Dataset Information

Detection of Synaptic Proteins in Microglia by Flow Cytometry.

ABSTRACT: A growing body of evidence indicates that microglia actively remove synapses in vivo, thereby playing a key role in synaptic refinement and modulation of brain connectivity. This phenomenon was mainly investigated in immunofluorescence staining and confocal microscopy. However, a quantification of synaptic material in microglia using these techniques is extremely time-consuming and labor-intensive. To address this issue, we aimed to quantify synaptic proteins in microglia using flow cytometry. With this approach, we first showed that microglia from the healthy adult mouse brain contain a detectable level of VGLUT1 protein. Next, we found more than two-fold increased VGLUT1 immunoreactivity in microglia from the developing brain (P15) as compared to adult microglia. These data indicate that microglia-mediated synaptic pruning mostly occurs during the brain developmental period. We then quantified the VGLUT1 staining in microglia in two transgenic models characterized by pathological microglia-mediated synaptic pruning. In the 5xFAD mouse model of Alzheimer's disease (AD) microglia exhibited a significant increase in VGLUT1 immunoreactivity before the onset of amyloid pathology. Moreover, conditional deletion of TDP-43 in microglia, which causes a hyper-phagocytic phenotype associated with synaptic loss, also resulted in increased VGLUT1 immunoreactivity within microglia. This work provides a quantitative assessment of synaptic proteins in microglia, under homeostasis, and in mouse models of disease.

SUBMITTER: Brioschi S

PROVIDER: S-EPMC7550663 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Detection of Synaptic Proteins in Microglia by Flow Cytometry.

Brioschi Simone S d'Errico Paolo P Amann Lukas S LS Janova Hana H Wojcik Sonja M SM Meyer-Luehmann Melanie M Rajendran Lawrence L Wieghofer Peter P Paolicelli Rosa C RC Biber Knut K

Frontiers in molecular neuroscience 20200929

A growing body of evidence indicates that microglia actively remove synapses <i>in vivo</i>, thereby playing a key role in synaptic refinement and modulation of brain connectivity. This phenomenon was mainly investigated in immunofluorescence staining and confocal microscopy. However, a quantification of synaptic material in microglia using these techniques is extremely time-consuming and labor-intensive. To address this issue, we aimed to quantify synaptic proteins in microglia using flow cytom ...[more]

PMID: 33132837

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Project description:The rapidly evolving nature of antibody drug development has resulted in technologies that generate vast numbers (hundreds to thousands) of lead antibody candidates during early discovery. These candidates must be rapidly pared down to identify the most drug-like candidates for in-depth analysis of their safety and efficacy, which can only be performed on a limited number of antibodies due to time and resource requirements. One key biophysical property of successful antibody therapeutics is high specificity, defined as low levels of nonspecific binding or polyspecificity. Although there has been some progress in developing assays for detecting antibody polyspecificity, most of these assays are limited by poor sensitivity or assay formats that require proprietary antibody surface display methods, and some of these assays use complex and poorly defined polyspecificity reagents. Here we report the PolySpecificity Particle (PSP) assay, a sensitive flow cytometry assay for evaluating antibody nonspecific interactions that overcomes previous limitations and can be used for evaluating diverse types of IgGs, multispecific antibodies and Fc-fusion proteins. Our approach uses micron-sized magnetic beads coated with Protein A to capture antibodies at extremely dilute concentrations (<0.02 mg/mL). Flow cytometry analysis of polyspecificity reagent binding to these conjugates results in sensitive detection of differences in nonspecific interactions for clinical-stage antibodies. Our PSP assay strongly discriminates between antibodies with different levels of polyspecificity using previously reported polyspecificity reagents that are either well-defined proteins or highly complex protein mixtures. Moreover, we also find that a unique reagent, namely ovalbumin, results in the best assay sensitivity and specificity. Importantly, our assay is much more sensitive than standard assays such as ELISAs. We expect that our simple, sensitive, and high-throughput PSP assay will accelerate the development of safe and effective antibody therapeutics.

Dataset Information

Detection of Synaptic Proteins in Microglia by Flow Cytometry.

Publications

Detection of Synaptic Proteins in Microglia by Flow Cytometry.

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