Microglial activation in schizophrenia: Is translocator 18 kDa protein (TSPO) the right marker?
Introduction
Genetic and epidemiological studies, as well as cross-sectional biomarker studies on blood and brain tissue, have shown an association between schizophrenia and the immune system (Benros et al., 2012; Ripke et al., 2014; Gandal et al., 2018; Hudson and Miller, 2018). It has therefore been hypothesized that activation of microglia, the main population of immune cells in the brain, is involved in schizophrenia pathogenesis (Howes and McCutcheon, 2017). Postmortem studies have addressed this hypothesis and analyzed the density and morphology of microglial cells in schizophrenia. Some studies reported the presence of microglial activation in schizophrenia (Radewicz et al., 2000; Wierzba-Bobrowicz, 2005), but this was not replicated by others (Brisch et al., 2017; Seredenina et al., 2017; van Kesteren et al., 2017).
Various studies applied positron emission tomography (PET) imaging with radioligands for the translocator 18 kDa protein (TSPO) receptor to measure microglial activation in patients with schizophrenia in vivo. Although the first studies showed increased binding of TSPO tracers (van Berckel et al., 2008; Doorduin et al., 2009), contradicting findings have been reported (Marques et al., 2018). A recent meta-analysis of individual participant data even showed a significant decreased binding of second generation TSPO tracers in first-episode psychosis patients (Plavén-Sigray et al., 2018). These results not only raise questions about the occurrence of microglial activation in schizophrenia patients, but also about the sensitivity and specificity of this method for detecting microglial activation in schizophrenia and other psychiatric disorders (Collste et al., 2017; Notter et al., 2018a, Notter et al., 2018b).
TSPO is a mitochondrial protein that is involved in a range of cellular functions, including steroidogenesis, proliferation and apoptosis (Gut et al., 2015). TSPO is expressed by many cell types, including myeloid immune cells, such as microglia and macrophages (Woods and Williams, 1996). In rodents, it has been shown that microglial activation leads to increased expression of TSPO and binding of TSPO PET ligands (Zhao et al., 2011; Bae et al., 2014; Karlstetter et al., 2014; Ory et al., 2015; Notter et al., 2018; Pannell et al., 2019). In humans, binding of TSPO tracers is increased in neuropathological conditions such as Alzheimer’s disease (Cagnin et al., 2001; Gulyás et al., 2009; Kreisl et al., 2013) and multiple sclerosis (Banati et al., 2000; Colasanti et al., 2016; Herranz et al., 2016; Nutma et al., 2019). From postmortem studies it is known that neuroinflammation and microglial activation are present in these conditions. In addition, it has been shown using autoradiography that the tracers bind primarily to activated microglia or macrophages in these diseases (Banati et al., 2000; Cagnin et al., 2001; Gulyás et al., 2009; Brackhan et al., 2016). PET imaging using TSPO tracers has therefore widely been used for various conditions to investigate microglial activation in vivo.
However, several papers have shown that TSPO expression in the central nervous system (CNS) is not restricted to microglia, since astrocytes, neurons and endothelial cells can also express TSPO (Woods and Williams, 1996; Cosenza-Nashat et al., 2009; Notter et al., 2018; Notter et al., 2018; Nutma et al., 2019). In addition, it is not clear whether TSPO is upregulated when human microglia are activated. In vitro experiments have addressed this question but showed heterogeneous results. Owen et al. found that TSPO expression and binding of TSPO ligands were not increased in activated human macrophages and cultured primary microglia, in contrast to activation of rodent macrophages and microglia (Narayan et al., 2017; Owen et al., 2017). Beaino et al. found that administration of lipopolysaccharide (LPS) and interferon-γ resulted in increased TSPO expression in adult microglia isolated from human white matter postmortem material (Beaino et al., 2017).
The aim of this study was to further explore whether TSPO expression is altered in schizophrenia and can be used as a marker to measure human microglial activation in this disease. We first analyzed the expression of TSPO and the relation with microglia, inflammatory and astrocytic markers in postmortem brain tissue of patients with schizophrenia and controls. We then analyzed whether TSPO is upregulated when human microglia are activated ex vivo. Previous studies on the regulation of TSPO expression were performed on human microglia that were cultured for at least five days before they were activated (Beaino et al., 2017; Narayan et al., 2017; Owen et al., 2017; Notter et al., 2018). However, culturing microglia drastically changes the phenotype of microglia and might influence their ability to regulate TSPO expression upon inflammatory conditions (Gosselin et al., 2017). Therefore, we also investigated the regulation of TSPO expression on isolated human primary microglia that were challenged with different pro- and anti-inflammatory compounds.
Section snippets
Human brain tissue
Fresh, frozen, and paraffin postmortem tissue of the medial frontal gyrus (MFG) and corpus callosum (CC) was provided by the Netherlands Brain Bank (hersenbank.nl). We selected the medial frontal gyrus (MFG) as region of interest (ROI) for the expression of TSPO in schizophrenia, since this region has frequently been associated with schizophrenia pathology (Smieskova et al., 2010; Fusar-Poli et al., 2011). To analyze the regulation of TSPO in isolated microglia, we additionally selected the CC
TSPO expression in postmortem brain tissue of schizophrenia patients and controls
We measured TSPO expression in grey and white matter of the MFG using qPCR and found similar expression levels between schizophrenia patients and controls (Supplementary Fig. 1; p-value grey 0.73; p-value white 0.81). Results were confirmed in the published microarray data of the Stanley Medical Research Institute on the dorsolateral prefrontal cortex from 34 patients with schizophrenia and 33 controls (median TSPO expression schizophrenia 7.75; controls 7.91; p-value 0.78). TSPO has been
Discussion
The aim of this study was to explore whether TSPO expression is altered in schizophrenia and can be used as a marker to measure human microglial activation in this disease. We did not find altered TSPO expression levels in postmortem brain tissue of schizophrenia patients. These results are in agreement with recent sizable transcriptome studies on postmortem brain tissue of schizophrenia patients (Birnbaum et al., 2018; Gandal et al., 2018), as well as the more recent TSPO PET studies that
Contributors
LDW, EMH and RSK designed the study. MAS, ML, JM and TvdD performed the experiments. MAS and LDW analyzed the data. MAS and LDW wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.
Role of the funding source
The study sponsors had no role in study design; the collection, analysis and interpretation of data and were not involved in preparation of the manuscript.
Conflicts of interest
The authors declare no potential conflict of interest.
Acknowledgements
This study was supported by a TOP subsidy (1212154) of the Netherlands Organization for Scientific Research (NWO), a 2014 NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation and the psychiatric donor program of the Netherlands Brain Bank (NBB-Psy), which is supported by the Netherlands Organization for Scientific Research (NWO). The authors thank the team of the Netherlands Brain Bank for their excellent services (www.brainbank.nl).
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