The microglial translocator protein (TSPO) in Alzheimer's disease reflects a phagocytic phenotype

Abstract

Translocator protein (TSPO) is a mitochondrial protein expressed by microglia, ligands for which are used as a marker of neuroinflammation in PET studies of Alzheimer's disease (AD). We previously showed increasing TSPO load in the cerebral cortex with AD progression, consistent with TSPO PET scan findings. Here, we aim to characterise the microglial phenotype associated with TSPO expression to aid interpretation of the signal generated by TSPO ligands in patients. Human post-mortem sections of temporal lobe (TL) and cerebellum (Cb) from cases classified by Braak group (0-II, III-IV, V-VI; each n = 10) were fluorescently double labelled for TSPO and microglial markers: Iba1, HLA-DR, CD68, MSR-A and CD64. Quantification was performed on scanned images using QuPath software to assess the microglial phenotype of TSPO. Qualitative analysis was also performed for TSPO with GFAP (astrocytes), CD31 (endothelial cells) and CD163 (perivascular macrophages) to characterise the cellular profile of TSPO. The percentage of CD68⁺TSPO⁺ double-labelled cells was significantly higher than for other microglial markers in both brain regions and in all Braak stages, followed by MSR-A⁺TSPO⁺ microglia. Iba1⁺TSPO⁺ cells were more numerous in the cerebellum than the temporal lobe, while CD64⁺TSPO⁺ cells were more numerous in the temporal lobe. No differences were observed for the other microglial markers. TSPO expression was also detected in endothelial cells, but not detected in astrocytes nor in perivascular macrophages. Our data suggest that TSPO is mainly related to a phagocytic profile of microglia (CD68⁺) in human AD, potentially highlighting the ongoing neurodegeneration.

Keywords: Alzheimer’s disease; Human; Inflammation; Microglia; Translocator protein.

Fig. 1

Detailed images of fluorescent double staining with microglial markers and TSPO. Single cell or cluster of cells positive for Iba1 (a–f), HLA-DR (g–l), CD68 (m–r), MSR-A (s–x) and CD64 (y–ad) (green) with TSPO⁺ cells (red) in the temporal lobe (a–c, g–i, m–o, s–u, y–aa) and cerebellum (d–f, j–l, p–r, v–x, ab–ad). Counterstained nuclei with DAPI (blue). Images are from Braak stage VI cases. Scale bars = 20 µm

Fig. 2

Fluorescent double labelling of microglial markers and TSPO in the temporal lobe. Images and quantification of Iba1⁺ (a–c), HLA-DR⁺(d–f), CD68⁺ (g–i), MSR-A⁺ (j–l) and CD64⁺ (m–o) microglial cells (green) with TSPO⁺ cells (red) normalised to corresponding microglial marker (%), presented by Braak group (0–II, III–IV, V–VI). Counterstained nuclei with DAPI (blue). Scale bars = 50 µm

Fig. 3

Fluorescent double labelling of microglial markers and TSPO in the cerebellum. Images and quantification of Iba1⁺ (a–c), HLA-DR⁺(d–f), CD68⁺ (g–i), MSR-A⁺ (j–l) and CD64⁺ (m–o) microglial cells (green) with TSPO⁺ cells (red) normalised to corresponding microglial marker (%), presented by Braak group (0–II, III–IV, V–VI). Counterstained nuclei with DAPI (blue). Scale bars = 50 µm

Fig. 4

Comparisons of microglia double labelled with TSPO and different microglial markers by Braak stage. Cell counts normalised to microglial marker cell count (%), including the whole post-mortem cohort in the temporal lobe (a) and cerebellum (b) and then separated by Braak stage in the temporal lobe (c, e, g) and cerebellum (d, f, h)

Fig. 5

Comparisons between temporal lobe (TL) and cerebellum (Cb). Double labelling cell counts normalised to microglial marker cell count, including the whole post-mortem cohort

Fig. 6

Fluorescent double staining of non-microglial markers and TSPO. GFAP⁺ astrocytes (green) (a–h), CD31⁺ endothelial cells (green) (i–p) and CD163⁺ perivascular macrophages (green) (q–x) with TSPO (red) in the temporal lobe (a–d, i–l, q–t) and cerebellum (e–h, m–p, u–x). Counterstained nuclei with DAPI (blue). Scale bars = 20 µm or 50 µm