H₂S (hydrogen sulfide) is a well known and pungent gas recently discovered to be synthesized enzymatically in mammalian and human tissues. In a relatively short period of time, H₂S has attracted substantial interest as an endogenous gaseous mediator and potential target for pharmacological manipulation. Studies in animals and humans have shown H₂S to be involved in diverse physiological and pathophysiological processes, such as learning and memory, neurodegeneration, regulation of inflammation and blood pressure, and metabolism. However, research is limited by the lack of specific analytical and pharmacological tools which has led to considerable controversy in the literature. Commonly used inhibitors of endogenous H₂S synthesis have been well known for decades to interact with other metabolic pathways or even generate NO (nitric oxide). Similarly, commonly used H₂S donors release H₂S far too quickly to be physiologically relevant, but may have therapeutic applications. In the present review, we discuss the enzymatic synthesis of H₂S and its emerging importance as a mediator in physiology and pathology. We also critically discuss the suitability of proposed ‘biomarkers’ of H₂S synthesis and metabolism, and highlight the complexities of the currently used pharmacological H₂S ‘donor’ molecules and ‘specific’ H₂S synthesis inhibitors in their application to studying the role of H₂S in human disease.

Significance: Alzheimer’s disease (AD) is the leading cause of dementia in the elderly. Although dysregulated hydrogen sulfide (H ₂ S) metabolism has been reported in AD, and H ₂ S donors are beneficial, molecular mechanisms underlying neuroprotective effects of H ₂ S are largely unknown. We now show that H ₂ S confers neuroprotection by sulfhydrating GSK3β to inhibit its activity, thereby preventing hyperphosphorylation of Tau, a key pathogenic event in AD. Administering H ₂ S donors improves motor and cognitive functions in a mouse model of AD.

The development of diabetic vascular complications is initiated, at least in part, by mitochondrial reactive oxygen species (ROS) production in endothelial cells. Hyperglycemia induces superoxide production in the mitochondria and initiates changes in the mitochondrial membrane potential that leads to mitochondrial dysfunction. Hydrogen sulfide (H2S) supplementation has been shown to reduce the mitochondrial oxidant production and shows efficacy against diabetic vascular damage in vivo. However, the half-life of H2S is very short and it is not specific for the mitochondria. We have therefore evaluated two novel mitochondria-targeted anethole dithiolethione and hydroxythiobenzamide H2S donors (AP39 and AP123 respectively) at preventing hyperglycemia-induced oxidative stress and metabolic changes in microvascular endothelial cells in vitro. Hyperglycemia (HG) induced significant increase in the activity of the citric acid cycle and led to elevated mitochondrial membrane potential. Mitochondrial oxidant production was increased and the mitochondrial electron transport decreased in hyperglycemic cells. AP39 and AP123 (30-300nM) decreased HG-induced hyperpolarisation of the mitochondrial membrane and inhibited the mitochondrial oxidant production. Both H2S donors (30-300nM) increased the electron transport at respiratory complex III and improved the cellular metabolism. Targeting H2S to mitochondria retained the cytoprotective effect of H2S against glucose-induced damage in endothelial cells suggesting that the molecular target of H2S action is within the mitochondria. Mitochondrial targeting of H2S also induced >1000-fold increase in the potency of H2S against hyperglycemia-induced injury. The high potency and long-lasting effect elicited by these H2S donors strongly suggests that these compounds could be useful against diabetic vascular complications.

Mitochondrial dysfunction is observed in many diseases. Targeting H ₂ S generation to mitochondria may be cytoprotective.