New chemical probe for visualising brain immune cells

Researchers in South Korea and Singapore have, for the first time, developed a chemical probe that enables live-imaging of a type of immune cells in the brain, known as microglia, in a live animal brain.

Age-dependent microglia labelling by CDr20. Three different aged mice (P180, P360 and P660) from wild-type or triple-transgenic mouse model of Alzheimer’s disease (3xTg-AD) mice were intravenously injected with CDr20. The red areas are the CDr20-labelled microglia, while the green areas are Dextran-FITC labelled blood vessels.

- Microglia – immune cells resident in the brain – have been found to play an important role in
neurological diseases. However, studying these cells in a live brain has been challenging.

- A collaboration of research institutes in South Korea and Singapore has developed a
fluorogenic marker that successfully allows scientists to visualise microglia at cellular
resolution in a live brain.

SINGAPORE, 26 April 2019 – Researchers in South Korea and Singapore have, for the first
time, developed a chemical probe that enables live-imaging of a type of immune cells in the
brain, known as microglia, in a live animal brain. This discovery, led by the Institute for Basic
Science (IBS) at Pohang University of Science and Technology (POSTECH), South Korea; the
Singapore Bioimaging Consortium (SBIC) and Singapore Immunology Network (SIgN) of the
Agency for Science, Technology and Research (A*STAR); and Duke-NUS Medical School,
Singapore, will enable critical imaging studies to help scientists understand the development of
brain diseases, such as stroke, autism, Alzheimer’s, and Parkinson’s disease.

Microglia are the brain’s primary resident immune cells. Although microglia were described a
century ago, these cells have only recently been found to play an important role in the
development of various neurological diseases. These discoveries have been aided by
technological advances in isolating these cells, and transgenic small animal systems that
express fluorescent proteins from microglia lineages, allowing for live imaging with light
microscopy. However, studying microglia in humans and primates has been extremely difficult.

Being able to study microglia separately from other cells in the brain is critical for understanding
brain development and disease. Although several biochemical markers and molecular imaging
tools have been developed to study these specialised cells, no current methods have enabled
the visualisation of microglia at cellular resolution in a live brain, which is clinically more relevant.

This collaborative study by two laboratories, led by Professor and Associate Director Young-Tae
Chang, from IBS, who is also affiliated with A*STAR’s SBIC, and Associate Professor Hyunsoo
Shawn Je, from Duke-NUS, identified a fluorescent marker for microglial cells, screened from a
library of potential probes. The researchers then undertook extensive ex vivo and in vivo studies
to demonstrate that one of the probes, named CDr20, could indeed label microglia in live cells.
Immunostaining was used to verify the selective staining of microglia. The authors then used
knock-out cell lines to identify the enzyme present in the microglia that ‘switched on’ the
fluorescence of the probe.

Prof Chang explained, “Through a thorough structure-activity relationships study, we developed
this high-performance fluorogenic chemical probe, CDr20, that can visualize microglia both in vitro
and in vivo. Using a genome-scale CRISPR-Cas9 knockout screen, we identified Ugt1a7c
as the functional target protein of CDr20 that activates the CDr20 fluorescence signal in microglia
through the enzymatic glucuronidation reaction. Our probe can also label human- and primatederived microglia,
so this will be extremely useful to study microglia function in higher mammals,
which is clinically more relevant.”

Fluorescent small molecules have become indispensable tools for biomedical research along
with the rapidly developing optical imaging technology. However, cell-type specific fluorescent
small molecules that are known to bind to molecular biomarkers are very rare.

“The Chang laboratory has extensive libraries of random fluorescent molecules and they utilize
massive-cell based screening to identify specific fluorescent small molecules that highlight brain
immune cells,” Assoc Prof Je said. “This small molecule is extremely selective to label microglia
and related lineages of brain-resident immune cells. We utilized state-of-art in vivo multiphoton
microscopy to image microglia in the brain upon intravenous dye injection, which has not been
possible before.”

The authors state that they will continue work to improve the functionality and utility of CDr20.
Furthermore, the Chang laboratory is screening new molecules that only label activated
microglia, which scientists suspect to play a role in neuroinflammation in neurodegenerative

Noting the collaboration involving A*STAR and Duke-NUS in Singapore, and IBS, POSTECH,
Ulsan National Institute of Science and Technology (UNIST), and Seoul National University in
South Korea, Prof Patrick Casey, Senior Vice Dean for Research at Duke-NUS, remarked, “This
work is an excellent example of multi-disciplinary collaboration among the best research
institutions in Singapore and South Korea. It provides an important contribution to the study of
microglia in the development of neurodegenerative disorders that can lead to future therapeutic

About Duke-NUS Medical School
Duke-NUS is a partnership between Duke University School of Medicine and the National
University of Singapore (NUS).

In 2005, with support from the Singapore government, NUS and Duke University, two academic
institutions with strong track records in research and education, committed to combine the unique
medical education curriculum at Duke University School of Medicine with the academic rigour
and rich resources offered by NUS, and to offer students an enriching and innovative medical
educational experience.

Duke-NUS is located on the main campus of the largest healthcare group in the country,
Singapore Health Services (SingHealth). This group collectively delivers multi-disciplinary care
among 42 clinical specialties across a large network of hospitals, national specialty centres and
polyclinics. Together, Duke-NUS and SingHealth constitute a leading, world class Academic
Medical Centre embodying the goal of delivering the highest levels of patient care, education and

For more information, please visit the website (link below)

For media enquiries, please contact:
Federico Graciano
Duke-NUS Medical School
Tel: +65 6601 3272
Email: [email protected]

Lekshmy Sreekumar
Duke-NUS Medical School
Tel: +65 65161138
Email: [email protected]

Published: 02 May 2019

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Reference/Note: Kim B, Fukuda M, Lee JY, Su D, Sanu S, Silvin A, Khoo ATT, Kwon T, Liu X, Chi W, Liu XG, Choi S, Wan DSY, Park SJ, Kim JS, Ginhoux F, Je HS and Chang YT (2019). Visualizing microglia with a fluorescence turn-on Ugt1a7c substrate. Angewandte Chemie. DOI: 10.1002/ange.201903058