Background: ER176 is a positron emission tomography (PET) tracer used to image neuroinflammation by binding to the translocator protein (TSPO). While TSPO is elevated in activated microglia, its expression across different cognitive states in the human brain remains poorly characterized. Our goal was to characterize the cellular expression of TSPO in the postmortem brain.

 

Methods: We performed multiplex immunofluorescence on amygdala and hippocampal sections from three postmortem cases with antemortem [C11] ER176 PET imaging. Subjects were genotyped for the TSPO rs6971 polymorphism and classified as high-affinity binders (G/G), mixed-affinity binders (A/G), or low-affinity binders (A/A). Cases are reported as cognitive status at time of imaging, neuropath diagnosis, and TSPO genotype: (1) cognitively unimpaired (CU), primary age related tauopathy (PART), A/G; (2) mild cognitive impairment (MCI), limbic predominant age-related TDP-43 encephalopathy (LATE), A/G; and (3) MCI, Alzheimer’s disease (AD), G/G. Sections were stained for TSPO, microglial marker IBA1, and nuclear marker DAPI.

 

Results: TSPO expression was detected in microglia, endothelial cells (including blood vessels and choroid plexus), astrocytes, and neurons (Figure 1). In microglia, TSPO did not fully overlap with IBA1-positive areas. In the CU/PART subject, neuronal TSPO staining was faint or absent (Figure 2), whereas the MCI/LATE and AD subjects showed more intense and frequent neuronal TSPO staining. Interestingly, the AD case with a high-affinity binding genotype (G/G) had fewer microglia overall, as identified by IBA1 staining, compared to mixed-affinity binding cases (A/G). Given that ER176 is used to image neuroinflammation via TSPO, this observation may reflect a paradox between binding affinity and microglial abundance, warranting further investigation.

 

Conclusions: This preliminary study demonstrates that TSPO is expressed across multiple cell types with variability in expression levels. These findings underscore the importance of further characterizing TSPO distribution in healthy and neurodegenerative conditions. Future work will expand multiplex immunofluorescence to additional cell markers and cases and explore correlations between TSPO staining and ER176 PET signal.