Characterisation of the human microglial profile identified with translocator protein (TSPO) and its role in the pathophysiology and progression of Alzheimer’s disease

Abstract

Neuroinflammation is one of the pathological hallmarks of Alzheimer’s disease (AD). Microglia are the main immune cell in the central nervous system and contribute to the development and severity of AD pathology. The need for novel methods to study reactive microglia are of the utmost importance, with accurate positron emission tomography (PET) methods becoming prevalent. Translocator protein (TSPO), expressed on microglial mitochondria, has been attributed to microglial reactivity and is commonly used as a PET ligand for this reason. However, clinicians’ interpretation of this ligand is somewhat conflicting as it is not known which functional or morphological subtypes of microglia are being identified. Therefore, this project aims to characterise which microglia TSPO identifies, and its importance in the context of AD. Furthermore, the cerebellum is used as a pseudo-reference region for PET scans in AD and this will be compared to the temporal lobe, an area of high AD pathology, to assess whether the cerebellum is appropriate to use for baseline measurements. Sixty cases from temporal lobe and cerebellum, split equally into three Braak groups (stages 0-II, stages III-IV and stages V-VI), were used for immunohistochemistry to assess TSPO expression, AD pathology (Aβ, hyperphosphorylated (p)Tau) and neuroinflammation (Iba1, HLA-DR and MSR-A), with half of the cases used for fluorescent double labelling of TSPO with microglial markers (Iba1, HLA-DR, CD68, MSR-A and CD64), astrocytes (GFAP), perivascular macrophages (CD163) and endothelial cells (CD31). Multiplex assays for thirty inflammatory proteins were performed to evaluate the neuroinflammatory microenvironment. As expected, Aβ, pTau and TSPO load increased as the disease progressed in the temporal lobe. In the cerebellum, Iba1, a marker of microglial motility, was significantly increased as AD progressed. There was a positive association found between TSPO and pTau in the temporal lobe. The inflammatory microenvironment showed increased IL15 only in the temporal lobe. The double fluorescent staining showed no significant change in cell count over the course of the disease for each individual set of double labelling in either brain region. However, when comparing the double labelling cell counts, the highest in both brain regions was CD68⁺TSPO⁺. Double staining with non-microglial markers displayed the absence of TSPO expression by astrocytes and perivascular macrophages, with some endothelial cells expressing TSPO. Overall, these findings suggest that TSPO expression may be related to a reactive/phagocytic phenotype with the higher expression in the more pathologically affected temporal lobe. The cerebellum exhibited a homeostatic environment with low levels of AD pathology, consistent lower TSPO expression, and presence of homeostatic microglia, meaning that this region remains appropriate to use as a pseudo-reference region for TSPO PET scans. Of note, the phagocytic profile of TSPO appeared to not be affected by disease stage or brain region.

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Identifiers

ORCID for Emma Garland: orcid.org/0000-0002-8501-4326

ORCID for Delphine Boche: orcid.org/0000-0002-5884-130X

ORCID for James Nicoll: orcid.org/0000-0002-9444-7246

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