Altered Sulfide Metabolism in Ethylmalonic Encephalopathy

Ethylmalonic Encephalopathy, EE, is an autosomal recessive, invariably fatal disorder characterized by early-onset brain failure, microangiopathy, chronic diarrhoea, defective cytochrome c oxidase (COX) in muscle and brain, and high excretion of ethylmalonic acid (EMA) in urine. ETHE1, a gene encoding a mitochondrial beta-lactamase-like, iron-coordinating metalloprotein, is mutated in EE. We generated an Ethe1-null mouse that manifested the EE cardinal features. We found that thiosulfate was excreted in massive amount in urines of both Ethe1-/- mice and EE patients. High thiosulfate (H2SSO3) and sulfide (H2S) levels were present in Ethe1-/- mouse tissues. Sulfide is a powerful inhibitor of COX and terminal beta-oxidation, with vasoactive and vasotoxic effects that could explain the microangiopathy in EE patients. Sulfide is detoxified by a mitochondrial pathway that includes a sulfur dioxygenase (SDO). SDO activity was absent in Ethe1-/- mice, whereas ETHE1 overexpression in HeLa cells and E. coli markedly increased it. Therefore, ETHE1 is a mitochondrial SDO involved in catabolism of sulfide, which accumulates to toxic levels in EE. An important question that warranted the PhD experimental work concerns the source of H2S in ETHE1 mutant patients, and how accumulated sulfide can act on the cytochrome c oxidase complex at molecular level. The presence of elevated levels of thiosulfate in several tissues of the Ethe1-/- mouse suggests endogenous production of H2S from catabolism of cysteine and other sulfur-containing organic compounds. H2S is also a major product of the intestinal bacterial flora, especially anaerobic species residing in the colon. The presence of a gradient of COX deficiency in luminal vs. cryptal colonocytes in Ethe1-/- colon mucosa suggests that a defect of ETHE1-SDO activity results in faulty detoxification of exogenously produced H2S. In order to achieve effective reduction of H2S production, it is crucial to clarify which are the sources of this compound in the body that can then constitute specific targets for therapy. Another important issue is to understand the organ-specific mechanisms, which lead to failure of some organs, such as the brain and the skeletal muscle, but not of others, such as the liver. These aims can be implemented through the creation and characterization of conditional tissue-specific KO animals. A further research line concerns the improvement of biochemical and molecular approaches for the diagnosis of EE.