Mutant Huntingtin Derails Cysteine Metabolism in Huntington's Disease at Both Transcriptional and Post-Translational Levels.

Cysteine is a semi-essential amino acid that not only plays an essential role as a component of protein synthesis, but also in the generation of numerous sulfur-containing molecules such as the antioxidant glutathione and coenzyme A. We previously showed that the metabolism of cysteine is dysregulated in Huntington's disease (HD), a neurodegenerative disorder triggered by the expansion of polyglutamine repeats in the protein huntingtin. In this study, we showed that cysteine metabolism is compromised at multiple levels in HD, both transcriptional and post-translational. Accordingly, restoring cysteine homeostasis may be beneficial in HD.

Hydrogen sulfide is neuroprotective in Alzheimer's disease by sulfhydrating GSK3ß and inhibiting Tau hyperphosphorylation.

Alzheimer's disease (AD), the most common cause of dementia and neurodegeneration in the elderly, is characterized by deterioration of memory and executive and motor functions. Neuropathologic hallmarks of AD include neurofibrillary tangles (NFTs), paired helical filaments, and amyloid plaques. Mutations in the microtubule-associated protein Tau, a major component of the NFTs, cause its hyperphosphorylation in AD. We have shown that signaling by the gaseous molecule hydrogen sulfide (H2S) is dysregulated during aging. H2S signals via a posttranslational modification termed sulfhydration/persulfidation, which participates in diverse cellular processes. Here we show that cystathionine γ-lyase (CSE), the biosynthetic enzyme for H2S, binds wild type Tau, which enhances its catalytic activity. By contrast, CSE fails to bind Tau P301L, a mutant that is present in the 3xTg-AD mouse model of AD. We further show that CSE is depleted in 3xTg-AD mice as well as in human AD brains, and that H2S prevents hyperphosphorylation of Tau by sulfhydrating its kinase, glycogen synthase kinase 3ß (GSK3ß). Finally, we demonstrate that sulfhydration is diminished in AD, while administering the H2S donor sodium GYY4137 (NaGYY) to 3xTg-AD mice ameliorates motor and cognitive deficits in AD.

Bilirubin Links Heme Metabolism to Neuroprotection by Scavenging Superoxide.

Bilirubin is one of the most frequently measured metabolites in medicine, yet its physiologic roles remain unclear. Bilirubin can act as an antioxidant in vitro, but whether its redox activity is physiologically relevant is unclear because many other antioxidants are far more abundant in vivo. Here, we report that depleting endogenous bilirubin renders mice hypersensitive to oxidative stress. We find that mice lacking bilirubin are particularly vulnerable to superoxide (O2⋅-) over other tested reactive oxidants and electrophiles. Whereas major antioxidants such as glutathione and cysteine exhibit little to no reactivity toward O2⋅-, bilirubin readily scavenges O2⋅-. We find that bilirubin's redox activity is particularly important in the brain, where it prevents excitotoxicity and neuronal death by scavenging O2⋅- during NMDA neurotransmission. Bilirubin's unique redox activity toward O2⋅- may underlie a prominent physiologic role despite being significantly less abundant than other endogenous and exogenous antioxidants.

Redox Mechanisms in Neurodegeneration: From Disease Outcomes to Therapeutic Opportunities.

SIGNIFICANCE: Once considered to be mere by-products of metabolism, reactive oxygen, nitrogen and sulfur species are now recognized to play important roles in diverse cellular processes such as response to pathogens and regulation of cellular differentiation. It is becoming increasingly evident that redox imbalance can impact several signaling pathways. For instance, disturbances of redox regulation in the brain mediate neurodegeneration and alter normal cytoprotective responses to stress. Very often small disturbances in redox signaling processes, which are reversible, precede damage in neurodegeneration. Recent Advances: The identification of redox-regulated processes, such as regulation of biochemical pathways involved in the maintenance of redox homeostasis in the brain has provided deeper insights into mechanisms of neuroprotection and neurodegeneration. Recent studies have also identified several post-translational modifications involving reactive cysteine residues, such as nitrosylation and sulfhydration, which fine-tune redox regulation. Thus, the study of mechanisms via which cell death occurs in several neurodegenerative disorders, reveal several similarities and dissimilarities. Here, we review redox regulated events that are disrupted in neurodegenerative disorders and whose modulation affords therapeutic opportunities. CRITICAL ISSUES: Although accumulating evidence suggests that redox imbalance plays a significant role in progression of several neurodegenerative diseases, precise understanding of redox regulated events is lacking. Probes and methodologies that can precisely detect and quantify in vivo levels of reactive oxygen, nitrogen and sulfur species are not available. FUTURE DIRECTIONS: Due to the importance of redox control in physiologic processes, organisms have evolved multiple pathways to counteract redox imbalance and maintain homeostasis. Cells and tissues address stress by harnessing an array of both endogenous and exogenous redox active substances. Targeting these pathways can help mitigate symptoms associated with neurodegeneration and may provide avenues for novel therapeutics. Antioxid. Redox Signal. 30, 1450-1499.

Regulators of the transsulfuration pathway.

The transsulfuration pathway is a metabolic pathway where transfer of sulfur from homocysteine to cysteine occurs. The pathway leads to the generation of several sulfur metabolites, which include cysteine, GSH and the gaseous signalling molecule hydrogen sulfide (H2 S). Precise control of this pathway is critical for maintenance of optimal cellular function and, therefore, the key enzymes of the pathway, cystathionine ß-synthase and cystathionine γ-lyase, are regulated at multiple levels. Disruption of the transsulfuration pathway contributes to the pathology of several conditions such as vascular dysfunction, Huntington's disease and during ageing. Treatment with donors of hydrogen sulfide and/or stimulation of this pathway have proved beneficial in several of these disorders. In this review, we focus on the regulation of the transsulfuration pathway pertaining to cysteine and H2 S, which could be targeted to develop novel therapeutics. LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

Cysteine Metabolism in Neuronal Redox Homeostasis.

Besides its essential role in protein synthesis, cysteine plays vital roles in redox homeostasis, being a component of the major antioxidant glutathione (GSH) and a potent antioxidant by itself. In addition, cysteine undergoes a variety of post-translational modifications that modulate several physiological processes. It is becoming increasingly clear that redox-modulated events play important roles not only in peripheral tissues but also in the brain where cysteine disposition is central to these pathways. Dysregulated cysteine metabolism is associated with several neurodegenerative disorders. Accordingly, restoration of cysteine balance has therapeutic benefits. This review discusses metabolic signaling pathways pertaining to cysteine disposition in the brain under normal and pathological conditions, highlighting recent findings on cysteine metabolism during aging and in neurodegenerative conditions such as Huntington's disease (HD) and molybdenum cofactor (MoCo) deficiency (MoCD) among others.

Golgi stress response reprograms cysteine metabolism to confer cytoprotection in Huntington's disease.

Golgi stress response is emerging as a physiologic process of comparable importance to endoplasmic reticulum (ER) and mitochondrial stress responses. However, unlike ER stress, the identity of the signal transduction pathway involved in the Golgi stress response has been elusive. We show that the Golgi stressor monensin acts via the PKR-like ER kinase/Activating Transcription Factor 4 pathway. ATF4 is the master regulator of amino acid metabolism, which is induced during amino acid depletion and other forms of stress. One of the genes regulated by ATF4 is the biosynthetic enzyme for cysteine, cystathionine γ-lyase (CSE), which also plays central roles in maintenance of redox homeostasis. Huntington's disease (HD), a neurodegenerative disorder, is associated with disrupted cysteine metabolism caused by depletion of CSE leading to abnormal redox balance and stress response. Thus, restoring CSE function and cysteine disposition may be beneficial in HD. Accordingly, we harnessed the monensin-ATF4-signaling cascade to stimulate CSE expression by preconditioning cells with monensin, which restores cysteine metabolism and an optimal stress response in HD. These findings have implications for treatment of HD and other diseases associated with redox imbalance and dysregulated ATF4 signaling.

Transcriptional control of amino acid homeostasis is disrupted in Huntington's disease.

Disturbances in amino acid metabolism, which have been observed in Huntington's disease (HD), may account for the profound inanition of HD patients. HD is triggered by an expansion of polyglutamine repeats in the protein huntingtin (Htt), impacting diverse cellular processes, ranging from transcriptional regulation to cognitive and motor functions. We show here that the master regulator of amino acid homeostasis, activating transcription factor 4 (ATF4), is dysfunctional in HD because of oxidative stress contributed by aberrant cysteine biosynthesis and transport. Consistent with these observations, antioxidant supplementation reverses the disordered ATF4 response to nutrient stress. Our findings establish a molecular link between amino acid disposition and oxidative stress leading to cytotoxicity. This signaling cascade may be relevant to other diseases involving redox imbalance and deficits in amino acid metabolism.

Allele-specific regulation of mutant Huntingtin by Wig1, a downstream target of p53.

p53 has been implicated in the pathophysiology of Huntington's disease (HD). Nonetheless, the molecular mechanism of how p53 may play a unique role in the pathology remains elusive. To address this question at the molecular and cellular biology levels, we initially screened differentially expressed molecules specifically dependent on p53 in a HD animal model. Among the candidate molecules, wild-type p53-induced gene 1 (Wig1) is markedly upregulated in the cerebral cortex of HD patients. Wig1 preferentially upregulates the level of mutant Huntingtin (Htt) compared with wild-type Htt. This allele-specific characteristic of Wig1 is likely to be explained by higher affinity binding to mutant Htt transcripts than normal counterpart for the stabilization. Knockdown of Wig1 level significantly ameliorates mutant Htt-elicited cytotoxicity and aggregate formation. Together, we propose that Wig1, a key p53 downstream molecule in HD condition, play an important role in stabilizing mutant Htt mRNA and thereby accelerating HD pathology in the mHtt-p53-Wig1 positive feedback manner.

Huntington's disease: Neural dysfunction linked to inositol polyphosphate multikinase.

Huntington's disease (HD) is a progressive neurodegenerative disease caused by a glutamine repeat expansion in mutant huntingtin (mHtt). Despite the known genetic cause of HD, the pathophysiology of this disease remains to be elucidated. Inositol polyphosphate multikinase (IPMK) is an enzyme that displays soluble inositol phosphate kinase activity, lipid kinase activity, and various noncatalytic interactions. We report a severe loss of IPMK in the striatum of HD patients and in several cellular and animal models of the disease. This depletion reflects mHtt-induced impairment of COUP-TF-interacting protein 2 (Ctip2), a striatal-enriched transcription factor for IPMK, as well as alterations in IPMK protein stability. IPMK overexpression reverses the metabolic activity deficit in a cell model of HD. IPMK depletion appears to mediate neural dysfunction, because intrastriatal delivery of IPMK abates the progression of motor abnormalities and rescues striatal pathology in transgenic murine models of HD.

Cystathionine γ-lyase deficiency mediates neurodegeneration in Huntington's disease.

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.

Golgi protein ACBD3 mediates neurotoxicity associated with Huntington's disease.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disease caused by the expansion of polyglutamine repeats in the gene for huntingtin (Htt). In HD, the corpus striatum selectively degenerates despite the uniform expression of mutant huntingtin (mHtt) throughout the brain and body. Striatal selectivity reflects the binding of the striatal-selective protein Rhes to mHtt to augment cytotoxicity, but molecular mechanisms underlying the toxicity have been elusive. Here, we report that the Golgi protein acyl-CoA binding domain containing 3 (ACBD3) mediates mHtt cytotoxicity via a Rhes/mHtt/ACBD3 complex. ACBD3 levels are markedly elevated in the striatum of HD patients, in a striatal cell line harboring polyglutamine repeats, and in the brains of HD mice. Moreover, ACBD3 deletion abolishes HD neurotoxicity, which is increased by ACBD3 overexpression. Enhanced levels of ACBD3 elicited by endoplasmic reticulum, mitochondrial, and Golgi stresses may account for HD-associated augmentation of ACBD3 and neurodegeneration.

Identification of a redox-sensitive cysteine in GCP60 that regulates its interaction with golgin-160.

Golgin-160 is ubiquitously expressed in vertebrates. It localizes to the cytoplasmic side of the Golgi and has a large C-terminal coiled-coil domain. The noncoiled-coil N-terminal head domain contains Golgi targeting information, a cryptic nuclear localization signal, and three caspase cleavage sites. Caspase cleavage of the golgin-160 head domain generates different fragments that can translocate to the nucleus by exposing the nuclear localization signal. We have previously shown that GCP60, a Golgi resident protein, interacts weakly with the golgin-160 head domain but has a strong interaction with one of the caspase-generated golgin-160 fragments (residues 140-311). This preferential interaction increases the Golgi retention of the golgin-160 fragment in cells overexpressing GCP60. Here we studied the interaction of golgin-160-(140-311) with GCP60 and identified a single cysteine residue in GCP60 (Cys-463) that is critical for the interaction of the two proteins. Mutation of the cysteine blocked the interaction in vitro and disrupted the ability to retain the golgin-160 fragment at the Golgi in cells. We also found that Cys-463 is redox-sensitive; in its reduced form, interaction with golgin-160 was diminished or abolished, whereas oxidation of the Cys-463 by hydrogen peroxide restored the interaction. In addition, incubation with a nitric oxide donor promoted this interaction in vitro. These findings suggest that nuclear translocation of golgin-160-(140-311) is a highly coordinated event regulated not only by cleavage of the golgin-160 head but also by the oxidation state of GCP60.

Insulin-stimulated exocytosis of GLUT4 is enhanced by IRAP and its partner tankyrase.

The glucose transporter GLUT4 and the aminopeptidase IRAP (insulin-responsive aminopeptidase) are the major cargo proteins of GSVs (GLUT4 storage vesicles) in adipocytes and myocytes. In the basal state, most GSVs are sequestered in perinuclear and other cytosolic compartments. Following insulin stimulation, GSVs undergo exocytic translocation to insert GLUT4 and IRAP into the plasma membrane. The mechanisms regulating GSV trafficking are not fully defined. In the present study, using 3T3-L1 adipocytes transfected with siRNAs (small interfering RNAs), we show that insulin-stimulated IRAP translocation remained intact despite substantial GLUT4 knockdown. By contrast, insulin-stimulated GLUT4 translocation was impaired upon IRAP knockdown, indicating that IRAP plays a role in GSV trafficking. We also show that knockdown of tankyrase, a Golgi-associated IRAP-binding protein that co-localizes with perinuclear GSVs, attenuated insulin-stimulated GSV translocation and glucose uptake without disrupting insulin-induced phosphorylation cascades. Moreover, iodixanol density gradient analyses revealed that tankyrase knockdown altered the basal-state partitioning of GLUT4 and IRAP within endosomal compartments, apparently by shifting both proteins toward less buoyant compartments. Importantly, the afore-mentioned effects of tankyrase knockdown were reproduced by treating adipocytes with PJ34, a general PARP (poly-ADP-ribose polymerase) inhibitor that abrogated tankyrase-mediated protein modification known as poly-ADP-ribosylation. Collectively, these findings suggest that physiological GSV trafficking depends in part on the presence of IRAP in these vesicles, and that this process is regulated by tankyrase and probably its PARP activity.

Mitotic phosphorylation of tankyrase, a PARP that promotes spindle assembly, by GSK3.

The assembly and function of mitotic spindles require poly(ADP-ribosyl)ation of spindle components by tankyrase, a poly(ADP-ribose) polymerase that aggregates to spindle poles during mitosis. Tankyrase itself is phosphorylated during mitosis, but the kinases involved remain undefined. Herein we report that mitotic phosphorylation of tankyrase is abrogated in cells treated with the GSK3 inhibitors LiCl and indirubin. Moreover, the electrophoretic mobility-shift of tankyrase arising from mitotic phosphorylation can be reproduced in vitro by GSK3-mediated phosphorylation. Lastly, mutagenesis study suggested that GSK3 in vitro phosphorylates tankyrase on S978, T982, S987, and S991, residues that comprise two adjacent copies of the canonical GSK3 phospho-acceptor motif [S/T]-X-X-X-[S/T]. Collectively, our data suggest that GSK3 contributes to mitotic tankyrase phosphorylation, raising the possibility that this phosphorylation might mediate some of the established roles of GSK3 in spindle assembly and mitotic progression.

GCP60 preferentially interacts with a caspase-generated golgin-160 fragment.

Golgin-160, a ubiquitous protein in vertebrates, localizes to the cytoplasmic face of the Golgi complex. Golgin-160 has a large coiled-coil C-terminal domain and a non-coiled-coil N-terminal ("head") domain. The head domain contains important motifs, including a nuclear localization signal, a Golgi targeting domain, and three aspartates that are recognized by caspases during apoptosis. Some of the caspase cleavage products accumulate in the nucleus when overexpressed. Expression of a non-cleavable form of golgin-160 impairs apoptosis induced by some pro-apoptotic stimuli; thus cleavage of golgin-160 appears to play a role in apoptotic signaling. We used a yeast two-hybrid assay to screen for interactors of the golgin-160 head and identified GCP60 (Golgi complex-associated protein of 60 kDa). Further analysis demonstrated that GCP60 interacts preferentially with one of the golgin-160 caspase cleavage fragments (residues 140-311). This strong interaction prevented the golgin-160 fragment from accumulating in the nucleus when this fragment and GCP60 were overexpressed. In addition, cells overexpressing GCP60 were more sensitive to apoptosis induced by staurosporine, suggesting that nuclear-localized golgin-160-(140-311) might promote cell survival. Our results suggest a potential mechanism for regulating the nuclear translocation and potential functions of golgin-160 fragments.

Tankyrase-1 overexpression reduces genotoxin-induced cell death by inhibiting PARP1.

Poly(ADP-ribose) polymerases or PARPs are a family of NAD(+)-dependent enzymes that modify themselves and other substrate proteins with ADP-ribose polymers. The founding member PARP 1 is localized predominantly in the nucleus and is activated by binding to DNA lesions. Excessive PARP 1 activation following genotoxin treatment causes NAD(+) depletion and cell death, whereas pharmacological PARP 1 inhibition protects cells from genotoxicity. This study investigates whether cellular viability and NAD(+) metabolism are regulated by tankyrase-1, a PARP member localized predominantly in the cytosol. Using a tetracycline-sensitive promoter to regulate tankyrase-1 expression in Madin-Darby canine kidney (MDCK) cells, we found that a 40-fold induction of tankyrase-1 (from 1,500 to 60,000 copies per cell) lowers steady-state NAD(+) levels but does not affect basal cellular viability. Moreover, the induction confers protection against the oxidative agent H(2)O(2) and the alkylating agent MNNG, genotoxins that kill cells by activating PARP 1. The cytoprotective effect of tankyrase-1 is not due to enhanced scavenging of oxidants or altered expression of Mcl-1, an anti-apoptotic molecule previously shown to be down-regulated by tankyrase-1 in CHO cells. Instead, tankyrase-1 appears to protect cells by preventing genotoxins from activating PARP 1-mediated reactions such as PARP 1 automodification and NAD(+) consumption. Our findings therefore indicate a cytoprotective function of tankyrase-1 mediated through altered NAD(+) homeostasis and inhibition of PARP 1 function.

JNK and tumor necrosis factor-alpha mediate free fatty acid-induced insulin resistance in 3T3-L1 adipocytes.

Lipid infusion and high fat feeding are established causes of systemic and adipose tissue insulin resistance. In this study, we treated 3T3-L1 adipocytes with a mixture of free fatty acids (FFAs) to investigate the molecular mechanisms underlying fat-induced insulin resistance. FFA treatment impaired insulin receptor-mediated signal transduction and decreased insulin-stimulated GLUT4 translocation and glucose transport. FFAs activated the stress/inflammatory kinases c-Jun N-terminal kinase (JNK) and IKKbeta, and the suppressor of cytokine signaling protein 3, increased secretion of the inflammatory cytokine tumor necrosis factor (TNF)-alpha, and decreased secretion of adiponectin into the medium. RNA interference-mediated down-regulation of JNK blocked JNK activation and prevented most of the FFA-induced defects in insulin action. Blockade of TNF-alpha signaling with neutralizing antibodies to TNF-alpha or its receptors or with a dominant negative TNF-alpha peptide had a partial effect to inhibit FFA-induced cellular insulin resistance. We found that JNK activation by FFAs was not inhibited by blocking TNF-alpha signaling, whereas the FFA-induced increase in TNF-alpha secretion was inhibited by RNA interference-mediated JNK knockdown. Together, these results indicate that 1) JNK can be activated by FFAs through TNF-alpha-independent mechanisms, 2) activated JNK is a major contributor to FFA-induced cellular insulin resistance, and 3) TNF-alpha is an autocrine/paracrine downstream effector of activated JNK that can also mediate insulin resistance.

Inhibition of Epstein-Barr virus OriP function by tankyrase, a telomere-associated poly-ADP ribose polymerase that binds and modifies EBNA1.

Tankyrase (TNKS) is a telomere-associated poly-ADP ribose polymerase (PARP) that has been implicated along with several telomere repeat binding factors in the regulation of Epstein-Barr virus origin of plasmid replication (OriP). We now show that TNKS1 can bind to the family of repeats (FR) and dyad symmetry regions of OriP by using a chromatin immunoprecipitation assay and DNA affinity purification. TNKS1 and TNKS2 bound to EBNA1 in coimmunoprecipitation experiments with transfected cell lysates and with purified recombinant proteins in vitro. Two RXXPDG-like TNKS-interacting motifs in the EBNA1 amino-terminal domain mediated binding with the ankyrin repeat domain of TNKS. Mutations of both motifs at EBNA1 G81 and G425 abrogated TNKS binding and enhanced EBNA1-dependent replication of OriP. Small hairpin RNA targeted knock-down of TNKS1 enhanced OriP-dependent DNA replication. Overexpression of TNKS1 or TNKS2 inhibited OriP-dependent DNA replication, while a PARP-inactive form of TNKS2 (M1045V) was compromised for this inhibition. We show that EBNA1 is subject to PAR modification in vivo and to TNKS1-mediated PAR modification in vitro. These results indicate that TNKS proteins can interact directly with the EBNA1 protein, associate with the FR region of OriP in vivo, and inhibit OriP replication in a PARP-dependent manner.

Identification of a tankyrase-binding motif shared by IRAP, TAB182, and human TRF1 but not mouse TRF1. NuMA contains this RXXPDG motif and is a novel tankyrase partner.

Tankyrase-1 and -2 are closely related poly(ADP-ribose) polymerases that use an ankyrin-repeat domain to bind diverse proteins, including TRF (telomere-repeat binding factor)-1, IRAP (insulin-responsive aminopeptidase), and TAB182 (182-kDa tankyrase-binding protein). TRF1 binding allows tankyrase to regulate telomere dynamics in human cells, whereas IRAP binding presumably allows tankyrase to regulate the targeting of IRAP. The mechanism by which tankyrase binds to diverse proteins has not been investigated. Herein we describe a novel RXXPDG motif shared by IRAP, TAB182, and human TRF1 that mediates their binding to tankyrases. Interestingly, mouse TRF1 lacks this motif and thus does not bind either tankyrase-1 or -2. Using the ankyrin domain of tankyrase as a bait in a yeast two-hybrid screen, we also found the RXXPDG motif in six candidate tankyrase partners, including the nuclear/mitotic apparatus protein (NuMA). We verified NuMA as an RXXPDG-mediated partner of tankyrase and suggest that this interaction contributes to the known colocalization of tankyrase and NuMA at mitotic spindle poles.