a neurodegenerative transgenic mouse model of Sandhoff disease (SD) and longitudinally compared TSPO levels to behavioral manifestations of disease and other neuropathological endpoints (neurodegeneration, reactive gliosis, ganglioside accumulation). This study confirmed TSPO upregulation prior to neurodegeneration in a brain region-dependent and disease course-dependent way. In brain regions with increased TSPO levels, there was a differential pattern of glial cell activation with astrocytes being activated earlier than microglia during the progression of disease. Immunofluorescent confocal imaging confirmed that TSPO colocalizes with both microglia and astrocyte markers, but the glial source of the TSPO response differs by brain region and age in SD mice.
We next wanted to gain insight into the function of TSPO in microglia. We previously demonstrated that TSPO ligands (TSPO-L) (1-100 nM) induced intracellular ROS production which was abrogated by NADPH oxidase (NOX2) inhibitors, thereby indicating an association between TSPO and NOX2. To further elucidate the relationship between TSPO and NOX, we determined the source of ROS production resulting from microglia exposure to TSPO-L. Intracellular and extracellular ROS production was inhibited by NOX inhibitors, but not by a mitochondria permeability transition pore inhibitor, indicating that the source of ROS production is from NOX and not from mitochondria. These findings were confirmed using the mitochondria specific ROS probe MitoSOX.
To further explore the TSPO-NOX2 association, we used 3 molecular approaches to examine protein-protein interactions under unstimulated or stimulated conditions (100 ng/mL lipopolysaccharide (LPS) for 18 hours) in primary microglia. 1) Co-immunoprecipitation (co-IP) revealed that the NOX2 subunits, gp91phox (gp91) and p22phox (p22), co-IP with TSPO supporting a protein-protein interaction. TSPO’s association with gp91 and p22 decreased with activation, but TSPO’s association with VDAC, a mitochondrial protein, remained constant. These findings suggest that microglia activation changes the dynamics of the TSPO-NOX2 interaction. 2) Confocal imaging and colocalization analysis of TSPO/gp91 or TSPO/p22 immunofluorescence confirmed that TSPO colocalizes with both NOX subunits. Under stimulated conditions, TSPO associated with gp91 and TSPO associated with p22, exhibit significantly decreased colocalization with VDAC suggesting a movement from the mitochondria to other cellular compartments. 3) Duolink Proximity Ligation Assay confirmed that TSPO interacts with p22, gp91 and VDAC. Our results suggest a novel TSPO-gp91-p22 interaction with VDAC in primary microglia that is disrupted by microglia activation and may be involved with redox homeostasis with significant implications for a new understanding of TSPO glial cell biology.
In summary, the present studies have strengthened the use of TSPO as a preclinical biomarker, confirmed its specific spatiotemporal upregulation in two cell types and have provided a new potential function of TSPO in microglia that has the possibility to revolutionize the TSPO field and to inform neurotoxicity assessments and neurological disease treatments.
- Loth_columbia_0054D_14904.pdf application/pdf 16.1 MB Download File
More About This Work
- Academic Units
- Environmental Health Sciences
- Thesis Advisors
- Guilarte, Tomas R.
- Ph.D., Columbia University
- Published Here
- October 18, 2018