No abstract

To monitor innate immune responses in the CNS, the 18 kDa Translocator protein (TSPO) is a frequently used target for PET imaging. The frequent assumption that increased TSPO expression in the human CNS reflects pro‐inflammatory activation of microglia has been extrapolated from rodent studies. However, TSPO expression does not increase in activated human microglia in vitro. Studies of multiple sclerosis (MS) lesions reveal that TSPO is not restricted to pro‐inflammatory microglia/macrophages, but also present in homeostatic or reparative microglia. Here, we investigated quantitative relationships between TSPO expression and microglia/macrophage phenotypes in white matter and lesions of brains with MS pathology. In white matter from brains with no disease pathology, normal appearing white matter (NAWM), active MS lesions and chronic active lesion rims, over 95% of TSPO+ cells are microglia/macrophages. Homeostatic microglial markers in NAWM and control tissue are lost/reduced in active lesions and chronic active lesion rims, reflecting cell activation. Nevertheless, pixel analysis of TSPO+ cells (n = 12,225) revealed that TSPO expression per cell is no higher in active lesions and chronic active lesion rims (where myeloid cells are activated) relative to NAWM and control. This data suggests that whilst almost all the TSPO signal in active lesions, chronic active lesion rims, NAWM and control is associated with microglia/macrophages, their TSPO expression predominantly reflects cell density and not activation phenotype. This finding has implications for the interpretation of TSPO PET signal in MS and other CNS diseases, and further demonstrates the limitation of extrapolating TSPO biology from rodents to humans.

Microglia, the resident myeloid cells in the central nervous system (CNS) play critical roles in shaping the brain during development, responding to invading pathogens, and clearing tissue debris or aberrant protein aggregations during ageing and neurodegeneration. The original concept that like macrophages, microglia are either damaging (pro-inflammatory) or regenerative (anti-inflammatory) has been updated to a kaleidoscope view of microglia phenotypes reflecting their wideranging roles in maintaining homeostasis in the CNS and, their contribution to CNS diseases, as well as aiding repair. The use of new technologies including single cell/nucleus RNA sequencing has led to the identification of many novel microglia states, allowing for a better understanding of their complexity and distinguishing regional variations in the CNS. This has also revealed differences between species and diseases, and between microglia and other myeloid cells in the CNS. However, most of the data on microglia heterogeneity have been generated on cells isolated from the cortex or whole brain, whereas white matter changes and differences between white and grey matter have been relatively understudied. Considering the importance of microglia in regulating white matter health, we provide a brief update on the current knowledge of microglia heterogeneity in the white matter, how microglia are important for the development of the CNS, and how microglial ageing affects CNS white matter homeostasis. We discuss how microglia are intricately linked to the classical white matter diseases such as multiple sclerosis and genetic white matter diseases, and their putative roles in neurodegenerative diseases in which white matter is also affected. Understanding the wide variety of microglial functions in the white matter may provide the basis for microglial targeted therapies for CNS diseases.

Microglial activation plays central roles in neuro-inflammatory and neurodegenerative diseases. Positron emission tomography (PET) targeting 18kDa Translocator Protein (TSPO) is widely used for localising inflammation in vivo, but its quantitative interpretation remains uncertain. For the first time, we show that TSPO gene and protein expression increases in activated microglia in mouse brain disease models postmortem, but does not change in a non-human primate (Callithrix jacchus) disease model or in common neurodegenerative and neuroinflammatory human diseases. We describe genetic divergence in the TSPO gene promoter that may be responsible for this, which is consistent with the hypothesis that the increase in TSPO in activated myeloid cells is unique to a subset of species within the Muroidea superfamily of rodents. These data emphasise that TSPO expression in human myeloid cells is related to different phenomena than in mice, and that TSPO PET reflects density of inflammatory cells rather than their activation state.

The lack of understanding as to the cellular and molecular basis of clinical and genetic heterogeneity in progressive multiple sclerosis (MS) has hindered the search for new effective therapies and biomarkers. Here, to address this gap, we analysed 740,000 single nuclei RNAseq profiles of 165 samples of white matter (WM) lesions, normal appearing WM, grey matter (GM) lesions and normal appearing GM from 55 MS patients and 28 controls. We find that gene expression changes in response to MS are highly cell-type specific in WM and GM lesions but are largely shared within an individual cell-type across lesions, following a continuum rather than discrete lesion-specific molecular programs. The major biological determinants of variability in gene expression in MS samples relate to individual patient effects, rather than to lesion types or other metadata. Using multi-omics factor analysis (MOFA+), we identify three subgroups of MS patients with distinct oligodendrocyte composition and WM glial gene expression signatures, suggestive of engagement of different pathological/regenerative processes. The discovery of these three patterns significantly advances our mechanistic understanding of progressive MS, provides a framework to use molecular biomarkers to stratify patients for best therapeutic approaches for progressive MS, and highlights the need for precision-medicine approaches to address heterogeneity among MS patients.

Most expression quantitative trait loci (eQTL) studies to date have been performed in heterogeneous brain tissues as opposed to specific cell types. To investigate the genetics of gene expression in adult human cell types from the central nervous system (CNS), we performed an eQTL analysis using single nuclei RNA-seq from 196 individuals in eight CNS cell types. We identified 6108 eGenes, a substantial fraction (43%, 2620 out of 6108) of which show cell-type specific effects, with strongest effects in microglia. Integration of CNS cell-type eQTLs with GWAS revealed novel relationships between expression and disease risk for neuropsychiatric and neurodegenerative diseases. For most GWAS loci, a single gene colocalized in a single cell type providing new clues into disease etiology. Our findings demonstrate substantial contrast in genetic regulation of gene expression among CNS cell types and reveal genetic mechanisms by which disease risk genes influence neurological disorders.

Microglial activation plays central roles in neuro-inflammatory and neurodegenerative diseases. Positron emission tomography (PET) targeting 18kDa Translocator Protein (TSPO) is widely used for localising inflammation in vivo, but its quantitative interpretation remains uncertain. We show that TSPO expression increases in activated microglia in mouse brain disease models but does not change in a non-human primate disease model or in common neurodegenerative and neuroinflammatory human diseases. We describe genetic divergence in the TSPO gene promoter, consistent with the hypothesis that the increase in TSPO expression in activated myeloid cells is unique to a subset of species within the Muroidea superfamily of rodents. We show that TSPO is mechanistically linked to classical pro-inflammatory myeloid cell function in rodents but not humans. These data emphasise that TSPO expression in human myeloid cells is related to different phenomena than in mice, and that TSPO PET reflects density of inflammatory cells rather than activation state.

Optic neuritis, a primary clinical manifestation commonly observed in multiple sclerosis (MS) is a major factor leading to permanent loss of vision. Despite decreased vision (optic neuritis), diplopia, and nystagmus, the immunopathology of the optic nerve in MS is unclear. Here, we have characterised the optic nerve pathology in a large cohort of MS cases (n=154), focusing on the immune responses in a sub-cohort of MS (n=30) and control (n=6) cases. Immunohistochemistry was used to characterise the myeloid (HLA-DR, CD68, Iba1, TMEM119, P2RY12) and adaptive immune cells (CD4, CD8, CD138) in the parenchyma, perivascular spaces, and meninges in optic nerve tissues from MS and control cases. Of the 154 MS cases, 122 (79%) reported visual problems of which 99 (81%) optic nerves showed evidence of damage. Of the 31 cases with no visual disturbances, 19 (61%) showed evidence of pathology. A pattern of myeloid cell activity and demyelination in the optic nerve was similar to white matter lesions in the brain and spinal cord. In the optic nerves, adaptive immune cells were more abundant in the meninges close to active and chronic active lesions, and significantly higher compared to the parenchyma. Similar to brain tissues in this Dutch cohort, B-cell follicles in the meninges were absent. Our study reveals that optic nerve pathology is a frequent event in MS and may occur in the absence of clinical symptoms.