Imaging neuroinflammation with TSPO: A new perspective on the cellular sources and subcellular localization

Abstract

Translocator Protein 18 kDa (TSPO), previously named Peripheral Benzodiazepine Receptor, is a well-validated and widely used biomarker of neuroinflammation to assess diverse central nervous system (CNS) pathologies in preclinical and clinical studies. Many studies have shown that in animal models of human neurological and neurodegenerative disease and in the human condition, TSPO levels increase in the brain neuropil, and this increase is driven by infiltration of peripheral inflammatory cells and activation of glial cells. Therefore, a clear understanding of the dynamics of the cellular sources of the TSPO response is critically important in the interpretation of Positron Emission Tomography (PET) studies and for understanding the pathophysiology of CNS diseases. Within the normal brain compartment, there are tissues and cells such as the choroid plexus, ependymal cells of the lining of the ventricles, and vascular endothelial cells that also express TSPO at even higher levels than in glial cells. However, there is a paucity of knowledge if these cell types respond and increase TSPO in the diseased brain. These cells do provide a background signal that needs to be accounted for in TSPO-PET imaging studies. More recently, there are reports that TSPO may be expressed in neurons of the adult brain and TSPO expression may be increased by neuronal activity. Therefore, it is essential to study this topic with a great deal of detail, methodological rigor, and rule out alternative interpretations and imaging artifacts. High levels of TSPO are present in the outer mitochondrial membrane. Recent studies have provided evidence of its localization in other cellular compartments including the plasma membrane and perinuclear regions which may define functions that are different from that in mitochondria. A greater understanding of the TSPO subcellular localization in glial cells and infiltrating peripheral immune cells and associated function(s) may provide an additional layer of information to the understanding of TSPO neurobiology. This review is an effort to outline recent advances in understanding the cellular sources and subcellular localization of TSPO in brain cells and to examine remaining questions that require rigorous investigation.

Keywords: Astrocytes; Confocal imaging; Microglia; Neuroinflammation; Positron emission tomography - immunofluorescence; Translocator protein 18 kDa (TSPO).

Fig. 1.

TSPO immunohistochemistry with diaminobenzidine (DAB) visualization in the hippocampus of wildtype and TSPO global knockout mice. A) TSPO immunostaining in a normal wildtype (WT) animal at the level of the hippocampus with high expression of TSPO in the ependymal cells of the lining of the ventricles (arrow 1), in the choroid plexus (arrow 2), in blood vessels throughout the brain neuropil, and in pyramidal neurons (PCL) of the hippocampus. B) Image of TSPO immunostaining at the same level of the hippocampus as in panel A but the brain tissue is from a global TSPO-KO mouse that was processed at the same time and under the same experimental conditions as in panel A. The image in the TSPO-KO tissue shows the complete loss of TSPO staining in the ependymal cells of the ventricles, blood vessels, and a nearly complete loss in the choroid plexus. However, there was essentially no loss in pyramidal neurons (PCL) of the hippocampus indicative of non-specific TSPO staining since the tissue is from a TSPO-KO mouse. C and D) are higher magnification images at the level of the CA3 region of the hippocampus. Arrows in Panel C point to TSPO staining of blood vessels in the WT tissue with the TSPO staining disappearing in blood vessels of the TSPO-KO mouse. E and F) are higher magnification images of the lateral ventricles. G and H) are images from the no primary or no secondary antibody controls. Brain slices from wildtype (WT) and TSPO knockout (KO) mice were processed using standard methods with the TSPO primary antibody (ab109497 Abcam) which has been extensively validated using multiple approaches including TSPO-KO cells and tissue (Loth et al., 2020 and supplementary materials within). Experimental methods are described in supplementary materials section.

Fig. 2.

Immunofluorescent confocal imaging of Purkinje cells and Bergmann glia in the cerebellar cortex. A) Top-view of a 3D condensed z-stack of double label immunofluorescence confocal imaging of Purkinje cells (calbindin-green) and Bergmann glia (GFAP-red). B and C) are the presentation of the individual channels. Bergmann glia cell bodies closely contact and wrap Purkinje cell soma and processes. One distinguishing anatomical feature of Bergmann glia is that they have processes that originate at their soma in the Purkinje cell layer and have a relatively straight and lengthy trajectory to the pial surface of the molecular layer (Reeber et al., 2018). On the other hand, Purkinje cell processes are shorter and radiate at all angles throughout the molecular layer. Green = Purkinje cell; Red = Bergmann glia. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3.

TSPO expression in the Purkinje cell layer of the cerebellum. A) Quadruple immunofluorescence confocal imaging represented in a top-down view of a 3D condensed z-stack of Purkinje cells (calbindin-gray color), Bergmann glia(GFAP-green color), microglia (Iba1-blue color) and TSPO (red color). The image shows the complex network of glial processes and cell bodies intimately touching the Purkinje cell soma and processes. B) Same image as panel A but the Bergmann glia and microglia channels are not presented to show the relationship of TSPO (red) with Purkinje cells (calbindin-green). Based on this view, in the yellow circles it appears that TSPO may be localized in Purkinje cells. C) In this image, only the TSPO signal is presented and enhanced to be able to visualize since TSPO levels are low in the normal brain [see LUT settings in supplementary Fig. 1]. D) This image again shows the Purkinje cell (calbindin-green) and TSPO (red) but in this image both signals are enhanced [see LUT settings in supplementary Fig. 1]. With signal enhancement one can see a perceived colocalization of TSPO with the Purkinje cell. However, analysis of the TSPO and Purkinje cell signals in the yellow circle using line intensity profiles indicates they do not colocalize as the peaks do not correspond with each other. E) The image provided is the same as D, but the microglia (blue – Iba1) signal is also added. One can see based on the intensity profile that TSPO colocalizes with microglia and not with the Purkinje cell. F) This image represents GFAP (green) staining of Bergmann glia process which in different areas colocalize with TSPO (red) generating a yellow color. Intensity profiles indicate that TSPO (red) colocalizes with Bergmann glia processes (green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4.

TSPO expression in the CA1 region of the hippocampus is not associated with neurons. A) Quadruple immunofluorescence confocal imaging represented in a top-down view of a 3D condensed z-stack of pyramidal neurons (NeuN-gray color), astrocytes (GFAP-green color), microglia (Iba1-blue color) and TSPO (red color). The image shows the complex network of glial processes and cell bodies intimately touching pyramidal neurons and processes. B1) This image is the same as in panel A, but now pyramidal neurons labeled with NeuN are displayed in green and TSPO in red. The microglia and astrocyte signal were removed to visualize the spatial relationship between TSPO and neurons. Areas of TSPO are noted in the yellow boxes and circles. There is a low level of TSPO expression that is outside or interspersed between pyramidal neurons. B2) This image is the same as in panel B1, but it represents a 67.5 degree rotation of the z-stack showing that the TSPO staining is not localized with neurons. C) Same image as B1 but now the astrocyte (blue signal) is added and the colocalization of TSPO with astrocyte in the yellow circle is analyzed. The left side of the image is the side view of the zstack and the bottom is the intensity profile (yellow line) indicating signal colocalization of TSPO and astrocyte. D) A similar approach was used in panel D as in panel C, but in this case, the TSPO signal inside the yellow circle appears to colocalized with the microglia marker Iba1 (blue). Again, the side view of the z-stack shows that the TSPO signal colocalizes with microglia which is confirmed in the intensity profile (yellow line) of the signals in the bottom of panel D. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5.

TSPO is highly expressed in blood vessels in the hippocampus and cerebellum of the normal mouse brain. A) Quadruple immunofluorescence confocal imaging represented in a top-down view of a 3D condensed z-stack of neurons in the dentate gyrus in the hippocampus (NeuN-cyan), blood vessels (CD31-blue), astrocytes (GFAP-green), and TSPO (red). The image shows the presence TSPO in a blood vessel in close contact with neurons in the granule cell layer. To the right is the side view of the z-stack showing the blood vessel with several TSPO positive puncta. B) the different panels show the individual signals showing that TSPO is present in blood vessels. C) Triple immunofluorescence confocal imaging represented in a top-down view of a 3D condensed z-stack of Purkinje cells (calbindin – green), blood vessels (CD31 – blue), and TSPO (red). Left panel shows the 3 signals and the right panel shows Purkinje cell and TSPO. Lower left panel shows the presence of TSPO in the blood vessel and the lower right panel is TSPO only. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)