A new Caenorhabditis elegans model to study copper toxicity in Wilson disease.

Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a copper (Cu) transporting ATPase whose trafficking from the Golgi to endo-lysosomal compartments drives sequestration of excess Cu and its further excretion from hepatocytes into the bile. Loss of ATP7B function leads to toxic Cu overload in the liver and subsequently in the brain, causing fatal hepatic and neurological abnormalities. The limitations of existing WD therapies call for the development of new therapeutic approaches, which require an amenable animal model system for screening and validation of drugs and molecular targets. To achieve this objective, we generated a mutant Caenorhabditis elegans strain with a substitution of a conserved histidine (H828Q) in the ATP7B ortholog cua-1 corresponding to the most common ATP7B variant (H1069Q) that causes WD. cua-1 mutant animals exhibited very poor resistance to Cu compared to the wild-type strain. This manifested in a strong delay in larval development, a shorter lifespan, impaired motility, oxidative stress pathway activation, and mitochondrial damage. In addition, morphological analysis revealed several neuronal abnormalities in cua-1 mutant animals exposed to Cu. Further investigation suggested that mutant CUA-1 is retained and degraded in the endoplasmic reticulum, similarly to human ATP7B-H1069Q. As a consequence, the mutant protein does not allow animals to counteract Cu toxicity. Notably, pharmacological correctors of ATP7B-H1069Q reduced Cu toxicity in cua-1 mutants indicating that similar pathogenic molecular pathways might be activated by the H/Q substitution and, therefore, targeted for rescue of ATP7B/CUA-1 function. Taken together, our findings suggest that the newly generated cua-1 mutant strain represents an excellent model for Cu toxicity studies in WD.

Effect of post-printing curing time on cytotoxicity of direct printed aligners: A pilot study.

INTRODUCTION: The aim of this in vitro study was to examine the potential impact of different curing times of 3D-printed orthodontic aligners on their cytotoxicity. METHOD: Some 60 samples of aligner material were directly 3D printed using Tera Harz TC-85 DAC resin and randomly allocated to three different curing time groups (14, 24 and 50 min). Zendura FLX samples were used as control. The samples were incubated in saliva for 14 days, and then the supernatant was collected. Human gingival fibroblasts (HGF-1)-CRL2014 were used to evaluate potential cytotoxicity. Furthermore, HGF-1 cells were plated on the samples as well as on a glass control sample. After 72 h of growth, their viability was tested. RESULTS: Compared with the glass, only the 50-min curing time markedly reduced fibroblast cell growth. Additionally, a negative linear trend was observed between curing time and fibroblast growth. In comparison with the aligner control group, all samples, including the aligner control samples, exhibited a significant reduction in the viability of human fibroblasts when exposed to saliva. CONCLUSIONS: 3D directly printed aligners showed a cytotoxic effect similar to that of thermoformed conventional aligners in terms of fibroblasts growth. A linear trend was found between curing time and cells growth, indicating that directly printed aligners could exhibit higher cytotoxicity if exposed to a longer curing time. This dependence on curing time underscores the importance of following a strict manufacturing process.

Pharmacoproteomics pinpoints HSP70 interaction for correction of the most frequent Wilson disease-causing mutant of ATP7B.

Pathogenic mutations in the copper transporter ATP7B have been hypothesized to affect its protein interaction landscape contributing to loss of function and, thereby, to hepatic copper toxicosis in Wilson disease. Although targeting mutant interactomes was proposed as a therapeutic strategy, druggable interactors for rescue of ATP7B mutants remain elusive. Using proteomics, we found that the frequent H1069Q substitution promotes ATP7B interaction with HSP70, thus accelerating endoplasmic reticulum (ER) degradation of the mutant protein and consequent copper accumulation in hepatic cells. This prompted us to use an HSP70 inhibitor as bait in a bioinformatics search for structurally similar Food and Drug Administration-approved drugs. Among the hits, domperidone emerged as an effective corrector that recovered trafficking and function of ATP7B-H1069Q by impairing its exposure to the HSP70 proteostatic network. Our findings suggest that HSP70-mediated degradation can be safely targeted with domperidone to rescue ER-retained ATP7B mutants and, hence, to counter the onset of Wilson disease.

Activation of Autophagy, Observed in Liver Tissues From Patients With Wilson Disease and From ATP7B-Deficient Animals, Protects Hepatocytes From Copper-Induced Apoptosis.

BACKGROUND & AIMS: Wilson disease (WD) is an inherited disorder of copper metabolism that leads to copper accumulation and toxicity in the liver and brain. It is caused by mutations in the adenosine triphosphatase copper transporting ß gene (ATP7B), which encodes a protein that transports copper from hepatocytes into the bile. We studied ATP7B-deficient cells and animals to identify strategies to decrease copper toxicity in patients with WD. METHODS: We used RNA-seq to compare gene expression patterns between wild-type and ATP7B-knockout HepG2 cells exposed to copper. We collected blood and liver tissues from Atp7b-/- and Atp7b+/- (control) rats (LPP) and mice; some mice were given 5 daily injections of an autophagy inhibitor (spautin-1) or vehicle. We obtained liver biopsies from 2 patients with WD in Italy and liver tissues from patients without WD (control). Liver tissues were analyzed by immunohistochemistry, immunofluorescence, cell viability, apoptosis assays, and electron and confocal microscopy. Proteins were knocked down in cell lines using small interfering RNAs. Levels of copper were measured in cell lysates, blood samples, liver homogenates, and subcellular fractions by spectroscopy. RESULTS: After exposure to copper, ATP7B-knockout cells had significant increases in the expression of 103 genes that regulate autophagy (including MAP1LC3A, known as LC3) compared with wild-type cells. Electron and confocal microscopy visualized more autophagic structures in the cytoplasm of ATP7B-knockout cells than wild-type cells after copper exposure. Hepatocytes in liver tissues from patients with WD and from Atp7b-/- mice and rats (but not controls) had multiple autophagosomes. In ATP7B-knockout cells, mammalian target of rapamycin (mTOR) had decreased activity and was dissociated from lysosomes; this resulted in translocation of the mTOR substrate transcription factor EB to the nucleus and activation of autophagy-related genes. In wild-type HepG2 cells (but not ATP7B-knockout cells), exposure to copper and amino acids induced recruitment of mTOR to lysosomes. Pharmacologic inhibitors of autophagy or knockdown of autophagy proteins ATG7 and ATG13 induced and accelerated the death of ATP7B-knockout HepG2 cells compared with wild-type cells. Autophagy protected ATP7B-knockout cells from copper-induced death. CONCLUSION: ATP7B-deficient hepatocytes, such as in those in patients with WD, activate autophagy in response to copper overload to prevent copper-induced apoptosis. Agents designed to activate this autophagic pathway might decrease copper toxicity in patients with WD.

TFEB Regulates ATP7B Expression to Promote Platinum Chemoresistance in Human Ovarian Cancer Cells.

ATP7B is a hepato-specific Golgi-located ATPase, which plays a key role in the regulation of copper (Cu) homeostasis and signaling. In response to elevated Cu levels, ATP7B traffics from the Golgi to endo-lysosomal structures, where it sequesters excess copper and further promotes its excretion to the bile at the apical surface of hepatocytes. In addition to liver, high ATP7B expression has been reported in tumors with elevated resistance to platinum (Pt)-based chemotherapy. Chemoresistance to Pt drugs represents the current major obstacle for the treatment of large cohorts of cancer patients. Although the mechanisms underlying Pt-tolerance are still ambiguous, accumulating evidence suggests that lysosomal sequestration of Pt drugs by ion transporters (including ATP7B) might significantly contribute to drug resistance development. In this context, signaling mechanisms regulating the expression of transporters such as ATP7B are of great importance. Considering this notion, we investigated whether ATP7B expression in Pt-resistant cells might be driven by transcription factor EB (TFEB), a master regulator of lysosomal gene transcription. Using resistant ovarian cancer IGROV-CP20 cells, we found that TFEB directly binds to the predicted coordinated lysosomal expression and regulation (CLEAR) sites in the proximal promoter and first intron region of ATP7B upon Pt exposure. This binding accelerates transcription of luciferase reporters containing ATP7B CLEAR regions, while suppression of TFEB inhibits ATP7B expression and stimulates cisplatin toxicity in resistant cells. Thus, these data have uncovered a Pt-dependent transcriptional mechanism that contributes to cancer chemoresistance and might be further explored for therapeutic purposes.

Full-length ATP7B reconstituted through protein trans-splicing corrects Wilson disease in mice.

Wilson disease (WD) is a genetic disorder of copper homeostasis, caused by deficiency of the copper transporter ATP7B. Gene therapy with recombinant adeno-associated vectors (AAV) holds promises for WD treatment. However, the full-length human ATP7B gene exceeds the limited AAV cargo capacity, hampering the applicability of AAV in this disease context. To overcome this limitation, we designed a dual AAV vector approach using split intein technology. Split inteins catalyze seamless ligation of two separate polypeptides in a highly specific manner. We selected a DnaE intein from Nostoc punctiforme (Npu) that recognizes a specific tripeptide in the human ATP7B coding sequence. We generated two AAVs expressing either the 5'-half of a codon-optimized human ATP7B cDNA followed by the N-terminal Npu DnaE intein or the C-terminal Npu DnaE intein followed by the 3'-half of ATP7B cDNA, under the control of a liver-specific promoter. Intravenous co-injection of the two vectors in wild-type and Atp7b -/- mice resulted in efficient reconstitution of full-length ATP7B protein in the liver. Moreover, Atp7b -/- mice treated with intein-ATP7B vectors were protected from liver damage and showed improvements in copper homeostasis. Taken together, these data demonstrate the efficacy of split intein technology to drive the reconstitution of full-length human ATP7B and to rescue copper-mediated liver damage in Atp7b -/- mice, paving the way to the development of a new gene therapy approach for WD.

Infantile ATP7B-Related End-Stage Liver Disease: An Exceptional Wilson Disease Phenotype From Consecutive Generations.

Wilson disease (WD) is a rare autosomal recessive disorder of copper metabolism typically presenting after 3 years of age. We describe a girl presenting with neonatal cholestasis rapidly progressing to end-stage liver disease. She presented hepatosplenomegaly, neurological impairment, Coombs-negative hemolytic anemia, central hypothyroidism. A patient-parents whole exome sequencing identified a homozygous state for ATP7B mutations causing WD in the proband (p.Gln7fs/p.His1069Gln) and her mother (p.His1069Gln/p.His1069Gln), who was then confirmed to have cirrhotic WD. A causative role of copper toxicity due to ATP7B loss of function was indicated by the presence of extrahepatic features of WD, consistent tests of copper metabolism-including a 7-fold increase in liver copper-and similarity of patient's liver gene expression profile and ultrastructure with that of WD models. This exceptionally early presentation could result from the combination of the ATP7B impairment and the intrauterine copper overload due to maternal undiagnosed WD.

Role of the cold shock domain protein A in the transcriptional regulation of HBG expression.

Impaired switching from fetal haemoglobin (HbF) to adult globin gene expression leads to hereditary persistence of fetal haemoglobin (HPFH) in adult life. This is of prime interest because elevated HbF levels ameliorate ß-thalassaemia and sickle cell anaemia. Fetal haemoglobin levels are regulated by complex mechanisms involving factors linked or not to the ß-globin gene (HBB) locus. To search for factors putatively involved in the expression of the γ-globin genes (HBG1, HBG2), we examined the reticulocyte transcriptome of three siblings who had different HbF levels and different degrees of ß-thalassaemia severity although they had the same ΗBA- and ΗΒB cluster genotypes. By mRNA differential display we isolated the cDNA coding for the cold shock domain protein A (CSDA), also known as dbpA, previously reported to interact in vitro with the HBG2 promoter. Expression studies performed in K562 and in primary erythroid cells showed an inverse relationship between HBG and CSDA expression levels. Functional studies performed by Chromatin Immunoprecipitation and reporter gene assays in K562 cells demonstrated that CSDA is able to bind the HBG2 promoter and suppress its expression. Therefore, our study demonstrated that CSDA is a trans-acting repressor factor of HBG expression and modulates the HPFH phenotype.

Synthetic Lethality Screening Identifies FDA-Approved Drugs that Overcome ATP7B-Mediated Tolerance of Tumor Cells to Cisplatin.

Tumor resistance to chemotherapy represents an important challenge in modern oncology. Although platinum (Pt)-based drugs have demonstrated excellent therapeutic potential, their effectiveness in a wide range of tumors is limited by the development of resistance mechanisms. One of these mechanisms includes increased cisplatin sequestration/efflux by the copper-transporting ATPase, ATP7B. However, targeting ATP7B to reduce Pt tolerance in tumors could represent a serious risk because suppression of ATP7B might compromise copper homeostasis, as happens in Wilson disease. To circumvent ATP7B-mediated Pt tolerance we employed a high-throughput screen (HTS) of an FDA/EMA-approved drug library to detect safe therapeutic molecules that promote cisplatin toxicity in the IGROV-CP20 ovarian carcinoma cells, whose resistance significantly relies on ATP7B. Using a synthetic lethality approach, we identified and validated three hits (Tranilast, Telmisartan, and Amphotericin B) that reduced cisplatin resistance. All three drugs induced Pt-mediated DNA damage and inhibited either expression or trafficking of ATP7B in a tumor-specific manner. Global transcriptome analyses showed that Tranilast and Amphotericin B affect expression of genes operating in several pathways that confer tolerance to cisplatin. In the case of Tranilast, these comprised key Pt-transporting proteins, including ATOX1, whose suppression affected ability of ATP7B to traffic in response to cisplatin. In summary, our findings reveal Tranilast, Telmisartan, and Amphotericin B as effective drugs that selectively promote cisplatin toxicity in Pt-resistant ovarian cancer cells and underscore the efficiency of HTS strategy for identification of biosafe compounds, which might be rapidly repurposed to overcome resistance of tumors to Pt-based chemotherapy.

Activity and Trafficking of Copper-Transporting ATPases in Tumor Development and Defense against Platinum-Based Drugs.

Membrane trafficking pathways emanating from the Golgi regulate a wide range of cellular processes. One of these is the maintenance of copper (Cu) homeostasis operated by the Golgi-localized Cu-transporting ATPases ATP7A and ATP7B. At the Golgi, these proteins supply Cu to newly synthesized enzymes which use this metal as a cofactor to catalyze a number of vitally important biochemical reactions. However, in response to elevated Cu, the Golgi exports ATP7A/B to post-Golgi sites where they promote sequestration and efflux of excess Cu to limit its potential toxicity. Growing tumors actively consume Cu and employ ATP7A/B to regulate the availability of this metal for oncogenic enzymes such as LOX and LOX-like proteins, which confer higher invasiveness to malignant cells. Furthermore, ATP7A/B activity and trafficking allow tumor cells to detoxify platinum (Pt)-based drugs (like cisplatin), which are used for the chemotherapy of different solid tumors. Despite these noted activities of ATP7A/B that favor oncogenic processes, the mechanisms that regulate the expression and trafficking of Cu ATPases in malignant cells are far from being completely understood. This review summarizes current data on the role of ATP7A/B in the regulation of Cu and Pt metabolism in malignant cells and outlines questions and challenges that should be addressed to understand how ATP7A and ATP7B trafficking mechanisms might be targeted to counteract tumor development.

Allele-specific transcriptional activity of the variable number of tandem repeats of the inducible nitric oxide synthase gene is associated with idiopathic achalasia.

BACKGROUND: Polymorphisms of genes involved in the regulation of the immune response are risk factors for achalasia, but their contribution to disease pathogenesis is unknown. Nitric oxide is involved both in immune function and inhibitory neurotransmission. OBJECTIVE: The objective of this article is to assess the association and the functional relevance of the CCTTT-inducible nitric oxide synthase (NOS2) gene promoter polymorphism in achalasia. METHODS: Genomic DNA was isolated from 181 achalasia patients and 220 controls. Genotyping of the (CCTTT)n repeats was performed by PCR and capillary electrophoresis, and data analyzed by considering the frequency of the different alleles. HT29 cells were transfected with iNOS luciferase promoter-reporter plasmids containing different (CCTTT)n. RESULTS: The alleles' distribution ranged from 7 to 18, with a peak frequency at 12 repeats. Analysis of the allele frequencies revealed that individuals carrying 10 and 13 CCTTT repeats were respectively less and more frequent in achalasia (OR 0.5, 95% CI 0.3-0.5 and OR 1.6, 95% CI 1-2.4, all p < 0.05). Long repeats were also significantly associated with an earlier onset of the disease (OR 1.69, 95% CI 1.13-2.53, p = 0.01). Transfection experiments revealed a similar allele-specific iNOS transcriptional activity. CONCLUSION: The functional polymorphism (CCTTT) of NOS2 promoter is associated with achalasia, likely by an allele-specific modulation of nitric oxide production.

HIF-2α regulates NANOG expression in human embryonic stem cells following hypoxia and reoxygenation through the interaction with an Oct-Sox cis regulatory element.

Low O2 tension is beneficial for human embryonic stem cell (hESC) maintenance but the mechanism of regulation is unknown. HIF-2α was found to bind directly to predicted hypoxic response elements (HREs) in the proximal promoter of OCT4, NANOG and SOX2 only in hESCs cultured under hypoxia (5% O2). This binding induced an array of histone modifications associated with gene transcription while a heterochromatic state existed at atmospheric O2. Interestingly, an enhanced euchromatic state was found when hESCs were exposed to hypoxia followed by 72 hours reoxygenation. This was sustained by HIF-2α which enhanced stemness by binding to an oct-sox cis-regulatory element in the NANOG promoter. Thus, these data have uncovered a novel role of HIF-2α as a direct regulator of key transcription factors controlling self-renewal in hESCs but also in the induction of epigenetic modifications ensuring a euchromatic conformation which enhances the regenerative potential of these cells.

Environmental oxygen tension regulates the energy metabolism and self-renewal of human embryonic stem cells.

Energy metabolism is intrinsic to cell viability but surprisingly has been little studied in human embryonic stem cells (hESCs). The current study aims to investigate the effect of environmental O2 tension on carbohydrate utilisation of hESCs. Highly pluripotent hESCs cultured at 5% O2 consumed significantly more glucose, less pyruvate and produced more lactate compared to those maintained at 20% O2. Moreover, hESCs cultured at atmospheric O2 levels expressed significantly less OCT4, SOX2 and NANOG than those maintained at 5% O2. To determine whether this difference in metabolism was a reflection of the pluripotent state, hESCs were cultured at 5% O2 in the absence of FGF2 for 16 hours leading to a significant reduction in the expression of SOX2. In addition, these cells consumed less glucose and produced significantly less lactate compared to those cultured in the presence of FGF2. hESCs maintained at 5% O2 were found to consume significantly less O2 than those cultured in the absence of FGF2, or at 20% O2. GLUT1 expression correlated with glucose consumption and using siRNA and chromatin immunoprecipitation was found to be directly regulated by hypoxia inducible factor (HIF)-2α at 5% O2. In conclusion, highly pluripotent cells associated with hypoxic culture consume low levels of O2, high levels of glucose and produce large amounts of lactate, while at atmospheric conditions glucose consumption and lactate production are reduced and there is an increase in oxidative metabolism. These data suggest that environmental O2 regulates energy metabolism and is intrinsic to the self-renewal of hESCs.

Enteric glial-derived S100B protein stimulates nitric oxide production in celiac disease.

BACKGROUND & AIMS: Enteric glia participates to the homeostasis of the gastrointestinal tract. In the central nervous system, increased expression of astroglial-derived S100B protein has been associated with the onset and maintaining of inflammation. The role of enteric glial-derived S100B protein in gastrointestinal inflammation has never been investigated in humans. In this study, we evaluated the expression of S100B and its relationship with nitric oxide production in celiac disease. METHODS: Duodenal biopsy specimens from untreated and on gluten-free diet patients with celiac disease and controls were respectively processed for S100B and inducible nitric oxide synthase (iNOS) protein expression and nitrite production. To evaluate the direct involvement of S100B in the inflammation, control biopsy specimens were exposed to exogenous S100B, and iNOS protein expression and nitrite production were measured. We also tested gliadin induction of S100B-dependent inflammation in cultured biopsy specimens deriving from on gluten-free diet patients in the absence or presence of the specific S100B antibody. RESULTS: S100B messenger RNA and protein expression, iNOS protein expression, and nitrite production were significantly increased in untreated patients but not in on gluten-free diet patients vs controls. Addition of S100B to control biopsy specimens resulted in a significant increase of iNOS protein expression and nitrite production. In celiac disease patients but not in controls biopsy specimens, gliadin challenge significantly increased S100B messenger RNA and protein expression, iNOS protein expression, and nitrite production, but these effects were completely inhibited by S100B antibody. CONCLUSIONS: Enteric glial-derived S100B is increased in the duodenum of patients with celiac disease and plays a role in nitric oxide production.