Long-term functional correction of cystathionine ß-synthase deficiency in mice by adeno-associated viral gene therapy.

Cystathionine ß-synthase (CBS) deficiency is a recessive inborn error of sulfur metabolism characterized by elevated blood levels of total homocysteine (tHcy). Patients diagnosed with CBS deficiency are currently treated by a combination of vitamin supplementation and restriction of foods containing the homocysteine precursor methionine, but the effectiveness of this therapy is limited due to poor compliance. A mouse model for CBS deficiency (Tg-I278T Cbs-/- ) was used to evaluate a potential gene therapy approach to treat CBS deficiency utilizing an AAVrh.10-based vector containing the human CBS cDNA downstream of the constitutive, strong CAG promoter (AAVrh.10hCBS). Mice were administered a single dose of virus and followed for up to 1 year. The data demonstrated a dose-dependent increase in liver CBS activity and a dose-dependent decrease in serum tHcy. Liver CBS enzyme activity at 1 year was similar to Cbs+/- control mice. Mice given the highest dose (5.6 × 1011 genomes/mouse) had mean serum tHcy decrease of 97% 1 week after injection and an 81% reduction 1 year after injection. Treated mice had either full- or substantial correction of alopecia, bone loss, and fat mass phenotypes associated with Cbs deficiency in mice. Our findings show that AAVrh.10-based gene therapy is highly effective in treating CBS deficiency in mice and supports additional pre-clinical testing for eventual use human trials.

Lessons Learned from Inherited Metabolic Disorders of Sulfur-Containing Amino Acids Metabolism.

The metabolism of sulfur-containing amino acids (SAAs) requires an orchestrated interplay among several dozen enzymes and transporters, and an adequate dietary intake of methionine (Met), cysteine (Cys), and B vitamins. Known human genetic disorders are due to defects in Met demethylation, homocysteine (Hcy) remethylation, or cobalamin and folate metabolism, in Hcy transsulfuration, and Cys and hydrogen sulfide (H2S) catabolism. These disorders may manifest between the newborn period and late adulthood by a combination of neuropsychiatric abnormalities, thromboembolism, megaloblastic anemia, hepatopathy, myopathy, and bone and connective tissue abnormalities. Biochemical features include metabolite deficiencies (e.g. Met, S-adenosylmethionine (AdoMet), intermediates in 1-carbon metabolism, Cys, or glutathione) and/or their accumulation (e.g. S-adenosylhomocysteine, Hcy, H2S, or sulfite). Treatment should be started as early as possible and may include a low-protein/low-Met diet with Cys-enriched amino acid supplements, pharmacological doses of B vitamins, betaine to stimulate Hcy remethylation, the provision of N-acetylcysteine or AdoMet, or experimental approaches such as liver transplantation or enzyme replacement therapy. In several disorders, patients are exposed to long-term markedly elevated Met concentrations. Although these conditions may inform on Met toxicity, interpretation is difficult due to the presence of additional metabolic changes. Two disorders seem to exhibit Met-associated toxicity in the brain. An increased risk of demyelination in patients with Met adenosyltransferase I/III (MATI/III) deficiency due to biallelic mutations in the MATIA gene has been attributed to very high blood Met concentrations (typically >800 µmol/L) and possibly also to decreased liver AdoMet synthesis. An excessively high Met concentration in some patients with cystathionine ß-synthase deficiency has been associated with encephalopathy and brain edema, and direct toxicity of Met has been postulated. In summary, studies in patients with various disorders of SAA metabolism showed complex metabolic changes with distant cellular consequences, most of which are not attributable to direct Met toxicity.

The Spectrum of Mutations of Homocystinuria in the MENA Region.

Homocystinuria is an inborn error of metabolism due to the deficiency in cystathionine beta-synthase (CBS) enzyme activity. It leads to the elevation of both homocysteine and methionine levels in the blood and urine. Consequently, this build-up could lead to several complications such as nearsightedness, dislocated eye lenses, a variety of psychiatric and behavioral disorders, as well as vascular system complications. The prevalence of homocystinuria is around 1/200,000 births worldwide. However, its prevalence in the Gulf region, notably Qatar, is exceptionally high and reached 1:1800. To date, more than 191 pathogenic CBS mutations have been documented. The majority of these mutations were identified in Caucasians of European ancestry, whereas only a few mutations from African-Americans or Asians were reported. Approximately 87% of all CBS mutations are missense and do not target the CBS catalytic site, but rather result in unstable misfolded proteins lacking the normal biological function, designating them for degradation. The early detection of homocystinuria along with low protein and methionine-restricted diet is the best treatment approach for all types of homocystinuria patients. Yet, less than 50% of affected individuals show a significant reduction in plasma homocysteine levels after treatment. Patients who fail to lower the elevated homocysteine levels, through high protein-restricted diet or by B6 and folic acid supplements, are at higher risk for cardiovascular diseases, neurodegenerative diseases, neural tube defects, and other severe clinical complications. This review aims to examine the mutations spectrum of the CBS gene, the disease management, as well as the current and potential treatment approaches with a greater emphasis on studies reported in the Middle East and North Africa (MENA) region.

Targeting Glutathione and Cystathionine ß-Synthase in Ovarian Cancer Treatment by Selenium-Chrysin Polyurea Dendrimer Nanoformulation.

Ovarian cancer is the main cause of death from gynecological cancer, with its poor prognosis mainly related to late diagnosis and chemoresistance (acquired or intrinsic) to conventional alkylating and reactive oxygen species (ROS)-generating drugs. We and others reported that the availability of cysteine and glutathione (GSH) impacts the mechanisms of resistance to carboplatin in ovarian cancer. Different players in cysteine metabolism can be crucial in chemoresistance, such as the cystine/glutamate antiporter system Xc (xCT) and the H2S-synthesizing enzyme cystathionine ß-synthase (CBS) in the pathway of cysteine catabolism. We hypothesized that, by disrupting cysteine metabolic flux, chemoresistance would be reverted. Since the xCT transporter is also able to take up selenium, we used selenium-containing chrysin (SeChry) as a plausible competitive inhibitor of xCT. For that, we tested the effects of SeChry on three different ovarian cancer cell lines (ES2, OVCAR3, and OVCAR8) and in two non-malignant cell lines (HaCaT and HK2). Results showed that, in addition to being highly cytotoxic, SeChry does not affect the uptake of cysteine, although it increases GSH depletion, indicating that SeChry might induce oxidative stress. However, enzymatic assays revealed an inhibitory effect of SeChry toward CBS, thus preventing production of the antioxidant H2S. Notably, our data showed that SeChry and folate-targeted polyurea dendrimer generation four (SeChry@PUREG4-FA) nanoparticles increased the specificity for SeChry delivery to ovarian cancer cells, reducing significantly the toxicity against non-malignant cells. Collectively, our data support SeChry@PUREG4-FA nanoparticles as a targeted strategy to improve ovarian cancer treatment, where GSH depletion and CBS inhibition underlie SeChry cytotoxicity.

Serine alleviates oxidative stress via supporting glutathione synthesis and methionine cycle in mice.

SCOPE: Serine lies at the central node linking biosynthetic flux from glycolysis to glutathione synthesis and one-carbon metabolic cycle which are closely related to antioxidant capacity. The present study was conducted to determine the effects of serine supplementation on oxidative stress and its relative mechanisms. METHODS AND RESULTS: Diquat treatment was performed to induce oxidative stress in mice and primary hepatocytes. The results showed that hepatic glutathione anti-oxidant systems were impaired and reactive oxygen species and homocysteine were increased in diquat-induced mice and hepatocytes, while such disadvantageous changes were diminished by serine supplementation both in vivo and in vitro. However, when cystathionine ß-synthase expression was inhibited by interference RNA in hepatocytes, the effects of serine supplementation on the improvement of glutathione synthesis and the alleviation of oxidative stress were diminished. Moreover, when hepatocytes were treated with cycloleucine, an inhibitor of methionine adenosyltransferase, the effects of serine supplementation on the improvement of methionine cycle and the alleviation of DNA hypomethylation and oxidative stress were also diminished. CONCLUSION: Our results indicated that serine supplementation alleviated oxidative stress via supporting glutathione synthesis and methionine cycle, mostly by condensing with homocysteine to synthesize cysteine and providing one-carbon units for homocysteine remethylation.

Homocystinuria: Therapeutic approach.

Homocystinuria is a disorder of sulfur metabolism pathway caused by deficiency of cystathionine ß-synthase (CBS). It is characterized by increased accumulation of homocysteine (Hcy) in the cells and plasma. Increased homocysteine results in various vascular and neurological complications. Present strategies to lower cellular and plasma homocysteine levels include vitamin B6 intake, dietary methionine restriction, betaine supplementation, folate and vitamin B12 administration. However, these strategies are inefficient for treatment of homocystinuria. In recent years, advances have been made towards developing new strategies to treat homocystinuria. These mainly include functional restoration to mutant CBS, enhanced clearance of Hcy from the body, prevention of N-homocysteinylation-induced toxicity and inhibition of homocysteine-induced oxidative stress. In this review, we have exclusively discussed the recent advances that have been achieved towards the treatment of homocystinuria. The review is an attempt to help clinicians in developing effective therapeutic strategies and designing novel drugs against homocystinuria.

Vitamin B6 nutritional status and cellular availability of pyridoxal 5'-phosphate govern the function of the transsulfuration pathway's canonical reactions and hydrogen sulfide production via side reactions.

The transsulfuration pathway (TS) acts in sulfur amino acid metabolism by contributing to the regulation of cellular homocysteine, cysteine production, and the generation of H2S for signaling functions. Regulation of TS pathway kinetics involves stimulation of cystathionine ß-synthase (CBS) by S-adenosylmethionine (SAM) and oxidants such as H2O2, and by Michaelis-Menten principles whereby substrate concentrations affect reaction rates. Although pyridoxal phosphate (PLP) serves as coenzyme for both CBS and cystathionine γ-lyase (CSE), CSE exhibits much greater loss of activity than CBS during PLP insufficiency. Thus, cellular and plasma cystathionine concentrations increase in vitamin B6 deficiency mainly due to the bottleneck caused by reduced CSE activity. Because of the increase in cystathionine, the canonical production of cysteine (homocysteine â†’ cystathionine â†’ cysteine) is largely maintained even during vitamin B6 deficiency. Typical whole body transsulfuration flux in humans is 3-7 µmol/h per kg body weight. The in vivo kinetics of H2S production via side reactions of CBS and CSE in humans are unknown but they have been reported for cultured HepG2 cells. In these studies, cells exhibit a pronounced reduction in H2S production capacity and rates of lanthionine and homolanthionine synthesis in deficiency. In humans, plasma concentrations of lanthionine and homolanthionine exhibit little or no mean change due to 4-wk vitamin B6 restriction, nor do they respond to pyridoxine supplementation of subjects in chronically low-vitamin B6 status. Wide individual variation in responses of the H2S biomarkers to such perturbations of human vitamin B6 status suggests that the resulting modulation of H2S production may have physiological consequences in a subset of people. Supported by NIH grant DK072398. This paper refers to data from studies registered at clinicaltrials.gov as NCT01128244 and NCT00877812.

Palm tocotrienol-rich fraction inhibits methionine-induced cystathionine ß-synthase in rat liver.

Oxidative stress plays an important role in cardiovascular diseases. The study investigated the effects of dietary palm tocotrienol-rich fraction on homocysteine metabolism in rats fed a high-methionine diet. Forty-two male Wistar rats were randomly assigned to six groups. Five groups were fed with high-methionine diet (1%) for 10 weeks. Groups 2 to 5 were also given dietary folate (8 mg/kg) and three doses of palm tocotrienol-rich fraction (30, 60 and 150 mg/kg) from week 6 to week 10. The last group was only given basal rat chow. High-methionine diet increased plasma homocysteine after 10 weeks, which was prevented by the supplementations of folate and high-dose palm tocotrienol-rich fraction. Hepatic S-adenosyl methionine (SAM) content was unaffected in all groups but S-adenosyl homocysteine (SAH) content was reduced in the folate group. Folate supplementation increased the SAM/SAH ratio, while in the palm tocotrienol-rich fraction groups, the ratio was lower compared with the folate. Augmented activity of hepatic cystathionine ß-synthase and lipid peroxidation content by high-methionine diet was inhibited by palm tocotrienol-rich fraction supplementations (moderate and high doses), but not by folate. The supplemented groups had lower hepatic lipid peroxidation than the high-methionine diet. In conclusion, palm tocotrienol-rich fraction reduced high-methionine-induced hyperhomocysteinaemia possibly by reducing hepatic oxidative stress in high-methionine-fed rats. It may also exert a direct inhibitory effect on hepatic cystathionine ß-synthase.

Methyl-donor supplementation in obese mice prevents the progression of NAFLD, activates AMPK and decreases acyl-carnitine levels.

Non-alcoholic fatty liver disease (NAFLD) results from increased hepatic lipid accumulation and steatosis, and is closely linked to liver one-carbon (C1) metabolism. We assessed in C57BL6/N mice whether NAFLD induced by a high-fat (HF) diet over 8 weeks can be reversed by additional 4 weeks of a dietary methyl-donor supplementation (MDS). MDS in the obese mice failed to reverse NAFLD, but prevented the progression of hepatic steatosis associated with major changes in key hepatic C1-metabolites, e.g. S-adenosyl-methionine and S-adenosyl-homocysteine. Increased phosphorylation of AMPK-α together with enhanced ß-HAD activity suggested an increased flux through fatty acid oxidation pathways. This was supported by concomitantly decreased hepatic free fatty acid and acyl-carnitines levels. Although HF diet changed the hepatic phospholipid pattern, MDS did not. Our findings suggest that dietary methyl-donors activate AMPK, a key enzyme in fatty acid ß-oxidation control, that mediates increased fatty acid utilization and thereby prevents further hepatic lipid accumulation.

Alleviation of alcoholic liver injury by betaine involves an enhancement of antioxidant defense via regulation of sulfur amino acid metabolism.

Previous studies suggested that the hepatoprotective activity of betaine is associated with its effects on sulfur amino acid metabolism. We examined the mechanism by which betaine prevents the progression of alcoholic liver injury and its therapeutic potential. Rats received a liquid ethanol diet for 6 wk. Ethanol consumption elevated serum triglyceride and TNFα levels, alanine aminotransferase and aspartate aminotransferase activities, and lipid accumulation in liver. The oxyradical scavenging capacity of liver was reduced, and expression of CD14, TNFα, COX-2, and iNOS mRNAs was induced markedly. These ethanol-induced changes were all inhibited effectively by betaine supplementation. Hepatic S-adenosylmethionine, cysteine, and glutathione levels, reduced in the ethanol-fed rats, were increased by betaine supplementation. Methionine adenosyltransferase and cystathionine γ-lyase were induced, but cysteine dioxygenase was down-regulated, which appeared to account for the increment in cysteine availability for glutathione synthesis in the rats supplemented with betaine. Betaine supplementation for the final 2 wk of ethanol intake resulted in a similar degree of hepatoprotection, revealing its potential therapeutic value in alcoholic liver. It is concluded that the protective effects of betaine against alcoholic liver injury may be attributed to the fortification of antioxidant defense via improvement of impaired sulfur amino acid metabolism.

Characterization of two pathogenic mutations in cystathionine beta-synthase: different intracellular locations for wild-type and mutant proteins.

Cystathionine ß-synthase (CBS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the condensation of homocysteine with serine to generate cystathionine. Homocystinuria is an autosomal recessive disorder commonly caused by a deficiency of CBS activity. Here, we characterized a novel CBS mutation (c.260C>A (p.T87N)) and a previously reported variant (c.700G>A (p.D234N)) found in Venezuelan homocystinuric patients, one nonresponsive and one responsive to vitamin B6. Both mutant proteins were expressed in vitro in prokaryotic and eukaryotic cells, finding lower soluble expression in HEK-293 cells (19% T87N and 23% D234N) compared to wild-type CBS. Residual activities obtained for the mutant proteins were 3.5% T87N and 43% D234N. Gel exclusion chromatography demonstrated a tendency of the T87N mutant to aggregate while the distribution of the D234N mutant was similar to wild-type enzyme. Using immunofluorescence microscopy, an unexpected difference in intracellular localization was observed between the wild-type and mutant proteins. While the T87N mutant exhibited a punctate appearance, the wild-type protein was homogeneously distributed inside the cell. Interestingly, the D234N protein showed both distributions. This study demonstrates that the pathogenic CBS mutations generate unstable proteins that are unable (T87N) or partially unable (D234N) to assemble into a functional enzyme, implying that these mutations might be responsible for the homocystinuria phenotype.