Development of a Fluorescently Labeled Ligand for Rapid Detection of DAT in Human and Mouse Peripheral Blood Monocytes

Gisela Andrea Camacho-Hernandez; Adithya Gopinath; Amarachi V Okorom; Habibeh Khoshbouei; Amy Hauck Newman

doi:10.1021/jacsau.3c00719

Development of a Fluorescently Labeled Ligand for Rapid Detection of DAT in Human and Mouse Peripheral Blood Monocytes

JACS Au. 2024 Jan 24;4(2):657-665. doi: 10.1021/jacsau.3c00719. eCollection 2024 Feb 26.

Authors

Gisela Andrea Camacho-Hernandez¹, Adithya Gopinath², Amarachi V Okorom¹, Habibeh Khoshbouei², Amy Hauck Newman¹

Affiliations

¹ Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse - Intramural Research Program, National Institutes of Health, Baltimore, Maryland 21224, United States.
² Department of Neuroscience, University of Florida College of Medicine, Gainesville, Florida 32611, United States.

Abstract

The dopamine transporter (DAT) is one of the key regulators of dopamine (DA) signaling in the central nervous system (CNS) and in the periphery. Recent reports in a model of Parkinson's disease (PD) have shown that dopamine neuronal loss in the CNS impacts the expression of DAT in peripheral immune cells. The mechanism underlying this connection is still unclear but could be illuminated with sensitive and high-throughput detection of DAT-expressing immune cells in the circulation. Herein, we have developed fluorescently labeled ligands (FLL) that bind to surface-expressing DAT with high affinity and selectivity. The diSulfoCy5-FLL (GC04-38) was utilized to label DAT in human and mouse peripheral blood mononuclear cells (PBMCs) that were analyzed via flow cytometry. Selective labeling was validated using DAT KO mouse PBMCs. Our studies provide an efficient and highly sensitive method using this novel DAT-selective FLL to advance our fundamental understanding of DAT expression and activity in PBMCs in health and disease and as a potential peripheral biomarker.