Claire F. Jessup

A Syntenic Cross Species Aneuploidy Genetic Screen Links RCAN1 Expression to β-Cell Mitochondrial Dysfunction in Type 2 Diabetes

Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic β-cell dysfunction. Reduced mitochondrial function is thought to be central to β-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in β-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis we profiled the glycaemic status of different DS mouse models. The Ts65Dn and Dp16 DS mouse lines were hyperglycemic, while Tc1 and Ts1Rhr mice were not, providing us with a region of chromosome 21 containing genes that cause hyperglycemia. We then examined whether any of these genes were upregulated in a set of ~5,000 gene expression changes we had identified in a large gene expression analysis of human T2D β-cells. This approach produced a single gene, RCAN1, as a candidate gene linking hyperglycemia and functional changes in T2D β-cells. Further investigations demonstrated that RCAN1 methylation is reduced in human T2D islets at multiple sites, correlating with increased expression. RCAN1 protein expression was also increased in db/db mouse islets and in human and mouse islets exposed to high glucose. Mice overexpressing RCAN1 had reduced in vivo glucose-stimulated insulin secretion and their β-cells displayed mitochondrial dysfunction including hyperpolarised membrane potential, reduced oxidative phosphorylation and low ATP production. This lack of β-cell ATP had functional consequences by negatively affecting both glucose-stimulated membrane depolarisation and ATP-dependent insulin granule exocytosis. Thus, from amongst the myriad of gene expression changes occurring in T2D β-cells where we had little knowledge of which changes cause β-cell dysfunction, we applied a trisomy 21 screening approach which linked RCAN1 to β-cell mitochondrial dysfunction in T2D.

Lentivirus-mediated gene transfer to the rat, ovine and human cornea

Gene therapy of the cornea shows promise for modulating corneal transplant rejection but the most appropriate vector for gene transfer has yet to be determined. We investigated a lentiviral vector (LV) for its ability to transduce corneal endothelium. A lentivector expressing enhanced yellow fluorescent protein (eYFP) under the control of the Simian virus type 40 early promoter (LV-SV40-eYFP) transduced 80–90% of rat, ovine and human corneal endothelial cells as detected by fluorescence microscopy. The kinetics of gene expression varied among species, with ovine corneal endothelium showing a relative delay in detectable reporter gene expression compared with the rat or human corneal endothelium. Vectors containing the myeloproliferative sarcoma virus promoter or the phosphoglycerate kinase promoter were not significantly more effective than LV-SV40-eYFP. The stability of eYFP expression in rat and ovine corneas following ex vivo transduction of the donor cornea was assessed following orthotopic corneal transplantation. Following transduction ex vivo, eYFP expression was maintained in corneal endothelial cells for at least 28 days after corneal transplantation in the sheep and >60 days in the rat. Thus, rat, ovine and human corneal endothelial cells were efficiently transduced by the LV, and gene expression appeared stable over weeks in vivo.

The β-Cell/EC Axis: How Do Islet Cells Talk to Each Other?

Within the pancreatic islet, the β-cell represents the ultimate biosensor. Its central function is to accurately sense glucose levels in the blood and consequently release appropriate amounts of insulin. As the only cell type capable of insulin production, the β-cell must balance this crucial workload with self-preservation and, when required, regeneration. Evidence suggests that the β-cell has an important ally in intraislet endothelial cells (ECs). As well as providing a conduit for delivery of the primary input stimulus (glucose) and dissemination of its most important effector (insulin), intraislet blood vessels deliver oxygen to these dense clusters of metabolically active cells. Furthermore, it appears that ECs directly impact insulin gene expression and secretion and β-cell survival. This review discusses the molecules and pathways involved in the crosstalk between β-cells and intraislet ECs. The evidence supporting the intraislet EC as an important partner for β-cell function is examined to highlight the relevance of this axis in the context of type 1 and type 2 diabetes. Recent work that has established the potential of ECs or their progenitors to enhance the re-establishment of glycemic control following pancreatic islet transplantation in animal models is discussed.

Gene therapy approaches to prolonging corneal allograft survival

Irreversible immunological rejection is the major cause of human corneal allograft failure and occurs despite the use of topical glucocorticoid immunosuppression. Systemic pharmacological interventions have not found widespread favour in corneal transplantation because of associated morbidities and inadequate demonstration of efficacy. Gene therapy offers tantalising prospects for improving corneal allograft survival, especially in those recipients at high risk of graft rejection. Donor corneas can be gene-modified ex vivo, while in storage prior to implantation, and the relative isolation of the transplanted cornea from the circulation decreases the risk of potential systemic complications. A wide variety of vectors have been found suitable for gene transfer to the cornea. The mechanisms involved in corneal graft rejection have been placed on a relatively secure footing over the past decade and in consequence a number of transgenes with promise for modulating rejection have been identified. However, relatively few studies have thus far demonstrated significant prolongation of corneal allograft survival after gene transfer to the donor cornea. In these instances, the therapeutic protein almost certainly acted at a proximal level in the afferent immune response, within the ocular environs.

Increased Expression of the Glucose-Responsive Gene, RCAN1, Causes Hypoinsulinemia, β-Cell Dysfunction, and Diabetes

RCAN1 is a chromosome 21 gene that controls secretion in endocrine cells, regulates mitochondrial function, and is sensitive to oxidative stress. Regulator of calcineurin 1 (RCAN1) is also an endogenous inhibitor of the protein phosphatase calcineurin, the inhibition of which leads to hypoinsulinemia and diabetes in humans and mice. However, the presence or the role of RCAN1 in insulin-secreting β-cells and its potential role in the pathogenesis of diabetes is unknown. Hence, the aim of this study is to investigate the presence of RCAN1 in β-cells and identify its role in β-cell function. RCAN1 is expressed in mouse islets and in the cytosol of pancreatic β-cells. We find RCAN1 is a glucose-responsive gene with a 1.5-fold increase in expression observed in pancreatic islets in response to chronic hyperglycemia. The overexpression of the human RCAN1.1 isoform in mice under the regulation of its endogenous promoter causes diabetes, age-associated hyperglycemia, reduced glucose tolerance, hypoinsulinemia, loss of β-cells, reduced β-cell insulin secretion, aberrant mitochondrial reactive oxygen species production, and the down-regulation of key β-cell genes. Our data therefore identifies a novel molecular link between the overexpression of RCAN1 and β-cell dysfunction. The glucose-responsive nature of RCAN1 provides a potential mechanism of action associated with the β-cell dysfunction observed in diabetes.

Gene therapy to improve pancreatic islet transplantation for Type 1 diabetes mellitus.

Pancreatic islet transplantation is a promising treatment option for Type 1 Diabetics, offering improved glycaemic control through restoration of insulin production and freedom from life-threatening hypoglycaemic episodes. Implementation of the Edmonton protocol in 2000, a glucocorticoid-free immunosuppressive regimen has led to improved islet transplantation success. >50% of islets are lost post-transplantation primarily through cytokine-mediated apoptosis, ischemia and hypoxia. Gene therapy presents a novel strategy to modify islets for improved survival post-transplantation. Current islet gene therapy approaches aim to improve islet function, block apoptosis and inhibit rejection. Gene transfer vectors include adenoviral, adeno-associated virus, herpes simplex virus vectors, retroviral vectors (including lentiviral vectors) and non-viral vectors. Adeno-associated virus is currently the best islet gene therapy vector, due to the vectors minimal immunogenicity and high safety profile. In animal models, using viral vectors to deliver genes conferring local immunoregulation, anti-apoptotic genes or angiogenic genes to islets can significantly improve islet survival in the early post-transplant period and influence long term engraftment. With recent improvements in gene delivery and increased understanding of the mechanisms underlying graft failure, gene therapy for islet transplantation has the potential to move closer to the clinic as a treatment for patients with Type 1 Diabetes.

Endothelial progenitor cells enhance islet engraftment, influence β-cell function, and modulate islet connexin 36 expression.

The success of pancreatic islet transplantation is limited by delayed engraftment and suboptimal function in the longer term. Endothelial progenitor cells (EPCs) represent a potential cellular therapy that may improve the engraftment of transplanted pancreatic islets. In addition, EPCs may directly affect the function of pancreatic β-cells. The objective of this study was to examine the ability of EPCs to enhance pancreatic islet transplantation in a murine syngeneic marginal mass transplant model and to examine the mechanisms through which this occurs. We found that cotransplanted EPCs improved the cure rate and initial glycemic control of transplanted islets. Gene expression data indicate that EPCs, or their soluble products, modulate the expression of the β-cell surface molecule connexin 36 and affect glucose-stimulated insulin release in vitro. In conclusion, EPCs are a promising candidate for improving outcomes in islet transplantation, and their mechanisms of action warrant further study.

Ultrastructural analysis, zinc transporters, glucose transporters and hormones expression in New world primate (Callithrix jacchus) and human pancreatic islets.

The New world primates (NWP) Callithrix jacchus separated from man approximately 50 million years ago and is a potential alternative small non-human primate model for diabetes research. Ultrastructure, and gene expression of pancreatic islets and the recently described diabetes auto antigenic zinc transporters families in human, NWP and pig pancreas were studied. Morphologically NWP islets were larger than pig islets and similar in size to human islets. NWP islets alpha cells had high dense core surrounded by a limiting membrane, beta cells by the mixed morphology of the granule core, and delta cells by moderate opaque core. Antibody staining for insulin, glucagon, somatostatin and Glucagon-like peptide-1 (GLP-1) showed that the distribution pattern of the different cell types within islets was comparable to pig and human islets. In all three species protein expression of zinc transporter ZnT8 was detected in most of the insulin producing beta cells whereas Zip14 expression was widely expressed in alpha and beta cells. In both human and NWP little or no expression of Glut2 was observed compared to Glut1 and glucokinase at the protein level, however the messenger RNA level of Glut2 was greater than Glut1 and glucokinase. In contrast all three glucose transporters were expressed in pig islets at the protein level. The expression of Zip14 in islets is reported for the first time. In conclusion NWP pancreatic islets express comparable islet cell types and distribution to humans and pigs. Importantly, marmosets have a similar glucose transporter profile to humans, making this non-endangered primate species a useful animal model for pancreatic biology.

In vitro adenovirus mediated gene transfer to the human cornea

Background/aims: Replication deficient adenovirus is an efficient vector for gene transfer to the cornea. The aim was to optimise the transduction of human corneal endothelium with adenoviral vectors and to measure transgene production from transduced corneas. Methods: Adenoviral vectors (AdV) encoding enhanced green fluorescent protein (eGFP) or a transgenic protein (scFv) were used to transfect 34 human corneas. Reporter gene expression was assessed after 72–96 hours of organ culture. The kinetics of scFv production was monitored in vitro for 1 month by flow cytometric analysis of corneal supernatants. Results: Transduction of human corneas with high doses (5×107–3×108 pfu) of AdV caused eGFP expression in 12–100% of corneal endothelial cells. Corneas were efficiently transduced following up to 28 days in cold storage. Very high AdV doses (2×109 pfu) reduced endothelial cell densities to 98 (SD 129) nuclei/mm2 (compared to 2114 (716) nuclei/mm2 for all other groups). Transgenic protein production peaked at 2.4 (0.9) μg/cornea/day at 2 weeks post-transduction, and decreased to 1.2 (0.4) μg/cornea/day by 33 days, at which time endothelial cell density had decreased to 431 (685) nuclei/mm2. Conclusion: Human corneas can be efficiently transduced by AdV following extended periods of cold storage, and transgene expression is maintained for at least 1 month in vitro.

RCAN1 Regulates Mitochondrial Function and Increases Susceptibility to Oxidative Stress in Mammalian Cells

Mitochondria are the primary site of cellular energy generation and reactive oxygen species (ROS) accumulation. Elevated ROS levels are detrimental to normal cell function and have been linked to the pathogenesis of neurodegenerative disorders such as Downs syndrome (DS) and Alzheimers disease (AD). RCAN1 is abundantly expressed in the brain and overexpressed in brain of DS and AD patients. Data from nonmammalian species indicates that increased RCAN1 expression results in altered mitochondrial function and that RCAN1 may itself regulate neuronal ROS production. In this study, we have utilized mice overexpressing RCAN1 (RCAN1ox) and demonstrate an increased susceptibility of neurons from these mice to oxidative stress. Mitochondria from these mice are more numerous and smaller, indicative of mitochondrial dysfunction, and mitochondrial membrane potential is altered under conditions of oxidative stress. We also generated a PC12 cell line overexpressing RCAN1 (PC12RCAN1). Similar to RCAN1ox neurons, PC12RCAN1 cells have an increased susceptibility to oxidative stress and produce more mitochondrial ROS. This study demonstrates that increasing RCAN1 expression alters mitochondrial function and increases the susceptibility of neurons to oxidative stress in mammalian cells. These findings further contribute to our understanding of RCAN1 and its potential role in the pathogenesis of neurodegenerative disorders such as AD and DS.