Fig 1: Model of TSPO-mediated ROS production in retinal phagocytes.a In the healthy retina, resident microglia populate the plexiform layers. With their long protrusions, they constantly scan their environment and phagocytose cell debris. Different insults in the RPE and photoreceptor layer rapidly alert microglia. Resident microglia transform into ameboid phagocytes, migrate to the lesion sites and recruit macrophages from the periphery. b In response to these pathological signals, microglia increase pro-inflammatory and pro-angiogenic cytokine expression to resolve neuroinflammation and promote tissue recovery. Reactive microglia also upregulate mitochondrial TSPO leading to increased cytosolic calcium levels, which is essential for NOX1-mediated ROS production. Chronic activation of microglia may be detrimental and promote retinal degeneration. Binding of the synthetic ligand XBD173 to TSPO limits inflammatory responses and inhibits the increase of cytosolic calcium levels thus preventing from ROS damage. XBD173 supports the conversion of reactive microglia towards a neuroprotective phenotype, limiting pathological CNV. BM Bruch’s membrane; OS outer segment; IS inner segment; ONL outer nuclear layer; OPL outer plexiform layer; INL inner nuclear layer; IPL inner plexiform layer; GCL ganglion cell layer; NFL nerve fiber layer.
Fig 2: Biological validation of high expression level of TSPO in BAT. (a) Western blot of BAT and WAT tissue extractions with anti-TSPO antibody (lane1–4: four duplicated BAT samples; lane5–8: four duplicated WAT samples), and the full length of blot could be found in SI Fig. 6b, (b) quantification of (a). Note higher TSPO level in BAT compared to WAT. (c) Histological staining of BAT (left) and WAT (right) tissue slices with mitochondria specific dye MitoTrack deep Red, and (d) immunohistological staining of BAT and WAT tissue with anti-TSPO antibody (green) and DAPI for nuclei (blue). Scale bar: 30 µm.
Fig 3: TSPO expression in grey matter lesions. Representative images of TSPO expression in control (A and D) and multiple sclerosis (B, C and E–I) in grey matter lesions; NAWM (B), and NAGM (E); leukocortical white matter (C), and grey matter (F); intracortical (G), subpial (H), and transcortical (I) lesions. Similar to white matter lesions leukocortical white matter lesions showed large TSPO+HLA-DR- cells (black arrowheads; C) which were GFAP+ astrocytes (C, inset). No differences were found in TSPO+ cells in grey matter lesions (J). No significant increase in TSPO+HLA-DR+ cells were found in grey matter lesions compared to control (K). Data are expressed as mean ± SEM. Scale bars in A–I = 50 µm. Insets are digitally zoomed to ×800. CON = control; GM = grey matter; LC = leukocortical; WM = white matter.
Fig 4: Schematic illustrating the non-cell-autonomous contributions of infiltrating retinal microglia to rod demise in inherited retinal degenerationsIn the rd10 mouse, rod photoreceptors bearing a mutation in the Pde6b protein experience cellular stress. A subset of rods undergoes apoptotic cell death (blue box) which is marked by the development of TUNEL, PI, and activated caspase-3 staining. Ramified microglia in the inner retina, sensing photoreceptor stress via unknown signals, infiltrate the outer nuclear layer (ONL) shortly after P18, expressing markers of activation (e.g., TSPO), pro-inflammatory cytokines (e.g., IL-1ß), and phagocytic molecules (e.g., CD68 and MFG-E8). A subset of stressed but viable rods increases the presentation of the cell-surface “eat-me signal” phosphatidylserine (PS, green), inducing direct cell–cell contact with infiltrating microglia via dynamic microglial processes. These contacts likely result in photoreceptor axon and process retraction, which is then followed by the phagocytosis of rod somata and the clearance of living cells (orange box). Application of the vitronectin receptor antagonist, cRGD, inhibits rod phagocytosis, and also decreases microglial activation, thus reducing the positive-feedback recruitment and activation of other nearby microglia. Reduction in microglial phagocytosis, via direct inhibition or via general microglial depletion, therefore results in decreased microglial clearance via this non-apoptotic mechanism. Activated, infiltrating microglia can additionally influence and potentiate the apoptotic route for rod death via IL-1ß secretion, possibly via direct signaling and/or the indirect activation of Müller cells.
Fig 5: TSPO-dependent Ca2+ regulation occurs at the OMM and requires VDAC1 and PKA. (a) Real-time qRT-PCR studies show mRNA levels of MCU, normalized to levels of VDAC in MEFs; n=3; P>0.05. (b) Real-time qRT-PCR studies show mRNA levels of MCU, normalized to levels of VDAC in MEFs with modulated MCU expression; n=7; ***P<0.001. (c) Real-time qRT-PCR studies show mRNA levels of TSPO, normalized to levels of VDAC in MEFs with modulated MCU expression, n=5. (d and e) Immunoblot of MCU in MEFs WT and knocked out for the gene with quantification in e. (f) Representative traces showing ATP-induced mitochondrial Ca2+ uptake in MEFs. (g) Graph showing the mean maximum [Ca2+]m in response to ATP (1 mM) in MEFs (n=3; ***P<0.001). (h) Representative traces showing ATP-induced Ca2+ transients in TSPO-silenced VDAC1-/- MEFs. (i) Bar chart showing the mean maximum [Ca2+]m in VDAC1-/- MEFs; n=4; **P<0.05. (j) Representative traces showing ATP-induced Ca2+ transients MEFs exposed to PKI
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