A Ratiometric Two-Photon Fluorescent Probe Reveals Reduction in Mitochondrial H₂S Production in Parkinson’s Disease Gene Knockout Astrocytes

Abstract

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 H2S 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 H2S, 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 H2S 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.

Fluorescent probes for hydrogen sulfide detection and bioimaging

“…Cho et al next reported another two-photon ratiometric probe 17 for H 2 S detection in mitochondria. 19 The detection mechanism of probe 17 was illustrated in Fig. 2b and d. 6-(Benzo[d]thiazol-2 0 -yl)-2-(methylamino)naphthalene was selected as the fluorophore, and 4-azidobenzyl carbamate was chosen as the H 2 S response site.…”

Direct imaging of biological sulfur dioxide derivatives in vivo using a two-photon phosphorescent probe

“…Recently, the Cho's and Kim's groups have reported some benzothiazole and benzimidazole-based compounds that have strong two-photon emission [28][29].…”

FRET ratiometric probes reveal the chiral-sensitive cysteine-dependent H2S production and regulation in living cells

“…The intensity-based fluorescence probes may suffer from multiple factors to influence the accurate analysis of H 2 S production and regulation in living cells. However, the ratiometric fluorescence probes provide built-in correction for environmental effects to improve the accuracy of bioimaging analysis [22][23][24][25][26][27] . The reported sulfide ratiometric probes are mainly based on two strategies: 1) a tuneable electronic system, after reaction with H 2 S, results in a spectroscopic alteration such as blue or red shift in emission.…”

“…The reported sulfide ratiometric probes are mainly based on two strategies: 1) a tuneable electronic system, after reaction with H 2 S, results in a spectroscopic alteration such as blue or red shift in emission. This method may suffer from the relative closeness of the two emission wavelengths from the probe change [22][23][24] . 2) the interruption of a large conjugate system by H 2 S addition can produce ratiometric probes with relatively large Stokes shifts.…”