Cystathionine protects against endoplasmic reticulum stress-induced lipid accumulation, tissue injury, and apoptotic cell death.

Cystathionine (R-S-(2-amino-2-carboxyethyl)-l-homocysteine) is a non-proteinogenic thioether containing amino acid. In mammals, cystathionine is formed as an intermediate of the transsulfuration pathway by the condensation of serine and homocysteine (Hcy) in a reaction catalyzed by cystathionine ß-synthase (CBS). Cystathionine is subsequently converted to cysteine plus ammonia and α-ketobutyrate by the action of cystathionine γ-lyase (CGL). Pathogenic mutations in CBS result in CBS-deficient homocystinuria (HCU) which, if untreated, results in mental retardation, thromboembolic complications and connective tissue disorders. Currently there is no known function for cystathionine other than serving as an intermediate in transsulfuration and to date, the possible contribution of the abolition of cystathionine synthesis to pathogenesis in HCU has not been investigated. Using both mouse and cell-culture models, we have found that cystathionine is capable of blocking the induction of hepatic steatosis and kidney injury, acute tubular necrosis, and apoptotic cell death by the endoplasmic reticulum stress inducing agent tunicamycin. Northern and Western blotting analysis indicate that the protective effects of cystathionine occur without any obvious alteration of the induction of the unfolded protein response. Our data constitute the first experimental evidence that the abolition of cystathionine synthesis may contribute to the pathology of HCU and that this compound has therapeutic potential for disease states where ER stress is implicated as a primary initiating pathogenic factor.

Long-term betaine therapy in a murine model of cystathionine beta-synthase deficient homocystinuria: decreased efficacy over time reveals a significant threshold effect between elevated homocysteine and thrombotic risk.

Classical homocystinuria (HCU) is caused by deficiency of cystathionine ß-synthase and is characterized by connective tissue disturbances, mental retardation and cardiovascular disease. Treatment for pyridoxine non-responsive HCU typically involves lowering homocysteine levels with a methionine-restricted diet and dietary supplementation with betaine. Compliance with the methionine-restricted diet is difficult and often poor. Investigating optimization of the efficacy of long-term betaine treatment in isolation from a methionine-restricted diet is precluded by ethical considerations regarding patient risk. The HO mouse model of HCU developed in our laboratory, exhibits constitutive expression of multiple pro-inflammatory cytokines and a hypercoagulative phenotype both of which respond to short-term betaine treatment. Investigation of the effects of long-term betaine treatment in the absence of methionine-restriction in HO HCU mice revealed that the ability of betaine treatment to lower homocysteine diminished significantly over time. Plasma metabolite analysis indicated that this effect was due at least in part, to reduced betaine-homocysteine S-methyltransferase (BHMT) mediated remethylation of homocysteine. Western blotting analysis revealed that BHMT protein levels are significantly repressed in untreated HCU mice but are significantly induced in the presence of betaine treatment. The observed increase in plasma homocysteine during prolonged betaine treatment was accompanied by a significant increase in the plasma levels of TNF-alpha and IL-1beta and reversion to a hypercoagulative phenotype. Our findings are consistent with a relatively sharp threshold effect between severely elevated plasma homocysteine and thrombotic risk in HCU and indicate that the HO mouse model can serve as a useful tool for both testing novel treatment strategies and examining the optimal timing and dosing of betaine treatment with a view toward optimizing clinical outcome.

Cystathionine beta-synthase null homocystinuric mice fail to exhibit altered hemostasis or lowering of plasma homocysteine in response to betaine treatment.

Cystathionine beta-synthase (CBS) deficient homocystinuria is an inherited metabolic defect that if untreated typically results in mental retardation, thromboembolism and a range of connective tissue disturbances. A knockout mouse model has previously been used to investigate pathogenic mechanisms in classical homocystinuria (Watanabe et al., PNAS 92 (1995) 1585-1589). This mouse model exhibits a semi-lethal phenotype and the majority of mice do not survive the early neonatal period. We report here that the birth incidence of cbs (-/-) mice produced from heterozygous crosses is non-Mendelian and not significantly improved by treatment with either the Hcy lowering compound betaine or the cysteine donor N-acetylcysteine. Betaine treatment did improve survival of cbs (-/-) mice and restored fertility to female cbs (-/-) mice but did so without significantly lowering Hcy levels. Surviving cbs (-/-) mice failed to show any alteration in coagulation parameters compared to wild-type controls. Moribund cbs (-/-) mice exhibited severe liver injury and hepatic fibrosis while surviving cbs (-/-) mice although less severely affected, still exhibited a level of severe liver injury that is not found in the human disease. The hepatopathy observed in this model may offer an explanation for the failure of cbs (-/-) mice to respond to betaine or exhibit a hypercoagulative phenotype. We conclude that although this model provides useful data on the biochemical sequelae of classical homocystinuria, it does not successfully recapitulate a number of important features of the human disease and its use for studying mechanisms in homocystinuria should be treated with caution as the hepatopathy produces changes which could influence the results.

A novel transgenic mouse model of CBS-deficient homocystinuria does not incur hepatic steatosis or fibrosis and exhibits a hypercoagulative phenotype that is ameliorated by betaine treatment.

Cystathionine beta-synthase (CBS) catalyzes the condensation of homocysteine (Hcy) and serine to cystathionine, which is then hydrolyzed to cysteine by cystathionine gamma-lyase. Inactivation of CBS results in CBS-deficient homocystinuria more commonly referred to as classical homocystinuria, which, if untreated, results in mental retardation, thromboembolic complications, and a range of connective tissue disorders. The molecular mechanisms that underlie the pathology of this disease are poorly understood. We report here the generation of a new mouse model of classical homocystinuria in which the mouse cbs gene is inactivated and that exhibits low-level expression of the human CBS transgene under the control of the human CBS promoter. This mouse model, designated "human only" (HO), exhibits severe elevations in both plasma and tissue levels of Hcy, methionine, S-adenosylmethionine, and S-adenosylhomocysteine and a concomitant decrease in plasma and hepatic levels of cysteine. HO mice exhibit mild hepatopathy but, in contrast to previous models of classical homocystinuria, do not incur hepatic steatosis, fibrosis, or neonatal death with approximately 90% of HO mice living for at least 6months. Tail bleeding determinations indicate that HO mice are in a hypercoagulative state that is significantly ameliorated by betaine treatment in a manner that recapitulates the disease as it occurs in humans. Our findings indicate that this mouse model will be a valuable tool in the study of pathogenesis in classical homocystinuria and the rational design of novel treatments.