A ratiometric two-photon fluorescent probe reveals reduction in mitochondrial H₂S production in Parkinson's disease gene knockout astrocytes

Hydrogen sulfide (H₂S) is a multifunctional signaling molecule that exerts neuroprotective effects in oxidative stress. In this article, we report a mitochondria-localized two-photon probe, SHS-M2, that can be excited by 750 nm femtosecond pulses and employed for ratiometric detection of H ₂S in live astrocytes and living brain slices using two-photon microscopy (TPM). SHS-M2 shows bright two-photon-excited fluorescence and a marked change in emission color from blue to yellow in response to H ₂S, low cytotoxicity, easy loading, and minimum interference from other biologically relevant species including reactive sulfur, oxygen, and nitrogen species, thereby allowing quantitative analysis of H₂S levels. Molecular TPM imaging with SHS-M2 in astrocytes revealed that there is a correlation between the ratiometric analysis and expression levels of cystathionine β-synthase (CBS), the major enzyme that catalyzes H ₂S production. In studies involving DJ-1, a Parkinson's disease (PD) gene, attenuated H₂S production in comparison with wild-type controls was observed in DJ-1-knockout astrocytes and brain slices, where CBS expression was decreased. These findings demonstrate that reduced H₂S levels in astrocytes may contribute to the development of PD and that SHS-M2 may be useful as a marker to detect a risk of neurodegenerative diseases, including PD.