Hydrogen sulfide (H2S) plays a key signalling role in human physiology. Deregulation of H2S production and/or its downstream actions have been implicated in the pathophysiology of several diseases. Therefore, understanding how the metabolism of this gasotransmitter is altered either under pathological conditions or following pharmacological administrations is crucial to identify novel drug targets for therapeutic interventions and evaluate the efficacy of treatments.
Endogenously synthesized by cytosolic enzymes, H2S is oxidatively catabolized in the mitochondrion, where H2S-derived electrons are transferred by sulfide:quinone oxidoreductase (SQR) to coenzyme Q. Relevant to human pathophysiology, low concentrations of coenzyme Q has been observed in Ataxia oculomotor apraxia- type 1 (AOA1), an autosomal recessive neurodegenerative disease caused by mutations in the gene encoding the DNA strand-break repair protein aprataxin (APTX). APTX function has been associated with both mitochondrial DNA repair and the transcriptional regulation of mitochondrial proteins. Hence, defects in APTX expression and/or function results in organelle dysfunction and oxidative stress.
Our working hypothesis is that the sulphide oxidation pathway is impaired in AOA1 due to secondary coenzyme Q deficiency, and its impairment contributes to the pathogenesis of the disease.
The present project aims at studying the metabolism of H2S either in APTX deficient cell models of AOA1 to understand its role in cellular dysfunction, and in colon cancer cells to comprehend the controversial effect of the N-acetylcysteine in cancer therapy, an antioxidant recently suggested to be involved in sulphide metabolism.
Research will focus on the quantification of the expression level and of the activity of enzymes involved in H2S synthesis and degradation as well as on the determination of the redox state and the bioenergetic parameters of the cells.
Presently, targeting the H2S metabolic enzymes has received a strong interest as a new strategy for therapeutic intervention in several diseases, especially in cancer in which alteration of the sulfide metabolic pathway is well documented [2]. However, this approach seems of great significance also for those disorders, in which no specific drug treatment exists. For this reason, in the present project we aim at investigating the role of H2S in the pathogenesis of the autosomal recessive genetic disorder AOA1 whose management is mainly supportive.
Several data indicate a possible link of APTX (dys)function to H2S metabolism. First of all, there is evidence that APTX deficiency causes a decrease of coenzyme Q [21], a lipid-soluble present in all cell membranes and involved in multiple metabolic functions. One of these functions is to shuttle electrons in the first reaction of the H2S oxidation pathway catalysed by SQR. Therefore, low concentrations of coenzyme Q, possibly associated to a decrease in SQR protein levels [11], can lead to H2S accumulation. This sulfide increase may contribute to the oxidative stress observed in APTX mutant cells and affect multiple physiological processes, possibly through modification of protein S-sulfhydration, or inhibition of the last complex of the respiratory chain at the highest concentration, thus blocking energy production.
Moreover, DNA repair enzymes, together with sulfide detoxifying ones, play a vital role in the protection of cells routinely exposed to sulfide, which under certain condition can directly induce DNA damage, even at low concentration [22, 23]. In addition to increasing the expression of some DNA repair enzyme [24], H2S supports formation and stabilization of mitochondrial DNA repair complexes [25], enhancing DNA repair capacity within mitochondria, critical for cellular energy metabolism and mitochondrial biogenesis.
Since APTX responds to DNA damage, especially in the mitochondrion, it is conceivable that also this DNA-repair protein is regulated by H2S or it plays a role in response to the H2S levels. However, these hypotheses have never been addressed. Preliminary results indicate a link between APTX and sulfide. Are the levels of the synthesizing and oxidising H2S enzymes altered in AOA1? Has the H2S concentration changed? Is H2S regulating the level and function of APTX? By answering these questions, we will contribute to shed some light on the pathogenesis of AOA1 and possibly other neurodegenerative disorders. The investigation on the role of the H2S metabolism impairment to the observed AOA1 cell dysfunction may help to characterise new biomarkers for the diagnosis of the disease and to identify targets for novel therapeutic approaches.
The development of drugs targeting the H2S metabolic enzymes is still in its infancy, yet some compounds has been tested in preclinical model of cancer [2]. After the discovery that various cancer cells upregulate their endogenous H2S production[13, 20] and utilize this mediator in autocrine and paracrine manner to stimulate proliferation, bioenergetics and tumor angiogenesis, inhibitors of the H2S synthesising enzymes have been developed [2] to decrease the gasotransmitter levels. On the other hand, pharmacological treatments that increase sulphide concentration can sustain cancer cell growth. Interestingly, NAC, an antioxidant used in cancer therapy as a complementary treatment, particularly to alleviate unwanted radiation- and chemotherapy-induced toxicity, was shown to alter the intracellular levels of sulfane sulphur [15], a major form of H2S storage in mammalian cells. The role NAC in cancer is still controversial because it is not clear if its administration ameliorates the toxic side effects of therapy without affecting its efficacy [19].
In this context the results of the biochemical assays performed after chronic treatment of colorectal adenocarcinoma SW480 cells with NAC will allow to understand the effect of this antioxidant on H2S metabolism, the impact of the "sulfur sulfur" species on the metabolic reprogramming of the tumor cells and how this can justify or not the effects that have been described in cancer therapy.