A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes

Summary

This protocol presents an in vitro live-imaging phagocytosis assay to measure the phagocytic capacity of astrocytes. Purified rat astrocytes and microglia are used along with pH indicator-conjugated synaptosomes. This method can detect real-time engulfment and degradation kinetics and provides a suitable screening platform to identify factors modulating astrocyte phagocytosis.

Abstract

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major immune cells and only phagocytes in the brain, recent studies have shown that astrocytes also participate in various phagocytic processes, such as developmental synapse elimination and clearance of amyloid beta plaques in Alzheimer's disease (AD). Despite these findings, the efficiency of astrocyte engulfment and degradation of their targets is unclear compared with that of microglia. This lack of information is mostly due to the lack of an assay system in which the kinetics of astrocyte- and microglia-mediated phagocytosis are easily comparable. To achieve this goal, we have developed a long-term live-imaging in vitro phagocytosis assay to evaluate the phagocytic capacity of purified astrocytes and microglia. In this assay, real-time detection of engulfment and degradation is possible using pH indicator-conjugated synaptosomes, which emit bright red fluorescence in acidic organelles, such as lysosomes. Our novel assay provides simple and effective detection of phagocytosis through live-imaging. In addition, this in vitro phagocytosis assay can be used as a screening platform to identify chemicals and compounds that can enhance or inhibit the phagocytic capacity of astrocytes. As synaptic pruning malfunction and pathogenic protein accumulation have been shown to cause mental disorders or neurodegenerative diseases, chemicals and compounds that modulate the phagocytic capacity of glial cells should be helpful in treating various neurological disorders.

Introduction

Glial cells, which refer to non-excitable cells in the brain, are the major cell type in the central nervous system (CNS). Previously, glial cells were regarded as mere supporting cells that mainly play passive roles in maintaining neuronal survival and basal synaptic properties. However, emerging evidence has revealed that glial cells play more active roles in various aspects of neurobiology, such as maintaining brain homeostasis, mediating synapse formation^1,2,3 and synapse elimination^4,5, and modulating synaptic pl....

Protocol

All methods described here have been approved by The Korea Advanced Institute of Science and Technology Institutional Animal Care and Use Committee (IACUC), KA2016-08.

1. Synaptosome Purification

NOTE: These procedures are adapted from a previously published paper¹⁹ with several modifications to improve the yield of purified synaptosomes (Figure 1).

1. Prepare discontinuous density gradient media.
   1. Place the following on ice: 3%, 10%, and 23% density gradient solutions in gradient buffer (bring to 250 mL of water with 109.54 g of sucrose, 6....

Results

In this in vitro phagocytosis assay with long-term live-imaging, we used synaptosomes from adult mouse brain homogenates, which were separated in the gradient solution between 23% gradient solution and 10% gradient solution by ultracentrifugation (Figure 3). After the preparation, synaptosomes exposed PS in their outer membrane (Figure 4), suggesting that they lost their function and could be recognized by PS receptors i.......

Discussion

In this article, we present methods for a long-term live-imaging in vitro phagocytosis assay using purified glial cells and pH indicator-conjugated synaptosomes. We show that compared with microglia, astrocytes possess different engulfment and degradation capacity during the phagocytosis of synaptosomes. In addition, our data suggest that astrocyte-secreted factors, which contain bridging molecules such as GAS6, ProteinS, and MEGE8, are essential for efficient PS-dependent phagocytosis of glial cells in the brai.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Synaptosome purification
Percoll	GE healthcare life sciences	17-0891-01
Quick Start Bradford Protein Assay Kit 2	BIO-RAD	5000202
pH indicator conjugation
Dimethyl sulfoxide(DMSO)	LPS solution	DMSO100
pHrodo red, succinimidyl ester	Molecula probes	P36600
Immunopanning
10X Earle’s balanced salt solution (EBSS)	Sigma	E7510
Bovine serum albumin	Bovogen	BSA025
Deoxyrebonuclease 1 (DNase)	Worthington	Is002007
(DMEM)	Gibco	11960-044
(dPBS)	Welgene	LB001-02
Fetal bovine serum (FBS)	Gibco	16000-044
Griffonia Simplicifolia Lectin(BSL-1)	Vector Labs	L-1100
Goat anti-mouse IgG+IgM(H+L)	Jackson ImmunoResearch	115-005-044
Goat anti-mouse IgM (μ-chain)	Jackson ImmunoResearch	115-005-020
Heparin-binding epidermal growth factor	Sigma	E4643
Human HepaCAM antibody	R&D systems	MAB4108
Integrin beta 5 monoclonal antibody (KN52)	eBioscience	14-0497-82
L-cysteine	Sigma	C7880
L-glutamate	Gibco	25030-081
N-acetly-L-cyteine (NAC)	Sigma	A8199
Neurobasal media	Gibco	21103-049
O4 hybridoma supernatant(mouse IgM)	Bansal et al.23
Papain	Worthington	Is003126
Penicillin/streptomycin	Gibco	15140-122
Pluristrainer 20 μm	PluriSelect	43-50020-03
Poly-D-lysine	Sigma	P6407
Progesterone	Sigma	P8783
Putrescine dihydrochloride	Sigma	P5780
Purified rat anti-mouse CD45	BD Pharmingen	550539
Purified mouse anti-rat CD45	BD Pharmingen	554875
Sodium pyruvate	Gibco	11360-070
Sodium selenite	Sigma	S5261
Transferrin	Sigma	T1147
Trypsin	Sigma	T9935
Trypsin inhibitor	Worthington	LS003086
Ultra-clear tube (Tube, Thinwall, Ultra-Clear)	Beckman Coulter	344059
Collect IP-ACM
Macrosep Advance Centrifugal Devices with Omega Membrane (10k)	PALL	MAP010C37
Macrosep Advance Centrifugal Devices with Omega Membrane (30k)	PALL	MAP030C37
Phagocytosis live imaging assay
Juli stage	NanoEntek
Time Series Analyzer V3 plugins	https://imagej.nih.gov/ij/plugins/time-series.html