Gursimran Chandhok

Wilson Disease Mutation Pattern with Genotype-Phenotype Correlations from Western India: Confirmation of p.C271* as a Common Indian Mutation and Identification of 14 Novel Mutations

Wilson disease (WD) is an autosomal recessive disorder resulting from mutations in the ATP7B gene, with over 600 mutations described. Identification of mutations has made genetic diagnosis of WD feasible in many countries. The heterogeneity of ATP7B mutants is, however, yet to be identified in the Indian population. We analyzed the mutational pattern of WD in a large region of Western India. We studied patients (n = 52) for ATP7B gene mutations in a cohort of families with WD and also in first‐degree relatives (n = 126). All 21 exon–intron boundaries of the WD gene were amplified and directly sequenced. We identified 36 different disease‐causing mutations (31 exonic and five intronic splice site variants). Fourteen novel mutations were identified. Exons 2, 8, 13, 14, and 18 accounted for the majority of mutations (86.4%). A previously recognized mutation, p.C271*, and the novel mutation p.E122fs, were the most common mutations with allelic frequencies of 20.2% and 10.6%, respectively. Frequent homozygous mutations (58.9%) and disease severity assessments allowed analysis of genotype–phenotype correlations. Our study significantly adds to the emerging data from other parts of India suggesting that p.C271* may be the most frequent mutation across India, and may harbor a moderate to severely disabling phenotype with limited variability.

The plant decapeptide OSIP108 prevents copper-induced apoptosis in yeast and human cells

We previously identified the Arabidopsis thaliana-derived decapeptide OSIP108, which increases tolerance of plants and yeast cells to oxidative stress. As excess copper (Cu) is known to induce oxidative stress and apoptosis, and is characteristic for the human pathology Wilson disease, we investigated the effect of OSIP108 on Cu-induced toxicity in yeast. We found that OSIP108 increased yeast viability in the presence of toxic Cu concentrations, and decreased the prevalence of Cu-induced apoptotic markers. Next, we translated these results to the human hepatoma HepG2 cell line, demonstrating anti-apoptotic activity of OSIP108 in this cell line. In addition, we found that OSIP108 did not affect intracellular Cu levels in HepG2 cells, but preserved HepG2 mitochondrial ultrastructure. As Cu is known to induce acid sphingomyelinase activity of HepG2 cells, we performed a sphingolipidomic analysis of OSIP108-treated HepG2 cells. We demonstrated that OSIP108 decreased the levels of several sphingoid bases and ceramide species. Moreover, exogenous addition of the sphingoid base dihydrosphingosine abolished the protective effect of OSIP108 against Cu-induced cell death in yeast. These findings indicate the potential of OSIP108 to prevent Cu-induced apoptosis, possibly via its effects on sphingolipid homeostasis.

Therapeutic Oligonucleotides Targeting Liver Disease: TTR Amyloidosis

The liver has become an increasingly interesting target for oligonucleotide therapy. Mutations of the gene encoding transthyretin (TTR), expressed in vast amounts by the liver, result in a complex degenerative disease, termed familial amyloid polyneuropathy (FAP). Misfolded variants of TTR are linked to the establishment of extracellular protein deposition in various tissues, including the heart and the peripheral nervous system. Recent progress in the chemistry and formulation of antisense (ASO) and small interfering RNA (siRNA) designed for a knockdown of TTR mRNA in the liver has allowed to address the issue of gene-specific molecular therapy in a clinical setting of FAP. The two therapeutic oligonucleotides bind to RNA in a sequence specific manner but exploit different mechanisms. Here we describe major developments that have led to the advent of therapeutic oligonucleotides for treatment of TTR-related disease.

The plant decapeptide OSIP108 prevents copper-induced toxicity in various models for Wilson disease.

BACKGROUND Wilson disease (WD) is caused by accumulation of excess copper (Cu) due to a mutation in the gene encoding the liver Cu transporter ATP7B, and is characterized by acute liver failure or cirrhosis and neuronal cell death. We investigated the effect of OSIP108, a plant derived decapeptide that prevents Cu-induced apoptosis in yeast and human cells, on Cu-induced toxicity in various mammalian in vitro models relevant for WD and in a Cu-toxicity zebrafish larvae model applicable to WD. METHODS The effect of OSIP108 was evaluated on viability of various cell lines in the presence of excess Cu, on liver morphology of a Cu-treated zebrafish larvae strain that expresses a fluorescent reporter in hepatocytes, and on oxidative stress levels in wild type AB zebrafish larvae. RESULTS OSIP108 increased not only viability of Cu-treated CHO cells transgenically expressing ATP7B and the common WD-causing mutant ATP7B(H1069Q), but also viability of Cu-treated human glioblastoma U87 cells. Aberrancies in liver morphology of Cu-treated zebrafish larvae were observed, which were further confirmed as Cu-induced hepatotoxicity by liver histology. Injections of OSIP108 into Cu-treated zebrafish larvae significantly increased the amount of larvae with normal liver morphology and decreased Cu-induced production of reactive oxygen species. CONCLUSIONS OSIP108 prevents Cu-induced toxicity in in vitro models and in a Cu-toxicity zebrafish larvae model applicable to WD. GENERAL SIGNIFICANCE All the above data indicate the potential of OSIP108 as a drug lead for further development as a novel WD treatment.

The Effect of Zinc and D-Penicillamine in a Stable Human Hepatoma ATP7B Knockout Cell Line

Mutations in the copper (Cu) transporter gene ATP7B, the primary cause of Wilson disease (WD), result in high liver Cu and death of hepatocytes. Cu chelators and zinc salts are the two most important drugs used in the treatment of WD patients; however, the molecular mechanisms of the drugs with regard to ATP7B expression have not been determined. A targeted knockout of ATP7B (KO) was established in the most widely used human hepatoma cell line, HepG2 for molecular studies of the pathogenesis and treatment of the disease. KO cells showed similar growth, Cu uptake, release, and gene expression as compared to parental cells. However, in the presence of Cu, morphological changes, oxidative stress, apoptosis, and loss of viability were observed. Induction of metallothionein (MT1X) after Cu exposure was significantly reduced in KO cells. Following zinc treatment, MT1X expression was strongly induced and a high percentage of KO cells could be rescued from Cu induced toxicity. D-penicillamine treatment had a minor effect on the viability of KO cells whereas the parental cell line showed a pronounced improvement. Combined treatment displayed a highly synergistic effect in KO cells. The data suggest that zinc has a previously unrecognized effect on the viability of hepatocytes that lack ATP7B due to a high induction of MT1X expression that compensates low gene expression after Cu exposure. A combination therapy that simultaneously targets at MT1X induction and Cu chelation improves the overall survival of hepatocytes for most efficient therapy of patients having WD.

Functional analysis and drug response to zinc and D-penicillamine in stable ATP7B mutant hepatic cell lines

AIM To study the effect of anti-copper treatment for survival of hepatic cells expressing different ATP7B mutations in cell culture. METHODS The most common Wilson disease (WD) mutations p.H1069Q, p.R778L and p.C271*, found in the ATP7B gene encoding a liver copper transporter, were studied. The mutations represent major genotypes of the United States and Europe, China, and India, respectively. A human hepatoma cell line previously established to carry a knockout of ATP7B was used to stably express WD mutants. mRNA and protein expression of mutant ATP7B, survival of cells, apoptosis, and protein trafficking were determined. RESULTS Low temperature increased ATP7B protein expression in several mutants. Intracellular ATP7B localization was significantly impaired in the mutants. Mutants were classified as high, moderate, and no survival based on their viability on exposure to toxic copper. Survival of mutant p.H1069Q and to a lesser extent p.C271* improved by D-penicillamine (DPA) treatment, while mutant p.R778L showed a pronounced response to zinc (Zn) treatment. Overall, DPA treatment resulted in higher cell survival as compared to Zn treatment; however, only combined Zn + DPA treatment fully restored cell viability. CONCLUSION The data indicate that the basic impact of a genotype might be characterized by analysis of mutant hepatic cell lines.

Evaluation of Therapeutic Oligonucleotides for Familial Amyloid Polyneuropathy in Patient-Derived Hepatocyte-Like Cells.

Familial amyloid polyneuropathy (FAP) is caused by mutations of the transthyretin (TTR) gene, predominantly expressed in the liver. Two compounds that knockdown TTR, comprising a small interfering RNA (siRNA; ALN-TTR-02) and an antisense oligonucleotide (ASO; IONIS-TTRRx), are currently being evaluated in clinical trials. Since primary hepatocytes from FAP patients are rarely available for molecular analysis and commercial tissue culture cells or animal models lack the patient-specific genetic background, this study uses primary cells derived from urine of FAP patients. Urine-derived cells were reprogrammed to induced pluripotent stem cells (iPSCs) with high efficiency. Hepatocyte-like cells (HLCs) showing typical hepatic marker expression were obtained from iPSCs of the FAP patients. TTR mRNA expression of FAP HLCs almost reached levels measured in human hepatocytes. To assess TTR knockdown, siTTR1 and TTR-ASO were introduced to HLCs. A significant downregulation (>80%) of TTR mRNA was induced in the HLCs by both oligonucleotides. TTR protein present in the cell culture supernatant of HLCs was similarly downregulated. Gene expression of other hepatic markers was not affected by the therapeutic oligonucleotides. Our data indicate that urine cells (UCs) after reprogramming and hepatic differentiation represent excellent primary human target cells to assess the efficacy and specificity of novel compounds.

Structure, function, and regulation of mitofusin‐2 in health and disease

Mitochondria are highly dynamic organelles that constantly migrate, fuse, and divide to regulate their shape, size, number, and bioenergetic function. Mitofusins (Mfn1/2), optic atrophy 1 (OPA1), and dynamin‐related protein 1 (Drp1), are key regulators of mitochondrial fusion and fission. Mutations in these molecules are associated with severe neurodegenerative and non‐neurological diseases pointing to the importance of functional mitochondrial dynamics in normal cell physiology. In recent years, significant progress has been made in our understanding of mitochondrial dynamics, which has raised interest in defining the physiological roles of key regulators of fusion and fission and led to the identification of additional functions of Mfn2 in mitochondrial metabolism, cell signalling, and apoptosis. In this review, we summarize the current knowledge of the structural and functional properties of Mfn2 as well as its regulation in different tissues, and also discuss the consequences of aberrant Mfn2 expression.

Organic cation transporter 3 mediates cisplatin and copper cross-resistance in hepatoma cells

Platinum-based drugs are first-line compounds in the treatment of many solid cancers. Major obstacles are tumors that become resistant and toxic side effects, both largely due to the expression of transporters that mediate the cellular processing of platinum. In this study, we addressed the establishment of cisplatin resistance in the absence of copper transporter ATP7B that has been previously found to be overexpressed in various resistant cells. Cisplatin sensitivity, induction of apoptosis, drug accumulation, and transporter gene expression were determined in hepatoma cell lines. Knockout or overexpression of copper transporter ATP7B did not affect cisplatin sensitivity. Cisplatin resistant cells showed a stably reduced cisplatin accumulation and a downregulation of organic cation transporter 3 (OCT3). In contrast, OCT3 overexpression could reverse resistance. Reduced MT1 expression was detected in the resistant cell line, however transient and highly dependent on the presence of cisplatin. Cross-resistance to copper was also associated with OCT3 downregulation. Our results suggest that a decreased level of OCT3 expression results in resistance to cisplatin and copper. OCT3 may represent a novel target for improved prognosis and anticancer therapy, including HCC.

Identification of survival-promoting OSIP108 peptide variants and their internalization in human cells.

The plant-derived decapeptide OSIP108 increases tolerance of yeast and human cells to apoptosis-inducing agents, such as copper and cisplatin. We performed a whole amino acid scan of OSIP108 and conducted structure-activity relationship studies on the induction of cisplatin tolerance (CT) in yeast. The use of cisplatin as apoptosis-inducing trigger in this study should be considered as a tool to better understand the survival-promoting nature of OSIP108 and not for purposes related to anti-cancer treatment. We found that charged residues (Arg, His, Lys, Glu or Asp) or a Pro on positions 4-7 improved OSIP108 activity by 10% or more. The variant OSIP108[G7P] induced the most pronounced tolerance to toxic concentrations of copper and cisplatin in yeast and/or HepG2 cells. Both OSIP108 and OSIP108[G7P] were shown to internalize equally into HeLa cells, but at a higher rate than the inactive OSIP108[E10A], suggesting that the peptides can internalize into cells and that OSIP108 activity is dependent on subsequent intracellular interactions. In conclusion, our studies demonstrated that tolerance/survival-promoting properties of OSIP108 can be significantly improved by single amino acid substitutions, and that these properties are dependent on (an) intracellular target(s), yet to be determined.