Overexpression of Cystathionine γ-Lyase Suppresses Detrimental Effects of Spinocerebellar Ataxia Type 3
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Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine (polyQ) disorder caused by a CAG repeat expansion in the ataxin-3 (ATXN3) gene resulting in toxic protein aggregation. Inflammation and oxidative stress are considered secondary factors contributing to the progression of this neurodegenerative disease. There is no cure that halts or reverses the progressive neurodegeneration of SCA3. Here we show that overexpression of cystathionine γ-lyase, a central enzyme in cysteine metabolism, is protective in a Drosophila model for SCA3. SCA3 flies show eye degeneration, increased oxidative stress, insoluble protein aggregates, reduced levels of protein persulfidation and increased activation of the innate immune response. Overexpression of Drosophila cystathionine γ-lyase restores protein persulfidation, decreases oxidative stress, dampens the immune response and improves SCA3-associated tissue degeneration. Levels of insoluble protein aggregates are not altered; therefore, the data implicate a modifying role of cystathionine γ-lyase in ameliorating the downstream consequence of protein aggregation leading to protection against SCA3-induced tissue degeneration. The cystathionine γ-lyase expression is decreased in affected brain tissue of SCA3 patients, suggesting that enhancers of cystathionine γ-lyase expression or activity are attractive candidates for future therapies.
Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is a rare progressive neurodegenerative disease and the most common dominantly inherited ataxia worldwide. SCA3 is a polyglutamine (polyQ) disorder caused by a CAG-trinucleotide repeat expansion encoding glutamine within the sequence of the ataxin-3 (ATXN3) gene. The length of the repeat expansion is directly related to the aggregation propensity of the ataxin3 protein and is inversely related to the age of onset of the disease. Protein aggregates are considered to be the cause for neuronal dysfunction and death, which is supported by several lines of evidence showing that aggregate prevention or increased (autophagic) clearance delays neuronal death and degeneration in multiple model systems (1, 2, 3, 4).
The pathophysiological sequel of neurodegeneration in SCA3 is not fully understood, although proteotoxic stress, transcriptional dysregulation, mitochondrial dysfunction, oxidative stress and inflammation have been implicated (5, 6, 7). To date, there are no disease-modifying treatments for polyQ diseases such as SCA3.
Cystathionine γ-lyase (CSE) is one of the central enzymes in cysteine and hydrogen sulfide metabolism (H2S). Homocysteine is a substrate for CSE leading to the production of H2S, α-ketobutyrate, ammonia, homolanthionine and cystathionine, with the latter serving as a CSE substrate to produce cysteine (8,9). Cysteine is also a substrate for CSE leading to the production of H2S, cystathionine and pyruvate (8,9). H2S and CSE are linked to aging and age-related pathologies (10, 11, 12, 13). H2S can act as an endogenous modulator of oxidative stress either by direct scavenging of reactive oxygen species (ROS) and nitrogen species (14) or through increasing the intracellular glutathione (GSH) pool (15,16). H2S also confers cytoprotection via suppression of inflammation (17) and by protecting mitochondrial function and integrity (17,18). Decreased levels of H2S in brain tissue are associated with neurodegenerative age-related diseases such as Parkinson’s (19), and administration of H2S has been shown protective in experimental models for this disease (20, 21, 22). Decreased levels of CSE have recently been observed in human Huntington disease tissues and in a mouse Huntington model (22). After addition of sodium hydrogen sulfide and l-cysteine, levels of protein persulfidation (also called protein S-sulfhydration) increased in a CSE-dependent manner in vitro (23), suggesting an influential effect of this type of posttranslational protein modification. Indeed, protein persulfidation has been demonstrated to mediate the activity of parkin, to serve as an antioxidant and to protect against cellular senescence (24, 25, 26). A possible link between SCA3, CSE and protein persulfidation remains to be determined as well as the potential neuroprotective effects of overexpressing the CSE enzyme directly in a neurodegenerative background.
Here, we investigated the possible protective role of CSE in a Drosophila model for SCA3. Drosophila was chosen because CSE is highly conserved between humans and flies (https://doi.org/flybase.org/blast), and an established Drosophila model for SCA3 is available (27,28). In the fly model for SCA3, a truncated version of the pathogenic human ATXN3 gene containing a multiple CAG repeat is expressed, and key features of SCA3 disease are present (27, 28, 29). This model is suitable because the CAG repeat, and not the mutated protein, is considered to be the disease-causing entity in SCA3 and in several other polyQ diseases as well (29,30). We used the Drosophila SCA3 model (also called SCA3 flies) to investigate the effects of CSE overexpression. We identified additional phenotypes in the SCA3 model, such as loss of tissue integrity in degenerative patches in the fly eye, increased activation of the innate immune response and decreased protein persulfidation. We found that transgene-mediated increased expression of CSE rescues these novel and also previously reported phenotypes of the fly SCA3 model. Rescue was also observed after addition of sodium thiosulfate, a drug approved by the U.S. Food and Drug Administration and a component of the transsulfuration pathway in which CSE plays a central role (9,31). The CSE-mediated rescue occurs independently of protein aggregate formation, indicating that rescue effects occur downstream of the formation of these toxic entities. We also found endogenous expression of CSE in brain areas that are affected in SCA3 patients. In addition a lower expression was observed in patient samples compared with controls. We present and discuss a possible role for CSE in polyQ disease pathology and treatment.
Materials and Methods
Below we provide a brief overview of the methods used for experiments presented in this article. For further details, please see the Supplementary Materials and Methods.
As wild-type control, the y1w1118 Drosophila line was used. Eip55E (Drosophila CSE)-overexpressing lines were generated in the laboratory. The GMR-GAL4 UAS-SCA3trQ78 fly stock was a gift from Nancy M Bonini (28,32). The detailed description of the Drosophila lines and fly food, backcrossing and supplementation of chemical compound information can be found in the Supplementary Materials and Methods.
Eye Degeneration Assay
To evaluate relative degeneration percentage, we used an eye scoring method that was previously described (33,34). Irregularly structured depigmented eyes without dark patches were defined as rough. The presence of one or more black patches along with the irregular structure and depigmentation was considered a degenerated rough eye. Each eye of 1-d-old flies was scored as a singular entity. We scored the total amount of degenerated eyes as opposed to the total amount of eyes (rough + degenerated). Total count of eyes scored per condition was between 100 and 1,000, depending on the number of progeny of a particular genotype.
Protein Persulfidation Assay
Molecular Biology Techniques
For a detailed description of quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), Western blot and protein oxidation analyses, and immunohistochemistry used in the current study, please see the Supplementary Materials and Methods.
All supplementary materials are available online at https://doi.org/www.molmed.org .
SCA3 Flies Show Increased Tissue Degeneration
Generation and Characterization of Various CSE Transgenic Lines
Overexpression of CSE Partially Rescues the Phenotype of SCA3 in Drosophila
To investigate a possible effect of CSE overexpression on the eye phenotype, we scored the percentage of degenerated eyes (i.e., when degenerated patches visualized in Figures 2C-C″ are present) 1 d after eclosion by using light microscopy. In the SCA3 background, CSE-overexpressing flies showed a significant decrease in the percentage of degenerative rough eyes (Figures 3C, D). Suppression of the SCA3-degenerative rough eye phenotype was observed in all CSE-overexpressing lines compared with their SCA3-expressing isogenic control lines. Similar results were obtained in both genetic backgrounds. The CSE3 line with the highest level of CSE overexpression reduced the number of degenerative eyes to a greater extent than the CSE2 line (Figure 3D). To further strengthen the rescue potential of CSE, we pharmacologically inhibited CSE with propargylglycine (PPG) as previously described (10). Supplementation of PPG to the fly food reversed the protective effect of CSE overexpression in the SCA3 background, as evidenced by an increased percentage of degenerated rough eyes (Figures 3C, D). Addition of PPG neither enhanced nor suppressed the percentage of degenerative eyes in the SCA3 background, strongly suggesting that the observed effect in the CSE-overexpressing background is due to inhibition of CSE and not due to other effects of PPG. Together, these results indicate that the rescuing potential is mediated by overexpression of CSE and is not influenced by the genetic background.
Overexpression of CSE Does Not Induce a Change in Levels of Insoluble Proteins
Overexpression of CSE Reduces Levels of Oxidative Damage of Proteins in SCA3 Flies
Oxidative stress is associated with the pathogenesis of SCA3 disease (40,41). Previously, it was demonstrated that CSE deficiency is linked to increased levels of oxidative stress (42). As a readout for oxidative stress, we used OxyBlot analysis as previously described (43). SCA3 flies showed increased levels of oxidized proteins (characteristically visible as multiple bands) compared with their isogenic non-SCA3 control lines (Figures 4C, D). The level of oxidized proteins was reduced in all three CSE overexpression lines in the SCA3 background compared with the isogenic controls (Figures 4C, D).
Overexpression of CSE Prevents SCA3-Associated Immune Induction
SCA3 Flies Show Reduced Levels of Protein Persulfidation
Decreased levels of CSE are associated with impaired neurological function (22). Here we demonstrated that increased levels of CSE are protective. Because CSE overexpression is associated with increased protein persulfidation (25,49), we hypothesized that, in a SCA3 background, protein persulfidation may be decreased and CSE overexpression may lead to restoration of this posttranslational modification.
Treatment with Sodium Thiosulfate Reduces Eye Degeneration in SCA3 Flies
To try to pharmacologically rescue SCA3-induced degeneration, we used sodium thiosulfate (STS). STS is a relatively stable nontoxic compound used in clinical settings to treat caliciphylaxis, extravasations during chemotherapy or cyanide poisoning (50), and is also a component of the transsulfuration pathway. Used as a substrate for rhodanaselike enzymes, thiosulfate could also be a source of targeted persulfidation, as recently proposed by Mishanina et al. (31). Therefore, we tested the effects of thiosulfate on SCA3 flies.
Endogenous CSE Is Present in Affected Brain Tissue of SCA3 Patients
To investigate a possible role of CSE in human SCA3 pathogenesis, we investigated the expression levels and localization of CSE in healthy tissue and in SCA3 disease tissue. To determine the presence and localization of CSE, we performed immunohistochemistry for CSE on postmortem pontine tissue of control individuals and SCA3 patients. From the sparse tissue available for this rare disease, pontine tissue was chosen to analyze the features of this disorder, because in this tissue several types of toxic protein aggregates are present with enough neurons preserved to allow immunohistochemical analysis (7) in contrast to other brain areas that are almost completely degenerated or that hardly show degeneration or protein aggregation (51,52). As control samples, postmortem tissue of individuals without a neurodegenerative or neuropsychiatric disease were used (Supplementary Table S1; n = 7). CSE protein expression was observed in vascular endothelium, neurons and astrocytes (Figures 7C-F). This localization pattern was not affected in pontine tissue of SCA3 patients (Figures 7G-J; Supplementary Figure S5). To investigate expression levels of CSE, we performed qRT-PCR analysis for CSE transcripts on pontine samples of SCA3 patients and control samples. qRT-PCR data revealed the presence mRNA levels of CSE in pontine tissue of control tissue (n = 7) and SCA3 (n = 6) patients; although, in the latter, levels were reduced (Figure 7K). Western blot analysis using an anti-CSE antibody (53) further confirmed the presence and decreased levels of endogenous CSE in pontine tissue of SCA3 patients compared with controls (Figures 7L, L′). Together, these data demonstrated that CSE is endogenously present but decreased expressed in affected brain areas of SCA3 patients.
We present evidence that CSE overexpression works protectively in a Drosophila model for the neurodegenerative disease SCA3. To our knowledge, protective effects by CSE overexpression in neurodegenerative animal models have not been described before. However, neuroprotective effects of H2S have been reported previously, not only in experimental models for Parkinson’s disease, (21), vascular dementia (54) and homocysteineinduced neurotoxicity, but also in in vitro models for oxidative stress in neurons (55) and Alzheimer’s disease (56). In an experimental mouse model for Parkinson’s disease, inhalation of H2S prevents the development of neurodegeneration and movements disorders (20).
The findings in Drosophila may be of clinical relevance because we observed that, in SCA3 patients, CSE expression is decreased in affected brain areas compared with controls. Recently, decreased levels of CSE were also demonstrated in striatal brain samples from patients with Huntington disease (22). CSE−/− mice showed impaired locomotor functions (22); therefore, it is possible that low levels of CSE negatively influence the progression of neurodegenerative phenotypes in Huntington disease and SCA3. This result is consistent with our findings showing that, in contrast to decreasing CSE levels, boosting CSE expression in a neurodegenerative background is beneficial. In contrast to our results, CSE protein levels were found unaltered in the cerebellum and cerebral cortex of one spinocerebellar ataxia patient (SCA subtype unknown) (22), suggesting that alteration of CSE levels in SCA patients may be confined to pontine tissue or may depend on the SCA subtype.
CSE is an essential enzyme in the transsulfuration pathway and plays a role in the endogenous production of cysteine (57) and H2S (58, 59, 60). Therefore, the beneficial effects of CSE can be mediated via cysteine, H2S or both. It is also possible that the increased expression of CSE catalyzes the formation of cysteine persulfides that can trans-persulfidate the proteins without any H2S being produced (61). The explanation of the beneficial effects by protein persulfidation is in agreement with our observations that this posttranslational modification is increased in CSE-overexpressing flies and restored in CSE-overexpressing flies in a SCA3 background. Moreover, it also explains the rescue effect of STS, assuming that the proposed effect of STS on protein persulfidation is correct (31). A protective effect of protein persulfidation has been demonstrated in other studies as well. For example, the activity of neuroprotective ubiquitin ligase parkin is regulated by protein persulfidation. Parkin persulfidation is markedly depleted in the brains of patients with Parkinson’s disease (24). Another study demonstrates that the capacity of H2S to protect against oxidative stress is executed via persulfidation (25,26). These findings together with our results suggest that boosting the transsulfuration pathway may contribute to neuroprotection via increased persulfidation of proteins.
Our results show that overexpression of CSE is associated with a dampening of the immune response and decreased levels of protein oxidation. This finding is consistent with previous findings because inflammation has been implicated as a critical mechanism responsible for the progressive nature of neurodegeneration (44,62), and there is an inverse link between an activated transsulfuration pathway and the immune response. In experimental models, H2S exerts antineuroinflammatory effects via inhibition of p38/Jun nuclear kinase and NF-κB signaling pathways (63), and the inhibition of CSE by PPG leads to increased inflammation (64). Furthermore, CSE has been shown to be a modulator of oxidative stress in mice (42). SCA3 is associated with oxidative stress because mutant ATXN3 is associated with a significantly reduced capability to counteract oxidative stress that contributes to neuronal cell death in SCA3 (65).
On the basis of the discussed results of others and our observations, we propose the following hypothetical model: PolyQ diseases lead to accumulation of toxic protein aggregates, and this somehow reduces levels of CSE and/or protein persulfidation. This result, in turn, contributes to increased oxidative stress and an augmented immune response leading to accelerated neurodegeneration. It is possible that overexpression of CSE induces protein persulfidation (via or independent from induced H2S and/or cysteine biosynthesis). Increased levels of protein persulfidation reduces the levels of oxidative stress and dampens the immune response, and, by this, the damaging effects of toxic protein aggregates are reduced and tissue integrity is better preserved.
Our data show that CSE levels are decreased in tissue of SCA3 patients. However, in our opinion, it is more important that CSE is still expressed in affected tissue and apparently in identical cell types, because this result allows a strategy to increase CSE expression or activity by pharmacological inventions to protect against tissue degeneration in SCA3. Little is known about the regulation of CSE, but there are substances available that are able to influence CSE activity or transcription. There is evidence that myeloid zinc finger 1 and specificity protein 1 transcription factor affect the transcription of CSE (66). Furthermore, studies suggest that CSE can be upregulated by bacterial endotoxin (66,67) and by nitric oxide (68). S-adenosylmethionine and pyridoxal-5′-phosphate stimulate CSE activity to increase H2S production (69,70). Alternatively to increasing CSE expression as a therapeutic option, rescue may be provoked by STS because our data show a protective effect of this compound as well, and it is tolerated by humans in high concentrations (71,72).
Our data indicate a modifying role of the transsulfuration pathway in SCA3 and suggest that this is mediated via protein persulfidation. The presence of CSE in SCA3-relevant brain regions, together with the protective effects of CSE overexpression in Drosophila, indicates the relevance for future research on developing clinically applicable activators of CSE or other members of the transsulfuration pathway. As the protective effects occur downstream of the formation of protein aggregates, it may be possible that activation of the transsulfuration pathway is protective for other polyglutamine expansion-induced diseases as well.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported by a VICI grant (to OCM Sibon) and a grant from the Jan Kornelis de Cock Foundation (to PM Snijder). Part of this work was performed at the UMCG Microscopy and Imaging Center (UMIC), sponsored by ZonMW grant 91111.006.
The authors express their gratitude to Martha Elwenspoek, Marian Bulthuis and Yi Xian Li for excellent technical support and to Bart Kanon and Jan Vonk for support and valuable advice.
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