TSPO co-occurrence with the microglial markers is primarily within the cell soma, with small amounts in the processes as observed with the microglial membrane markers (Iba1, HLA-DR, MSR-A and CD64) (Fig. 1). For CD68+TSPO+ staining, co-occurrence can be seen as both markers have a punctate pattern (Fig. 1m–r).

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

Quantification of the percentage of TSPO+ cells which co-express Iba1 (Iba1+TSPO+) showed no change in the temporal lobe (P = 0.5912) or cerebellum (P = 0.3123) over the course of the disease (Fig. 2c and 3c). This was also the case for: HLA-DR+TSPO+ cells (temporal lobe P = 0.9782; cerebellum P = 0.5178) (Figs. 2f and 3f), CD68+TSPO+ cells (temporal lobe P = 0.5171; cerebellum P = 0.7394) (Figs. 2i and 3i), MSR-A+TSPO+ cells (temporal lobe P = 0.3713; cerebellum P = 0.7106) (Fig. 2l and 3l), and CD64+TSPO+ cells (temporal lobe P = 0.6781; cerebellum P = 0.5976) (Figs. 2o and 3o). There were no changes in the percentage of single (Supplementary Fig. 1) or double cell counts (Supplementary Fig. 2) normalised to total cells across the Braak stages for any of the microglial markers, except for Iba1+TSPO+ cells in the cerebellum (P = 0.0377) (Supplementary Fig. 2). To confirm that our data were not affected by the overall number of cells changing over the course of disease, we performed analysis of the nuclei count across the Braak stages and found no significant difference (Supplementary Fig. 3).

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

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

To understand which marker, if any, had increased association with TSPO, comparisons were performed between the co-occurrence of TSPO and the different microglial markers, firstly independently of the Braak stage. The proportion of CD68+TSPO+ cells were the highest represented double-stained cells in both brain regions (temporal lobe: median 43.44%; cerebellum: median 42.15%), with their percentage significantly higher than Iba1+TSPO+ (P < 0.0001), HLA-DR+TSPO+ (P < 0.0001) and CD64+TSPO+ (P < 0.0001) cells in the temporal lobe, and higher than HLA-DR+TSPO+ (P = 0.0012) and CD64+TSPO+ (P < 0.0001) cells in the cerebellum (Fig. 4a and b). The second highest double-stained cell population was MSR-A+TSPO+ cells in both regions (temporal lobe: median 23.62%; cerebellum: median 20.00%) which was significantly higher than Iba1+TSPO+ (P < 0.0001), HLA-DR+TSPO+ (P = 0.0345) and CD64+TSPO+ (P < 0.0001) cells in the temporal lobe, and CD64+TSPO+ (P < 0.0001) cells in the cerebellum (Fig. 4a and b). The proportion of Iba1+TSPO+ cells was significantly higher than CD64+TSPO+ cells (P < 0.0001) in the cerebellum but not in the temporal lobe (Fig. 4b). Iba1+TSPO+ cells were the lowest population in the temporal lobe (median 5.59%) and CD64+TSPO+ cells the lowest in the cerebellum (median 3.53%).

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)

When the cases were split by Braak stage group, a similar pattern of difference was seen.

In the temporal lobe: Braak stage 0–II exhibited the highest cell population for CD68+TSPO+ (median 66.9%), which was significantly higher than Iba1+TSPO+ (P = 0.0004) and CD64+TSPO+ (P = 0.0013) cells (Fig. 4c). The next highest double-stained cell percentage was MSR-A+TSPO+ (median 24.47%), which was significantly higher than Iba1+TSPO+ cells (P = 0.0251) (Fig. 4c). The lowest cell population was Iba1+TSPO+ (median 2.89%) (Fig. 4c).

In Braak stage III–IV, CD68+TSPO+ was the highest represented cells (median 37.43%) and was significantly higher than Iba1+TSPO+ (P = 0.0205) and CD64+TSPO+ (P = 0.005) cells (Fig. 4e). Then, MSR-A+TSPO+ cells were the next most prevalent (median 29.91%) and were significantly more than CD64+TSPO+ cells (P = 0.0166) (Fig. 4e). The lowest percentage of double-stained cells in this Braak group were CD64+TSPO+ cells (median 5.84%) (Fig. 4e). In Braak stage V–VI, the highest cell population remained CD68+TSPO+ (median 57.99%), which was significantly higher than Iba1+TSPO+ (P = 0.0017), HLA-DR+TSPO+ (P = 0.0282) and CD64+TSPO+ (P = 0.005) cells (Fig. 4g).

In the cerebellum: Braak stage 0–II displayed CD68+TSPO+ with the highest cell percentage (median 43.17%) and was significantly higher than CD64+TSPO+ (P = 0.0007) (Fig. 4d). MSR-A+TSPO+ was the next highest cell population (median 18.33%), which was significantly higher than CD64+TSPO+ cells (P = 0.044) (Fig. 4d) and was the lowest population in this group (median 3.86%) (Fig. 4d).

Braak stage III–IV exhibited a similar pattern with CD68+TSPO+ cells being the highest (median 35.71%), significantly higher than HLA-DR+TSPO+ (P = 0.0168) and CD64+TSPO+ (P = 0.0006) cells (Fig. 4f).

In Braak stage V–VI, there was a significant difference between CD68+TSPO+ and CD64+TSPO+ (P = 0.0178) cells with CD68+TSPO+ being the most prevalent (median 24.29%) and CD64+TSPO+ the lowest cell population (median 4.57%) (Fig. 4h).

Interestingly, when comparing the percentage of cells in the temporal and cerebellum for the double labelling of each set of staining, there was no significant difference for HLA-DR+TSPO+, CD68+TSPO+ or MSR-A+TSPO+ cell percentages (Fig. 5b, c, d). This provides further evidence that while CD68+TSPO+ was the highest compared to other markers, this was independent of brain region. There were significantly more Iba1+TSPO+ cells in the cerebellum than the temporal lobe (P = 0.0003) (Fig. 5a), and a significantly higher percentage of CD64+TSPO+ cells in the temporal lobe compared to the cerebellum. (Fig. 5e).

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

Qualitative analysis of the non-microglial markers showed very little, if any, expression of TSPO in GFAP+ astrocytes or CD163+ perivascular macrophages (Fig. 6a–h and q–x). Co-occurrence of CD31+ endothelial cells with TSPO was detected (Fig. 6i–p). GFAP+ astrocytes were star shaped and abundant in the grey matter of the temporal lobe (Fig. 6d), while in the cerebellum, they appeared mainly clustered around the junction between granular and molecular layers of the grey matter (Fig. 6h). The TSPO staining in conjunction with GFAP was minimal within the astrocytes (Fig. 6a–h). The endothelial staining was predominantly located surrounding the lumen of the blood vessel with some co-occurrence of TSPO (Fig. 6i–p). Of note, TSPO expression was present throughout the whole of the blood vessel wall and not just localised to the intraluminal area (Fig. 6i–p), likely associated with smooth muscle cells. There was no co-occurrence of TSPO with CD163+ perivascular macrophages (Fig. 6q–x).

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