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Biochimie xxx (2014) 1e5

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Research paper

Functional expression of electron transport chain complexes in mouse rod outer segments Daniela Calzia a, *, Greta Garbarino b, Federico Caicci c, Lucia Manni c, Simona Candiani b, Silvia Ravera a, Alessandro Morelli a, Carlo Enrico Traverso d, Isabella Panfoli a a

Department of Pharmacy, DIFAR-Biochemistry Lab, University of Genova, Italy DISTAV, University of Genova, Italy Department of Biology, Università di Padova, Italy d Clinica Oculistica, DINOGMI, University of Genova, Genova, Italy b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 November 2013 Accepted 14 February 2014 Available online xxx

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. Ó 2014 Elsevier Masson SAS. All rights reserved.

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

1. Introduction Vertebrate photoreceptors carry out light capture thanks to the presence of the visual pigment Rhodopsin (Rh) [1]. Rods, associated with achromatic vision, are composed of an inner part, the Inner Segment (IS), containing mitochondria, and an Outer Segment (OS). The OS is an elongated stack of membrane disks [2,1] surrounded by the plasma membrane, connected to IS by a cilium. The proposed mechanism of energy supply for phototransduction of glycolytic ATP and phosphocreatine diffusion from the IS to the OS [3e5] has been challenged [6,7]. New findings point out to the existence of an extramitochondrial aerobic metabolism in bovine OS disks, which would better account for the ATP need of the light stimulated photoreceptor [8e10]. Our previous proteomic and functional studies showed the presence and activity in purified * Corresponding author. DIFAR-Biochemistry Lab., University of Genova, Viale Benedetto XV, 3 16132 Genova, Italy. Tel.: þ39 (0) 10 353 7397; fax: þ39 (0) 10 353 8153. E-mail address: [email protected] (D. Calzia).

bovine rod OS disks, of the ETC complexes I to IV, F1-Fo ATP synthase, and of the enzymes of the Tricarboxylic Acid Cycle [8e10]. Disks consume oxygen in the presence of various respiring substrates [9]. The main function of the ETC complexes IeV is to produce ATP through oxidative phosphorylation (OXPHOS), but it has been recognized as one of the major cellular generators of reactive oxygen intermediates (ROI) [11e13]. Respiratory chain defects can affect any organ, at any age [14] but usually, neurological manifestations are prevalent [15]. Oxidative stress is a risk factor for age related macular degeneration (AMD) [16,17]. Any impairment in the OXPHOS can increase ROI production and therefore oxidative stress [6]. In the present study we report for the first time the in situ activity of ETC complexes I, II, and IV in rod OS on unfixed mouse retinal sections by histochemical assay. The presence of ND4L subunit of ETC I and IV subunit of ETC IV in mouse OS was also confirmed by Transmission Electron Microscopy (TEM) analysis on mouse retinal sections. Besides confirming that the four ETC are expressed and catalytically active in mouse rod OS, like bovine ones, data may represent a reliable method to detect ETC dysfunction in oxidative stress related retinal pathologies in mouse models of disease.

http://dx.doi.org/10.1016/j.biochi.2014.02.007 0300-9084/Ó 2014 Elsevier Masson SAS. All rights reserved.

Please cite this article in press as: D. Calzia, et al., Functional expression of electron transport chain complexes in mouse rod outer segments, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.02.007

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D. Calzia et al. / Biochimie xxx (2014) 1e5

2. Materials and methods

3. Results

2.1. Materials

3.1. Immunogold on mouse retinal sections

Tissue-Tek OCT compound was purchased from Bio-Optica. All other chemicals and enzymes were purchased from Sigma Aldrich (St. Louis, MO, USA).

Our previous results on bovine rod OS prompted us to verify whether proteins of the mitochondrial OXPHOS are true components of the OS also in mouse retinas. To this end, immunogold TEM imaging was performed on mouse retinal sections. Panel A reports labelling with an Ab against Rhodopsin (Rh), (5 nm diameter gold particles). Rh signal is intense in OS disk while it is absent on resin, as reported in Inset (representing the enlarged vision of squared area in Panel A). Panels BeF show retinal sections labelled with an Ab against the ND4L subunit of ETC complex I (10 nm diameter gold particles). ND4L signal is present in rod OS (see Panel D and its enlarged OS detail in panel E and F). Labelling appears specific because it is absent in nuclei and cytoplasm (panel B) but present in the IS mitochondria (Panel C). In Panels GeK retinal sections labelled with Ab anti subunit IV of ETC complex IV (COX IV) (10 nm diameter gold particles) are reported. COX IV signal is present in the OS, as shown in Panel I and its magnification (Panels J and K), in the IS mitochondria (Panel H), but not in nuclei and cytoplasm (Panel G).

2.2. Sample preparation Eyes were excised from adult male Swiss mice (weighing 25 g; Charles River, Calco, Italy) housed at constant temperature (22 C) and relative humidity (50%) under a regular lightedark schedule. Water and standard mouse fodder were freely available. For immunogold assays, eyes were filled with fixative (4% paraformaldehyde and 0.1% glutaraldehyde in PBS buffer solution) for 1.5 h and then washed overnight with 50 mM NH4Cl, dehydrated and embedded in LR White Resin followed by polymerization at 58 C. Ultrathin sections (80 nm) were placed on Formvar-coated nickel grids and used the next day for immunogold labelling. For histochemical reactions unfixed eyes were directly cryoprotected o.n. in 30% sucrose in PBS and in Tissue-Tek OCT (Electron Microscopy Sciences, Fort Washington, PA) and then cut transversally by cryostat Frigocut 2800E (Reichert-Jung, Germany) at 12 mm thickness. Animal manipulations were conducted in conformity with institutional guidelines, in accordance with the European legislation (European Communities Directive of 24 November 1986,86/ 609/EEC) and with the NIH Guide for the Care and Use of Laboratory Animals. 2.3. TEM immunogold microscopy of mouse retinas For immunostaining of sections, the postembedding immunogold method was applied. Sections were first treated with blocking solution (10% goat serum, 0.1% Tween 20, PBS 1), then incubated with mouse monoclonal anti-rhodopsin (diluted 1:200) (Sigma Aldrich, St. Louis, MO, USA) or rabbit polyclonal anti ND4L subunit of ETC complex I (diluted 1:25) (Abcam Cambridge, UK) or rabbit polyclonal anti subunit IV of ETC complex IV (diluted 1:25) (Abcam Cambridge, UK) overnight at 4 C. Ab binding was detected using a secondary Ab (goat anti-rabbit IgG (Sigma Aldrich, St. Louis, MO, USA) (diluted 1:100), or goat anti-mouse IgG (British BioCell International) (diluted 1:100)) coupled to gold particles (10 nm diameter for anti-rabbit, and 5 nm diameter for anti-mouse). Sections were analyzed at an FEI Tecnai G2 transmission electron microscope operating at 100 kV. In negative controls, instead of the specific primary Ab, the preimmune serum was applied to the sections. The images were acquired with TIA Fei software Cam, collected and typeset in Corel Draw X3. Controls were performed by omitting primary Ab, which resulted in absence of crossreactivity (data not shown). 2.4. Histochemical reactions for ETC activity Transversal sections were incubated in: 0.8 mM NADH, 1.3 mM nitro blue tetrazolium (NBT) in PBS for Complex I assay; 200 mM of succinic acid, 0.2 mM phenazine methosulfate (PMS), 1.5 mM NBT, in PBS for Complex II assay; 50 mM phosphate (pH 7.4), 0.75 mg/ml DAB (3,30 diaminobenzidine), 0.75 mg/ml cytochrome c for Complex IV assay. Histochemical reactions were performed at 37 C in the dark for 1e2 h and checked every 30 min. Incubation was stopped in 0.1 M phosphate buffer once clear differentiation between highly reactive and nonreactive portions could be discerned. Control sections were incubated with PBS only in absence of substrate.

3.2. Activity of ETC I, II and IV on mouse retinal sections Activity of the Electron Transport Chain (ETC) complexes I, II, IV was detected in unfixed transversal sections by histochemical reactions. Sections were transversally cut following the section plane showed in Fig. 2, Panel F, including the area containing only sclera, pigment epithelium (RPE) and rod outer segment (OS) layers, as confirmed by haematoxylin and eosin staining of these sections (Fig. 2 Panel D). Activity of ETC I, II and IV could be clearly detected in both pigmented epithelium mitochondria and rod OS layers (Fig. 2, Panels A, B, C) with respect to negative controls (i.e: transversal retinal section incubated in PBS (Fig. 1 Panel E)). Violet and brown colour in Fig. 2 panel A and B represent the product of the reduction reaction of NBT to NBT-formazan with the parallel oxidation of NADH and succinate by ETC complex I and ETC complex II, respectively. Cytochrome c oxidase activity oxidises DAB, producing a brown precipitate, shown in Fig. 2 Panel C. 4. Discussion New findings on rod OS metabolism have been recently reported [8e10]. The present and previous data suggest that the mammalian OS, theta is devoid of mitochondria, is a site of extramitochondrial aerobic metabolism. Histochemical assays of ETC complexes I, II and IV on transversal not-fixed mouse retinal sections (Fig. 2 Panels A, B and C) show that ETC are active in both OS and RPE (retinal pigmented epithelium) layers, in comparison to control sections incubated in the absence of substrates (Fig. 2 Panel E). Activities were observed in situ on retinal sections, ruling out the possibility of a contamination. Previously activity of ETC IV was reported in mitochondria of RPE, photoreceptor IS, outer plexiform layer, and inner plexiform layer, but not in rod OS on rat [18] and human [19] longitudinal retinal sections. The ETC I and IV were found in different regions of mouse retina but not in rod OS by using histochemical reactions on fixed sections [20e22]. Immunogold shows the presence of Rh with the ETC proteins ND4L (a subunit of ETC I) and COX subunit IV in the rod OS on mouse retinal sections (Fig. 1). The ETC I, II and IV appear true components of the mouse OS, as its bovine counterpart. The present histochemical protocol was developed for the visualization of the ETC complex activity in situ in the rod OS. It differs from previous ones as far as the treatment of samples



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Fig. 1. Transmission Electron Microscopy (TEM) on mouse retina. A. Rods outer segments (OSs) labelled with antibody (Ab) anti-Rh (large, 5 nm width gold particles). Squared area is enlarged in inset. Note the presence of intense labelling on discs, the absence on resin. BeF: Rods labelled with anti-ND4L (10 nm width gold particles). B. Detail of a nucleus (n) and close cytoplasm. Note the absence of labelling. C. Rod mitochondria (mt) of IS. Arrowheads: labelling; nm: nuclear membrane. D. Outer segments (OSs). EeF. Detail of labelling on discs belonging to outer segments. E represents the squared area D. GeK: Rods labelled antibodies anti subunit IV of cytochrome c oxidase (10 nm width gold particles). G. Detail of a nucleus (n) and close cytoplasm. Note the absence of labelling. H. Rod mitochondria (mt) of IS. Arrowheads: labelling; nm: nuclear membrane. I. Outer segments (OSs). J, K. Detail of labelling on discs belonging to outer segments. J represents the squared area I.

(unfixed retinas), sectioning plane (transversal cutting of rod OS), incubation time and concentration of substrates are concerned. This allows the ETC proteins to be fully active and to better access the substrate. The protocol can be applied for the investigation of individual ETC complex activities, as well as for sequential doublestaining of ETC complexes IeIV. This may be useful to study functional ETC impairment (generating oxidative stress) in murine models of human retinal degeneration [23].

In the mouse OS, as in mitochondrial inner membranes, the ETC proteins would transfer electrons to O2 building up a proton electrochemical potential difference [9]. This in turn would be used by FoF1-ATP synthase to produce ATP [24]. Traditionally, the OXPHOS is thought to occur exclusively in the mitochondria, however an extra-mitochondrial aerobic ATP synthesis in rod OS would more efficiently respond to the energetic need of the phototransduction, as we have previously discussed [9,10]. The


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Fig. 2. Activity of Electron Transport Chain (ETC) I, II, IV on unfixed frontal retinal sections. A. Violet signal represents ETC I activity due to the reduction of NBT in NBT-formazan in presence of NADH. B. Brown signal represents ETC II activity in the presence of succinate. C. Brown precipitate represents ETC IV activity due to the DAB oxidation in the presence of Cytochrome c. D. Haematoxylin and eosin staining of not fixed retinal sections. E. Not fixed retinal section incubated with PBS only, as control sample. A, B, C were acquired with 200 magnification. D, E were acquired with 100 magnification. F. Schematic representation of transversal section plane used for histochemical analysis on not fixed mouse eye. Figures are representative of 8 different experiments.

ETC and ATP synthase would be transferred to the OS from the inner mitochondrial membranes by a fusion process that remains to be investigated. Mitochondria are intrinsically dynamic organelle [25], that were shown to display specific contact sites with the ER. These domains were named mitochondriaeER associated membranes (MAMs), an interface at which, among other new roles an eterologous fusion may be hypothesized [26]. The present data would offer a justification to the relationship among oxidative stress and retinal diseases such as diabetic retinopathy [27], glaucoma [28], retinitis pigmentosa [29e31], photoreceptor cell death after retinal detachment [32] and age-related macular degeneration (AMD) [33e37]. Notably, a dysfunctional ETC is a major source of ROI [38e40]. OS contain a high level of polyunsaturated fatty acid, such as docosahexaenoic acid (DHA) [41] vulnerable to lipid peroxidation [42e44]. In turn, disk membrane lipid peroxidation would be associated with impairment of the protein functions therein located, in particular, the ETC, that are extremely sensitive to the membrane environment. Oxidative stress would act in synergy with the antioxidant capacity decline in ageing [45]. The RPE/ photoreceptor complex is a highly oxidative micro-environment due to its location between the sensory retina and the choroid. It can be affected by oxidative stress because of the presence of chromophores (lipofuscin, melanin, Rhodopsin) which are capable of generating ROI [46e49] and moreover cytochromes, as here reported. ROI can react with lipids, protein and DNA [50e53] but also induce damage to FeeS centres of mitochondrial proteins among which some subunits of ETC complexes I, II and III as well as aconitase [54e56]. Considering the presence of aconitase in the OS [8,10], its inactivation may result in TCA impairment with an energy charge failure. Dysfunctional cytochromes might release

free iron triggering redox reactions. AMD in particular, is a leading cause of blindness in the developed world. Consistently, treatment with antioxidants (vitamins A,C,E, Lutein and zeaxanthin) reduces the risk of vision loss in AMD [57,33]. Interestingly, polyphenols (resveratrol, quercetin, curcumin), known inhibitors of ATP synthase, display a beneficial effect on retinal diseases [58,59].

Abbreviations AMD, age-related macular degeneration; ATP synthase, F1FoATP synthase; ETC, electron transport chain; COX, cytochrome c oxydaseoxidase; DR, diabetic retinopathy; Rod IS, rod inner segment; Rod OS, rod outer segment; OXPHOS, oxidative phosphorylation; PUFA, polyunsaturated fatty acids; RD, death after retinal detachment; ROI, reactive oxygen intermediates; ROP, retinopathy of prematurity; SD, standard deviation; TEM, transmission electron microscopy.

Funding This work was supported by the Athenaeum Research Projects from University of Genoa [Grant CUP D31J11001610005] for IP.

Acknowledgements We thank Mario Pestarino for his invaluable contribution. Authors declare no competing financial interests.



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