39 Translocator protein expression in multiple sclerosis Introduction The 18 kDa translocator protein (TSPO) is an outer mitochondrial membrane protein that has attracted increasing interest for its use as a PET imaging target to visualise inflammation in the brain. TSPO is expressed in many tissues, including the brain, and has been suggested to be involved in mitochondrial “household” functions, although its exact functions are unknown1. TSPO PET signal is markedly upregulated in neurodegenerative and neuroinflammatory diseases including multiple sclerosis2-6, Alzheimer’s disease7,8, Parkinson’s disease9, viral encephalitis10,11, amyotrophic lateral sclerosis12, Huntington’s disease13 and frontotemporal dementia14 and thus has become recognised as a marker of in vivo neuroinflammation15-17. A limitation of these applications has been uncertainty regarding the interpretation of increased signal; many of the studies have widely assumed that increased signal reflects activated microglia, while ignoring the potential contributions of astrocytes and other cell types 2,11,12,15,18-30. Although recent studies using animal models of neurodegenerative diseases have shown astrocytic TSPO31-44, only a few have examined astrocytic expression of TSPO in the human CNS45, and these descriptions have been qualitative rather than quantitative46-48. In experimental autoimmune encephalomyelitis, and in cuprizone, two animal models of multiple sclerosis, increased TSPO expression has been described in astrocytes and microglia44,49. Increased microglial TSPO expression is associated with pro-inflammatory markers in rodents50. Basedonanimalmodel studies, TSPOhas been suggestedas a therapeutic target for modulation of the pathogenic microglial phenotypes44,51. However, in humans, TSPO is not upregulated in either pro-inflammatory macrophages52 or primary microglia53. These in vitro data are consistent with the finding that monocytes isolated from people with multiple sclerosis show lower TSPO expression compared to healthy controls54. Additionally, microglia/ macrophages in multiple sclerosis lesions adopt an intermediate phenotype, and the classical M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes probably represent extreme states only found in vitro55. Thus, in contrast to the pattern of TSPO expression in rodent models, it may not be so specifically associated with pro-inflammatory microglia in multiple sclerosis56. For interpretation of TSPO PET studies of brain inflammation in multiple sclerosis and for exploration of potential therapeutic targeting of TSPO, it is thus crucial to determine whether specific microglial markers are associated with TSPO expression and to what extent these cells contribute to the TSPO PET signal in neuroinflammatory diseases in vivo. Here we have performed a quantitative neuropathological study in a large cohort of multiple sclerosis brain and spinal cord tissues to characterise the cell types and identify the phenotypes of microglia expressing TSPO in lesions in the white and grey matter. We report three important findings. First, we confirm previous data showing that TSPO expression is increased in active and chronic active lesions, and that HLA-DR+ microglia are the cell type responsible for the majority of the signal. However, astrocytes expressing TSPO in the centre of chronic active and in inactive lesions also are important contributors to the total number of TSPO+ cells. Second, we show that, in humans, TSPO reports on microglia density rather than relative microglial activation and polarization. Finally, we confirm a strong, direct relationship between TSPO expression and TSPO radioligand binding in brain tissue ex vivo. Therefore, we show that whilst expression of TSPO reflects activated microglia, it is misleading to characterise TSPO as a marker restricted to pro-inflammatory microglia.
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