Academic journal article Genetics

Genetic Complexity in a Drosophila Model of Diabetes-Associated Misfolded Human Proinsulin

Academic journal article Genetics

Genetic Complexity in a Drosophila Model of Diabetes-Associated Misfolded Human Proinsulin

Article excerpt

MODEL organisms are widely employed in mechanistic studies of human Mendelian disease (Bedell et al. 1997a,b; Chintapalli et al. 2007; Lieschke and Currie 2007; Ocorr et al. 2007; Passador-Gurgel et al. 2007; Schlegel and Stainier 2007; Lessing and Bonini 2009). They are likewise an important resource for investigating the genetic underpinnings of continuously varying quantitative traits (Palsson and Gibson 2004; Telonis-Scott et al. 2005; Wang et al. 2005, 2006; Dworkin and Gibson 2006; Bergland et al. 2008; Gibson and Reed 2008; Ayroles et al. 2009; Dworkin et al. 2009; Goering et al. 2009; Mackay et al. 2009, 2010, 2011). Numerous models of human disease have been established in the fly (reviewed in Pandey and Nichols 2011), including transgenic models of diseases ranging from neurodegeneration/retinal degeneration (Bilen and Bonini 2005; Ryoo et al. 2007; Lessing and Bonini 2009; Yu and Bonini 2011) to cancer (Rudrapatna et al. 2012). Success with genetic screens to identify suppressors and enhancers of disease when mutants are overexpressed in a developing tissue, such as the eye-antennal imaginal disc, suggested to us that it might be possible to generate a fly model of misfolded insulin-associated diabetes.

A number of dominant mutations in human proinsulin have been identified in patients with permanent neonatal diabetes (Stoy et al. 2007, 2010). One class of these involves mutations leading to an unpaired cysteine. The mutation of Cys-96 to Tyr-hINSC96Y-abolishes a disulfide bridge between the A and B chains of the polypeptide, causing proinsulin to misfold and accumulate in the endoplasmic reticulum (ER). Induction of the unfolded protein response (UPR), caused by ER stress, ultimately leads to pancreatic b-cell death (Oyadomari et al. 2002; Hartley et al. 2010). Mutant insulininduced diabetes may also be a model for the more common type 2 (adult onset) form of diabetes, where increased demand for insulin overwhelms the pathways regulating protein folding and trafficking. In this case, the accumulation of misfolded wild-type proinsulin in the ER is hypothesized to trigger pathways that respond to loss of proteostatic control (Oyadomari et al. 2002; Scheuner and Kaufman 2008).

Many signaling mechanisms regulating proteostasis-the dynamics of protein expression and turnover including folding, processing, transport, regulation, and degradation-are conserved between fly and human (Geminard et al. 2009; Karpac and Jasper 2009; Haselton and Fridell 2010; Biteau et al. 2011). Misfolded alleles of rhodopsin, for example, cause age-related retinal degeneration in both species. In the fly model, overexpression of ninaE (a mutant allele of the fly ortholog of human rhodopsin-1) in the eye-antennal imaginal disc induces ER stress-associated UPR and proapoptotic signaling, resulting in adult-onset eye degeneration (Ryoo et al. 2007; Kang and Ryoo 2009; Mendes et al. 2009; Kang et al. 2012). Strongly conserved signaling mechanisms in these pathways led us to reason that overexpression of mutant human preproinsulin (hINSC96Y) in the fly would likewise unleash UPR and cell death, thus recapitulating biological processes acting in the human form of the disease.

To test this prediction we created a transgenic model of permanent neonatal diabetes in the fly by expressing hINSC96Y under regulatory control of the UAS-Gal4 system. We drove hINS expression in larval/pupal imaginal discs, precursors of adult structures, and measured the loss of adult tissue, expected if the mutant activated cell death pathways. We also examined phenotypes in flies expressing wild-type human preproinsulin (hINSWT) as a control. Here we describe phenotypic characteristics of this Mendelian model of disease, including sex-specific differences, dosage, environmental sensitivity, and reorganization of gene expression.

In addition, we examined dominant and partially dominant genetic variation in disease severity by crossing a panel of inbred lines derived from a natural population sample [Drosophila Genetic Reference Panel (DGRP)] (Mackay et al. …

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