The International Journal of Biochemistry & Cell Biology
Drosophila type XV/XVIII collagen mutants manifest integrin mediated mitochondrial dysfunction, which is improved by cyclosporin A and losartan
Graphical abstract
Highlights
► We found a novel role of basement membrane in mitochondrial homeostasis. ► Mitochondrial dysfunction in flies lacking collagen XV/XVIII homologue. ► Mutants’ phenotypes were improved by mitochondrial protection. ► Collagen XV/XVIII participates in renin–angiotensin system. ► We provide the possible therapeutic strategies for multiplexin-related diseases.
Introduction
Basement membranes are thin layers of extracellular matrix (ECM), which provides scaffolds to cells to form tissues and to perform physiological functions. They are composed of various complex polymers of laminins, collagen types IV, XV, XVIII, nidogens and heparan/chondroitin sulfate proteoglycans (HS/CSPGs) such as perlecans and syndecans (Blumberg et al., 1987, Hynes, 2012). They are widely shared among metazoans, implying that they are essential elements to establish multicellularity. In fact, mutations or knockout mice of these genes cause various developmental defects or abnormalities in tissue maintenance: e.g. Alport syndrome (COL4A5), hemorrhage in brain and porencephaly (COL4A1), muscular dystrophy, epidermolysis bullosa (Laminin) (Van Agtmael and Bruckner-Tuderman, 2010, Wiradjaja et al., 2010, Yurchenco and Patton, 2009).
Vertebrate collagen types XV and XVIII are paralogous molecules, grouped together as a distinct group “multiplexin (multiple triple-helix domains and interruptions) collagens” within the large collagen family (Oh et al., 1994). They are structurally characterized by HS/CS side chains and a central collagenous triple helical region with multiple interruptions flanked by an N-terminal thrombospondin type 1 repeat and a C-terminal endostatin domain (Dong et al., 2003, Halfter et al., 1998, Li et al., 2000, Seppinen and Pihlajaniemi, 2011). Mutations in human collagen type XVIII lead to Knobloch syndrome 1 (KNO1), characterized by myopia, cataracts, vitreoretinal degeneration and retinal detachment, with occipital encephalocele [OMIM#267750] (Sertié et al., 2000). Similar conditions were observed in mice lacking Col18a1, suggesting that collagen type XVIII is important for maintaining normal ocular structure (Fukai et al., 2002, Marneros and Olsen, 2005). Although no human diseases have been identified so far, collagen type XV is believed to be involved in some diseases that manifest mild skeletal/cardiovascular myopathy and neuropathy from the observations of mice lacking Col15a1 (Eklund et al., 2001, Rasi et al., 2010a, Rasi et al., 2010b). In addition, collagen types XV and XVIII are unlikely to have an overlapped function (Ylikärppä et al., 2003b). In invertebrate genome, there is one multiplexin collagen orthologous gene: cle-1 in Caenorhabditis elegans and multiplexin (mp) in Drosophila. Analyses of these mutants show that multiplexin collagens participates in axon guidance, cell migration and diffusion of Wnt/Wingless proteins (Ackley et al., 2001, Ackley et al., 2003, Meyer and Moussian, 2009, Momota et al., 2011). Thus, the biological roles of multiplexin collagens are appreciated as structural components as collagen, as well as HS/CSPG molecules, endowing physical strength and modulation of local diffusive signaling in tissues. However, they are not sufficient to explain the pathogenesis of KNO1 patients and mice models, especially, of pleiotropic manifestations that appear later in life or after an extensive load (Eklund et al., 2001, Fukai et al., 2002, Marneros and Olsen, 2005, Rasi et al., 2010a, Rasi et al., 2010b, Ylikärppä et al., 2003a, Ylikärppä et al., 2003b).
To elucidate these mechanisms, we investigated Drosophila hypomorphic mutants of Multiplexin (Mp), the only orthologue of vertebrate multiplexin and evaluated its validity as fly models to study the pathogenesis of human related diseases. In the previous report, we demonstrated that mutants displayed neural defects and altered basement membrane ultrastructures, reminiscent phenotypes of Col18a1−/− mice (Momota et al., 2011). Here we demonstrate that mutants also manifested progressive deterioration of muscular function and myopathic skeletal/cardiac muscles, reminiscent phenotypes of Col15a1−/− mice. In addition, we demonstrate that this age-advanced effect is attributable to mitochondrial degeneration associated with the abolished focal adhesion signaling of βPS integrin and enhanced reactive oxygen species (ROS) production. Notably, mutants’ mitochondria manifested abnormal cristae, swollen appearances and diffuse outer membranes, signs of enhanced mitochondrial permeability (Irwin et al., 2003, Telfer et al., 2010, Uribe-Carvajal et al., 2011). These observations prompted us to test the effect of cyclosporin A, an inhibitor of mitochondrial permeability transition pore (mPTP). On the other hand, we attempted to suppress ROS production. In mammals, blockade of angiotensin II type 1 receptor (AT1R) had been shown not only to attenuate ROS production driven by NADPH oxidase but also to improve mitochondrial function (de Cavanagh et al., 2009). In Drosophila, no apparent AT1R orthologue was identified in our database search, however, the renin–angiotensin system (RAS) had been shown to exist in invertebrates (Salzet et al., 2001, Salzet and Verger-Bocquet, 2001) and a fly homologue of vertebrate angiotensin-converting enzyme 2 (ACE2), called ACER, had been shown to play a critical role in heart development (Crackower et al., 2002). Therefore, we assumed that the equivalent system also exists in Drosophila and tested the effect of losartan, an AT1R blocker. Importantly, both of these drugs effectively improved mutants’ phenotype. These results suggest that poor binding of Mp to β integrin causes unregulated mPTP opening and AT1R activity, leading to mitochondrial dysfunctions that would worsen the manifestations of multiplexin-related diseases. Thus, we provide the pharmacological evidences for the potential therapeutic applications of these drugs in patients and propose that multiplexin collagens may participate in some unidentified autosomal recessive mitochondrial diseases.
Section snippets
Fly culture
Flies were raised at room temperature on corn meal diet. Flies were anesthetized by carbon dioxide gas prior to dissection. Fly stocks used were mpf07253/f07253 and mpf03008/f03008 (Exelixis collection at Harvard) (Momota et al., 2011, Thibault et al., 2004) and Oregon-R (DGRC, Kyoto, Japan) as wild type controls. Losartan (1.0 mM: LKT laboratories, St. Paul, MN) or Cyclosporin A (50 μM: Wako, Osaka, Japan) were mixed in the food. The concentrations of these drugs were based on the estimation
Mutants manifest mild myopathy with abnormal mitochondria
The piggyBack inserted alleles mpf07253 and mpf03008 were characterized as hypomorphic alleles with trace of mp gene products in adults (Momota et al., 2011). Even though the homozygous mutants are viable and fertile, we have found that mpf07253/f07253 mutants exhibited lower survivability than wild type flies (Supplementary material, Fig. S1). We asked whether the mutants’ muscles were weak and susceptible to exercise as observed in Col15a1 knockout mice. To test this hypothesis, we took
Discussion
We present a novel role of basement membrane in mitochondria, a critical player in metabolism. Multiplexin collagens represent ancient collagen molecules that are widely shared among metazoans (Exposito et al., 2002, Hynes, 2012) and its multiple isoforms are ubiquitously expressed in various tissues (Ackley et al., 2001, Meyer and Moussian, 2009, Momota et al., 2011, Myers et al., 1996, Tomono et al., 2002). Therefore, it is tempting to hypothesize that the machinery to regulate mitochondria
Acknowledgements
We are grateful to HM and MBB lab members for helpful discussions. Hirotoshi Ri and Wataru Uegami for cryosections. The Central Research Laboratory in Okayama University Medical School for technical assistance in electron microscopy. The monoclonal antibody against βPS (CF. 6G11) developed by Brower et al. was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242, USA.
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