Enzyme-driven process explains how NeuO selectively stains living neurons, solving a decade-old mystery

The secret behind NeuO (neuronal selective fluorescent probe), which accurately identifies and fluorescently stains neurons only, has finally been unveiled. A research team led by Professor Young-Tae Chang from POSTECH’s Department of Chemistry and Dr. Beomsue Kim from the Korea Brain Research Institute’s Neural Circuit Research Group has elucidated the mechanism of NeuO’s selective staining of neurons. This research has been published in the journal Angewandte Chemie International Edition.

NeuO is a unique fluorescent substance that stains only living neurons across various animals, from mice to humans. This molecule, first developed in 2015, is already commercially available and widely utilized by researchers worldwide. However, the principle behind how NeuO selectively targets brain neurons to emit fluorescence had long remained a mystery, which has limited NeuO’s applications.

To unveil this secret, the research team established four hypotheses: the hypothesis that NeuO directly binds to neuronal proteins, the hypothesis that it can enter only neurons through specific channels in the cell membrane, the hypothesis that NeuO enters all cells but is actively pumped out of non-neuronal cells, and the hypothesis that NeuO’s chemical structure is modified by a specific enzyme only within neurons.

After testing each hypothesis one by one, NeuO’s selective staining mechanism was explained by the fourth hypothesis. The research team discovered that NeuO undergoes special chemical changes exclusively within neurons by the enzyme PAK6 (serine/threonine-protein kinase PAK6, a protein phosphorylation enzyme).

This chemical reaction called phosphorylation is a process in which phosphate groups bind to proteins or molecules. Through this phosphorylation process, NeuO becomes more fluorescent and remains inside the neurons without escaping the cells. In other words, NeuO is transformed into a ‘light-emitting molecule’ when it encounters specific enzymes within neurons and accumulates inside the cells, thereby enhancing its fluorescence.

Professor Young-Tae Chang and Dr. Beomsue Kim, who led the research, stated, “This research goes beyond the academic achievement of solving NeuO’s selective staining mechanism, which remained an unsolved mystery for 10 years, and opens the path for more precise tracking and study of living neurons in the brain based on cell-specific enzyme activity. This will also be tremendously helpful in developing neuronal staining methods for diagnosing degenerative brain diseases such as Alzheimer’s disease and Parkinson’s disease.”