Prof. Jerome Lejeune’s clinical observations that Down syndrome (DS) had features both comparable and opposite (mirroring) to homocystinuria, led him to propose that in DS, an alteration in the equilibrium between homocysteine catabolism and cysteine production must occur. Following this hypothesis research has been oriented towards Cystathionine-β-synthase, a gene located on the chromosome 21. Evidence has shown that there is an increase in CBS enzyme activity in cells and tissues from individuals with DS, and nowadays, many researchers recognise that CBS might be implicated in the cognitive impairment related to DS.

Cystathionine-beta-synthase (CBS)

CBS is the first and rate-limiting enzyme in the reverse transsulfuration pathway (Figure 1) in which L-cysteine is generated from methionine. CBS catalyses the conversion of L-homocysteine into L-cystathionine by displacing the OH group of L-serine with the thiol of L-homocysteine in a β-replacement reaction to form L-cystathionine, which is subsequently hydrolysed by cystathionine γ-lyase (CSE) into L-cysteine.

Figure 1. Role of CBS and other enzymes in the regulation of mammalian sulfur amino acid metabolism. Abbreviations: Methionine (Met), ATP by methionine adenosyltransferase (MAT), S-adenosylmethionine (SAM), methyltransferases (MT), S-adenosylhomocysteine (SAH), SAH hydrolase (SAHH), homocysteine (Hcy), betaine homocysteine methyltransferase (BHMT), ubiquitous methionine synthase (MS), methyl tetrahydrofolate (methyl-THF), cystathionine beta-synthase (CBS), serine (Ser), cystathionine (Cth), cystathionine gamma-lyase (CSE), cysteine (Cys). Reproduced with permission (Zuhra K. et al., Biomolecules. 2020).

CBS is a pyridoxal 5’-phosphate (PLP, the active form of vitamin B6)-dependent enzyme. Human CBS is a homotetrameric enzyme of 63-kDa subunits. Each subunit binds, in addition to its two substrates (homocysteine and serine), heme (iron(II)-centered porphyrin), the role of which is not fully understood, and S-adenosyl-methionine (SAM), that acts as an allosteric activator. CBS is expressed in liver, pancreas, brain, heart, kidney and lung. Its expression is primarily regulated port-transcriptionally, but modifications at the messenger RNA level, are observed under certain (patho)-physiological changes.

Besides its important role in the metabolism of sulphur-containing aminoacids, CBS is involved is the production of hydrogen sulphide (H2S), an endogenous signalling gasotransmitter involved in the modulation of multiple physiological responses in the vascular, immune and nervous systems.

CBS in Down syndrome:

Due to its localisation on chromosome 21, the CBS gene is overexpressed in patients with Down syndrome. Evidence suggests that the increased activity of CBS alters homocysteine metabolism, and indeed overproduction of H2S has been documented in individuals with Down syndrome. Two decades ago and based on his own clinical findings, P. Kamoun put forward the hypothesis that CBS-derived overproduction of H2S, might be responsible for some of the pathophysiological aspects of Down syndrome (Kamoun P., 2001), in part, via inhibition of mitochondrial Complex IV and impairment of ATP generation, which, in turn, produces a global energetic deficit in Down syndrome individuals. Some studies indeed demonstrated that DS cells exhibit impaired mitochondrial function. Yet, it wasn’t until the multiple roles of H2S as a biological regulator were demonstrated, that the “Kamoun hypothesis” was revisited. Recently, the hypothesis was experimentally confirmed by the team of Csaba Szabo, which evaluated the effect of CBS silencing/inhibition on the proliferation, mitochondrial oxygen consumption and Complex IV activity in DS fibroblasts. The team confirmed that 1) CBS protein and H2S levels were elevated in DS cells, 2) that mitochondrial metabolism is impaired in these cells, and 3) demonstrated that CBS inhibition or downregulation improves bioenergetic functions, restores mitochondrial Complex IV activity and consequently improve viability and proliferation rate in DS cells (Panagaki et al 2019).

Figure 2. Here, as postulated by the team of C. Szabo: The bell-shaped role of CBS expression and H2S biosynthesis in the regulation of cell viability in health and disease. CBS inhibition can impair cancer cell viability by reducing the formation of H2S which the cancer cells use as a cytoprotective factor and bioenergetic “fuel”. CBS inhibition can also improve cell viability, for instance in Down syndrome, by normalizing the toxic overproduction of H2S. Reproduced with permission (Zuhra K. et al., Biomolecules. 2020).

As a biological modulator, gasotransmitter or signalling molecule, H2S has been implicated among others in long-term potentiation in the hippocampus, neuroprotection processes, neurotransmission, hyperpolarization of neuronal cell membranes, oxidative stress response and neuronal excitability, the impact of higher than normal H2S levels is expected to be deleterious for neural development, brain homeostasis and cognition. Recently, it has been shown by the group of Yann Herault (Marechal D. et al., 2019), that CBS overdosage is necessary and sufficient to induce cognitive impairment in a mouse model of DS which are trisomic for CBS (Dp1Yah).

Much work is left to be done to fully decipher the role of CBS/H2S in the neurodevelopmental and cognitive phenotypes of DS, and the Jerome Lejeune Foundation is actively sponsoring several projects with this aim. Including development of models and the search for pharmacological inhibitors with therapeutic potential.

Animal models:

To analyse the possible role of CBS in Down syndrome, there are several lines of transgenic mice expressing the human CBS gene described in Herault et al, Disease Models & Mechanisms 2017. Novel rat models to evaluate the function of CBS are currently being developed by the lab of Yann Herault in the IGBMC.

Cell models:

The enzymatic activity of CBS and the outcome of inhibition/downregulation of the enzyme, has been studied in fibroblasts of Down syndrome patients. Currently, human induced pluripotent stem cells (iPSC) and iPSC-derived neuronal cells, are routinely used by teams supported by the Foundation, to study CBS/H2S activity and its impact on cellular homeostasis.  

Translational research in the field of CBS inhibition/ Pharmacological Inhibitors:

Interest in the pharmacological inhibition of CBS/H2S pathway, has only emerged in the last decade, when a role of CBS/H2S in cancer and in Down syndrome became evident. Before that, only activation/upregulation of the pathway was of interest for the treatment of homocystinuria.

Progress in this field is expected to stimulate further work to identify clinically useful and highly selective inhibitors of CBS. Extensive work on this subject is currently ongoing and is being sponsored by the Jerome Lejeune Foundation.

CBS is also involved in other pathologies:

Homocystinuria: Caused by mutations in the protein sequence that abolish CBS activity. This disease is accompanied by mental retardation, connective tissue disturbance and thromboembolic disorders. The prevalence is as high as 1/6000. The different mutations cause misfolding of the protein subunits, disruption of oligomers assembly and protein aggregation, resulting in inactivity of the enzyme.

Human lung adenocarcinoma: There is in the adenocarcinoma the presence of a tissue overexpression of CBS, CSE, and MPST compared with adjacent lung tissue, and H2S activates mitochondrial DNA repair (Szczesny et al., 2016)

Colorectal cancer, ovarian cancer, and breast cancer: Role of CBS/H2S in cell proliferation and cellular bioenergetics (Bhattacharyya et al., 2013; Sen et al., 2015; Szabo et al., 2013, Zuhra et al Biomolecules 2020)

Glioma: CBS silenced glioma exhibited increased depth of invasion, vascular density, and cell proliferation with increased levels of VEGF and HIF2α expression, suggesting that CBS/H2S may suppress glioma (Takano et al., 2014).

Some major (non-exclusive) publications on CBS and Down syndrome

  1. Augsburger, F.; Szabo, C. Potential role of the 3-mercaptopyruvate sulfurtransferase (3-MST)—hydrogensulfide (H2S) pathway in cancer cells. Pharmacol. Res. 2020, 154, 104083.
  2. Butler, C., Knox, A.J., Bowersox, J. et al. The Production of Transgenic Mice Expressing Human Cystathionine Beta-Synthase to Study Down Syndrome. Behav Genet 36429–438 (2006).
  3. Eto K. and Kimura H. (2002) A novel enhancing mechanism for hydrogen sulfide-producing activity of cystathionine beta-synthase. J. Biol. Chem. 277:42680–42685
  4. Herault, Y., Delabar, J.M., Fisher, E.M.C., Tybulewicz, V.L.J., Yu, E. and Brault, V. (2017) Rodent models in Down syndrome research: impact and future opportunities. Dis. Model Mech., 10, 1165–1186
  5. Kamoun P. (2001) Mental retardation in Down syndrome: a hydrogen sulfide hypothesis. Med. Hypotheses 57:389–392
  6. Kamoun P., Belardinelli M. C., Chabli A., Lallouchi K. and Chadefaux-Vekemans B. (2003) Endogenous hydrogen sulfide overproduction in Down syndrome. Am. J. Med. Genet. 116A:310–311
  7. Kimura, H. (2002) Hydrogen sulfide as a neuromodulator.Mol. Neurobiol., 26, 13–19
  8. Kimura, H. (2015). Signaling molecules: Hydrogen sulfide and polysulfide.Antioxidants and Redox Signaling, 22, 362–376.
  9. Kimura H. Signalling by hydrogen sulfide and polysulfides via protein Ssulfuration. Br J Pharmacol.2020;177:720–733.
  10. Kraus J. P., Oliveriusova J., Sokolova J., Kraus E., Vicek C., De Franchis R., MacLean K. N., Bao L., Bukoskova G., Patterson D., Paces V., Ansorge W. and Kozich V. (1998). The human cystathionine beta-synthase (CBS) gene: complete sequence, alternative splicing, and polymorphisms. Genomics 52:312–324
  11. Lejeune, J. 1975. Reflexions sur la debilite de l’intelligence des enfants trisomiques 21. Pont. Acad. Sci. 3:1-12.
  12. Lejeune J., Rethore M. O., de Blois M. C., Maunoury-Burolla C., Mir M., Nicolle L., Borowy F., Borghi E. and Recan D. (1986). Metabolism of monocarbons and trisomy 21: sensitivity to methotrexate. Ann. Genet. 29:16–19
  13. Lejeune J. (1990). Pathogenesis of mental deficiency in trisomy 21. Am. J. Med. Genet. Supp. 7:20–30
  14. MacLean K. N., Kraus E. and Kraus J. P. (2004). The dominant role of Sp1 in regulating the cystathionine beta synthase −1a and −1b promoters facilitates potential tissue-specific regulation by Kruppel-like factors. J. Biol. Chem. 279:8558–8566
  15. Marechal D, Brault V, Leon A, Martin D, Lopes Pereira P, Loaëc N, Birling MC, Friocourt G, Blondel M, Herault Y. Cbs overdosage is necessary and sufficient to induce cognitive phenotypes in mouse models of Down syndrome and interacts genetically with Dyrk1a. Hum Mol Genet. 2019 May 1;28(9):1561-1577.
  16. Munke M., Kraus J. P., Ohura T. and Francke U. (1988). The gene for cystathionine beta-synthase (CBS) maps to the subtelomeric region on human chromosome 21q and to proximal mouse chromosome 17. Am. J. Hum. Genet. 42:550–550
  17. Panagaki, T.; Randi, E.B.; Augsburger, F.; Szabo, C. Overproduction of H2S, generated by CBS, inhibits mitochondrial Complex IV and suppresses oxidative phosphorylation in Down syndrome. Proc. Natl. Acad. Sci. USA 2019, 116, 18769–18771
  18. Peeters M. A., Poon A., Zipursky A., and Lejeune J. (1986). Toxicity of leukaemia therapy in children with Down syndrome. Lancet, 1279, November 29, 1986
  19. Reeves, R. H., J. D. Gearhart, and J. W. Littlefield. 1986. Genetic basis for a mouse model of Down syndrome. Brain Res. Bull. 16:803-814.
  20. Robert K., Vialard F., Thiery E., Toyama K., Sinet P.-M., Janel N. and London J. (2003). Expression of the cystathionine beta synthase gene during mouse development and immunolocalization in the adult brain. J. Histochem. Cytochem. 51:363–371
  21. Roizen N. J. and Patterson D. (2003). Down’s syndrome. Lancet, 361:1281–1289
  22. Sinet, P. M., J. Couturier, B. Dutrillaux, M. Poissonnier, 0. Raoul, M.-O. Rethore, D. Allard, J. Lejeune, and H. Jerome. 1976. Trisomie 21 et superoxyde dismutase-1 (IPO-A): tentative de localisation sur la sous bande 21q22.1. Exp. Cell. Res. 97:47-55.
  23. Szabo, C. A timeline of hydrogen sulfide (H2S) research: From environmental toxin to biological mediator.Biochem. Pharmacol. 2018, 149, 5–1
  24. Szabo C. The re-emerging pathophysiological role of the cystathionine-β-synthase – hydrogen sulfide system in Down syndrome. FEBS J. 2020 Aug;287(15):3150-3160.
  25. Zhong H, Yu H, Chen J, Sun J, Guo L, Huang P, Zhong Y. Hydrogen Sulfide and Endoplasmic Reticulum Stress: A Potential Therapeutic Target for Central Nervous System Degeneration Diseases. Front Pharmacol. 2020 May 14;11:702.
  26. Zuhra, K.; Augsburger, F.; Majtan, T.; Szabo, C. Cystathionine-β-synthase: Molecular Regulation and Pharmacological Inhibition. Biomolecules 2020, 10, 697.