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. 2014 May 1;509(7498):96-100.
doi: 10.1038/nature13136. Epub 2014 Mar 26.

Cystathionine γ-Lyase Deficiency Mediates Neurodegeneration in Huntington's Disease

Free PMC article

Cystathionine γ-Lyase Deficiency Mediates Neurodegeneration in Huntington's Disease

Bindu D Paul et al. Nature. .
Free PMC article


Huntington's disease is an autosomal dominant disease associated with a mutation in the gene encoding huntingtin (Htt) leading to expanded polyglutamine repeats of mutant Htt (mHtt) that elicit oxidative stress, neurotoxicity, and motor and behavioural changes. Huntington's disease is characterized by highly selective and profound damage to the corpus striatum, which regulates motor function. Striatal selectivity of Huntington's disease may reflect the striatally selective small G protein Rhes binding to mHtt and enhancing its neurotoxicity. Specific molecular mechanisms by which mHtt elicits neurodegeneration have been hard to determine. Here we show a major depletion of cystathionine γ-lyase (CSE), the biosynthetic enzyme for cysteine, in Huntington's disease tissues, which may mediate Huntington's disease pathophysiology. The defect occurs at the transcriptional level and seems to reflect influences of mHtt on specificity protein 1, a transcriptional activator for CSE. Consistent with the notion of loss of CSE as a pathogenic mechanism, supplementation with cysteine reverses abnormalities in cultures of Huntington's disease tissues and in intact mouse models of Huntington's disease, suggesting therapeutic potential.


Extended Data Figure 1
Extended Data Figure 1. CSE expression is not altered in the brain in amyotrophic lateral sclerosis, multiple sclerosis and spinocerebellar ataxia
a, Western blots show that CSE expression in the motor cortex of samples from controls and patients with amyotrophic lateral sclerosis (ALS) showing substantial neurodegeneration in the motor cortex are similar. Extracts were prepared from the motor cortex and analysed for CSE expression using anti-CSE antibodies and β-actin as a loading control. b, Expression of CSE is not altered in the corpus callosum of patients with multiple sclerosis (MS), where multiple lesions, demyelination and decrease in oligodendrocytes was observed in the corpus callosum of the brain. c, d, Levels of CSE do not change in the cerebral cortex (c) or cerebellum (d) of patients with spinocerebellar ataxia (SCA). Neuropathological analysis of the brains of these patients revealed severe neuronal loss and gliosis in the cerebellum.
Extended Data Figure 2
Extended Data Figure 2. Cse−/− mice are more vulnerable to stress induced by 3-nitropropionic acid
Wild-type and Cse−/− male mice at 8 months of age were injected with a single dose of 3-nitropropionic acid (3-NP) (100 mg kg−1), and lysates were prepared 24 h later from the striatum and cortex and analysed for oxidative stress. a, b, Striata (a) and cortex (b) of Cse−/− mice show elevated protein oxidation as measured by protein carbonylation, which is more pronounced in the striatum. n = 3 (means ± s.e.m.). c, d, Cse−/− mice also show augmented levels of protein nitration in the striatum (c) and cortex (d) in comparison with wild-type mice. Note the increased basal level of protein oxidation in the Cse−/− mice.
Figure 1
Figure 1. CSE is expressed in the brain and is depleted in Huntington’s disease
a, CSE expression is detectable in whole-brain lysates. WT, wild type. b, CSE is expressed in different regions of the brain. c, Cse−/− mice show a limb-clasping phenotype. d, Striatal STHdhQ111/Q111 have decreased expression of CSE. e, Quantification of d. n = 3 (means ± s.e.m.); ***P < 0.001. f, Expression of CSE is decreased significantly in all brain regions of 13-week-old R6/2 mice analysed except in the cerebellum. g, Relative quantification of f. n = 6 (means ± s.e.m.) for cortex, striatum and cerebellum; n = 3 (means ± s.e.m.) for other regions; ***P < 0.001; **P < 0.01; NS, not significant. h, Expression of CSE is decreased in striata of Q175 mice. i, Quantification of h. n = 3 (means ± s.e.m.); ***P < 0.001. j, Post-mortem striatal brain samples (P1–P6) from patients with Huntington’s disease show a decrease in CSE expression. k, Relative quantification of j. n = 5 (means ± s.e.m.) for controls; n = 6 (means ± s.e.m.) for samples from patients with Huntington’s disease (HD); ***P < 0.001. l, CSE depletion is increased with the severity of the disease. C1 and C2 are controls;HD1–HD4 are samples from patients with Huntington’s disease. m, Relative quantification of HD grades. n = 3 (means ± s.e.m.) for HD2; n = 4 (means ± s.e.m.) for normal, HD3 and HD4; *P < 0.05 (control versus HD2 and HD2 versus HD3); ***P < 0.001 (control versus HD4). n, CSE expression is decreased in the liver and pancreas. o, Relative quantification of n. n = 3 (means ± s.e.m.); ***P < 0.001.
Figure 2
Figure 2. Decreased CSE activity and growth in striatal Q111 cells
a, The reverse transsulphuration pathway leading to the production of cysteine from methionine. CSE produces cysteine from cystathionine generated by CBS. Cysteine and homocysteine are substrates for the production of H2S. b, Decreased H2S production in Q111 cells in comparison with Q7 cells. n = 3 (means ± s.e.m.); ***P < 0.001. c, Decreased cysteine synthesis in Q111 cells. n = 3 (means ± s.e.m.); ***P < 0.001. d, Impaired growth of Q111 cells in cysteine-free medium as monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Q111 cells underwent cell death, which was rescued by supplementation with l-cysteine. n = 3 (means ± s.e.m.); ***P < 0.001. e, Growth retardation of Q111 cells in cysteine-free medium is rescued by the transfection of CSE construct as assessed by the MTT assay. **P < 0.01. f, Decreased cysteine levels in striata of R6/2 mice. n = 3 (means ± s.e.m.); *P < 0.05. g, Decreased synthesis of H2S in striata of R6/2 mice. n = 3 (means ± s.e.m.); ***P < 0.001. h, Decreased synthesis of H2S in striata of Q175 mice. n = 3 (means ± s.e.m.); *P < 0.05.
Figure 3
Figure 3. CSE is depleted at the transcriptional level in Huntington’s disease
a, CSE is not sequestered in the insoluble pellet fraction by mutant huntingtin. b, CSE is not differentially degraded in Q7 and Q111 cells after treatment with the proteasome inhibitor MG132. c, CSE mRNA is decreased in Q111 cells, as revealed by RT–PCR using β-actin as the internal control. d, Reduction of CSE expression in Q111 cells verified by real-time quantitative PCR. n = 3 (means ± s.e.m.); ***P < 0.001. e, CSE promoter activity is repressed in Q111 cells as revealed by luciferase assays using a CSE-luc reporter construct and an endogenous internal control. n = 4 (means ± s.e.m.); ***P < 0.001. f, Overexpression of the transcription factor Sp1 and its co-activator TAF4 rescues CSE expression. Empty vector controls are denoted by C. g, Relative quantification of f. n = 3 (means ± s.e.m.); **P < 0.01. h, Overexpression of Sp1 and TAF4 elevates CSE transcript levels as revealed by quantitative PCR. n = 3 (means ± s.e.m.); **P < 0.01. i, Overexpression of Sp1/TAF4 rescues lethality of Q111 cells in cysteine-free media as measured by the MTT assay. n = 3 (means ± s.e.m.); ***P < 0.001.
Figure 4
Figure 4. CSE protects against oxidative stress, and cysteine supplementation delays neurodegeneration
a, Striatal Q111 cells are more vulnerable to oxidative stress induced by 0.1 mM H2O2 as monitored by MTT assays, effects that are rescued by CSE overexpression. n = 3 (means ± s.e.m.); **P < 0.01. b, Cse−/− mice have impaired motor function. WT, Cse−/− homozygous and Cse+/− heterozygous mice were placed on an accelerating rotarod, and latency to fall was recorded. Both the Cse−/− homozygous and Cse+/− heterozygous mice were impaired in their motor functions; the homozygous knockout mice showed the greatest deficits. n = 5 (means ± s.e.m.) for WT, n = 4 (means ± s.e.m.) for Cse+/− heterozygous and n = 11 for Cse−/− homozygous; ***P < 0.001 (WT versus Cse−/−); **P < 0.01 (WT versus Cse+/− and Cse+/− versus Cse−/−). c, Cysteine supplementation delays motor symptoms in R6/2 mice. Mice were placed on regular diet or a cysteine-supplemented diet along with 20 mM N-acetylcysteine in the drinking water, and clasping phenotype was monitored. n = 8 (means ± s.e.m.); ***P < 0.001. See also Supplementary Videos 1–3. d, Cysteine supplementation improves performance on an accelerating rotarod in R6/2 mice. n = 8 (means ± s.e.m.) for WT; n = 6 (means ± s.e.m.) for R6/2; n = 7 (means ± s.e.m.) for R6/2 + cysteine; *P < 0.05. e, Grip strength is improved in R6/2 mice placed on a cysteine-supplemented diet. *P < 0.05. f, Decrease in brain masses of R6/2 mice is ameliorated by cysteine treatment. g, Striatal atrophy is decreased in R6/2 mice treated with cysteine as assessed by Nissl staining of coronal sections of the brain. h, Striatal volume of R6/2 mice on a cysteine supplemented diet is larger than in the untreated R6/2 mice. n = 3 (means ± s.e.m.) for WT, n = 4 (means ± s.e.m.) for untreated R6/2 mice and R6/2 mice treated with cysteine. *P < 0.05. i, Cysteine supplementation prolongs survival in R6/2 mice (Kaplan–Meier analysis). R6/2 mice (n = 7 per group) were treated as in f, and survival in weeks was monitored. Statistical analysis was conducted with the log-rank (Mantel–Cox) test; P = 0.004.

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