Inflammatory Blood Biomarkers Are Associated with Long-Term Clinical Disease Severity in Parkinson's Disease.

An altered immune response has been identified as a pathophysiological factor in Parkinson's disease (PD). We aimed to identify blood immunity-associated proteins that discriminate PD from controls and that are associated with long-term disease severity in PD patients. Immune response-derived proteins in blood plasma were measured using Proximity Extension Technology by OLINK in a cohort of PD patients (N = 66) and age-matched healthy controls (N = 52). In a selection of 30 PD patients, we evaluated changes in protein levels 7-10 years after the baseline and assessed correlations with motor and cognitive assessments. Data from the Parkinson's Disease Biomarkers Program (PDBP) cohort and the Parkinson's Progression Markers Initiative (PPMI) cohort were used for independent validation. PD patients showed an altered immune response compared to controls based on a panel of four proteins (IL-12B, OPG, CXCL11, and CSF-1). The expression levels of five inflammation-associated proteins (CCL23, CCL25, TNFRSF9, TGF-alpha, and VEGFA) increased over time in PD and were partially associated with more severe motor and cognitive symptoms at follow-up. Increased CCL23 levels were associated with cognitive decline and the APOE4 genotype. Our findings provide further evidence for an altered immune response in PD that is associated with disease severity in PD over a long period of time.

Distinct gene expression in demyelinated white and grey matter areas of patients with multiple sclerosis.

Demyelination of the central nervous system is a prominent pathological hallmark of multiple sclerosis and affects both white and grey matter. However, demyelinated white and grey matter exhibit clear pathological differences, most notably the presence or absence of inflammation and activated glial cells in white and grey matter, respectively. In order to gain more insight into the differential pathology of demyelinated white and grey matter areas, we micro-dissected neighbouring white and grey matter demyelinated areas as well as normal-appearing matter from leucocortical lesions of human post-mortem material and used these samples for RNA sequencing. Our data show that even neighbouring demyelinated white and grey matter of the same leucocortical have a distinct gene expression profile and cellular composition. We propose that, based on their distinct expression profile, pathological processes in neighbouring white and grey matter are likely different which could have implications for the efficacy of treating grey matter lesions with current anti-inflammatory-based multiple sclerosis drugs.

Exploring reported genes of microglia RNA-sequencing data: Uses and considerations.

The advent of RNA-sequencing techniques has made it possible to generate large, unbiased gene expression datasets of tissues and cell types. Several studies describing gene expression data of microglia from Alzheimer's disease or multiple sclerosis have been published, aiming to generate more insight into the role of microglia in these neurological diseases. Though the raw sequencing data are often deposited in open access databases, the most accessible source of data for scientists is what is reported in published manuscripts. We observed a relatively limited overlap in reported differentially expressed genes between various microglia RNA-sequencing studies from multiple sclerosis or Alzheimer's diseases. It was clear that differences in experimental set up influenced the number of overlapping reported genes. However, even when the experimental set up was very similar, we observed that overlap in reported genes could be low. We identified that papers reporting large numbers of differentially expressed microglial genes generally showed higher overlap with other papers. In addition, though the pathology present within the tissue used for sequencing can greatly influence microglia gene expression, often the pathology present in samples used for sequencing was underreported, leaving it difficult to assess the data. Whereas reanalyzing every raw dataset could reduce the variation that contributes to the observed limited overlap in reported genes, this is not feasible for labs without (access to) bioinformatic expertise. In this study, we thus provide an overview of data present in manuscripts and their supplementary files and how these data can be interpreted.

Viscoelastic properties of white and gray matter-derived microglia differentiate upon treatment with lipopolysaccharide but not upon treatment with myelin.

BACKGROUND: The biomechanical properties of the brain have increasingly been shown to relate to brain pathology in neurological diseases, including multiple sclerosis (MS). Inflammation and demyelination in MS induce significant changes in brain stiffness which can be linked to the relative abundance of glial cells in lesions. We hypothesize that the biomechanical, in addition to biochemical, properties of white (WM) and gray matter (GM)-derived microglia may contribute to the differential microglial phenotypes as seen in MS WM and GM lesions. METHODS: Primary glial cultures from WM or GM of rat adult brains were treated with either lipopolysaccharide (LPS), myelin, or myelin+LPS for 24 h or left untreated as a control. After treatment, microglial cells were indented using dynamic indentation to determine the storage and loss moduli reflecting cell elasticity and cell viscosity, respectively, and subsequently fixed for immunocytochemical analysis. In parallel, gene expression of inflammatory-related genes were measured using semi-quantitative RT-PCR. Finally, phagocytosis of myelin was determined as well as F-actin visualized to study the cytoskeletal changes. RESULTS: WM-derived microglia were significantly more elastic and more viscous than microglia derived from GM. This heterogeneity in microglia biomechanical properties was also apparent when treated with LPS when WM-derived microglia decreased cell elasticity and viscosity, and GM-derived microglia increased elasticity and viscosity. The increase in elasticity and viscosity observed in GM-derived microglia was accompanied by an increase in Tnfα mRNA and reorganization of F-actin which was absent in WM-derived microglia. In contrast, when treated with myelin, both WM- and GM-derived microglia phagocytose myelin decrease their elasticity and viscosity. CONCLUSIONS: In demyelinating conditions, when myelin debris is phagocytized, as in MS lesions, it is likely that the observed differences in WM- versus GM-derived microglia biomechanics are mainly due to a difference in response to inflammation, rather than to the event of demyelination itself. Thus, the differential biomechanical properties of WM and GM microglia may add to their differential biochemical properties which depend on inflammation present in WM and GM lesions of MS patients.

Dynamic indentation reveals differential viscoelastic properties of white matter versus gray matter-derived astrocytes upon treatment with lipopolysaccharide.

Astrocytes in white matter (WM) and gray matter (GM) brain regions have been reported to have different morphology and function. Previous single cell biomechanical studies have not differentiated between WM- and GM-derived samples. In this study, we explored the local viscoelastic properties of isolated astrocytes and show that astrocytes from rat brain WM-enriched areas are ~1.8 times softer than astrocytes from GM-enriched areas. Upon treatment with pro-inflammatory lipopolysaccharide, GM-derived astrocytes become significantly softer in the nuclear and the cytoplasmic regions, where the F-actin network appears rearranged, whereas WM-derived astrocytes preserve their initial mechanical features and show no alteration in the F-actin cytoskeletal network. We hypothesize that the flexibility in biomechanical properties of GM-derived astrocytes may contribute to promote regeneration of the brain under neuroinflammatory conditions.

Regulation of microglial TMEM119 and P2RY12 immunoreactivity in multiple sclerosis white and grey matter lesions is dependent on their inflammatory environment.

Multiple Sclerosis (MS) is the most common cause of acquired neurological disability in young adults, pathologically characterized by leukocyte infiltration of the central nervous system, demyelination of the white and grey matter, and subsequent axonal loss. Microglia are proposed to play a role in MS lesion formation, however previous literature has not been able to distinguish infiltrated macrophages from microglia. Therefore, in this study we utilize the microglia-specific, homeostatic markers TMEM119 and P2RY12 to characterize their immunoreactivity in MS grey matter lesions in comparison to white matter lesions. Furthermore, we assessed the immunological status of the white and grey matter lesions, as well as the responsivity of human white and grey matter derived microglia to inflammatory mediators. We are the first to show that white and grey matter lesions in post-mortem human material differ in their immunoreactivity for the homeostatic microglia-specific markers TMEM119 and P2RY12. In particular, whereas immunoreactivity for TMEM119 and P2RY12 is decreased in the center of WMLs, immunoreactivity for both markers is not altered in GMLs. Based on data from post-mortem human microglia cultures, treated with IL-4 or IFNγ+LPS and on  counts of CD3+ or CD20+ lymphocytes in lesions, we show that downregulation of TMEM119 and P2RY12  immunoreactivity in MS lesions corresponds with the presence of lymphocytes and lymphocyte-derived cytokines within the parenchyma but not in  the meninges. Furthermore, the presence of TMEM119+ and partly P2RY12+ microglia in pre-active lesions as well as in  the rim of active white and grey matter lesions, in addition to TMEM119+ and P2RY12+ rod-like microglia in subpial grey matter lesions suggest that blocking the entrance of lymphocytes into the CNS of MS patients may not interfere with all possible effects of TMEM119+ and P2RY12+ microglia in both white and grey matter MS lesions.