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Morelli AM, Scholkmann F. Should the standard model of cellular energy metabolism be reconsidered? Possible coupling between the pentose phosphate pathway, glycolysis and extra-mitochondrial oxidative phosphorylation. Biochimie 2024; 221:99-109. [PMID: 38307246 DOI: 10.1016/j.biochi.2024.01.018] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2023] [Revised: 01/17/2024] [Accepted: 01/30/2024] [Indexed: 02/04/2024]
Abstract
The process of cellular respiration occurs for energy production through catabolic reactions, generally with glucose as the first process step. In the present work, we introduce a novel concept for understanding this process, based on our conclusion that glucose metabolism is coupled to the pentose phosphate pathway (PPP) and extra-mitochondrial oxidative phosphorylation in a closed-loop process. According to the current standard model of glycolysis, glucose is first converted to glucose 6-phosphate (glucose 6-P) and then to fructose 6-phosphate, glyceraldehyde 3-phosphate and pyruvate, which then enters the Krebs cycle in the mitochondria. However, it is more likely that the pyruvate will be converted to lactate. In the PPP, glucose 6-P is branched off from glycolysis and used to produce NADPH and ribulose 5-phosphate (ribulose 5-P). Ribulose 5-P can be converted to fructose 6-P and glyceraldehyde 3-P. In our view, a circular process can take place in which the ribulose 5-P produced by the PPP enters the glycolysis pathway and is then retrogradely converted to glucose 6-P. This process is repeated several times until the complete degradation of glucose 6-P. The role of mitochondria in this process is to degrade lipids by beta-oxidation and produce acetyl-CoA; the function of producing ATP appears to be only secondary. This proposed new concept of cellular bioenergetics allows the resolution of some previously unresolved controversies related to cellular respiration and provides a deeper understanding of metabolic processes in the cell, including new insights into the Warburg effect.
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Affiliation(s)
| | - Felix Scholkmann
- Neurophotonics and Biosignal Processing Research Group, Biomedical Optics Research Laboratory, Department of Neonatology, University Hospital Zurich, University of Zurich, Zurich, Switzerland.
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Ravera S, Bertola N, Puddu A, Bruno S, Maggi D, Panfoli I. Crosstalk between the Rod Outer Segments and Retinal Pigmented Epithelium in the Generation of Oxidative Stress in an In Vitro Model. Cells 2023; 12:2173. [PMID: 37681906 PMCID: PMC10487269 DOI: 10.3390/cells12172173] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2023] [Revised: 08/21/2023] [Accepted: 08/22/2023] [Indexed: 09/09/2023] Open
Abstract
Dysfunction of the retinal pigment epithelium (RPE) is associated with several diseases characterized by retinal degeneration, such as diabetic retinopathy (DR). However, it has recently been proposed that outer retinal neurons also participate in the damage triggering. Therefore, we have evaluated the possible crosstalk between RPE and photoreceptors in priming and maintaining oxidative damage of the RPE. For this purpose, we used ARPE-19 cells as a model of human RPE, grown in normal (NG, 5.6 mM) or high glucose (HG, 25 mM) and unoxidized (UOx) or oxidized (Ox) mammalian retinal rod outer segments (OSs). ARPE-19 cells were efficient at phagocytizing rod OSs in both NG and HG settings. However, in HG, ARPE-19 cells treated with Ox-rod OSs accumulated MDA and lipofuscins and displayed altered LC3, GRP78, and caspase 8 expression compared to untreated and UOx-rod-OS-treated cells. Data suggest that early oxidative damage may originate from the photoreceptors and subsequently extend to the RPE, providing a new perspective to the idea that retinal degeneration depends solely on a redox alteration of the RPE.
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Affiliation(s)
- Silvia Ravera
- Department of Experimental Medicine, Università di Genoa, Via De Toni 14, 16132 Genova, Italy
| | - Nadia Bertola
- Molecular Pathology Unit, IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi 10, 16132 Genova, Italy
| | - Alessandra Puddu
- Department of Internal Medicine and Medical Specialties, University of Genova, Viale Benedetto XV 6, 16132 Genova, Italy
| | - Silvia Bruno
- Department of Experimental Medicine, Università di Genoa, Via De Toni 14, 16132 Genova, Italy
| | - Davide Maggi
- Department of Internal Medicine and Medical Specialties, University of Genova, Viale Benedetto XV 6, 16132 Genova, Italy
| | - Isabella Panfoli
- Department of Pharmacy-(DIFAR), Università di Genova, Viale Benedetto XV 3, 16132 Genova, Italy
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The Flavone Cirsiliol from Salvia x jamensis Binds the F 1 Moiety of ATP Synthase, Modulating Free Radical Production. Cells 2022; 11:cells11193169. [PMID: 36231131 PMCID: PMC9562182 DOI: 10.3390/cells11193169] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2022] [Revised: 08/25/2022] [Accepted: 10/04/2022] [Indexed: 11/16/2022] Open
Abstract
Several studies have shown that mammalian retinal rod outer segments (OS) are peculiar structures devoid of mitochondria, characterized by ectopic expression of the molecular machinery for oxidative phosphorylation. Such ectopic aerobic metabolism would provide the chemical energy for the phototransduction taking place in the OS. Natural polyphenols include a large variety of molecules having pleiotropic effects, ranging from anti-inflammatory to antioxidant and others. Our goal in the present study was to investigate the potential of the flavonoid cirsiliol, a trihydroxy-6,7-dimethoxyflavone extracted from Salvia x jamensis, in modulating reactive oxygen species production by the ectopic oxidative phosphorylation taking place in the OS. Our molecular docking analysis identified cirsiliol binding sites inside the F1 moiety of the nanomotor F1Fo-ATP synthase. The experimental approach was based on luminometry, spectrophotometry and cytofluorimetry to evaluate ATP synthesis, respiratory chain complex activity and H2O2 production, respectively. The results showed significant dose-dependent inhibition of ATP production by cirsiliol. Moreover, cirsiliol was effective in reducing the free radical production by the OS exposed to ambient light. We report a considerable protective effect of cirsiliol on the structural stability of rod OS, suggesting it may be considered a promising compound against oxidative stress.
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Morelli AM, Chiantore M, Ravera S, Scholkmann F, Panfoli I. Myelin sheath and cyanobacterial thylakoids as concentric multilamellar structures with similar bioenergetic properties. Open Biol 2021; 11:210177. [PMID: 34905702 PMCID: PMC8670949 DOI: 10.1098/rsob.210177] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
There is a surprisingly high morphological similarity between multilamellar concentric thylakoids in cyanobacteria and the myelin sheath that wraps the nerve axons. Thylakoids are multilamellar structures, which express photosystems I and II, cytochromes and ATP synthase necessary for the light-dependent reaction of photosynthesis. Myelin is a multilamellar structure that surrounds many axons in the nervous system and has long been believed to act simply as an insulator. However, it has been shown that myelin has a trophic role, conveying nutrients to the axons and producing ATP through oxidative phosphorylation. Therefore, it is tempting to presume that both membranous structures, although distant in the evolution tree, share not only a morphological but also a functional similarity, acting in feeding ATP synthesized by the ATP synthase to the centre of the multilamellar structure. Therefore, both molecular structures may represent a convergent evolution of life on Earth to fulfill fundamentally similar functions.
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Affiliation(s)
| | - Mariachiara Chiantore
- Department of Earth, Environment and Life Sciences, University of Genova, Genova, Italy
| | - Silvia Ravera
- Experimental Medicine Department, University of Genova, Genova, Italy
| | - Felix Scholkmann
- Biomedical Optics Research Laboratory, Department of Neonatology, University Hospital Zurich, University of Zurich, Zurich, Switzerland
| | - Isabella Panfoli
- Experimental Medicine Department, University of Genova, Genova, Italy
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Yan WM, Long P, Chen MZ, Wei DY, Wang JC, Zhang ZM, Zhang L, Chen T. Retinal neovascularization induced by mutant Vldlr gene inhibited in an inherited retinitis pigmentosa mouse model: an in-vivo study. Int J Ophthalmol 2021; 14:990-997. [PMID: 34282382 PMCID: PMC8243196 DOI: 10.18240/ijo.2021.07.05] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2020] [Accepted: 03/29/2021] [Indexed: 11/23/2022] Open
Abstract
AIM To explore whether the retinal neovascularization (NV) in a genetic mutant mice model could be ameliorated in an inherited retinitis pigmentosa (RP) mouse, which would help to elucidate the possible mechanism and prevention of retinal NV diseases in clinic. METHODS The Vldlr -/- mice, the genetic mutant mouse model of retinal NV caused by the homozygous mutation of Vldlr gene, with the rd1 mice, the inherited RP mouse caused by homozygous mutation of Pde6b gene were bred. Intercrossing of the above two mice led to the birth of the F1 hybrids, further inbreeding of which gave birth to the F2 offspring. The ocular genotypes and phenotypes of the mice from all generations were examined, with the F2 offspring grouped according to the genotypes. RESULTS The rd1 mice exhibited the RP phenotype of outer retinal degeneration and loss of retinal function. The Vldlr -/- mice exhibited the phenotype of retinal NV obviously shown by the fundus fluorescein angiography. The F1 hydrides, with the heterozygote genotype, exhibited no phenotypes of RP or retinal NV. The F2 offspring with homozygous genotypes were grouped into four subgroups. They were the F2-I mice with the wild-type Pde6b and Vldlr genes (Pde6b+/+ -Vldlr+/+ ), which had normal ocular phenotypes; the F2-II mice with homozygous mutant Vldlr gene (Pde6b+/+ -Vldlr-/- ), which exhibited the retinal NV phenotype; the F2-III mice with homozygous mutant Pde6b gene (Pde6b-/- -Vldlr+/+ ), which exhibited the RP phenotype. Specifically, the F2-IV mice with homozygous mutant Vldlr and Pde6b gene (Pde6b-/- -Vldlr-/- ) showed only the RP phenotype, without the signs of retinal NV. CONCLUSION The retinal NV can be inhibited by the RP phenotype, which implies the role of a hyperoxic state in treating retinal NV diseases.
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Affiliation(s)
- Wei-Ming Yan
- Department of Ophthalmology, the 900 Hospital of Joint Logistic Support Forceo of PLA (Clinical Medical College of Fujian Medical University, Dongfang Hopsital Affiliated to Xiamen University), Fuzhou 350025, Fujian Province, China
- Center of Clinical Aerospace Medicine, Air Force Medical University, Xi'an 710032, Shaanxi Province, China
| | - Pan Long
- Department of Ophthalmology, the West General Hospital of Chinese PLA, Chengdu 610083, Sichuan Province, China
| | - Mei-Zhu Chen
- Department of Ophthalmology, the 900 Hospital of Joint Logistic Support Forceo of PLA (Clinical Medical College of Fujian Medical University, Dongfang Hopsital Affiliated to Xiamen University), Fuzhou 350025, Fujian Province, China
| | - Dong-Yu Wei
- Center of Clinical Aerospace Medicine, Air Force Medical University, Xi'an 710032, Shaanxi Province, China
| | - Jian-Cong Wang
- BeiJing HealthOLight Technology Co., Ltd., Beijing 10010, China
| | - Zuo-Ming Zhang
- Center of Clinical Aerospace Medicine, Air Force Medical University, Xi'an 710032, Shaanxi Province, China
| | - Lei Zhang
- Shaanxi Eye Hospital, Xi'an People's Hospital (Xi'an Fourth Hospital), Affiliated Guangren Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710004, Shaanxi Province, China
| | - Tao Chen
- Center of Clinical Aerospace Medicine, Air Force Medical University, Xi'an 710032, Shaanxi Province, China
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Ravera S, Bartolucci M, Calzia D, Morelli AM, Panfoli I. Efficient extra-mitochondrial aerobic ATP synthesis in neuronal membrane systems. J Neurosci Res 2021; 99:2250-2260. [PMID: 34085315 DOI: 10.1002/jnr.24865] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2020] [Revised: 04/29/2021] [Accepted: 05/10/2021] [Indexed: 11/09/2022]
Abstract
The nervous system displays high energy consumption, apparently not fulfilled by mitochondria, which are underrepresented therein. The oxidative phosphorylation (OxPhos) activity, a mitochondrial process that aerobically provides ATP, has also been reported also in the myelin sheath and the rod outer segment (OS) disks. Thus, commonalities and differences between the extra-mitochondrial and mitochondrial aerobic metabolism were evaluated in bovine isolated myelin (IM), rod OS, and mitochondria-enriched fractions (MIT). The subcellular fraction quality and the absence of contamination fractions have been estimated by western blot analysis. Oxygen consumption and ATP synthesis were stimulated by conventional (pyruvate + malate or succinate) and unconventional (NADH) substrates, observing that oxygen consumption and ATP synthesis by IM and rod OS are more efficient than by MIT, in the presence of both kinds of respiratory substrates. Mitochondria did not utilize NADH as a respiring substrate. When ATP synthesis by either sample was assayed in the presence of 10-100 µM ATP in the assay medium, only in IM and OS it was not inhibited, suggesting that the ATP exportation by the mitochondria is limited by extravesicular ATP concentration. Interestingly, IM and OS but not mitochondria appear able to synthesize ATP at a later time with respect to exposure to respiratory substrates, supporting the hypothesis that the proton gradient produced by the electron transport chain is buffered by membrane phospholipids. The putative transfer mode of the OxPhos molecular machinery from mitochondria to the extra-mitochondrial structures is also discussed, opening new perspectives in the field of neurophysiology.
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Affiliation(s)
- Silvia Ravera
- Department of Experimental Medicine, University of Genoa, Genoa, Italy
| | - Martina Bartolucci
- Laboratory of Mass Spectrometry - Core Facilities, Istituto Giannina Gaslini, Genoa, Italy.,Department of Pharmacy, Biochemistry Lab., University of Genoa, Genoa, Italy
| | - Daniela Calzia
- Department of Pharmacy, Biochemistry Lab., University of Genoa, Genoa, Italy
| | | | - Isabella Panfoli
- Department of Pharmacy, Biochemistry Lab., University of Genoa, Genoa, Italy
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Friesacher A, Lopez Torres LT, Valmaggia C, Rüesch R, Todorova MG. Linking the Presence of Macular Oedema to Structural and Functional Alterations in Retinitis Pigmentosa. Klin Monbl Augenheilkd 2021; 238:418-427. [PMID: 33853187 DOI: 10.1055/a-1389-5416] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
OBJECTIVE To investigate the association between the central retinal thickness (CRT), the retinal nerve fibre layer thickness (RNFL), and the functional alterations in retinitis pigmentosa (RP) patients. METHODS Forty-three patients with typical RP and nineteen age-matched controls, who underwent SD-OCT (macular and optic disc OCT protocols) and electrophysiology, were included. The RP group was divided into two subgroups: with clinical appearance of macular oedema (ME-RP; 30 eyes) and without macular oedema (no-ME; 44 eyes). Central retinal thickness OCT data were averaged in three zones (zone 1 [0°-3°], zone 2 [3°-8°], and zone 3 [8°-15°]) and were evaluated in relation to the RNFL thickness and electrophysiological data. RESULTS The ME-RP group showed increased CRT (zone 1) and RNFL thickness compared to the controls and no-ME-RP (p ≤ 0.002). The no-ME-RP group had reduced CRT thickness (all zones; p ≤ 0.018) compared to the controls and ME-RP, whereas the RNFL thickness in the no-ME-RP group was reduced only compared to the ME-RP group (p < 0.001). The ME-RP group showed significantly more attenuated functional responses than the no-ME-RP patients. A significant positive interaction was found between the CRT (zones 1 and 2) and the RNFL thickness within ME-RP (p ≤ 0.010). Significant negative interactions were found between CRT, RNFL thickness, and functional findings within ME-RP (p ≤ 0.049). CONCLUSION The presence of macular oedema correlated well with increased RNFL thickness and residual function in RP patients. Such association provides evidence of an underlying transneuronal mechanism of retinal degeneration. Simultaneous monitoring of CRT and RNFL thickness may help in the future to evaluate the progression of the disease and the efficacy of treatments in RP patients.
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Affiliation(s)
- Anna Friesacher
- Department of Ophthalmology (Chairman Prof. Dr. med. Ch. Valmaggia), Cantonal Hospital St. Gallen, St. Gallen, Switzerland.,Department of Ophthalmology (Chairman Prof. Dr. med. H. Scholl), University of Basel, Basel, Switzerland
| | - Lisette T Lopez Torres
- Department of Ophthalmology (Chairman Prof. Dr. med. H. Scholl), University of Basel, Basel, Switzerland
| | - Christophe Valmaggia
- Department of Ophthalmology (Chairman Prof. Dr. med. Ch. Valmaggia), Cantonal Hospital St. Gallen, St. Gallen, Switzerland.,Department of Ophthalmology (Chairman Prof. Dr. med. H. Scholl), University of Basel, Basel, Switzerland
| | - Reinhard Rüesch
- Department of Ophthalmology (Chairman Prof. Dr. med. Ch. Valmaggia), Cantonal Hospital St. Gallen, St. Gallen, Switzerland
| | - Margarita G Todorova
- Department of Ophthalmology (Chairman Prof. Dr. med. Ch. Valmaggia), Cantonal Hospital St. Gallen, St. Gallen, Switzerland.,Department of Ophthalmology (Chairman Prof. Dr. med. H. Scholl), University of Basel, Basel, Switzerland
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Murenu E, Kostidis S, Lahiri S, Geserich AS, Imhof A, Giera M, Michalakis S. Metabolic Analysis of Vitreous/Lens and Retina in Wild Type and Retinal Degeneration Mice. Int J Mol Sci 2021; 22:ijms22052345. [PMID: 33652907 PMCID: PMC7956175 DOI: 10.3390/ijms22052345] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Revised: 02/19/2021] [Accepted: 02/23/2021] [Indexed: 02/06/2023] Open
Abstract
Photoreceptors are the light-sensing cells of the retina and the major cell type affected in most inherited retinal degenerations. Different metabolic pathways sustain their high energetic demand in physiological conditions, particularly aerobic glycolysis. The principal metabolome of the mature retina has been studied, but only limited information is available on metabolic adaptations in response to key developmental events, such as eye opening. Moreover, dynamic metabolic changes due to retinal degeneration are not well understood. Here, we aimed to explore and map the ocular metabolic dynamics induced by eye opening in healthy (wild type) or Pde6b-mutant (retinal degeneration 1, Rd1) mice, in which photoreceptors degenerate shortly after eye opening. To unravel metabolic differences emerging before and after eye opening under physiological and pathophysiological conditions, we performed nuclear magnetic resonance (NMR) spectroscopy-based metabolome analysis of wild type and Rd1 retina and vitreous/lens. We show that eye opening is accompanied by changes in the concentration of selected metabolites in the retina and by alterations in the vitreous/lens composition only in the retinal degeneration context. As such, we identify NAcetylaspartate as a potential novel vitreous/lens marker reflecting progressive retinal degeneration. Thus, our data can help elucidating mechanisms underlying key events in retinal physiology and reveal changes occurring in pathology, while highlighting the importance of the vitreous/lens in the characterization of retinal diseases.
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Affiliation(s)
- Elisa Murenu
- Department of Ophthalmology, Ludwig-Maximilians-Universität München, Mathildenstraße 8, 80336 Munich, Germany;
- Department of Pharmacy, Ludwig-Maximilians Universität München, Butenandtstr. 7, 81377 Munich, Germany;
| | - Sarantos Kostidis
- Leiden University Medical Center, Center for Proteomics & Metabolomics, P.O. Box 9600, 2300 RC Leiden, The Netherlands; (S.K.); (M.G.)
| | - Shibojyoti Lahiri
- Biomedical Center Munich-Molecular Biology, Ludwig-Maximilians-Universität München, Großhaderner Strasse 9, 82152 Planegg-Martinsried, Germany; (S.L.); (A.I.)
| | - Anna S. Geserich
- Department of Pharmacy, Ludwig-Maximilians Universität München, Butenandtstr. 7, 81377 Munich, Germany;
| | - Axel Imhof
- Biomedical Center Munich-Molecular Biology, Ludwig-Maximilians-Universität München, Großhaderner Strasse 9, 82152 Planegg-Martinsried, Germany; (S.L.); (A.I.)
| | - Martin Giera
- Leiden University Medical Center, Center for Proteomics & Metabolomics, P.O. Box 9600, 2300 RC Leiden, The Netherlands; (S.K.); (M.G.)
| | - Stylianos Michalakis
- Department of Ophthalmology, Ludwig-Maximilians-Universität München, Mathildenstraße 8, 80336 Munich, Germany;
- Department of Pharmacy, Ludwig-Maximilians Universität München, Butenandtstr. 7, 81377 Munich, Germany;
- Correspondence: ; Tel.: +49-89-2180-77325
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della Volpe Waizel M, Scholl HPN, Valmaggia C, Todorova MG. Retinal vessel oximetry in children with inherited retinal diseases. Acta Ophthalmol 2021; 99:52-60. [PMID: 32573052 DOI: 10.1111/aos.14466] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2019] [Accepted: 04/21/2020] [Indexed: 12/22/2022]
Abstract
BACKGROUND Retinal vessel oximetry (RO) has been used to show altered metabolic function in patients with inherited retinal diseases (IRDs). The aim of this study was to investigate RO parameters of children with IRDs and presumed IRD carriers (pIRDc) and to compare them to controls. METHODS In this cross-sectional cohort study, 142 eyes from 71 Caucasian subjects were included: 40 eyes with IRDs, 26 eyes with pIRDc and 76 control eyes. The oxygen saturation was measured with the Retinal Vessel Analyser (IMEDOS Systems UG, Jena, Germany). Mean oxygen saturations in the peripapillary retinal arterioles (A-SO2 ; %) and venules (V-SO2 ; %) were estimated, and their difference (A-V SO2 ; %) was calculated. In addition, we evaluated the mean diameter in all major retinal arterioles (D-A; μm) and venules (D-V; μm). anova-based linear mixed-effects models were calculated with SPSS® . RESULTS In general, children suffering from IRDs differed from controls when the A-SO2 and A-V SO2 were taken into account: both the A-SO2 and the A-V SO2 were significantly increased (p = 0.012). In subgroup analyses, children suffering from rod-cone dystrophy (RCD) presented an A-SO2 increase (99.12 ± 8.24%) when compared to controls (91.33 ± 10.34%, p = 0.014) and pIRDc (92.37 ± 6.57%, p = 0.065). For V-SO2 significant changes in RCD (67.42 ± 9.19%) were found in comparison with controls (58.24 ± 11.74%, p < 0.041), pIRDc (56.67 ± 7.16%, p = 0.007), cone-rod dystrophies (CRD, 52.17 ± 5.32%, p < 0.001) and inherited macular dystrophies (IMD, 55.74 ± 6.96%, p = 0.004), In addition, A-V SO2 was decreased in RCD (31.69 ± 3.92%) when measured against CRD (41.9 ± 8.87%, p = 0.017) or IMD (39.52 ± 8.95%, p = 0.059). CONCLUSION In general, we found that children with IRDs presented early metabolic changes. Within IRDs, children with RCD showed more affected metabolic changes. Thus, RO may support early screening to rule out IRDs in children, and more precisely may help to differentiate those suffering from RCD.
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Affiliation(s)
- Maria della Volpe Waizel
- Department of Ophthalmology University of Basel Basel Switzerland
- Institute of Molecular and Clinical Ophthalmology Basel (IOB) Basel Switzerland
| | - Hendrik P. N. Scholl
- Department of Ophthalmology University of Basel Basel Switzerland
- Institute of Molecular and Clinical Ophthalmology Basel (IOB) Basel Switzerland
| | - Christophe Valmaggia
- Department of Ophthalmology University of Basel Basel Switzerland
- Department of Ophthalmology Cantonal Hospital St. Gallen St. Gallen Switzerland
| | - Margarita G. Todorova
- Department of Ophthalmology University of Basel Basel Switzerland
- Department of Ophthalmology Cantonal Hospital St. Gallen St. Gallen Switzerland
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Ravera S, Esposito A, Degan P, Caicci F, Manni L, Liguori A, Bisio A, Iobbi V, Schito A, Traverso CE, Panfoli I. The diterpene Manool extracted from Salvia tingitana lowers free radical production in retinal rod outer segments by inhibiting the extramitochondrial F 1 F o ATP synthase. Cell Biochem Funct 2021; 39:528-535. [PMID: 33472276 DOI: 10.1002/cbf.3618] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2020] [Revised: 11/17/2020] [Accepted: 12/28/2020] [Indexed: 11/05/2022]
Abstract
Uncontrolled oxidative stress production, especially in the outer retina is one of the causes of retinal degenerations. Mitochondria are considered the principal source of oxidative stress. However, a Reactive Oxygen Intermediates (ROI) production in the retinal photoreceptor layer seems to depend also on the expression of an extramitochondrial oxidative phosphorylation (OxPhos) machinery in the rod outer segments (OS). In fact, OS conduct aerobic metabolism, producing ATP through oxygen consumption, although it is devoid of mitochondria. As diterpenes display an antioxidant effect, we have evaluated the effect Manool, extracted from Salvia tingitana, on the extramitochondrial OxPhos and the ROI production in the retinal rod OS. Results confirm that the OxPhos machinery is ectopically expressed in the OS and that F1 Fo -ATP synthase is a target of Manool, which inhibited the OS ATP synthesis, binding the F1 moiety with high affinity, as analysed by molecular docking. Moreover, the overall slowdown of OxPhos metabolism reduced the ROI production elicited in the OS by light exposure, in vitro. In conclusion, data are consistent with the antioxidant properties of Salvia spp., suggesting its ability to lower oxidative stress production, a primary risk factor for degenerative retinal diseases. SIGNIFICANCE OF THE STUDY: Here we show that Manool, a diterpene extracted from Salvia tingitana has the potential to lower the free radical production by light-exposed rod outer segments in vitro, by specifically targeting the rod OS F1 Fo -ATP synthase belonging to the extramitochondrial OxPhos expressed on the disk membrane. The chosen experimental model allowed to show that the rod OS is a primary producer of oxidative stress linked to the pathogenesis of degenerative retinal diseases. Data are also consistent with the antioxidant and anti-inflammatory action of Salvia spp., suggesting a beneficial effect also in vivo.
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Affiliation(s)
- Silvia Ravera
- Dipartimento di Medicina Sperimentale, Università di Genova, Genova, Italy
| | - Alfonso Esposito
- Centro di Biologia Integrata (CIBIO), Università di Trento, Trento, Italy
| | - Paolo Degan
- UOC Mutagenesi, IRCCS Policlinico San Martino -IST (Istituto Nazionale per la Ricerca sul Cancro), Genova, Italy
| | - Federico Caicci
- Dipartimento di Biologia, Università di Padova, Padova, Italy
| | - Lucia Manni
- Dipartimento di Biologia, Università di Padova, Padova, Italy
| | - Anna Liguori
- Dipartimento di Farmacia (DIFAR), Università di Genova, Genova, Italy
| | - Angela Bisio
- Dipartimento di Farmacia (DIFAR), Università di Genova, Genova, Italy
| | - Valeria Iobbi
- Dipartimento di Farmacia (DIFAR), Università di Genova, Genova, Italy
| | - Anna Schito
- Dipartimento di Scienze Chirurgiche e Diagnostiche Integrate (DISC), Sezione di Microbiologia, Università di Genova, Genova, Italy
| | | | - Isabella Panfoli
- Dipartimento di Farmacia (DIFAR), Università di Genova, Genova, Italy
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Ravera S, Esposito A, Degan P, Caicci F, Calzia D, Perrotta E, Manni L, Bisio A, Iobbi V, Schito A, Traverso CE, Panfoli I. Sclareol modulates free radical production in the retinal rod outer segment by inhibiting the ectopic f 1f o-atp synthase. Free Radic Biol Med 2020; 160:368-375. [PMID: 32853720 DOI: 10.1016/j.freeradbiomed.2020.08.014] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/04/2020] [Revised: 08/15/2020] [Accepted: 08/18/2020] [Indexed: 01/06/2023]
Abstract
We have previously shown that the retinal rod outer segments (OS) produce reactive oxygen species in the function of illumination in vitro, establishing a relationship among the extra-mitochondrial oxidative phosphorylation and phototransduction. This source of oxidative stress in the OS can be modulated by polyphenols, acting as inhibitors of F1Fo-ATP synthase. The present study aimed at exploring whether sclareol, a diterpene, interacts with F1Fo-ATP synthase mitigating the light-induced free radical production in the rod OS. Characterization of bovine retinal sections was conducted by immunogold analysis. Reactive oxygen intermediates production, oxygen consumption, the activity of the four respiratory complexes and ATP synthesis were evaluated in purified bovine rod OS. Molecular docking analyses were also conducted. Sclareol reduced free radical production by light-exposed rod OS. Such antioxidant effect was associated with an inhibition of the respiratory complexes and oxygen consumption (OCR), in coupled conditions. Sclareol also inhibited the rod OS ATP synthetic ability. Since the inhibitor effect on respiratory complexes and OCR is not observed in uncoupled conditions, it is supposed that the modulating effect of sclareol on the ectopic oxidative phosphorylation in the rod OS targets specifically the F1Fo-ATP synthase. This hypothesis is confirmed by the in silico molecular docking analyses, which shows that sclareol binds the F1 moiety of ATP synthase with high affinity. In conclusion, a beneficial effect of sclareol can be envisaged as a modulator of oxidative stress in the photoreceptor, a risk factor for the degenerative retinal diseases, suggestive of its potential beneficial action also in vivo.
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Affiliation(s)
- Silvia Ravera
- Dipartimento di Medicina Sperimentale, Università di Genoa, Via De Toni 14, 16132, Genova, Italy
| | - Alfonso Esposito
- Centro di Biologia Integrata (CIBIO), Università di Trento, Via Sommarive, 9, Povo, 38123, Trento, Italy
| | - Paolo Degan
- UOC Mutagenesi, IRCCS Policlinico San Martino -IST, Istituto Nazionale per La Ricerca Sul Cancro), Largo Rosanna Benzi 10, 16132, Genova, Italy
| | - Federico Caicci
- Dipartimento di Biologia, Università di Padova, Via U. Bassi 58/B, 35121, Padova, Italy
| | - Daniela Calzia
- Dipartimento di Farmacia (DIFAR), Università di Genova, V.le Benedetto XV 3, 16132, Genova, Italy
| | - Eleonora Perrotta
- Dipartimento di Medicina Sperimentale, Università di Genoa, Via De Toni 14, 16132, Genova, Italy
| | - Lucia Manni
- Dipartimento di Biologia, Università di Padova, Via U. Bassi 58/B, 35121, Padova, Italy
| | - Angela Bisio
- Dipartimento di Farmacia (DIFAR), Università di Genova, V.le Benedetto XV 3, 16132, Genova, Italy
| | - Valeria Iobbi
- Dipartimento di Farmacia (DIFAR), Università di Genova, V.le Benedetto XV 3, 16132, Genova, Italy
| | - Anna Schito
- Dipartimento di Scienze Chirurgiche e Diagnostiche Integrate (DISC), Sezione di Microbiologia, Università di Genova, Largo Rosanna Benzi 8, 16145, Genova, Italy
| | - Carlo Enrico Traverso
- Clinica Oculistica, (DINOGMI) Università di Genova, V.le Benedetto XV 6, 16132, Genova, Italy
| | - Isabella Panfoli
- Dipartimento di Farmacia (DIFAR), Università di Genova, V.le Benedetto XV 3, 16132, Genova, Italy.
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12
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Ravera S, Caicci F, Degan P, Maggi D, Manni L, Puddu A, Nicolò M, Traverso CE, Panfoli I. Inhibitory Action of Antidiabetic Drugs on the Free Radical Production by the Rod Outer Segment Ectopic Aerobic Metabolism. Antioxidants (Basel) 2020; 9:E1133. [PMID: 33203090 PMCID: PMC7696108 DOI: 10.3390/antiox9111133] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2020] [Revised: 11/04/2020] [Accepted: 11/12/2020] [Indexed: 12/17/2022] Open
Abstract
Rod outer segments (OS) express the FoF1-ATP synthase and the respiratory chain, conducting an ectopic aerobic metabolism that produces free radicals in vitro. Diabetic retinopathy, a leading cause of vision loss, is associated with oxidative stress in the outer retina. Since metformin and glibenclamide, two anti-type 2 diabetes drugs, target the respiratory complexes, we studied the effect of these two drugs, individually or in association, on the free radical production in purified bovine rod OS. ATP synthesis, oxygen consumption, and oxidative stress production were assayed by luminometry, oximetry and flow cytometry, respectively. The expression of FoF1-ATP synthase was studied by immunogold electron microscopy. Metformin had a hormetic effect on the OS complex I and ATP synthetic activities, being stimulatory at concentrations below 1 mM, and inhibitory above. Glibenclamide inhibited complexes I and III, as well as ATP production in a concentration-dependent manner. Maximal concentrations of both drugs inhibited the ROI production by the light-exposed OS. Data, consistent with the delaying effect of these drugs on the onset of diabetic retinopathy, suggest that a combination of the two drugs at the beginning of the treatment might reduce the oxidative stress production helping the endogenous antioxidant defences in avoiding retinal damage.
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Affiliation(s)
- Silvia Ravera
- Dipartimento di Medicina Sperimentale, Università di Genoa, Via De Toni 14, 16132 Genova, Italy;
| | - Federico Caicci
- Dipartimento di Biologia, Università di Padova, via U. Bassi 58/B, 35121 Padova, Italy; (F.C.); (L.M.)
| | - Paolo Degan
- U.O. Mutagenesis and Preventive Oncology, IRCCS Ospedale Policlinico San Martino, L.go R. Benzi, 10, 16132 Genova, Italy;
| | - Davide Maggi
- Department of Internal Medicine and Medical Specialties, University of Genova, 16132 Genova, Italy; (D.M.); (A.P.)
| | - Lucia Manni
- Dipartimento di Biologia, Università di Padova, via U. Bassi 58/B, 35121 Padova, Italy; (F.C.); (L.M.)
| | - Alessandra Puddu
- Department of Internal Medicine and Medical Specialties, University of Genova, 16132 Genova, Italy; (D.M.); (A.P.)
| | - Massimo Nicolò
- Clinica Oculistica (DINOGMI), Università di Genova, V.le Benedetto XV 6, 16132 Genova, Italy; (M.N.); (C.E.T.)
- Fondazione per la Macula onlus, Università di Genova, V.le Benedetto XV 6, 16132 Genova, Italy
| | - Carlo E. Traverso
- Clinica Oculistica (DINOGMI), Università di Genova, V.le Benedetto XV 6, 16132 Genova, Italy; (M.N.); (C.E.T.)
| | - Isabella Panfoli
- Dipartimento di Farmacia (DIFAR), Università di Genova, V.le Benedetto XV 3, 16132 Genova, Italy
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13
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Morelli AM, Ravera S, Panfoli I. The aerobic mitochondrial ATP synthesis from a comprehensive point of view. Open Biol 2020; 10:200224. [PMID: 33081639 PMCID: PMC7653358 DOI: 10.1098/rsob.200224] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Most of the ATP to satisfy the energetic demands of the cell is produced by the F1Fo-ATP synthase (ATP synthase) which can also function outside the mitochondria. Active oxidative phosphorylation (OxPhos) was shown to operate in the photoreceptor outer segment, myelin sheath, exosomes, microvesicles, cell plasma membranes and platelets. The mitochondria would possess the exclusive ability to assemble the OxPhos molecular machinery so to share it with the endoplasmic reticulum (ER) and eventually export the ability to aerobically synthesize ATP in true extra-mitochondrial districts. The ER lipid rafts expressing OxPhos components is indicative of the close contact of the two organelles, bearing different evolutionary origins, to maximize the OxPhos efficiency, exiting in molecular transfer from the mitochondria to the ER. This implies that its malfunctioning could trigger a generalized oxidative stress. This is consistent with the most recent interpretations of the evolutionary symbiotic process whose necessary prerequisite appears to be the presence of the internal membrane system inside the eukaryote precursor, of probable archaeal origin allowing the engulfing of the α-proteobacterial precursor of mitochondria. The process of OxPhos in myelin is here studied in depth. A model is provided contemplating the biface arrangement of the nanomotor ATP synthase in the myelin sheath.
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Affiliation(s)
- Alessandro Maria Morelli
- Pharmacy Department (DIFAR), Biochemistry Laboratory, University of Genova, Viale Benedetto XV, 3, 16132 Genova, Italy
| | - Silvia Ravera
- Experimental Medicine Department (DIMES), University of Genova, Via De Toni, 14, 16132 Genova, Italy
| | - Isabella Panfoli
- Pharmacy Department (DIFAR), Biochemistry Laboratory, University of Genova, Viale Benedetto XV, 3, 16132 Genova, Italy
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14
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Bruschi M, Bartolucci M, Petretto A, Calzia D, Caicci F, Manni L, Traverso CE, Candiano G, Panfoli I. Differential expression of the five redox complexes in the retinal mitochondria or rod outer segment disks is consistent with their different functionality. FASEB Bioadv 2020; 2:315-324. [PMID: 32395704 PMCID: PMC7211042 DOI: 10.1096/fba.2019-00093] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2019] [Revised: 11/27/2019] [Accepted: 03/05/2020] [Indexed: 12/28/2022] Open
Abstract
PURPOSE The retinal rod outer segment (OS) disk membranes, devoid of mitochondria, conducts oxidative phosphorylation (OxPhos). This study aimed at identifying which proteins expressed in the retinal rod OS disks determined the considerable adenosine-5'-triphosphate production and oxygen consumption observed in comparison with retinal mitochondria. PROCEDURES Characterization was conducted by immunogold transmission electron microscopy on retinal sections. OxPhos was studied by oximetry and luminometry. The proteomes of OS disks and mitochondria purified from bovine retinas were studied by mass spectrometry. Statistical and bioinformatic analyses were conducted by univariate, multivariate, and machine learning methods. RESULTS Weighted gene coexpression network analysis identified two protein expression profile modules functionally associated with either retinal mitochondria or disk samples, in function of a strikingly different ability of each sample to utilized diverse substrate for F1Fo-ATP synthase. The OS disk proteins correlated better than mitochondria with the tricarboxylic acids cycle and OxPhos proteins. CONCLUSIONS The differential enrichment of the expression profile of the OxPhos proteins in the disks versus mitochondria suggests that these proteins may represent a true proteome component of the former, with different functionality. These findings may shed new light on the pathogenesis of rod-driven retinal degenerative diseases.
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Affiliation(s)
- Maurizio Bruschi
- Laboratory of Molecular NephrologyIstituto Giannina GasliniGenoaItaly
| | - Martina Bartolucci
- Laboratory of Mass Spectrometry‐Core FacilitiesIstituto Giannina GasliniGenovaItaly
| | - Andrea Petretto
- Laboratory of Mass Spectrometry‐Core FacilitiesIstituto Giannina GasliniGenovaItaly
| | - Daniela Calzia
- Dipartimento di Farmacia‐DIFARUniversità di GenovaGenoaItaly
| | | | - Lucia Manni
- Department of BiologyUniversità di PadovaPadovaItaly
| | - Carlo Enrico Traverso
- Clinica Oculistica, (Di.N.O.G.M.I.) Università Department of Intensive Care di GenovaIRCCS Azienda Ospedaliera Universitaria San Martino‐ISTGenoaItaly
| | - Giovanni Candiano
- Laboratory of Molecular NephrologyIstituto Giannina GasliniGenoaItaly
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15
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Photobiomodulation Mediates Neuroprotection against Blue Light Induced Retinal Photoreceptor Degeneration. Int J Mol Sci 2020; 21:ijms21072370. [PMID: 32235464 PMCID: PMC7177783 DOI: 10.3390/ijms21072370] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2020] [Revised: 03/21/2020] [Accepted: 03/27/2020] [Indexed: 12/12/2022] Open
Abstract
Potent neuroprotective effects of photobiomodulation with 670 nm red light (RL) have been demonstrated in several models of retinal disease. RL improves mitochondrial metabolism, reduces retinal inflammation and oxidative cell stress, showing its ability to enhance visual function. However, the current knowledge is limited to the main hypothesis that the respiratory chain complex IV, cytochrome c oxidase, serves as the primary target of RL. Here, we demonstrate a comprehensive cellular, molecular, and functional characterization of neuroprotective effects of 670 nm RL and 810 nm near-infrared light (NIRL) on blue light damaged murine primary photoreceptors. We show that respiratory chain complexes I and II are additional PBM targets, besides complex IV, leading to enhanced mitochondrial energy metabolism. Accordingly, our study identified mitochondria related RL- and NIRL-triggered defense mechanisms promoting photoreceptor neuroprotection. The observed improvement of mitochondrial and extramitochondrial respiration in both inner and outer segments is linked with reduced oxidative stress including its cellular consequences and reduced mitochondria-induced apoptosis. Analysis of regulatory mechanisms using gene expression analysis identified upregulation α-crystallins that indicate enhanced production of proteins with protective functions that point to the rescued mitochondrial function. The results support the hypothesis that energy metabolism is a major target for retinal light therapy.
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16
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Morelli AM, Ravera S, Calzia D, Panfoli I. An update of the chemiosmotic theory as suggested by possible proton currents inside the coupling membrane. Open Biol 2020; 9:180221. [PMID: 30966998 PMCID: PMC6501646 DOI: 10.1098/rsob.180221] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Understanding how biological systems convert and store energy is a primary purpose of basic research. However, despite Mitchell's chemiosmotic theory, we are far from the complete description of basic processes such as oxidative phosphorylation (OXPHOS) and photosynthesis. After more than half a century, the chemiosmotic theory may need updating, thanks to the latest structural data on respiratory chain complexes. In particular, up-to date technologies, such as those using fluorescence indicators following proton displacements, have shown that proton translocation is lateral rather than transversal with respect to the coupling membrane. Furthermore, the definition of the physical species involved in the transfer (proton, hydroxonium ion or proton currents) is still an unresolved issue, even though the latest acquisitions support the idea that protonic currents, difficult to measure, are involved. Moreover, FoF1-ATP synthase ubiquitous motor enzyme has the peculiarity (unlike most enzymes) of affecting the thermodynamic equilibrium of ATP synthesis. It seems that the concept of diffusion of the proton charge expressed more than two centuries ago by Theodor von Grotthuss is to be taken into consideration to resolve these issues. All these uncertainties remind us that also in biology it is necessary to consider the Heisenberg indeterminacy principle, which sets limits to analytical questions.
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Affiliation(s)
- Alessandro Maria Morelli
- 1 Pharmacy Department, Biochemistry Lab, University of Genova , Viale Benedetto XV 3, 16132 Genova , Italy
| | - Silvia Ravera
- 2 Experimental Medicine Department, University of Genova , Via De Toni 14, 16132 Genova , Italy
| | - Daniela Calzia
- 1 Pharmacy Department, Biochemistry Lab, University of Genova , Viale Benedetto XV 3, 16132 Genova , Italy
| | - Isabella Panfoli
- 2 Experimental Medicine Department, University of Genova , Via De Toni 14, 16132 Genova , Italy
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17
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Fu Z, Sun Y, Cakir B, Tomita Y, Huang S, Wang Z, Liu CH, S. Cho S, Britton W, S. Kern T, Antonetti DA, Hellström A, E.H. Smith L. Targeting Neurovascular Interaction in Retinal Disorders. Int J Mol Sci 2020; 21:E1503. [PMID: 32098361 PMCID: PMC7073081 DOI: 10.3390/ijms21041503] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2020] [Revised: 02/13/2020] [Accepted: 02/21/2020] [Indexed: 02/07/2023] Open
Abstract
The tightly structured neural retina has a unique vascular network comprised of three interconnected plexuses in the inner retina (and choroid for outer retina), which provide oxygen and nutrients to neurons to maintain normal function. Clinical and experimental evidence suggests that neuronal metabolic needs control both normal retinal vascular development and pathological aberrant vascular growth. Particularly, photoreceptors, with the highest density of mitochondria in the body, regulate retinal vascular development by modulating angiogenic and inflammatory factors. Photoreceptor metabolic dysfunction, oxidative stress, and inflammation may cause adaptive but ultimately pathological retinal vascular responses, leading to blindness. Here we focus on the factors involved in neurovascular interactions, which are potential therapeutic targets to decrease energy demand and/or to increase energy production for neovascular retinal disorders.
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Affiliation(s)
- Zhongjie Fu
- Department of Ophthalmology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA; (Z.F.); (Y.S.); (B.C.); (Y.T.); (S.H.); (Z.W.); (C.-H.L.); (S.S.C.); (W.B.)
- Manton Center for Orphan Disease, Boston Children’s Hospital, Boston, MA 02115, USA
| | - Ye Sun
- Department of Ophthalmology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA; (Z.F.); (Y.S.); (B.C.); (Y.T.); (S.H.); (Z.W.); (C.-H.L.); (S.S.C.); (W.B.)
| | - Bertan Cakir
- Department of Ophthalmology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA; (Z.F.); (Y.S.); (B.C.); (Y.T.); (S.H.); (Z.W.); (C.-H.L.); (S.S.C.); (W.B.)
| | - Yohei Tomita
- Department of Ophthalmology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA; (Z.F.); (Y.S.); (B.C.); (Y.T.); (S.H.); (Z.W.); (C.-H.L.); (S.S.C.); (W.B.)
| | - Shuo Huang
- Department of Ophthalmology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA; (Z.F.); (Y.S.); (B.C.); (Y.T.); (S.H.); (Z.W.); (C.-H.L.); (S.S.C.); (W.B.)
| | - Zhongxiao Wang
- Department of Ophthalmology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA; (Z.F.); (Y.S.); (B.C.); (Y.T.); (S.H.); (Z.W.); (C.-H.L.); (S.S.C.); (W.B.)
| | - Chi-Hsiu Liu
- Department of Ophthalmology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA; (Z.F.); (Y.S.); (B.C.); (Y.T.); (S.H.); (Z.W.); (C.-H.L.); (S.S.C.); (W.B.)
| | - Steve S. Cho
- Department of Ophthalmology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA; (Z.F.); (Y.S.); (B.C.); (Y.T.); (S.H.); (Z.W.); (C.-H.L.); (S.S.C.); (W.B.)
| | - William Britton
- Department of Ophthalmology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA; (Z.F.); (Y.S.); (B.C.); (Y.T.); (S.H.); (Z.W.); (C.-H.L.); (S.S.C.); (W.B.)
| | - Timothy S. Kern
- Center for Translational Vision Research, Gavin Herbert Eye Institute, Irvine, CA 92697, USA;
| | - David A. Antonetti
- Kellogg Eye Center, Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI 48105, USA;
| | - Ann Hellström
- Section for Ophthalmology, Department of Clinical Neuroscience, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, 405 30 Göteborg, Sweden;
| | - Lois E.H. Smith
- Department of Ophthalmology, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA; (Z.F.); (Y.S.); (B.C.); (Y.T.); (S.H.); (Z.W.); (C.-H.L.); (S.S.C.); (W.B.)
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18
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Ravera S, Signorello MG, Bartolucci M, Ferrando S, Manni L, Caicci F, Calzia D, Panfoli I, Morelli A, Leoncini G. Extramitochondrial energy production in platelets. Biol Cell 2018. [PMID: 29537672 DOI: 10.1111/boc.201700025] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
BACKGROUND INFORMATION Energy demand in human platelets is very high, to carry out their functions. As for most human cells, the aerobic metabolism represents the primary energy source in platelets, even though mitochondria are negligibly represented. Following the hypothesis that other structures could be involved in chemical energy production, in this work, we have investigated the functional expression of an extramitochondrial aerobic metabolism in platelets. RESULTS Oximetric and luminometric analyses showed that platelets consume large amounts of oxygen and produce ATP in the presence of common respiring substrates, such as pyruvate + malate or succinate, although morphological electron microscopy analysis showed that these contain few mitochondria. However, evaluation of the anaerobic glycolytic metabolism showed that only 13% of consumed glucose was converted to lactate. Interestingly, the highest OXPHOS activity was observed in the presence of NADH, not a readily permeant respiring substrate for mitochondria. Also, oxygen consumption and ATP synthesis fuelled by NADH were not affected by atractyloside, an inhibitor of the adenine nucleotide translocase, suggesting that these processes may not be ascribed to mitochondria. Functional data were confirmed by immunofluorescence microscopy and Western blot analyses, showing a consistent expression of the β subunit of F1 Fo -ATP synthase and COXII, a subunit of Complex IV, but a low signal of translocase of the inner mitochondrial membrane (a protein not involved in OXPHOS metabolism). Interestingly, the NADH-stimulated oxygen consumption and ATP synthesis increased in the presence of the physiological platelets agonists, thrombin or collagen. CONCLUSIONS Data suggest that in platelets, aerobic energy production is mainly driven by an extramitochondrial OXPHOS machinery, originated inside the megakaryocyte, and that this metabolism plays a pivotal role in platelet activation. SIGNIFICANCE This work represents a further example of the existence of an extramitochondrial aerobic metabolism, which can contribute to the cellular energy balance.
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Affiliation(s)
- Silvia Ravera
- Department of Pharmacy, Biochemistry Lab, University of Genova, Genova, 16132, Italy
| | | | - Martina Bartolucci
- Department of Pharmacy, Biochemistry Lab, University of Genova, Genova, 16132, Italy
| | - Sara Ferrando
- Dipartimento di Scienze della Terra, dell'Ambiente e della Vita (DISTAV), University of Genoa, Genoa, 16132, Italy
| | - Lucia Manni
- Department of Biology, Università di Padova, Padova, Italy
| | | | - Daniela Calzia
- Department of Pharmacy, Biochemistry Lab, University of Genova, Genova, 16132, Italy
| | - Isabella Panfoli
- Department of Pharmacy, Biochemistry Lab, University of Genova, Genova, 16132, Italy
| | - Alessandro Morelli
- Department of Pharmacy, Biochemistry Lab, University of Genova, Genova, 16132, Italy
| | - Giuliana Leoncini
- Department of Pharmacy, Biochemistry Lab, University of Genova, Genova, 16132, Italy
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19
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Calzia D, Garbarino G, Caicci F, Pestarino M, Manni L, Traverso CE, Panfoli I, Candiani S. Evidence of Oxidative Phosphorylation in Zebrafish Photoreceptor Outer Segments at Different Larval Stages. J Histochem Cytochem 2018; 66:497-509. [PMID: 29547350 DOI: 10.1369/0022155418762389] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Previous studies on purified bovine rod outer segments (OS) disks pointed to Oxidative Phosphorylation (OXPHOS) as being the most likely mechanism involved in ATP production, as yet not fully understood, to support the first phototransduction steps. Bovine and murine rod OS disks, devoid of mitochondria, would house respiratory chain complexes I to IV and ATP synthase, similar to mitochondria. Zebrafish ( Danio rerio) is a well-suited animal model to study vertebrate embryogenesis as well as the retina, morphologically and functionally similar to its human counterpart. The present article reports fluorescence and Transmission Electron Microscopy colocalization analyses of respiratory complexes I and IV and ATP synthase with zpr3, the rod OS marker, in adult and larval zebrafish retinas. MitoTracker Deep Red 633 staining and assays of complexes I and III-IV activity suggest that those proteins are active in OS. Results show that an extramitochondrial aerobic metabolism is active in the zebrafish OS at 4 and 10 days of larval development, as well as in adults, suggesting that it is probably maintained during embryogenesis. Data support the hypothesis of an extramitochondrial aerobic metabolism in the OS of zebrafish.
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Affiliation(s)
- Daniela Calzia
- Department of Pharmacy-DIFAR-Biochemistry and Physiology Lab, University of Genoa, Genova, Italy
| | - Greta Garbarino
- Dipartimento di Scienze della Terra, dell'Ambiente e della Vita (DISTAV), University of Genoa, Genova, Italy
| | | | - Mario Pestarino
- Dipartimento di Scienze della Terra, dell'Ambiente e della Vita (DISTAV), University of Genoa, Genova, Italy
| | - Lucia Manni
- Department of Biology, Università di Padova, Padova, Italy
| | - Carlo Enrico Traverso
- Clinica Oculistica, (Di.N.O.G.M.I.), University of Genoa, IRCCS Azienda Ospedaliera Universitaria San Martino-IST, Genova, Italy
| | - Isabella Panfoli
- Department of Pharmacy-DIFAR-Biochemistry and Physiology Lab, University of Genoa, Genova, Italy
| | - Simona Candiani
- Dipartimento di Scienze della Terra, dell'Ambiente e della Vita (DISTAV), University of Genoa, Genova, Italy
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20
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Bojinova RI, Schorderet DF, Valmaggia C, Türksever C, Schoetzau A, Todorova MG. Higher retinal vessel oxygen saturation: investigating its relationship with macular oedema in retinitis pigmentosa patients. Eye (Lond) 2018; 32:1209-1219. [PMID: 29507331 DOI: 10.1038/s41433-018-0043-1] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2017] [Revised: 01/20/2018] [Accepted: 01/28/2018] [Indexed: 01/18/2023] Open
Abstract
PURPOSE Primary objective-to investigate the effect of retinal vessel oxygen saturation (SO2) on macular oedema (ME) in retinitis pigmentosa (RP) patients. Secondary objective-to link the presence of ME to metabolic (oxygen saturation of retinal vessels, SO2), functional (multifocal electroretinography, mfERG) and structural (Spectral Domain Optical Coherent Tomography, SD-OCT) alterations in RP. DESIGN Prospective, cross-sectional, non-interventional study. SUBJECTS Patients with typical RP (N = 37) and controls (N = 19), who underwent retinal vessel Oximetry (RO), SD-OCT and mfERG, were included. METHODS A computer-based program of the retinal vessel analyser unit (IMEDOS Systems UG, Jena, Germany) was used to measure SO2. We evaluated the mean SO2, in all major retinal arterioles (oxygen saturation in retinal arterioles, A-SO2, %) and venules (oxygen saturation in retinal venules, V-SO2, %). MfERG responses were averaged in zones (zone 1 (0-3°), zone 2 (3-8°) and zone 3 (8-15°)) and compared to corresponding areas of the OCT. The effect of ME on SO2 was evaluated dividing the RP in two subgroups: with clinical appearance of ME (ME-RP) and without it (no-ME-RP). MAIN OUTCOME MEASURES Parallel recording and juxtaposition of metabolic (SO2) to structural (OCT) and functional-(mfERG) measures. Mean ( ± SD) A-SO2 and V-SO2 were higher in no-ME-RP (96.77% (±6.31) and 59.93% (±7.76)) and even higher in the ME-RP (99.82% (±6.21) and 65.63% (±7.63)), compared to controls (93.15% (±3.76) and 53.77% (±3.70), p ≤ 0.006). RESULTS The subgroup ME-RP differed significantly from the subgroup no-ME-RP by increased A-SO2 and V-SO2, p ≤ 0.026. The presence of ME confirmed a different relationship between the altered SO2 and the vessel diameters, against the functional and structural parameters. CONCLUSION Based on our results, the presence of macular oedema indicates a tendency toward greater alteration of the metabolic function in RP patients.
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Affiliation(s)
- Rossiana I Bojinova
- Department of Ophthalmology, University of Basel, Mittlere Strasse 91, Basel, CH-4031, Switzerland.,University of Montreal, 495 Prince Arthur West, Montreal, H2X1T4, Canada
| | - Daniel F Schorderet
- IRO-Institute for Research in Ophthalmology, Sion, Switzerland.,Department of Ophthalmology, University of Lausanne, Lausanne, Switzerland.,School of Life Sciences, Federal Institute of Technology, Lausanne, Switzerland
| | | | - Cengiz Türksever
- Department of Ophthalmology, University of Basel, Mittlere Strasse 91, Basel, CH-4031, Switzerland.,VISTA Klinik, Binningen, Baselland, Switzerland
| | - Andreas Schoetzau
- Department of Ophthalmology, University of Basel, Mittlere Strasse 91, Basel, CH-4031, Switzerland
| | - Margarita G Todorova
- Department of Ophthalmology, University of Basel, Mittlere Strasse 91, Basel, CH-4031, Switzerland.
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Calzia D, Degan P, Caicci F, Bruschi M, Manni L, Ramenghi LA, Candiano G, Traverso CE, Panfoli I. Modulation of the rod outer segment aerobic metabolism diminishes the production of radicals due to light absorption. Free Radic Biol Med 2018; 117:110-118. [PMID: 29378336 DOI: 10.1016/j.freeradbiomed.2018.01.029] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/07/2017] [Revised: 01/17/2018] [Accepted: 01/23/2018] [Indexed: 12/19/2022]
Abstract
Oxidative stress is a primary risk factor for both inflammatory and degenerative retinopathies. Our previous data on blue light-irradiated retinas demonstrated an oxidative stress higher in the rod outer segment (OS) than in the inner limb, leading to impairment of the rod OS extra-mitochondrial aerobic metabolism. Here the oxidative metabolism and Reactive Oxygen Intermediates (ROI) production was evaluated in purified bovine rod OS in function of exposure to different illumination conditions. A dose response was observed to varying light intensities and duration in terms of both ROI production and ATP synthesis. Pretreatment with resveratrol, inhibitor of F1Fo-ATP synthase, or metformin, inhibitor of the respiratory complex I, significantly diminished the ROI production. Metformin also diminished the rod OS Complex I activity and reduced the maximal OS response to light in ATP production. Data show for the first time the relationship existing in the rod OS between its -aerobic- metabolism, light absorption, and ROI production. A beneficial effect was exerted by metformin and resveratrol, in modulating the ROI production in the illuminated rod OS, suggestive of their beneficial action also in vivo. Data shed new light on preventative interventions for cone loss secondary to rod damage due to oxidative stress.
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Affiliation(s)
- Daniela Calzia
- Dipartimento di Farmacia-DIFAR,-Biochemistry Lab., University of Genoa, V.le Benedetto XV 3, 16132 Genova, Italy.
| | - Paolo Degan
- UOC Mutagenesi, IRCCS AOU San Martino - IST (Istituto Nazionale per la Ricerca sul Cancro), Genova, Italy
| | - Federico Caicci
- Department of Biology, Università di Padova, via U. Bassi 58/B, 35121 Padova, Italy
| | - Maurizio Bruschi
- Laboratory of Pathophysiology of Uremia, Istituto Giannina Gaslini, Genova, Italy
| | - Lucia Manni
- Department of Biology, Università di Padova, via U. Bassi 58/B, 35121 Padova, Italy
| | - Luca A Ramenghi
- Neonatal Intensive Care Unit, U.O.S. Malattie Metaboliche, V.le Benedetto XV 6, Genova, Italy
| | - Giovanni Candiano
- Neonatal Intensive Care Unit, U.O.S. Malattie Metaboliche, V.le Benedetto XV 6, Genova, Italy
| | - Carlo Enrico Traverso
- Clinica Oculistica, (DINOGMI) University of Genoa, V.le Benedetto XV 6, Genova, Italy
| | - Isabella Panfoli
- Dipartimento di Farmacia-DIFAR,-Biochemistry Lab., University of Genoa, V.le Benedetto XV 3, 16132 Genova, Italy
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22
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Taurino F, Gnoni A. Systematic review of plasma-membrane ecto-ATP synthase: A new player in health and disease. Exp Mol Pathol 2018; 104:59-70. [DOI: 10.1016/j.yexmp.2017.12.006] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2017] [Revised: 12/15/2017] [Accepted: 12/31/2017] [Indexed: 02/07/2023]
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Bruschi M, Petretto A, Caicci F, Bartolucci M, Calzia D, Santucci L, Manni L, Ramenghi LA, Ghiggeri G, Traverso CE, Candiano G, Panfoli I. Proteome of Bovine Mitochondria and Rod Outer Segment Disks: Commonalities and Differences. J Proteome Res 2018; 17:918-925. [PMID: 29299929 DOI: 10.1021/acs.jproteome.7b00741] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The retinal rod outer segment (OS) is a stack of disks surrounded by the plasma membrane, housing proteins related to phototransduction, as well as mitochondrial proteins involved in oxidative phosphorylation (OxPhos). This prompted us to compare the proteome of bovine OS disks and mitochondria to assess the significant top gene signatures of each sample. The two proteomes, obtained by LTQ-Orbitrap Velos mass spectrometry, were compared by statistical analyses. In total, 4139 proteins were identified, 2045 of which overlapping in the two sets. Nonhierarchical Spearman's correlogram revealed that the groups were clearly discriminated. Partial least square discriminant plus support vector machine analysis identified the major discriminative proteins, implied in phototransduction and lipid metabolism, respectively. Gene Ontology analysis identified top gene signatures of the disk proteome, enriched in vesiculation, glycolysis, and OxPhos proteins. The tricarboxylic acid cycle and the electron transport proteins were similarly enriched in the two samples, but the latter was up regulated in disks. Data suggest that the mitochondrial OxPhos proteins may represent a true OS proteome component, outside the mitochondrion. This knowledge may help the scientific community in the further studies of retinal physiology and pathology.
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Affiliation(s)
| | | | - Federico Caicci
- Department of Biology, Università di Padova , 35121 Padova, Italy
| | | | - Daniela Calzia
- Dipartimento di Farmacia-DIFAR, Università di Genova , 16132 Genoa, Italy
| | | | - Lucia Manni
- Department of Biology, Università di Padova , 35121 Padova, Italy
| | | | | | - Carlo E Traverso
- Clinica Oculistica, (Di.N.O.G.M.I.) Università Department of Intensive Care di Genova, IRCCS Azienda Ospedaliera Universitaria San Martino-IST , 16132 Genoa, Italy
| | | | - Isabella Panfoli
- Dipartimento di Farmacia-DIFAR, Università di Genova , 16132 Genoa, Italy
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24
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Todorova MG. Metabolic, structural and functional alterations in patients with inherited diseases of the retina. Acta Ophthalmol 2017. [DOI: 10.1111/aos.13524] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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25
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Bojinova RI, Türksever C, Schötzau A, Valmaggia C, Schorderet DF, Todorova MG. Reduced metabolic function and structural alterations in inherited retinal dystrophies: investigating the effect of peripapillary vessel oxygen saturation and vascular diameter on the retinal nerve fibre layer thickness. Acta Ophthalmol 2017; 95:252-261. [PMID: 27682439 DOI: 10.1111/aos.13247] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2015] [Accepted: 07/31/2016] [Indexed: 12/13/2022]
Abstract
PURPOSE To evaluate the relationship between the peripapillary metabolic alterations [retinal vessel Oximetry (RO)] and the structural findings [retinal vessel diameter and retinal nerve fibre layer thickness (RNFL)] in patients with inherited retinal dystrophies (IRD). METHODS Patients with IRD [24 patients with rod-cone dystrophy (RCD), 15 patients with cone-rod dystrophy, 13 patients with inherited maculopathy] and 18 age-matched controls, who underwent RO imaging and spectral domain optical coherence tomography, were included. The average and quadrant oxygen saturation in all four major peripapillary retinal arterioles (A-SO2 ) and venules (V-SO2 ) were measured, and their difference (A-V SO2 ) was calculated. The corresponding retinal vessel diameter of these arterioles (D-A) and venules (D-V) was measured. The data were compared to the peripapillary RNFL thickness within the IRD subgroups and to the data obtained in the controls. RESULTS In general, patients with IRD had higher average V-SO2 values when compared to controls (p ≤ 0.029). Rod-cone dystrophy (RCD) patients differed from controls, but also from patients with other IRDs, when the average and quadrant oxygen saturation values (A-SO2 and V-SO2 ) were evaluated (p ≤ 0.026). Within the RCD group, the correlations of RNFL thickness to V-SO2 , A-V SO2 , D-A and D-V were significant (p ≤ 0.030), thus indicating a different relationship between the RNFL thickness and the examined parameters, when compared to the other groups. CONCLUSION It becomes evident from our combined metabolic-structural approach that a prediction model, to identify which individual is at risk of developing a photoreceptor degeneration of RCD type, can be proposed. It will take into account the peripapillary retinal oxygen saturation, the retinal vessel diameter and the RNFL thickness values.
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Affiliation(s)
- Rossiana I. Bojinova
- Department of Ophthalmology; University of Basel; Basel Switzerland
- McGill University; Montreal Canada
| | - Cengiz Türksever
- Department of Ophthalmology; University of Basel; Basel Switzerland
| | - Andreas Schötzau
- Department of Ophthalmology; University of Basel; Basel Switzerland
| | | | - Daniel F. Schorderet
- IRO - Institute for Research in Ophthalmology; Sion Switzerland
- Department of Ophthalmology; University of Lausanne; Lausanne Switzerland
- School of Life Sciences; Federal Institute of Technology; Lausanne Switzerland
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26
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Preserved functional and structural integrity of the papillomacular area correlates with better visual acuity in retinitis pigmentosa. Eye (Lond) 2016; 30:1310-1323. [PMID: 27494084 DOI: 10.1038/eye.2016.136] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2015] [Accepted: 05/05/2016] [Indexed: 12/25/2022] Open
Abstract
PurposeLinking multifocal electroretinography (mfERG) and optical coherence tomography (OCT) findings with visual acuity in retinitis pigmentosa (RP) patients.DesignProspective, cross-sectional, nonintervention study.SubjectsPatients with typical RP and age-matched controls, who underwent SD-OCT (spectral domain OCT) and mfERG, were included.MethodsMfERG responses were averaged in three zones (zone 1 (0°-3°), zone 2 (3°-8°), and zone 3 (8°-15°)). Baseline-to-trough- (N1) and trough-to-peak amplitudes (N1P1) of the mfERG were compared with corresponding areas of the OCT. The papillomacular area (PMA) was analyzed separately. Correlations between best-corrected visual acuity (BCVA, logMAR) and each parameter were determined.Main outcome measuresComparing structural (OCT) and functional (mfERG) measures with the BCVA.ResultsIn RP patients, the N1 and N1P1 responses showed positive association with the central retinal thickness outside zone 1 (P≤0.002), while the central N1 and the N1P1 responses in zones 1, 2, and 3-with the BCVA (P≤0.007). The integrity of the IS/OS line on OCT showed also a positive association with the BCVA (P<0.001). Isolated analysis of the PMA strengthened further the structure-function association with the BCVA (P≤0.037). Interactions between the BCVA and the OCT, respectively, the mfERG parameters were more pronounced in the RP subgroup without macular edema (P≤0.020).ConclusionIn RP patients, preserved structure-function of PMA, measured by mfERG amplitude and OCT retinal thickness, correlated well with the remaining BCVA. The subgroup analyses revealed stronger links between the examined parameters, in the RP subgroup without appearance of macular edema.
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27
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Impairment of extramitochondrial oxidative phosphorylation in mouse rod outer segments by blue light irradiation. Biochimie 2016; 125:171-8. [DOI: 10.1016/j.biochi.2016.03.016] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2015] [Accepted: 03/30/2016] [Indexed: 01/30/2023]
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Why do premature newborn infants display elevated blood adenosine levels? Med Hypotheses 2016; 90:53-6. [PMID: 27063086 DOI: 10.1016/j.mehy.2016.03.007] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2016] [Accepted: 03/07/2016] [Indexed: 11/27/2022]
Abstract
Our preliminary data show high levels of adenosine in the blood of very low birth weight (VLBW) infants, positively correlating to their prematurity (i.e. body weight class). This prompted us to look for a mechanism promoting such impressive adenosine increase. We hypothesized a correlation with oxygen challenge. In fact, it is recognized that either oxygen lack or its excess contribute to the pathogenesis of the injuries of prematurity, such as retinopathy (ROP) and periventricular white matter lesions (PWMI). The optimal concentration of oxygen for resuscitation of VLBW infants is currently under revision. We propose that the elevated adenosine blood concentrations of VLBW infants recognizes two sources. The first could be its activity-dependent release from unmyelinated brain axons. Adenosine in this respect would be an end-product of the hypometabolic VLBW newborn unmyelinated axon intensely firing in response to the environmental stimuli consequent to premature birth. Adenosine would be eventually found in the blood due to blood-brain barrier immaturity. In fact, adenosine is the primary activity-dependent signal promoting differentiation of premyelinating oligodendrocyte progenitor cells (OPC) into myelinating cells in the Central Nervous System, while inhibiting their proliferation and inhibiting synaptic function. The second, would be the ecto-cellular ATP synthesized by the endothelial cell plasmalemma exposed to ambient oxygen concentrations due to premature breathing, especially in lung. ATP would be rapidly transformed into adenosine by the ectonucleotidase activities such as NTPDase I (CD39), and NT5E (CD73). An ectopic extra-mitochondrial aerobic ATP synthetic ability was reported in many cell plasma-membranes, among which endothelial cells. The potential implications of the cited hypotheses for the neonatology area would be great. The amount of oxygen administration for reviving of newborns would find a molecular basis for its assessment. VLBW infants may be regarded as those in which premature exposure to ambient oxygen concentrations and oxidative stress causes a premature functioning of the extra-mitochondrial oxidative phosphorylation primarily in axons and endothelium. Adenosine may become a biomarker of prematurity risk, whose implications further studies may assess.
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Bruschi M, Santucci L, Ravera S, Candiano G, Bartolucci M, Calzia D, Lavarello C, Inglese E, Ramenghi LA, Petretto A, Ghiggeri GM, Panfoli I. Human urinary exosome proteome unveils its aerobic respiratory ability. J Proteomics 2016; 136:25-34. [PMID: 26850698 DOI: 10.1016/j.jprot.2016.02.001] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2016] [Revised: 01/28/2016] [Accepted: 02/01/2016] [Indexed: 12/13/2022]
Abstract
UNLABELLED Exosomes are 40-100-nm vesicles released by most cell types after fusion of multivesicular endosomes with the plasma membrane. Exosomes, ubiquitary in body fluids including urines, contain proteins and RNA species specific of the tissue of origin. Exosomes from urine have been extensively studied as a promising reservoir for disease biomarkers. Here, we report the proteome analysis of urinary exosomes compared to urinoma, studied by Orbitrap mass spectrometry. A discovery approach was utilized on the sample. 3429 proteins were present, with minimal overlapping among exosome and urinoma. 959 proteins (28%) in exosome and 1478 proteins (43%) in urinoma were exclusively present in only one group. By cytoscape analysis, the biological process gene ontology was correlated to their probability (P ≤ 0.05) to be functional. This was never studied before and showed a significant clustering around metabolic functions, in particular to aerobic ATP production. Urinary exosomes carry out oxidative phosphorylation, being able to synthesize ATP and consume oxygen. A previously unsuspected function emerges for human urinary exosomes as bioactive vesicles that consume oxygen to aerobically synthesize ATP. Determination of normal human urine proteome can help generate the healthy urinary protein database for comparison, useful for various renal diseases. BIOLOGICAL SIGNIFICANCE The findings reported represent a significant advance in the understanding of the healthy human urinary proteome. The methodology utilized to analyze the collection of proteomic data allowed the assessment of the unique composition of urinary exosomes with respect to urinoma and to elucidate the presence in the former of molecular pathways previously unknown. The paper has the potential to impact its field of research, due to the biological relevance of the metabolic capacity of urinary exosomes, which may represent their important general feature.
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Affiliation(s)
- Maurizio Bruschi
- Division of Nephrology, Dialysis, and Transplantation and Laboratory on Pathophysiology of Uremia, Istituto Giannina Gaslini, Genoa, Italy
| | - Laura Santucci
- Division of Nephrology, Dialysis, and Transplantation and Laboratory on Pathophysiology of Uremia, Istituto Giannina Gaslini, Genoa, Italy
| | - Silvia Ravera
- Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy
| | - Giovanni Candiano
- Division of Nephrology, Dialysis, and Transplantation and Laboratory on Pathophysiology of Uremia, Istituto Giannina Gaslini, Genoa, Italy
| | - Martina Bartolucci
- Laboratory of Mass Spectrometry - Core Facilities, Istituto Giannina Gaslini, Genova, Italy
| | - Daniela Calzia
- Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy
| | - Chiara Lavarello
- Laboratory of Mass Spectrometry - Core Facilities, Istituto Giannina Gaslini, Genova, Italy
| | - Elvira Inglese
- Laboratory of Mass Spectrometry - Core Facilities, Istituto Giannina Gaslini, Genova, Italy
| | - Luca A Ramenghi
- Neonatal Intensive Care Unit, Istituto Giannina Gaslini, Genoa, Italy
| | - Andrea Petretto
- Laboratory of Mass Spectrometry - Core Facilities, Istituto Giannina Gaslini, Genova, Italy
| | - Gian Marco Ghiggeri
- Division of Nephrology, Dialysis, and Transplantation and Laboratory on Pathophysiology of Uremia, Istituto Giannina Gaslini, Genoa, Italy
| | - Isabella Panfoli
- Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy.
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Panfoli I, Ravera S, Podestà M, Cossu C, Santucci L, Bartolucci M, Bruschi M, Calzia D, Sabatini F, Bruschettini M, Ramenghi LA, Romantsik O, Marimpietri D, Pistoia V, Ghiggeri G, Frassoni F, Candiano G. Exosomes from human mesenchymal stem cells conduct aerobic metabolism in term and preterm newborn infants. FASEB J 2015; 30:1416-24. [PMID: 26655706 DOI: 10.1096/fj.15-279679] [Citation(s) in RCA: 55] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2015] [Accepted: 11/23/2015] [Indexed: 01/13/2023]
Abstract
Exosomes are secreted nanovesicles that are able to transfer RNA and proteins to target cells. The emerging role of mesenchymal stem cell (MSC) exosomes as promoters of aerobic ATP synthesis restoration in damaged cells, prompted us to assess whether they contain an extramitochondrial aerobic respiration capacity. Exosomes were isolated from culture medium of human MSCs from umbilical cord of ≥37-wk-old newborns or between 28- to 30-wk-old newborns (i.e.,term or preterm infants). Characterization of samples was conducted by cytofluorometry. Oxidative phosphorylation capacity was assessed by Western blot analysis, oximetry, and luminometric and fluorometric analyses. MSC exosomes express functional respiratory complexes I, IV, and V, consuming oxygen. ATP synthesis was only detectable in exosomes from term newborns, suggestive of a specific mechanism that is not completed at an early gestational age. Activities are outward facing and comparable to those detected in mitochondria isolated from term MSCs. MSC exosomes display an unsuspected aerobic respiratory ability independent of whole mitochondria. This may be relevant for their ability to rescue cell bioenergetics. The differential oxidative metabolism of pretermvs.term exosomes sheds new light on the preterm newborn's clinical vulnerability. A reduced ability to repair damaged tissue and an increased capability to cope with anoxic environment for preterm infants can be envisaged.-Panfoli, I., Ravera, S., Podestà, M., Cossu, C., Santucci, L., Bartolucci, M., Bruschi, M., Calzia, D., Sabatini, F., Bruschettini, M., Ramenghi, L. A., Romantsik, O., Marimpietri, D., Pistoia, V., Ghiggeri, G., Frassoni, F., Candiano, G. Exosomes from human mesenchymal stem cells conduct aerobic metabolism in term and preterm newborn infants.
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Affiliation(s)
- Isabella Panfoli
- *Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy
| | - Silvia Ravera
- *Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy
| | - Marina Podestà
- *Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy
| | - Claudia Cossu
- *Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy
| | - Laura Santucci
- *Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy
| | - Martina Bartolucci
- *Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy
| | - Maurizio Bruschi
- *Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy
| | - Daniela Calzia
- *Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy
| | - Federica Sabatini
- *Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy
| | - Matteo Bruschettini
- *Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy
| | - Luca Antonio Ramenghi
- *Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy
| | - Olga Romantsik
- *Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy
| | - Danilo Marimpietri
- *Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy
| | - Vito Pistoia
- *Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy
| | - Gianmarco Ghiggeri
- *Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy
| | - Francesco Frassoni
- *Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy
| | - Giovanni Candiano
- *Dipartimento di Farmacia, Laboratorio di Biochimica, Università di Genova, Genoa, Italy; and Laboratorio Cellule Staminali Post-Natali e Terapie Cellulari, Laboratory of Pathophysiology of Uremia, Neonatal Intensive Care Unit, and Laboratorio Oncologia, Istituto Giannina Gaslini, Genoa, Italy
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Lopez Torres LT, Türksever C, Schötzau A, Orgül S, Todorova MG. Peripapillary retinal vessel diameter correlates with mfERG alterations in retinitis pigmentosa. Acta Ophthalmol 2015; 93:e527-33. [PMID: 25809154 DOI: 10.1111/aos.12707] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2014] [Accepted: 02/06/2015] [Indexed: 12/25/2022]
Abstract
PURPOSE To investigate relationship between the peripapillary retinal vessel diameter and the residual retinal function, measured by mfERG, in patients with retinitis pigmentosa (RP). PATIENTS AND METHODS A cross-sectional study based on 23 patients with RP (43 eyes) and 20 controls (40 eyes) was performed. Retinal vessel diameters were measured using a computer-based program of the retinal vessel analyser (RVA; IMEDOS Systems UG, Jena, Germany). We evaluated the mean diameter in all four major retinal arterioles (D-A) and venules (D-V) within 1.0-1.5 optic disc diameters from the disc margin. The data were compared with the N1 amplitudes (measured from the baseline to the trough of the first negative wave), with the N1P1 amplitudes (measured from the trough of the first negative wave to the peak of the first positive wave) of the mfERG overall response and with the mfERG responses averaged in zones [zone 1 (0°-3°), zone 2 (3°-8°), zone 3 (8°-15°) and zone 4 (15°-24°)]. RESULTS Mean (±SD) D-A and D-V were narrower in patients with RP [84.86 μm (±13.37 μm) and 103.35 μm (±13.65 μm), respectively] when compared to controls [92.81 μm (±11.49 μm) and 117.67 μm (±11.93 μm), respectively; the p-values between groups were p = 0.003 for D-A and p < 0.001 for D-V, linear mixed-effects model]. The RP group revealed clear differences compared to the controls: D-A and D-V became narrower with reduced mfERG responses. D-V correlated significantly with the overall mfERG N1 amplitudes (p = 0.013) and with N1P1 amplitudes (p = 0.016). D-V correlated with the mfERG amplitudes averaged in zones: (zone 2, 3 and 4; p ≤ 0.040) and N1P1 mfERG amplitudes (zones 1, 2, 3 and 4; p ≤ 0.013). CONCLUSIONS Peripapillary retinal vessel diameter is reduced in RP proportionally to functional alterations.
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Affiliation(s)
| | - Cengiz Türksever
- Department of Ophthalmology; University of Basel; Basel Switzerland
| | - Andreas Schötzau
- Department of Ophthalmology; University of Basel; Basel Switzerland
| | - Selim Orgül
- Department of Ophthalmology; University of Basel; Basel Switzerland
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Bartolucci M, Ravera S, Garbarino G, Ramoino P, Ferrando S, Calzia D, Candiani S, Morelli A, Panfoli I. Functional Expression of Electron Transport Chain and FoF1-ATP Synthase in Optic Nerve Myelin Sheath. Neurochem Res 2015; 40:2230-41. [PMID: 26334391 DOI: 10.1007/s11064-015-1712-0] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2015] [Revised: 08/23/2015] [Accepted: 08/27/2015] [Indexed: 12/15/2022]
Abstract
Our previous studies reported evidence for aerobic ATP synthesis by myelin from both bovine brainstem and rat sciatic nerve. Considering that the optic nerve displays a high oxygen demand, here we evaluated the expression and activity of the five Respiratory Complexes in myelin purified from either bovine or murine optic nerves. Western blot analyses on isolated myelin confirmed the expression of ND4L (subunit of Complex I), COX IV (subunit of Complex IV) and β subunit of F1Fo-ATP synthase. Moreover, spectrophotometric and in-gel activity assays on isolated myelin, as well as histochemical activity assays on both bovine and murine transversal optic nerve sections showed that the respiratory Complexes are functional in myelin and are organized in a supercomplex. Expression of oxidative phosphorylation proteins was also evaluated on bovine optic nerve sections by confocal and transmission electron microscopy. Having excluded a mitochondrial contamination of isolated myelin and considering the results form in situ analyses, it is proposed that the oxidative phosphorylation machinery is truly resident in optic myelin sheath. Data may shed a new light on the unknown trophic role of myelin sheath. It may be energy supplier for the axon, explaining why in demyelinating diseases and neuropathies, myelin sheath loss is associated with axonal degeneration.
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Affiliation(s)
- Martina Bartolucci
- Biochemistry Laboratory, Department of Pharmacy (DIFAR), University of Genova, Viale Benedetto XV, 3, 16132, Genoa, Italy
| | - Silvia Ravera
- Biochemistry Laboratory, Department of Pharmacy (DIFAR), University of Genova, Viale Benedetto XV, 3, 16132, Genoa, Italy.
| | - Greta Garbarino
- Department of Earth, Environmental and Life Sciences, (DISTAV), University of Genova, C.so Europa 26, 16132, Genoa, Italy
| | - Paola Ramoino
- Department of Earth, Environmental and Life Sciences, (DISTAV), University of Genova, C.so Europa 26, 16132, Genoa, Italy
| | - Sara Ferrando
- Department of Earth, Environmental and Life Sciences, (DISTAV), University of Genova, C.so Europa 26, 16132, Genoa, Italy
| | - Daniela Calzia
- Biochemistry Laboratory, Department of Pharmacy (DIFAR), University of Genova, Viale Benedetto XV, 3, 16132, Genoa, Italy
| | - Simona Candiani
- Department of Earth, Environmental and Life Sciences, (DISTAV), University of Genova, C.so Europa 26, 16132, Genoa, Italy
| | - Alessandro Morelli
- Biochemistry Laboratory, Department of Pharmacy (DIFAR), University of Genova, Viale Benedetto XV, 3, 16132, Genoa, Italy
| | - Isabella Panfoli
- Biochemistry Laboratory, Department of Pharmacy (DIFAR), University of Genova, Viale Benedetto XV, 3, 16132, Genoa, Italy
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Fernández-Sánchez L, Lax P, Noailles A, Angulo A, Maneu V, Cuenca N. Natural Compounds from Saffron and Bear Bile Prevent Vision Loss and Retinal Degeneration. Molecules 2015; 20:13875-93. [PMID: 26263962 PMCID: PMC6332441 DOI: 10.3390/molecules200813875] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2015] [Revised: 07/23/2015] [Accepted: 07/28/2015] [Indexed: 12/13/2022] Open
Abstract
All retinal disorders, regardless of their aetiology, involve the activation of oxidative stress and apoptosis pathways. The administration of neuroprotective factors is crucial in all phases of the pathology, even when vision has been completely lost. The retina is one of the most susceptible tissues to reactive oxygen species damage. On the other hand, proper development and functioning of the retina requires a precise balance between the processes of proliferation, differentiation and programmed cell death. The life-or-death decision seems to be the result of a complex balance between pro- and anti-apoptotic signals. It has been recently shown the efficacy of natural products to slow retinal degenerative process through different pathways. In this review, we assess the neuroprotective effect of two compounds used in the ancient pharmacopoeia. On one hand, it has been demonstrated that administration of the saffron constituent safranal to P23H rats, an animal model of retinitis pigmentosa, preserves photoreceptor morphology and number, the capillary network and the visual response. On the other hand, it has been shown that systemic administration of tauroursodeoxycholic acid (TUDCA), the major component of bear bile, to P23H rats preserves cone and rod structure and function, together with their contact with postsynaptic neurons. The neuroprotective effects of safranal and TUDCA make these compounds potentially useful for therapeutic applications in retinal degenerative diseases.
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Affiliation(s)
- Laura Fernández-Sánchez
- Departament of Physiology, Genetics and Microbiology, University of Alicante, 03690 Alicante, Spain.
| | - Pedro Lax
- Departament of Physiology, Genetics and Microbiology, University of Alicante, 03690 Alicante, Spain.
| | - Agustina Noailles
- Departament of Physiology, Genetics and Microbiology, University of Alicante, 03690 Alicante, Spain.
| | - Antonia Angulo
- Department of Optics, Pharmacology and Anatomy, University of Alicante, 03690 Alicante, Spain.
| | - Victoria Maneu
- Department of Optics, Pharmacology and Anatomy, University of Alicante, 03690 Alicante, Spain.
| | - Nicolás Cuenca
- Departament of Physiology, Genetics and Microbiology, University of Alicante, 03690 Alicante, Spain.
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Bruschi M, Ravera S, Santucci L, Candiano G, Bartolucci M, Calzia D, Lavarello C, Inglese E, Petretto A, Ghiggeri G, Panfoli I. The human urinary exosome as a potential metabolic effector cargo. Expert Rev Proteomics 2015; 12:425-32. [DOI: 10.1586/14789450.2015.1055324] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
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Calzia D, Oneto M, Caicci F, Bianchini P, Ravera S, Bartolucci M, Diaspro A, Degan P, Manni L, Traverso CE, Panfoli I. Effect of polyphenolic phytochemicals on ectopic oxidative phosphorylation in rod outer segments of bovine retina. Br J Pharmacol 2015; 172:3890-903. [PMID: 25917043 DOI: 10.1111/bph.13173] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2014] [Revised: 03/24/2015] [Accepted: 04/20/2015] [Indexed: 12/25/2022] Open
Abstract
BACKGROUND AND PURPOSE The rod outer segments (OS) of the retina are specialized organelles where phototransduction takes place. The mitochondrial electron transport complexes I-IV, cytochrome c and Fo F1 -ATP synthase are functionally expressed in the OS disks. Here, we have studied the effect of some polyphenolic compounds acting as inhibitors of mitochondrial ATPase/synthase activity on the OS ectopic Fo F1 - ATP synthase. The mechanism of apoptosis in the OS was also investigated studying the expression of cytochrome c, caspase 9 and 3 and Apaf-1. EXPERIMENTAL APPROACH We prepared OS from fresh bovine retinae. Semi-quantitative Western blotting, confocal and electron microscopy, and cytofluorimetry were used along with biochemical analyses such as oximetry, ATP synthesis and hydrolysis. KEY RESULTS Resveratrol and curcumin plus piperine inhibited ATP synthesis and oxygen consumption in the OS. Epigallocatechin gallate and quercetin inhibited ATP hydrolysis and oxygen consumption in the OS. Malondialdehyde and hydrogen peroxide were produced in respiring OS in the presence of substrates. Cytochrome c was located inside the disk membranes. Procaspase 9 and 3, as well as Apaf-1 were expressed in the OS. CONCLUSIONS AND IMPLICATIONS These polyphenolic phytochemicals modulated the Fo F1 -ATP synthase activity of the the OS reducing production of reactive oxygen intermediates by the OS ectopic electron transport chain. Polyphenols decrease membrane peroxidation and cytochrome c release from disks, preventing the induction of caspase-dependent apoptosis in the OS Such effects are relevant in the design of protection against functional impairment of the OS following oxidative stress from exposure to intense illumination.
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Affiliation(s)
- Daniela Calzia
- Department of Pharmacy-DIFAR, Biochemistry and Physiology Laboratory, University of Genoa, Genova, Italy
| | - Michele Oneto
- Department of Pharmacy-DIFAR, Biochemistry and Physiology Laboratory, University of Genoa, Genova, Italy
| | | | - Paolo Bianchini
- Department of Nanophysics, Istituto Italiano di Tecnologia, Genova, Italy
| | - Silvia Ravera
- Department of Pharmacy-DIFAR, Biochemistry and Physiology Laboratory, University of Genoa, Genova, Italy
| | - Martina Bartolucci
- Department of Pharmacy-DIFAR, Biochemistry and Physiology Laboratory, University of Genoa, Genova, Italy
| | - Alberto Diaspro
- Department of Nanophysics, Istituto Italiano di Tecnologia, Genova, Italy
| | - Paolo Degan
- UOC Mutagenesi, IRCCS Azienda Ospedaliera Universitaria San Martino - Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
| | - Lucia Manni
- Department of Biology, Università di Padova, Padova, Italy
| | - Carlo Enrico Traverso
- Clinica Oculistica, Di.N.O.G.M.I., University of Genoa, IRCCS Azienda Ospedaliera Universitaria San Martino - Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
| | - Isabella Panfoli
- Department of Pharmacy-DIFAR, Biochemistry and Physiology Laboratory, University of Genoa, Genova, Italy
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Ravera S, Bartolucci M, Cuccarolo P, Litamè E, Illarcio M, Calzia D, Degan P, Morelli A, Panfoli I. Oxidative stress in myelin sheath: The other face of the extramitochondrial oxidative phosphorylation ability. Free Radic Res 2015; 49:1156-64. [PMID: 25971447 DOI: 10.3109/10715762.2015.1050962] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Oxidative phosphorylation (OXPHOS) is not only the main source of ATP for the cell, but also a major source of reactive oxygen species (ROS), which lead to oxidative stress. At present, mitochondria are considered the organelles responsible for the OXPHOS, but in the last years we have demonstrated that it can also occur outside the mitochondrion. Myelin sheath is able to conduct an aerobic metabolism, producing ATP that we have hypothesized is transferred to the axon, to support its energetic demand. In this work, spectrophotometric, cytofluorimetric, and luminometric analyses were employed to investigate the oxidative stress production in isolated myelin, as far as its respiratory activity is concerned. We have evaluated the levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), markers of lipid peroxidation, as well as of hydrogen peroxide (H2O2), marker of ROS production. To assess the presence of endogenous antioxidant systems, superoxide dismutase, catalase, and glutathione peroxidase activities were assayed. The effect of certain uncoupling or antioxidant molecules on oxidative stress in myelin was also investigated. We report that isolated myelin produces high levels of MDA, 4-HNE, and H2O2, likely through the pathway composed by Complex I-III-IV, but it also contains active superoxide dismutase, catalase, and glutathione peroxidase, as antioxidant defense. Uncoupling compounds or Complex I inhibitors increase oxidative stress, while antioxidant compounds limit ROS generation. Data may shed new light on the role of myelin sheath in physiology and pathology. In particular, it can be presumed that the axonal degeneration associated with myelin loss in demyelinating diseases is related to oxidative stress caused by impaired OXPHOS.
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Affiliation(s)
- S Ravera
- Department of Pharmacy (DIFAR), Biochemistry Laboratory, University of Genova , Genova , Italy
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Funk RHW, Schumann U, Engelmann K, Becker KA, Roehlecke C. Blue light induced retinal oxidative stress: Implications for macular degeneration. World J Ophthalmol 2014; 4:29-34. [DOI: 10.5318/wjo.v4.i3.29] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/13/2014] [Revised: 03/19/2014] [Accepted: 06/11/2014] [Indexed: 02/05/2023] Open
Abstract
A number of studies have shown that oxidative stress can be harmful for the retina. The real causal circumstances that lead to degenerative diseases like age related macular degeneration remain obscure. Whether light induced radical stress is a direct interaction of light with photoreceptors or a secondary mechanism within the pigment epithelium or choroid is in discussion. Among the molecular mechanisms involved are production of reactive oxygen species (ROS), secondary lipid peroxidation, protein oxidation and DNA-damage. The initial trigger to write this review was first a recent finding of our group that the photoreceptor outer segments produce great amounts of ROS and second the detection of ectopic enzymes of the respiratory chain localized there - in addition to the hitherto known ROS sources like the visual pigments with their intermediates and the photoreceptor mitochondria harbouring the respiratory chain.
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Calzia D, Garbarino G, Caicci F, Manni L, Candiani S, Ravera S, Morelli A, Traverso CE, Panfoli I. Functional expression of electron transport chain complexes in mouse rod outer segments. Biochimie 2014; 102:78-82. [PMID: 24565809 DOI: 10.1016/j.biochi.2014.02.007] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2013] [Accepted: 02/14/2014] [Indexed: 10/25/2022]
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.
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Affiliation(s)
- Daniela Calzia
- Department of Pharmacy, DIFAR-Biochemistry Lab, University of Genova, Italy.
| | | | | | - Lucia Manni
- Department of Biology, Università di Padova, Italy
| | | | - Silvia Ravera
- Department of Pharmacy, DIFAR-Biochemistry Lab, University of Genova, Italy
| | - Alessandro Morelli
- Department of Pharmacy, DIFAR-Biochemistry Lab, University of Genova, Italy
| | | | - Isabella Panfoli
- Department of Pharmacy, DIFAR-Biochemistry Lab, University of Genova, Italy
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Antioxidant drug therapy approaches for neuroprotection in chronic diseases of the retina. Int J Mol Sci 2014; 15:1865-86. [PMID: 24473138 PMCID: PMC3958826 DOI: 10.3390/ijms15021865] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2013] [Revised: 01/18/2014] [Accepted: 01/21/2014] [Indexed: 12/31/2022] Open
Abstract
The molecular pathways contributing to visual signal transduction in the retina generate a high energy demand that has functional and structural consequences such as vascularization and high metabolic rates contributing to oxidative stress. Multiple signaling cascades are involved to actively regulate the redox state of the retina. Age-related processes increase the oxidative load, resulting in chronically elevated levels of oxidative stress and reactive oxygen species, which in the retina ultimately result in pathologies such as glaucoma or age-related macular degeneration, as well as the neuropathic complications of diabetes in the eye. Specifically, oxidative stress results in deleterious changes to the retina through dysregulation of its intracellular physiology, ultimately leading to neurodegenerative and potentially also vascular dysfunction. Herein we will review the evidence for oxidative stress-induced contributions to each of the three major ocular pathologies, glaucoma, age-related macular degeneration, and diabetic retinopathy. The premise for neuroprotective strategies for these ocular disorders will be discussed in the context of recent clinical and preclinical research pursuing novel therapy development approaches.
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40
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Panfoli I, Ravera S, Bruschi M, Candiano G, Morelli A. Proteomics unravels the exportability of mitochondrial respiratory chains. Expert Rev Proteomics 2014; 8:231-9. [DOI: 10.1586/epr.11.1] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
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Hypothesis of lipid-phase-continuity proton transfer for aerobic ATP synthesis. J Cereb Blood Flow Metab 2013; 33:1838-42. [PMID: 24084698 PMCID: PMC3851912 DOI: 10.1038/jcbfm.2013.175] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/21/2013] [Revised: 09/09/2013] [Accepted: 09/12/2013] [Indexed: 12/12/2022]
Abstract
The basic processes harvesting chemical energy for life are driven by proton (H(+)) movements. These are accomplished by the mitochondrial redox complex V, integral membrane supramolecular aggregates, whose structure has recently been described by advanced studies. These did not identify classical aqueous pores. It was proposed that H(+) transfer for oxidative phosphorylation (OXPHOS) does not occur between aqueous sources and sinks, where an energy barrier would be insurmountable. This suggests a novel hypothesis for the proton transfer. A lipid-phase-continuity H(+) transfer is proposed in which H(+) are always bound to phospholipid heads and cardiolipin, according to Mitchell's hypothesis of asymmetric vectorial H(+) diffusion. A phase separation is proposed among the proton flow, following an intramembrane pathway, and the ATP synthesis, occurring in the aqueous phase. This view reminiscent of Grotthus mechanism would better account for the distance among the Fo and F1 moieties of FoF1-ATP synthase, for its mechanical coupling, as well as the necessity of a lipid membrane. A unique active role for lipids in the evolution of life can be envisaged. Interestingly, this view would also be consistent with the evidence of an OXPHOS outside mitochondria also found in non-vesicular membranes, housing the redox complexes.
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Tricarboxylic acid cycle-sustained oxidative phosphorylation in isolated myelin vesicles. Biochimie 2013; 95:1991-8. [DOI: 10.1016/j.biochi.2013.07.003] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2013] [Accepted: 07/02/2013] [Indexed: 12/11/2022]
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43
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Roehlecke C, Schumann U, Ader M, Brunssen C, Bramke S, Morawietz H, Funk RHW. Stress reaction in outer segments of photoreceptors after blue light irradiation. PLoS One 2013; 8:e71570. [PMID: 24039718 PMCID: PMC3770596 DOI: 10.1371/journal.pone.0071570] [Citation(s) in RCA: 78] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2012] [Accepted: 07/07/2013] [Indexed: 12/26/2022] Open
Abstract
The retina is prone to oxidative stress from many factors which are also involved in the pathogenesis of degenerative diseases. In this study, we used the application of blue light as a physiological stress factor. The aim of this study was to identify the major source of intracellular ROS that mediates blue light-induced detrimental effects on cells which may lead to cytotoxicity. We hypothesized that outer segments are the major source of blue light induced ROS generation. In photoreceptors, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzymes and the recently found respiratory chain complexes may represent a major source for reactive oxygen species (ROS), beside mitochondria and chromophores. Therefore, we investigated this hypothesis and analysed the exact localization of the ROS source in photoreceptors in an organotypic culture system for mouse retinas. Whole eyeball cultures were irradiated with visible blue light (405 nm) with an output power of 1 mW/cm2. Blue light impingement lead to an increase of ROS production (detected by H2DCFDA in live retinal explants), which was particularly strong in the photoreceptor outer segments. Nox-2 and Nox-4 proteins are sources of ROS in blue light irradiated photoreceptors; the Nox inhibitor apocynin decreased ROS stimulated by blue light. Concomitantly, enzyme SOD-1, a member of the antioxidant defense system, indicator molecules of protein oxidation (CML) and lipid oxidation (MDA and 4-HNE) were also increased in the outer segments. Interestingly, outer segments showed a mitochondrial-like membrane potential which was demonstrated using two dyes (JC-1 and TMRE) normally exclusively associated with mitochondria. As in mitochondria, these dyes indicated a decrease of the membrane potential in hypoxic states or cell stress situations. The present study demonstrates that ROS generation and oxidative stress occurs directly in the outer segments of photoreceptors after blue light irradiation.
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Affiliation(s)
- Cora Roehlecke
- Institute of Anatomy, Technische Universität (TU) Dresden, Dresden, Germany
- * E-mail:
| | - Ulrike Schumann
- Institute of Anatomy, Technische Universität (TU) Dresden, Dresden, Germany
| | - Marius Ader
- Center for Regenerative Therapies Dresden (CRTD) DFG – Cluster of Excellence, Biotechnology Center, Dresden, Germany
| | - Coy Brunssen
- Division of Vascular Endothelium and Microcirculation, Department of Medicine III, TU Dresden, Dresden, Germany
| | - Silvia Bramke
- Institute of Anatomy, Technische Universität (TU) Dresden, Dresden, Germany
| | - Henning Morawietz
- Center for Regenerative Therapies Dresden (CRTD) DFG – Cluster of Excellence, Biotechnology Center, Dresden, Germany
- Division of Vascular Endothelium and Microcirculation, Department of Medicine III, TU Dresden, Dresden, Germany
| | - Richard H. W. Funk
- Institute of Anatomy, Technische Universität (TU) Dresden, Dresden, Germany
- Center for Regenerative Therapies Dresden (CRTD) DFG – Cluster of Excellence, Biotechnology Center, Dresden, Germany
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Calzia D, Candiani S, Garbarino G, Caicci F, Ravera S, Bruschi M, Manni L, Morelli A, Traverso CE, Candiano G, Tacchetti C, Panfoli I. Are rod outer segment ATP-ase and ATP-synthase activity expression of the same protein? Cell Mol Neurobiol 2013; 33:637-49. [PMID: 23568658 PMCID: PMC11497934 DOI: 10.1007/s10571-013-9926-7] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2012] [Accepted: 03/12/2013] [Indexed: 12/15/2022]
Abstract
Vertebrate retinal rod outer segments (OS) consist of a stack of disks surrounded by the plasma membrane, where phototransduction takes place. Energetic metabolism in rod OS remains obscure. Literature described a so-called Mg(2+)-dependent ATPase activity, while our previous results demonstrated the presence of oxidative phosphorylation (OXPHOS) in OS, sustained by an ATP synthetic activity. Here we propose that the OS ATPase and ATP synthase are the expression of the same protein, i.e., of F1Fo-ATP synthase. Imaging on bovine retinal sections showed that some OXPHOS proteins are expressed in the OS. Biochemical data on bovine purified rod OS, characterized for purity, show an ATP synthase activity, inhibited by classical F1Fo-ATP synthase inhibitors. Moreover, OS possess a pH-dependent ATP hydrolysis, inhibited by pH values below 7, suggestive of the functioning of the inhibitor of F1 (IF1) protein. WB confirmed the presence of IF1 in OS, substantiating the expression of F1Fo ATP synthase in OS. Data suggest that the OS F1Fo ATP synthase is able to hydrolyze or synthesize ATP, depending on in vitro or in vivo conditions and that the role of IF1 would be pivotal in the prevention of the reversal of ATP synthase in OS, for example during hypoxia, granting photoreceptor survival.
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Affiliation(s)
| | | | | | - Federico Caicci
- Dipartimento di Biologia, Università di Padova, Padua, Italy
| | | | - Maurizio Bruschi
- Laboratorio di Fisiopatologia dell’Uremia, Istituto G. Gaslini, Genoa, Italy
| | - Lucia Manni
- Dipartimento di Biologia, Università di Padova, Padua, Italy
| | | | | | - Giovanni Candiano
- Laboratorio di Fisiopatologia dell’Uremia, Istituto G. Gaslini, Genoa, Italy
| | - Carlo Tacchetti
- IFOM Centro di Oncologia cellulare e Ultrastruttura, Dipartimento di Medicina Sperimentale, Università degli Studi di Genova, Genoa, Italy
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Calzia D, Barabino S, Bianchini P, Garbarino G, Oneto M, Caicci F, Diaspro A, Tacchetti C, Manni L, Candiani S, Ravera S, Morelli A, Enrico Traverso C, Panfoli I. New findings in ATP supply in rod outer segments: insights for retinopathies. Biol Cell 2013; 105:345-58. [PMID: 23659850 DOI: 10.1111/boc.201300003] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2013] [Accepted: 05/03/2013] [Indexed: 01/10/2023]
Abstract
BACKGROUND INFORMATION The rod outer segment (OS) is the specialised organelle where phototransduction takes place. Our previous proteomic and biochemical analyses on purified rod disks showed the functional expression of the respiratory chain complexes I-IV and F1 Fo -ATP synthase in OS disks, as well as active soluble tricarboxylic acid cycle enzymes. Here, we focussed our study on the whole OS that contains the cytosol and plasma membrane and disks as native flattened saccules, unlike spherical osmotically intact disks. RESULTS OS were purified from bovine retinas and characterised for purity. Oximetry, ATP synthesis and cytochrome c oxidase (COX) assays were performed. The presence of COX and F₁F₀-ATP synthase (ATP synthase) was assessed by semi-quantitative Western blotting, immunofluorescence or confocal laser scanning microscopy on whole bovine retinas and bovine retinal sections and by immunogold transmission electron microscopy (TEM) of purified OS or bovine retinal sections. Both ATP synthase and COX are catalytically active in OS. These are able to consume oxygen (O₂) in the presence of pyruvate and malate. CLSM analyses showed that rhodopsin autofluorescence and MitoTracker Deep Red 633 fluorescence co-localise on rod OS. Data are confirmed by co-localisation studies of ATP synthase with Rh in rod OS by immunofluorescence and TEM in bovine retinal sections. CONCLUSIONS Our data confirm the expression and activity of COX and ATP synthase in OS, suggestive of the presence of an extra-mitochondrial oxidative phosphorylation in rod OS, meant to supply ATP for the visual transduction. In this respect, the membrane rich OS environment would be meant to absorb both light and O₂. The ability of OS to manipulate O₂ may shed light on the pathogenesis of many retinal degenerative diseases ascribed to oxidative stress, as well as on the efficacy of the treatment with dietary supplements, presently utilised as supporting therapies.
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Affiliation(s)
- Daniela Calzia
- Department of Pharmacy-DIFAR, Biochemistry and Physiology Lab, University of Genoa, Genova, Italy
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Ravera S, Nobbio L, Visigalli D, Bartolucci M, Calzia D, Fiorese F, Mancardi G, Schenone A, Morelli A, Panfoli I. Oxydative phosphorylation in sciatic nerve myelin and its impairment in a model of dysmyelinating peripheral neuropathy. J Neurochem 2013; 126:82-92. [PMID: 23578247 DOI: 10.1111/jnc.12253] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2012] [Revised: 03/13/2013] [Accepted: 03/14/2013] [Indexed: 11/30/2022]
Abstract
Myelin sheath is the proteolipid membrane wrapping the axons of CNS and PNS. We have shown data suggesting that CNS myelin conducts oxidative phosphorylation (OXPHOS), challenging its role in limiting the axonal energy expenditure. Here, we focused on PNS myelin. Samples were: (i) isolated myelin vesicles (IMV) from sciatic nerves, (ii) mitochondria from primary Schwann cell cultures, and (iii) sciatic nerve sections, from wild type or Charcot-Marie-Tooth type 1A (CMT1A) rats. The latter used as a model of dys-demyelination. O₂ consumption and activity of OXPHOS proteins from wild type (Wt) or CMT1A sciatic nerves showed some differences. In particular, O₂ consumption by IMV from Wt and CMT1A 1-month-old rats was comparable, while it was severely impaired in IMV from adult affected animals. Mitochondria extracted from CMT1A Schwann cell did not show any dysfunction. Transmission electron microscopy studies demonstrated an increased mitochondrial density in dys-demyelinated axons, as to compensate for the loss of respiration by myelin. Confocal immunohistochemistry showed the expression of OXPHOS proteins in the myelin sheath, both in Wt and dys-demyelinated nerves. These revealed an abnormal morphology. Taken together these results support the idea that also PNS myelin conducts OXPHOS to sustain axonal function.
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Panfoli I, Calzia D, Bruschi M, Oneto M, Bianchini P, Ravera S, Petretto A, Diaspro A, Candiano G. Functional expression of oxidative phosphorylation proteins in the rod outer segment disc. Cell Biochem Funct 2013; 31:532-8. [PMID: 23322616 DOI: 10.1002/cbf.2943] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2012] [Revised: 11/06/2012] [Accepted: 11/28/2012] [Indexed: 11/09/2022]
Abstract
The rod Outer Segment (OS) disc, an organelle devoid of mitochondria, is specialized in phototransduction, a process requiring a continual chemical energy supply. We have shown that OS discs express functional mitochondrial electron transport chains, Fo F1 -ATP synthase and the tricarboxylic acid cycle enzymes, all mitochondrial features. Here, we focus on oxygen consumption and adenosine triphosphate (ATP) synthesis by OS discs analysing electron transport chain I-III-IV and II-II-IV pathways, supported by reduced nicotinamide adenine dinucleotide and succinate, respectively. Interestingly, respiratory capacity of discs was measurable also in the presence of 3-hydroxy-butyrrate, a typical metabolic substrate for the brain. Data were supported by a two-dimensional electrophoresis analyses conducted as our previous one, but focused to those mitochondrial proteins that are involved in oxidative phosphorylation. Carbonic anhydrase was also found active in OS discs. Moreover, colocalization of Rhodopsin with respiratory complex I and ATP synthase seems a further step in the characterization of some proteins typical of the mitochondrial inner membranes that are expressed in the rod discs. The existence of oxygen utilization in the outer retina, likely supplying ATP for phototransduction, may shed light on some retinal pathologies related to oxidative stress of the outer retina.
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Affiliation(s)
- Isabella Panfoli
- DIFAR-Biochemistry Lab., University of Genoa, 16132 Genova, Italy.
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Panfoli I, Calzia D, Ravera S, Morelli AM, Traverso CE. Extra-mitochondrial aerobic metabolism in retinal rod outer segments: new perspectives in retinopathies. Med Hypotheses 2012; 78:423-7. [PMID: 22284635 DOI: 10.1016/j.mehy.2011.12.012] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2011] [Revised: 12/06/2011] [Accepted: 12/29/2011] [Indexed: 01/11/2023]
Abstract
Vertebrate retinal rods are photoreceptors for dim-light vision. They display extreme sensitivity to light thanks to a specialized subcellular organelle, the rod outer segment. This is filled with a stack of membranous disks, expressing the proteins involved in visual transduction, a very energy demanding process. Our previous proteomic and biochemical studies have shed new light on the chemical energy processes that supply ATP to the outer segment, suggesting the presence of an extra-mitochondrial aerobic metabolism in rod outer segment, devoid of mitochondria, which would account for a quantitatively adequate ATP supply for phototransduction. Here the functional presence of an oxidative phosphorylation in the rod outer limb is examined for its relationship to many physiological and pathological data on the rod outer segment. We hypothesize that the rod outer limb is at risk of oxidative stress, in any case of impairment in the respiratory chain functioning, or of blood supply. In fact, the electron transfer chain is a major source of reactive O(2) species, known to produce severe alteration to the membrane lipids, especially those of the outer segment that are rich in polyunsaturated fatty acids. We propose that the disk membrane may become the target of reactive oxygen species that may be released by the electron transport chain under pathologic conditions. For example, during aging reactive oxygen species production increases, while cellular antioxidant capacity decreases. Also the apoptosis of the rod observed after exposure to bright or continuous illumination can be explained considering that an overfunctioning of phototransduction may damage the disk membrane to a point at which cytochrome c escapes from the intradiskal space, where it is presently supposed to be, activating a putative caspase 9 and the apoptosome. A pathogenic mechanism for many inherited and acquired retinal degenerations, representing a major problem in clinical ophthalmology, is proposed: a number of rod pathologies would be promoted by impairment of energy supply and/or oxidative stress in the rod outer segment. In conclusion we suppose that the damaging role of oxygen, be it hypoxia or hyperoxia invoked in most of the blinding diseases, acquired and even hereditary is to be seeked for inside the photoreceptor outer segment that would conceal a potential for cell death that is still to be recognized.
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Affiliation(s)
- I Panfoli
- DIPTERIS - University of Genova, Corso Europa 26, 16132 Genova, Italy.
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Raimondo F, Morosi L, Chinello C, Perego R, Bianchi C, Albo G, Ferrero S, Rocco F, Magni F, Pitto M. Protein profiling of microdomains purified from renal cell carcinoma and normal kidney tissue samples. ACTA ACUST UNITED AC 2012; 8:1007-16. [DOI: 10.1039/c2mb05372a] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
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Abstract
Nervous system is a great oxygen consumer, but the site of oxygen absorption has remained elusive. Four proteomic studies have shown that the respiratory complexes I to V may be expressed in isolated myelin. Myelin is an outgrowth of glial cells, surrounding many axons in multiple spires both in peripheral and central nervous system. Recent quantitative analyses strongly support the daring hypothesis that myelin is functional in aerobic ATP production, to supply the neuron with chemical energy. A vision of myelin sheath as a structure devoted to the oxygen absorbance for glucose combustion in nervous system thank to its enormous surface, would be also supported by an impressive series of characteristics and properties of myelin that do not presently find an explanation, all of which are herein examined.
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Affiliation(s)
- Alessandro Morelli
- Department of Biology, Faculty of Sciences, University of Genova, Viale Benedetto XV-3, 16132 Genoa, Italy.
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