Biochimie. 2014 Jul;102:78-82. doi: 10.1016/j.biochi.2014.02.007.

Functional expression of electron transport chain complexes in mouse rod outer segments.

Calzia D1, Garbarino G2, Caicci F3, Manni L3, Candiani S2, Ravera S4, Morelli A4, Traverso CE5, Panfoli I4.
  • 1Department of Pharmacy, DIFAR-Biochemistry Lab, University of Genova, Italy. Electronic address: dcalzia@gmail.com.
  • 2DISTAV, University of Genova, Italy.
  • 3Department of Biology, Università di Padova, Italy.
  • 4Department of Pharmacy, DIFAR-Biochemistry Lab, University of Genova, Italy.
  • 5Clinica Oculistica, DINOGMI, University of Genova, Genova, Italy.

 

Abstract

Rod photoreceptors efficiently carry out phototransduction cascade, an energetically costly process. Our recent data in bovine rod outer segment (OS) demonstrated that ATP for phototransduction is produced by an extramitochondrial oxidative phosphorylation, thanks to the expression of the Electron Transport Chain (ETC) complexes and of F1Fo ATP synthase in disks. Here we have focused on mouse retinas, reporting the activity of ETC complexes I, II, IV assayed directly on unfixed mouse eye sections, as well as immunogold TEM analysis of fixed mouse eye sections to verify the presence of ND4L subunit of ETC complex I and subunit IV of ETC complex IV in rod OS. Data suggest the presence of functional ETC in mouse rod OS, like their bovine counterpart. The protocol here developed for in situ assay of the ETC complexes activity represents a reliable method for the detection of ETC dysfunction in mice models of retinal pathologies. In fact, the ETC is a major source of reactive oxygen intermediates, and oxidative stress, especially when ectopically expressed in the OS. In turn, oxidative stress contributes to many retinal pathologies, such as diabetic retinopathy, age related macular degeneration, photoreceptor death after retinal detachment and some forms of retinitis pigmentosa.

KEYWORDS: Electron transport chain protein; Retinal diseases; Retinal sections; Rod outer segments; Transmission electron microscopy

PMID: 24565809

 

SUPPLEMENTARY

In vertebrates visual transduction is carried out by photoreceptor cells: cones and rods. The rod is composed by an Outer Segment (OS) consisting of a stack of floating membranous disks surrounded by plasma membrane and an Inner Segment (IS), containing nucleus and cytosolic organelle . Disks house proteins involved into the first steps of phototrasduction. Energy supply to support vision in the OS is still poorly understood. Anaerobic glycolysis may provide enough ATP in dark adapted rod but not under illumination conditions. The hypothesis that the ATP can diffuse from IS to OS seems unlikely. Although OS are devoid of mitochondria, our recent proteomic and biochemical analysis showed the expression and activity of ectopic Electron Transport Chain (ETC) [1] complexes I-IV and F1Fo-ATP synthase in disks and of Krebs Cycle enzymes in the rod OS [2], which perform the complete glucose oxidation and can therefore aerobically produce ATP for phototranduction.

 

Calzia et al_Figure 1. copia

Figure 1. Activity of ETC complexes I, II, III and IV in purified bovine OS. Activity of ETC I is expressed as IU/mg of total protein (µmol of reduced ferricyanide/min/mg). In the presence of 30 µM rotenone, activity decreased by about 60%. Activity of ETC II is expressed as IU/mg (µmol of reduced DCPI/min/mg of total protein). Activity of ETC III is expressed as IU/mg µmol of reduced cytochrome c /min/mg of total protein. In the presence of 50µM antimycin A, activity decreased by about 72%. Activity of ETC IV is expressed as IU/mg (µmol of oxidized cytochrome c/min/mg of total proteins). In the presence of 0.5mM KCN, activity decreased by about 99%. Each point, representative of four separate experiments, is the mean±S.D.

 

Confirmative of these findings, Figure 1 reports the activity in purified bovine OS of the ETC complexes I to IV. Activities are specific, as they are inhibited by specific inhibitors. Preventing any possible artifacts occurring during OS purification, the paper featured therein reported the in situ histochemical assay of the activity of ETC Complexes I, II and IV on unfixed retinal sections, revealing bona fide resident activities, in the OS. Moreover, the assay can be optimized by selecting a particular sectioning plane of the eye ball that permits to cut rod OS transversally, and appropriate incubation times and substrate concentrations. In this way IS visualization, where mitochondria are present, is excluded avoiding color saturation of colorimetric reactions and substrate removal by the IS mitochondria ETC activity. By contrast, as shown in Figure 2, longitudinal retinal sections display poor ETC complex I and II activity staining in the OS while in IS reaction are almost saturated. Being the ETC dysfunction a major source of free radical production our data would offer a new instrument to investigate a possible OS ETC impairment in oxidative stress-related retinal diseases in mouse animal models. Disk membranes are prone to lipid peroxidation due to the presence of high concentrations of polyunsatured fatty acid (PUFA). This can lead to the impairment of membrane associated proteins, such as the ETC complexes. Being ETC complexes I, III and IV organized into a supercomplex [3] Figure 3 reported the hypothesis that membrane alteration due to oxidative stress may cause the supercomplex impairment and proton gradient dissipation, accompanied by inactivation of F1Fo-ATP synthase. Deficient ATP supply can eventually cause rod death. Recently, morphological alterations of the OS organization and lamellar structure disruption as well as oxidative stress production both in the OS and in the IS were described in a retinal disease animal model obtained irradiating retinas with blue light [4]. Oxidative stress is a risk factor for many retinal diseases, such as diabetic retinopathy, glaucoma, retinitis pigmentosa and age-related macular degeneration. We have recently shown that antioxidant polyphenols known to be inhibitors of synthetic/hydrolytic activity of mitochondrial F1Fo-ATP synthase also act on the OS extramitochondrial ATP synthase. This suggests a molecular mode of action of polyphenols in reducing reactive oxygen species production through modulation of the ectopic F1Fo-ATP synthase in the OS disks [5]. Moreover the presence of caspase-dependent cell death machinery in the OS may be the ultimate cause of the rod apoptosis in case of stressor stimuli such as exposure to bright light, or continuous ambient light.

 

Calzia et al_Figure 2. copia

Figure 2. Activity of ETC Complex I, II, IV on unfixed longitudinal retinal sections. A. Violet signal represents ETC I activity. B. Brown signal represents ETC II activity in the presence of succinate. Images were acquired with 200x magnification in A and 400X in B. Figures are representative of 8 different experiments.

 

 

Calzia et al_Figure 3. copia

Figure 3 Graphical representation of functional ectopic ETC complexes and F1Fo-ATP synthase in the rod OS. Under stressor stimuli like continuous light exposure a hyperfunctioning of the ETC causes increased reactive oxygen species production as well as lipid peroxidation and membrane damage. Proton gradient dissipation eventually leads to ATP synthase inhibition and energy depletion in OS.

 

References:

[1]      I. Panfoli, D. Calzia, P. Bianchini, S. Ravera, A. Diaspro, G. Candiano, et al., Evidence for aerobic metabolism in retinal rod outer segment disks, International Journal of Biochemistry and Cell Biology. 41 (2009) 2555–2565.

[2]      I. Panfoli, D. Calzia, S. Ravera, M. Bruschi, C. Tacchetti, S. Candiani, et al., Extramitochondrial tricarboxylic acid cycle in retinal rod outer segments, Biochimie. 93 (2011) 1565–1575.

[3]      M.L. Genova, A. Baracca, A. Biondi, G. Casalena, M. Faccioli, A.I. Falasca, et al., Is supercomplex organization of the respiratory chain required for optimal electron transfer activity?, Biochim Biophys Acta. 1777 (2008) 740–746.

[4]      C. Roehlecke, U. Schumann, M. Ader, C. Brunssen, S. Bramke, H. Morawietz, et al., Stress reaction in outer segments of photoreceptors after blue light irradiation, PLoS One. 8 (2013) e71570.

[5]      D. Calzia, S. Barabino, P. Bianchini, G. Garbarino, M. Oneto, F. Caicci, et al., New findings in ATP supply in rod outer segments: insights for retinopathies., Biology of the Cell / under the Auspices of the European Cell Biology Organization. 105 (2013) 345–58.

 

 

 

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