Imaging immune cells in the brain

Researchers in South Korea and Singapore have 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.

Described in the journal Angewandte Chemie, their discovery 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, and 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.

Led by Professor Young-Tae Chang from the Institute for Basic Science (IBS) at Pohang University of Science and Technology (POSTECH) and Associate Professor Hyunsoo Shawn Je, from Duke-NUS Medical School, researchers identified a fluorescent marker for microglial cells, screened from a library of potential probes, and 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, and the authors used knockout cell lines to identify the enzyme present in the microglia that ‘switched on’ the fluorescence of the probe.

“Through a thorough structure-activity relationships study, we developed this high-performance fluorogenic chemical probe, CDr20, that can visualise microglia both in vitro and in vivo,” Prof Chang said. “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 primate-derived 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 utilise 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 utilised state-of-the-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 disorders.

“This work is an excellent example of multidisciplinary collaboration among the best research institutions in Singapore and South Korea,” said Professor Patrick Casey, Senior Vice Dean for Research at Duke-NUS. “It provides an important contribution to the study of microglia in the development of neurodegenerative disorders that can lead to future therapeutic approaches.”

Image caption: 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.

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