in-vitro-quantitative-imaging-assay-for-phagocytosis-dead

In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson&#39;s disease and Alzheimer&#39;s disease. ...

University of Oxford

Cerebral small vessel disease (SVD) is a major contributor to stroke, cognitive impairment and dementia with limited therapeutic interventions. There is a critical need to provide mechanistic insight and improve translation between pre-clinical research and the clinic. A 2-day workshop was held which brought together experts from several disciplines in cerebrovascular disease, dementia and cardiovascular biology, to highlight current advances in these fields, explore synergies and scope for development. These proceedings provide a summary of key talks at the workshop with a particular focus on animal models of cerebral vascular disease and dementia, mechanisms and approaches to improve translation. The outcomes of discussion groups on related themes to identify the gaps in knowledge and requirements to advance knowledge are summarized.

Small vessels, dementia and chronic diseases - molecular mechanisms and pathophysiology.

Dickkopf-related protein 1 (Dkk1) is a secreted protein ligand of low-density lipoprotein receptor-related protein 6 (LRP6), which antagonises canonical Wnt signalling. Elevated Dkk1 levels have been linked to Alzheimer's disease (AD), with protein blockade protective in pre-clinical AD models, suggesting inhibitors of Dkk1-LRP6 binding may have therapeutic utility against AD. Cell-based Dkk1-LRP6 assays reported in the literature use either modified Dkk1 protein and/or do not possess suitable throughput for drug screening. Here we report a novel immunocytochemical-based assay utilising high-content imaging (HCI) and automated data analysis suitable for the screening of protein and small-molecule inhibitors of Dkk1-LRP6 binding.

A novel high-content imaging-based technique for measuring binding of Dickkopf-1 to low-density lipoprotein receptor-related protein 6.

When trying to identify new potential therapeutic protein targets, access to data and knowledge is increasingly important. In a field where new resources and data sources become available every day, it is crucial to be able to take a step back and look at the wider picture in order to identify potential drug targets. While this task is routinely performed by bespoke literature searches, it is often time-consuming and lacks uniformity when comparing multiple targets at one time. To address this challenge, we developed TargetDB, a tool that aggregates public information available on given target(s) (links to disease, safety, 3D structures, ligandability, novelty, etc.) and assembles it in an easy to read output ready for the researcher to analyze. In addition, we developed a target scoring system based on the desirable attributes of good therapeutic targets and machine learning classification system to categorize novel targets as having promising or challenging tractrability. In this manuscript, we present the methodology used to develop TargetDB as well as test cases.

TargetDB: A target information aggregation tool and tractability predictor.

Excessive and dysregulated inflammation is known to contribute to disease progression. HSP90 is an intracellular chaperone known to regulate inflammatory processes including the NLRP3 inflammasome and secretion of the pro-inflammatory cytokine interleukin(IL)-1β. Here, primarily using an in vitro inflammasome ASC speck assay, and an in vivo model of murine peritonitis, we tested the utility of HSP90 inhibitors as anti-inflammatory molecules. We report that the HSP90 inhibitor EC144 effectively inhibited inflammatory processes including priming and activation of NLRP3 in vitro and in vivo. A specific inhibitor of the β HSP90 isoform was ineffective suggesting the importance of the α isoform in inflammatory signalling. EC144 inhibited IL-1β and IL-6 in vivo when administered orally, and was brain-penetrant. These data suggest that HSP90 inhibitors may be useful for targeting inflammation in diverse diseases that are worsened by the presence of inflammation.

Inhibition of the NLRP3 inflammasome by HSP90 inhibitors.

TREM2 is a microglial cell surface receptor, with risk mutations linked to Alzheimer's disease (AD), including R47H. TREM2 signalling via SYK aids phagocytosis, chemotaxis, survival, and changes to microglial activation state. In AD mouse models, knockout (KO) of TREM2 impairs microglial clustering around amyloid and prevents microglial activation. The R47H mutation is proposed to reduce TREM2 ligand binding. We investigated cell phenotypes of the R47H mutant and TREM2 KO in a model of human microglia, and compared their transcriptional signatures, to determine the mechanism by which R47H TREM2 disrupts function.

TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages.

Article	Year
Small vessels, dementia and chronic diseases - molecular mechanisms and pathophysiology. Clinical science (London, England : 1979)\| PubMed ID: 29712883	2018
A novel high-content imaging-based technique for measuring binding of Dickkopf-1 to low-density lipoprotein receptor-related protein 6. Journal of pharmacological and toxicological methods\| PubMed ID: 30476623	2019
TargetDB: A target information aggregation tool and tractability predictor. PloS one\| PubMed ID: 32877430	2020
Inhibition of the NLRP3 inflammasome by HSP90 inhibitors. Immunology\| PubMed ID: 32954500	2021
TREM2 Alzheimer's variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages. Alzheimer's research & therapy\| PubMed ID: 33198789	2020

John B. Davis

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