Name: a novel in vitro live-imaging assay of astrocyte-mediated phagocytosis using ph indicator-conjugated synaptosomes
Uploaded: 2018-02-05T13:30:00
Description: Watch this Scientific Journal Video about A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes at JoVE.com

The authors thank Yeon-Joo Jung for her experimental support during synaptosome purification and Jungjoo Park for images of synaptosomes with PS exposure. In addition, we thank all members in Chung&#39;s laboratory for helpful discussion. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (NRF-2016M3C7A1905391 and NRF-2016R1C1B3006969) (W.-S. C).

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).
<ol>
	<li>Prepare discontinuous density gradient media.
	<ol>
...

<ol>
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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...

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 formation1,2,3 and synapse elimination4,5, and modulating synaptic plasticity6,7. Glial cells in the CNS include astrocytes, microglia, and oligodendrocytes. Among these cells, astrocytes and microglia have been shown to play phagocytic roles by engulfing synapses4,5, apoptotic cells8, neural debris9, and pathogenic proteins, such as amyloid beta plaques10,11. In the developing brain, astrocytes eliminate synapses in the dorsal lateral geniculate nucleus (dLGN) through MERTK- and MEGF10-dependent phagocytosis4. Similarly, microglia also eliminate C1q-coated synapses during developmental stages through the classical complement cascade5. Interestingly, it has been suggested that defects in synapse pruning can be one of the initiators of several neurological disorders. For example, it has been shown that mutations in complement component 4 (C4), which increases complement-mediated synapse pruning by microglia, are strongly associated with the prevalence of schizophrenia in humans12. A recent paper has also shown that the classical complement pathway is hyperactivated in the initiation stages of AD and induces early synapse loss in this disease13.
Compared with microglia-mediated phagocytosis, whether astrocyte-mediated phagocytosis contributes to the initiation and progression of various neurological disorders is less clear. However, a recent paper suggests that factors that alter the rate of normal synapse pruning by astrocytes may disrupt brain homeostasis and contribute to AD susceptibility and pathology14. The rate of synapse pruning by astrocytes is powerfully controlled by ApoE isomers, with a protective allele for AD (ApoE2) strongly enhancing the rate and a risk allele for AD (ApoE4) significantly lowering the rate. Moreover, transgenic mice expressing ApoE4 accumulated much more synaptic C1q than control or ApoE2 mice14. These data suggest that impaired astrocyte-mediated phagocytosis in the early AD brain may induce the accumulation of senescent C1q-coated synapses/synaptic debris that activates complement-mediated microglial phagocytosis, driving synaptic degeneration. The impaired phagocytic capacity of astrocytes in ApoE4 carriers may also contribute to the uncontrolled accumulation of amyloid beta plaques in AD-affected brains.
In addition, it has been shown that glial cells in the aged Drosophila brain lose their phagocytic capacity due to decreased translation of Draper, a homolog of Megf10 that astrocytes use for phagocytosing synapses. Restoring Draper levels rescued the phagocytic capacity of glial cells, which efficiently cleared damaged axonal debris in the aged brain to a similar extent as that in the young brain, indicating that aging-induced alterations in the phagocytic capacity of astrocytes may contribute to disruption of brain homeostasis15.
Based on these new findings, modulating the phagocytic capacity of astrocytes may be an attractive therapeutic strategy to prevent and treat various neurological disorders. In this regard, there have been several attempts to enhance the phagocytic capacity of astrocytes, for example, by inducing acidification of lysosomes with acidic nanoparticles16 and overexpressing transcription factor EB (TFEB), which can enhance lysosome biogenesis17. Despite these attempts, it is still unclear how astrocytes and microglial cells differ in their phagocytic kinetics and whether we should increase or decrease their phagocytic capacities in various diseases.
In this paper, we present a novel in vitro assay for detecting the phagocytic capacity of astrocytes in real-time. The data show different kinetics of engulfment and degradation in astrocytes and microglia. Astrocyte-conditioned medium (ACM), which contains secreted factors from astrocytes, is essential for effective phagocytosis of both astrocytes and microglia. Furthermore, Megf10, a phagocytic receptor in astrocytes and a homolog of Ced-1 and Draper, plays critical roles in astrocyte-mediated phagocytosis8,18.

<table>
	<tbody>
		<tr>
			<td>Synaptosome purification</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE healthcare life sciences</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick Start Bradford Protein Assay Kit 2</td>
			<td>BIO-RAD</td>
			<td>5000202</td>
			<td></td>
		</tr>
		<tr>
			<td>pH indicator conjugation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide(DMSO)</td>
			<td>LPS solution</td>
			<td>DMSO100</td>
			<td></td>
		</tr>
		<tr>
			<td>pHrodo red, succinimidyl ester</td>
			<td>Molecula probes</td>
			<td>P36600</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunopanning</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X Earle&#8217;s balanced salt solution (EBSS)</td>
			<td>Sigma</td>
			<td>E7510</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Bovogen</td>
			<td>BSA025</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyrebonuclease 1 (DNase)</td>
			<td>Worthington</td>
			<td>Is002007</td>
			<td></td>
		</tr>
		<tr>
			<td>(DMEM)</td>
			<td>Gibco</td>
			<td>11960-044</td>
			<td></td>
		</tr>
		<tr>
			<td>(dPBS)</td>
			<td>Welgene</td>
			<td>LB001-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Griffonia Simplicifolia Lectin(BSL-1)</td>
			<td>Vector Labs</td>
			<td>L-1100</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG+IgM(H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgM (&#956;-chain)</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin-binding epidermal growth factor</td>
			<td>Sigma</td>
			<td>E4643</td>
			<td></td>
		</tr>
		<tr>
			<td>Human HepaCAM antibody</td>
			<td>R&#38;D systems</td>
			<td>MAB4108</td>
			<td></td>
		</tr>
		<tr>
			<td>Integrin beta 5 monoclonal antibody (KN52)</td>
			<td>eBioscience</td>
			<td>14-0497-82</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma</td>
			<td>C7880</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamate</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetly-L-cyteine (NAC)</td>
			<td>Sigma</td>
			<td>A8199</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>O4 hybridoma supernatant(mouse IgM)</td>
			<td>Bansal et al.23</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>Is003126</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluristrainer 20 &#956;m</td>
			<td>PluriSelect</td>
			<td>43-50020-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified rat anti-mouse CD45</td>
			<td>BD Pharmingen</td>
			<td>550539</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified mouse anti-rat CD45</td>
			<td>BD Pharmingen</td>
			<td>554875</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T9935</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Worthington</td>
			<td>LS003086</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear tube (Tube, Thinwall, Ultra-Clear)</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Collect IP-ACM</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Macrosep Advance Centrifugal Devices with Omega Membrane (10k)</td>
			<td>PALL</td>
			<td>MAP010C37</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrosep Advance Centrifugal Devices with Omega Membrane (30k)</td>
			<td>PALL</td>
			<td>MAP030C37</td>
			<td></td>
		</tr>
		<tr>
			<td>Phagocytosis live imaging assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Juli stage</td>
			<td>NanoEntek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Time Series Analyzer V3 plugins</td>
			<td>https://imagej.nih.gov/ij/plugins/time-series.html</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel in vitro live-imaging assay of astrocyte-mediated phagocytosis using ph indicator-conjugated synaptosomes

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&#39;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.

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...

Watch this Scientific Journal Video about A Novel In Vitro Live-imaging Assay of Astrocyte-mediated Phagocytosis Using pH Indicator-conjugated Synaptosomes at JoVE.com

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

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.

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

Astrocytes are the major cell type in the brain and directly contact synapses and blood vessels. Although microglial cells have been considered the major ...

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Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Live Imaging of Drosophila Larval Neuroblasts

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Novel Assay for Cold Nociception in Drosophila Larvae

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...