Tissue-specific expression of the ATPase inhibitory factor 1 and its role in neuronal function

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(1)Universidad Autónoma de Madrid Programa de Doctorado en Biociencias Moleculares. Tissue-specific expression of the ATPase inhibitory factor 1 and its role in neuronal function. Pau B. Esparza Moltó Madrid, 2020.

(2) Departamento de Biología Molecular Facultad de Ciencias Universidad Autónoma de Madrid. Tissue-specific expression of the ATPase inhibitory factor 1 and its role in neuronal function. A dissertation submitted by Pau Bernat Esparza Moltó BSc in Biochemistry and Biomedical Sciences for the degree of Doctor of Philosophy in Molecular Biosciences of Universidad Autónoma de Madrid, 2020. Advisor: José M. Cuezva Departamento de Biología Molecular Centro de Biología Molecular “Severo Ochoa” (UAM-CSIC).

(3) This work has been supported by a predoctoral fellowship from Center for Biomedical Research Network in Rare Diseases (CIBERER) and from Fundación La Caixa (Obra Social La Caixa, LCF/BQ/ES15/1036002). The project was funded by the Spanish Ministerio de Economía y Competitividad (Grants SAF2013-41945R and SAF2016-75916-R), CIBERER (CB06/07/0017) and Fundación Ramón Areces. The international research stay was supported by The Company of Biologists (DMMTF-180215), IUBMB Wood-Whelan Research Fellowship and the European Molecular Biology Organization (EMBO STF-7759)..

(4) Declaration This dissertation is the result of the work that Pau Bernat Esparza Moltó has performed under my supervision. Pau joined my laboratory at Centro de Biología Molecular “Severo Ochoa” in September 1st, 2014 to develop his PhD Thesis and ended his project in December 1st, 2019. The work he has performed qualifies him to obtain the PhD in Molecular Biosciences. During his stay in my laboratory, Pau has received a fellowship from Center for Biomedical Research Network in Rare Diseases (CIBERER) (from September 1st, 2014 to August 31st, 2015) and from “la Caixa” Foundation for PhD studies in Spain (from November 1st, 2015 to October 31st, 2019).. Madrid, December 1st, 2019,. Prof. José M. Cuezva Full professor and Chair Department of Molecular Biology Universidad Autónoma de Madrid.

(5) A la meua família, A Sheila.

(6) Agradecimientos En primer lugar, quiero agradecer a mi director de tesis, el Prof. José M. Cuezva, por darme la oportunidad de realizar este trabajo en su laboratorio y haberme guiado durante estos años de formación como científico. Pepe, muchas gracias por estar ahí y por tener tu puerta siempre abierta. Muchas gracias a todos los compañeros del laboratorio, con los que he compartido numerosos momentos. He podido contar con vosotros tanto dentro como fuera del trabajo. Sin duda, habéis hecho que esta etapa de mi vida haya sido mucho más agradable. Gracias a Fulvio, por tu amistad y tus reflexiones sobre la vida y la ciencia. A Cris (Yoli), por ser ese contrapunto a mi orden y mis manías. A Cris (Núñez), por tenerlo todo controlado y llevar los ratones a rajatabla, y a Brenda, por estar dispuesta a ayudar con todo. A Formen, por tus buenos consejos y ayuda. A Ana (Afamada), por tu alegría y tu inestimable aportación a la acetilación y la autofagia en este laboratorio. A Laura, por tu ayuda cuando lo he necesitado. A Sonia por tu vitalidad y ser la nueva DJ del laboratorio. Y a Cris (Formen), por tu energía y tu disposición a ayudar. No me puedo olvidar de las últimas incorporaciones al laboratorio: Inés y Juan; estoy seguro de que con vosotros no decaerá la ciencia ni la vida social del laboratorio. He tenido la fortuna de haber conocido a mucha gente que ha estado en el laboratorio y a la que también quiero expresar mi más sincero agradecimiento. Margarita, muchas gracias por haber estado ahí, en mis inicios y echarme una mano cuando más lo necesitaba. También Noelia, Carmen, Estefanía, Lucía (Pila), Lucía García, Sandra, Bea Soldevilla, Javi y María. Igualmente Marek, y los estudiantes Adrián y Bea. Muchas gracias a los vecinos de los laboratorios 320 y 321, con los que hemos compartido reactivos, consejos y piques en la fiesta de primavera. Gracias Araceli por tu más sincero apoyo y por haberme escuchado cuando me hacía falta. También muchas gracias a Beatriz Pardo, Laura, Inés, Irene, Paloma y Paula, por guiarme en el mundo de la neurona y su metabolismo. A Marta, Sofía y Álvaro del 320 por los anticuerpos y los buenos momentos. No puedo dejar de agradecer a Enrique, por sus visitas, su ayuda y su interés por nosotros. En este proyecto hemos abordado distintas cuestiones y esto no hubiera sido posible sin la colaboración con otros grupos del CBM. Así, quiero agradecer a Marta P. Pereira por haberme enseñado y apoyado en los estudios de comportamiento y las inyecciones estereotáxicas. Pero mucho más importante, por contribuir a que entrara en el mundo de la neurobiología. Muchas gracias también al Dr. José A. Esteban y a su laboratorio, especialmente a Carla y Sergio, por enseñarme a hacer los estudios de electrofisiología y a tener paciencia con estos. No puedo dejar de citar los Servicios del CBM (Animalario, Genómica, Proteómica, Microscopía Óptica y Confocal, Microscopía Electrónica y los servicios administrativos) y del CNB (Histología y Criopreservación), que han sido. i.

(7) fundamentales para el desarrollo de este trabajo. Ni tampoco a Mada, a quien agradezco su ayuda con las burocracias que acarrea la tesis. I would like to thank Prof. Gerald Shadel for accepting me in his lab at The Salk Institute and supporting my work during my stay. I would also like to extend my gratitude to my colleagues at Shadelab for welcoming me and contributing to make my experience in San Diego something I will always remember. Gracias a la gente que ha hecho de mi etapa en Madrid una parte muy bonita de mi vida. Muchas gracias, Alberto y Jony por haber sido mi segunda familia en Madrid y por haber podido contar con vosotros. En estos 5 años me he sentido en casa. Santos, eres un gran amigo y no sabes cuánto agradezco haberte conocido. Muchas gracias también al resto del CaboPalos team por esos buenos momentos a mitad de semana. No puc deixar darrere als meus amics de sempre. Moltes gràcies, Josep, Eric, Javi, Ángel U., Àngel, Jaume i Héctor, per haver estat prop de mi en la distància i per haver-me preguntat pels ratolins. Moltes gràcies a la meua família. Mare, pare, moltes gràcies per haver fet ser qui sóc i per recolzar-me i animar-me a seguir en el món de la ciència. A tu també, Guillem, per haver crescut junts i haver gaudit temps junts. Gran part d’aquest treball ha sigut gràcies a vosaltres. També vull agrair als meus iaios per la seua estima incondicional i als meus oncles i cosins pel seu suport en tot moment. Finalment, moltes gràcies, Sheila per estar sempre aquí, fer-me costat i compartir somnis amb mi. Gràcies per animar-me en els moments complicats, celebrar els bons i enriquir-me com a persona.. ii.

(8) Resumen La mitocondria es un orgánulo esencial para la fisiología celular ya que proporciona la mayor parte de la energía necesaria para las funciones celulares y es un nodo clave en el metabolismo intermediario, la ejecución de muerte celular y la señalización intracelular. La ATP sintasa es un complejo mitocondrial muy relevante que integra estas funciones: sintetiza ATP por fosforilación oxidativa, participa en la permeabilización de la membrana mitocondrial interna en la muerte celular y está implicada en señalización mitocondrial. Un regulador clave de la enzima es el factor inhibidor 1 de la ATPasa (IF1), que inhibe sus actividades catalíticas cuando está unido a ella. En este trabajo hemos caracterizado la expresión específica de tejido de IF1 en tejidos humanos y de ratón, y hemos cuantificado el contenido del inhibidor y de la ATP sintasa en los mismos. Hemos encontrado diferencias importantes en la expresión de IF1 en tejidos de ambas especies y además, que algunos tejidos con alta demanda energética contienen un exceso molar del inhibidor con respecto a la enzima. En estos tejidos, una fracción relevante de IF1 está unido a la ATP sintasa, mientras que otra fracción está fosforilada y, por tanto, no puede unirse. Además, la expresión de IF1 está controlada fundamentalmente a niveles post-transcripcionales, especialmente en el corazón de ratón, donde la traducción del mRNA de IF1 está reprimida por la proteína LRPPRC. Para estudiar la relevancia fisiológica de IF1 en la regulación de la actividad de la ATP sintasa en condiciones normales, hemos desarrollado modelos celulares y de ratón con expresión diferencial de IF1. En concreto, se ha generado un ratón knockout condicional de IF1 en neuronas y un ratón transgénico que sobreexpresa la proteína humana en el mismo tipo celular. La expresión de IF1 en neuronas del cerebro define la actividad de la ATP sintasa, demostrando que una fracción de la enzima está inhibida por IF1 en condiciones fisiológicas normales. Además, la expresión de IF1 regula la respiración, el potencial de membrana y la producción de especies reactivas del oxígeno (ROS) en la mitocondria de neurona. La inactivación de IF1 reduce los niveles de oligómeros de ATP sintasa y altera la estructura de las crestas mitocondriales, lo que apoya el papel que tienen los oligómeros de la enzima en la arquitectura mitocondrial. Análisis transcriptómicos y proteómicos del hipocampo sugieren la existencia de diferencias en la función sináptica y cognitiva entre ratones con distintos niveles de IF1. De hecho, los ratones que sobreexpresan IF1 tienen mayor transmisión sináptica y memoria a largo plazo en comparación con los ratones knockout y controles. El tratamiento con MitoQ, un antioxidante mitocondrial, impide la aparición de estos cambios, respaldando el papel de las ROS mitocondriales en función sináptica y en memoria. En resumen, IF1 es una proteína clave en la regulación de la estructura y actividad mitocondriales para la función neuronal. Por tanto, el módulo IF1/ATP sintasa puede tener potencial terapéutico para enfermedades y condiciones que cursan con problemas cognitivos.. v.

(9) Summary Mitochondria are crucial organelles in cellular physiology that provide most of the energy necessary to sustain cellular activities and are critical hubs in intermediary metabolism, cell death execution and intracellular signaling. The ATP synthase is a primary mitochondrial complex that integrates these functions: provides ATP by oxidative phosphorylation, is responsible for the permeabilization of the inner mitochondrial membrane for cell death execution and is involved in mitochondrial signaling. A major regulator of the enzyme is the ATPase inhibitory factor 1 (IF1), which inhibits its catalytic activities when bound to the enzyme. In this study, we have characterized the tissue-specific expression of IF1 in human and mouse tissues, and quantitated the content of the inhibitor and of the ATP synthase. We have found relevant differences in IF1 expression between human and mouse tissues. Moreover, in some high-energy demanding tissues, the molar content of the inhibitor exceeds that of the ATP synthase. In these tissues, a relevant fraction of IF1 is bound to the enzyme, whereas the other fraction is phosphorylated and hence unable to bind to it. In addition, control of IF1 expression is exerted primarily at post-transcriptional levels, especially in mouse heart, where translation of IF1 mRNA is repressed by leucine rich pentatricopeptide repeat containing protein (LRPPRC). To study the physiological relevance of IF1 in the regulation of the ATP synthase under normal conditions, we have developed cellular and mouse models with differential expression of the inhibitor. In particular, we have generated a conditional IF1-knockout mouse in forebrain neurons and a transgenic mouse overexpressing human IF1 in the same cell type. The expression of IF1 in neurons defines the activity of the ATP synthase, confirming that a fraction of the brain enzyme is inhibited under normal physiological conditions. Moreover, IF1 expression also regulates mitochondrial respiration, membrane potential and the production of reactive oxygen species (ROS) in primary neuronal cultures. Genetic ablation of IF1 reduces the levels of oligomeric assemblies of the ATP synthase and alters mitochondrial cristae structure, supporting the role of oligomers of the enzyme in shaping mitochondrial architecture. Transcriptomic and proteomic studies of the hippocampus suggested differences in synaptic and cognitive functions upon differential expression of IF1. Indeed, we have found that mice overexpressing IF1 in neurons exhibit enhanced synaptic transmission and long-term memory, when compared to knockout and control mice. Importantly, the mitochondrially targeted antioxidant MitoQ prevents these changes, supporting the role of mitochondrial ROS in synaptic function and cognition. Overall, these findings underpin the relevance of IF1 in the organization of mitochondrial structure and function for brain homeostasis. Therefore, the ATP synthase/IF1 module shows promising therapeutic potential for pathologies and conditions involving cognitive impairment.. vii.

(10) Index Agradecimientos ...................................................................................................................... i Resumen................................................................................................................................. v Summary................................................................................................................................ vii Index ....................................................................................................................................... 1 Table and Figure Index........................................................................................................... 7 Abbreviations .......................................................................................................................... 9 Introduction ........................................................................................................................... 13 1. Mitochondria are central organelles in cellular physiology ........................................... 13 2. Mitochondrial structure and dynamics .......................................................................... 13 2.1. Mitochondrial compartments .................................................................................. 13 2.2. Mitochondrial dynamics.......................................................................................... 16 3. Organization and regulation of the oxidative phosphorylation system ......................... 16 3.1. Components of the electron transport chain .......................................................... 17 3.2. The mitochondrial ATP synthase: structure, assembly and expression ................ 19 3.3. Regulation of oxidative phosphorylation ................................................................ 20 3.4. Production of reactive oxygen species in mitochondria......................................... 22 3.5. Supramolecular organization of the OXPHOS complexes .................................... 24 4. Mitochondrial functions ................................................................................................. 25 4.1. Mitochondria are biosynthetic hubs and the gate to cell death.............................. 25 4.2. Mitochondria are signaling hubs in hormetic responses........................................ 27 5. IF1 is a master regulator of the ATP synthase ............................................................. 29 5.1. Regulation of the expression and activity of IF1 .................................................... 32 5.2. IF1 is overexpressed in most prevalent human carcinomas ................................. 33 5.3. Signaling pathways and cellular programs modulated by IF1 ............................... 34 Objectives ............................................................................................................................. 39 Materials and Methods ......................................................................................................... 43 1. Materials........................................................................................................................ 43 1.1. Human tissue samples ........................................................................................... 43 1.2. Cell lines ................................................................................................................. 43 1.

(11) 1.3. Animals ................................................................................................................... 43 1.4. Bacterial strains ...................................................................................................... 44 1.5. Plasmids ................................................................................................................. 44 1.6. Oligonucleotides ..................................................................................................... 46 1.7. Radioisotopes......................................................................................................... 48 1.8. Antibodies ............................................................................................................... 48 1.9. Chemical compounds............................................................................................. 52 2. Methods......................................................................................................................... 52 2.1. Methods in Molecular Biology ................................................................................ 52 2.1.1. Cloning strategies ............................................................................................ 52 2.1.2. Expression and purification of recombinant proteins ...................................... 53 2.1.3. Isolation and purification of DNA ..................................................................... 54 2.1.4. Purification of RNA and reverse transcription ................................................. 54 2.1.5. Polymerase chain reaction (PCR) ................................................................... 54 2.1.6. Agarose gel electrophoresis ............................................................................ 56 2.1.7. In vitro transcription and translation assays .................................................... 56 2.2. Methods in Biochemistry ........................................................................................ 57 2.2.1. Protein extraction from biological samples and quantification ........................ 57 2.2.2. Protein electrophoresis and Western blot analysis ......................................... 57 2.2.3. 2D-gel electrophoresis..................................................................................... 58 2.2.4. Determination of tissue protein carbonylation ................................................. 58 2.2.5. Isolation of mitochondria from tissue samples ................................................ 59 2.2.6. Blue native gels ............................................................................................... 59 2.2.7. Determination of ATP hydrolytic activity .......................................................... 59 2.3. Methods in Cell Biology.......................................................................................... 60 2.3.1. Cell culture ....................................................................................................... 60 2.3.2. Preparation and culture of primary mouse embryonic fibroblasts................... 60 2.3.3. Preparation and culture of primary mouse hippocampal neurons .................. 60 2.3.4. Transient transfection of cell lines and transduction of MEFs......................... 61 2.3.5. Immunofluorescence in fixed cells .................................................................. 61. 2.

(12) 2.3.6. Measurement of oxygen consumption rate with Seahorse Analyzer .............. 62 2.3.7. Determination of glycolytic flux ........................................................................ 62 2.3.8. Determination of mitochondrial membrane potential by flow cytometry in MEFs ................................................................................................................................... 63 2.3.9. Imaging of membrane potential and mtROS production in neurons ............... 63 2.4. Methods in animals ................................................................................................ 63 2.4.1. General housing and animal care conditions .................................................. 63 2.4.2. Organ and serum collection from mice............................................................ 64 2.4.3. Antibody production ......................................................................................... 64 2.4.4. Immunohistochemistry..................................................................................... 64 2.4.5. Transcriptomic analysis of the hippocampus of IF1-KO, control and TG mice ................................................................................................................................... 64 2.4.6. Proteomic analysis of the hippocampus of IF1-KO, control and TG mice ...... 65 2.4.7. Targeted metabolomic analysis of the hippocampus of IF1-KO, control and TG mice............................................................................................................................ 67 2.4.8. Determination of ATP and GSH in the hippocampus ...................................... 68 2.4.9. Electron microscopy ........................................................................................ 68 2.4.10. Electrophysiology........................................................................................... 69 2.4.11. Behavior ......................................................................................................... 70 2.4.12. Treatment of mice with the antioxidant MitoQ ............................................... 72 2.4.13. In vivo determination of mitochondrial ROS production ................................ 72 2.5. Statistical analyses ................................................................................................. 72 Results .................................................................................................................................. 75 1. Tissue-specific expression and post-transcriptional regulation of IF1 in human and mouse tissues ................................................................................................................... 75 1.1. IF1 is differentially expressed in human tissues .................................................... 75 1.2. One isoform of IF1 is expressed in mouse tissues ................................................ 77 1.3. Generation of a polyclonal antibody against mouse IF1........................................ 79 1.4. Mouse tissues can be divided according to high or low expression of IF1............ 80 1.5. IF1 expression is post-translationally repressed in mouse heart .......................... 84 1.6. A fraction of IF1 is phosphorylated in mouse tissues with high IF1 content.......... 84. 3.

(13) 1.7. IF1 protein levels decrease in some tissues from aged mice ................................ 87 2. Development of IF1-KO cells ........................................................................................ 88 2.1. CRISPR/Cas-mediated knockout of IF1 in Hepa 1-6 cells does not affect respiration ....................................................................................................................................... 88 2.2. Development of tissue-specific conditional IF1-KO mice by site-specific recombination ................................................................................................................ 90 2.3. IF1-KO MEFs show impaired induction of the autophagic flux.............................. 92 3. Development of mouse lines expressing different doses of IF1 in neurons................. 95 4. Differential expression of IF1 in neurons modulates synaptic transmission and memory ........................................................................................................................................... 97 4.1. Transcriptomic profiling of the hippocampus of IF1-KO, control and TG mice...... 97 4.2. Proteomic analysis of the hippocampus of IF1-KO, control and TG mice ........... 100 4.3. Metabolic reprogramming in the hippocampus of IF1-KO, control and TG mice 102 4.4. Differential expression of IF1 controls mitochondrial respiration and mtROS production in neurons .................................................................................................. 104 4.5. IF1 overexpression induces the expression of antioxidant enzymes in the hippocampus ............................................................................................................... 106 4.6. IF1 expression controls the organization of the OXPHOS system and mitochondrial ultrastructure................................................................................................................ 107 4.7. IF1 overexpression enhances basal synaptic transmission ................................ 110 4.8. Differential expression of IF1 affects basal exploratory activity and memory in mice ..................................................................................................................................... 113 5. mtROS guide the positive effects of IF1 overexpression in synaptic function and memory ......................................................................................................................................... 115 5.1. Quenching mtROS blunts the enhanced long-term memory of TG mice ............ 115 5.2. mtROS promote the induction of the antioxidant response in the hippocampus 116 5.3. Quenching mtROS production reduces the enhanced synaptic transmission of TG mice ............................................................................................................................. 118 6. IF1 overexpression in neurons promotes exploratory activity and long-term memory in aged mice ........................................................................................................................ 118 Discussion........................................................................................................................... 123 1. Tissue-specific expression and post-transcriptional regulation of IF1 in human and mouse tissues ................................................................................................................. 123 4.

(14) 2. Differential expression of IF1 in neurons in vivo modulates energy metabolism, signaling and neuronal function ..................................................................................................... 128 3. The ATP synthase/IF1 module represents a target for therapy in neuronal pathology ......................................................................................................................................... 136 Conclusiones ...................................................................................................................... 141 Conclusions ........................................................................................................................ 143 References.......................................................................................................................... 147 Appendix I. Supplementary information ............................................................................. 167 1. Supplementary Materials and Methods ...................................................................... 167 1.1. Analysis of CRISPR/Cas off-target activity in IF1-KO cells ................................. 167 1.2. Confirmation of additional hits of the transcriptome of the hippocampus of IF1-KO, control and transgenic mice ........................................................................................ 167 2. Supplementary Figures ............................................................................................... 169 Appendix II. Publications .................................................................................................... 173. 5.

(15) Table and Figure Index Table 1. Plasmids ................................................................................................................. 45 Table 2. Oligonucleotides used in the present study ........................................................... 47 Table 3. Antibodies used in the present study ..................................................................... 52 Supplementary Table S1. Oligonucleotides used in the analysis of CRISPR/Cas off-target activity and of the transcriptomic results .............................................................................168. Figure 1. Mitochondrial organization and dynamics ............................................................. 15 Figure 2. Schematic of the oxidative phosphorylation system ............................................. 17 Figure 3. ROS production and supramolecular organization of the electron transport chain .............................................................................................................................................. 23 Figure 4. IF1 binds to the ATP synthase and exerts structural, metabolic and signaling functions................................................................................................................................ 30 Figure 5. Human tissues can be divided according to IF1 expression ................................ 76 Figure 6. Expression of the mRNAs encoding IF1, β-F1 and HSP60 in human tissues ..... 76 Figure 7. Quantification of IF1 expression in human tissues ............................................... 78 Figure 8. Only one IF1 isoform is expressed in mouse tissues ........................................... 79 Figure 9. Development of a polyclonal antibody against mouse IF1 ................................... 80 Figure 10. Energy demanding tissues express high levels of IF1 protein ........................... 81 Figure 11. Mouse liver, heart and muscle show very low expression of IF1 ....................... 82 Figure 12. Quantification of IF1 expression in mouse tissues ............................................. 83 Figure 13. Post-transcriptional repression of IF1 expression in mouse heart ..................... 85 Figure 14. Phosphorylated IF1 is present in tissues with high IF1 content ......................... 86 Figure 15. The IF1/β-F1 ratio decreases in some tissues from aged mice ......................... 88 Figure 16. Generation and metabolic characterization of IF1-KO Hepa 1-6 cells by CRISPR/Cas ......................................................................................................................... 89 Figure 17. Strategy for the development of tissue-specific conditional IF1-KO mice .......... 90 Figure 18. Generation of Atpif1-floxed mice by ubiquitous deletion of the selection cassette .............................................................................................................................................. 91 Figure 19. Development of IF1-KO mouse embryonic fibroblasts (MEFs) .......................... 93 Figure 20. Metabolic characterization of IF1-KO mouse embryonic fibroblasts (MEFs) ..... 94 Figure 21. Generation of mouse lines expressing different levels of IF1 in neurons........... 96 Figure 22. Transcriptome of the hippocampus of IF1-KO, CRL and TG mice..................... 98 Figure 23. Functional analysis of the transcriptome suggests differences in pathways related to synaptic function and learning .......................................................................................... 99 Figure 24. Proteome of the hippocampus of IF1-KO and IF1-overexpressing mice ......... 101. 7.

(16) Figure 25. Functional analysis of the proteome suggests differences in pathways related to mitochondrial and synaptic functions ................................................................................. 102 Figure 26. Activation of AMPK in transgenic mice suggests IF1-induced metabolic reprogramming in the hippocampus ................................................................................... 103 Figure 27. Metabolomic analysis of the hippocampus of IF1-KO, CRL and TG mice ....... 104 Figure 28. IF1-mediated inhibition of the ATP synthase controls mitochondrial respiration and ROS production in hippocampal neurons........................................................................... 105 Figure 29. Overexpression of IF1 triggers the activation of the antioxidant response in neurons ............................................................................................................................... 106 Figure 30. IF1 contributes to the superassembly of the ATP synthase ............................. 107 Figure 31. Alterations in mitochondrial ultrastructure and organization in response to differential expression of IF1 .............................................................................................. 109 Figure 32. Transgenic mice overexpressing IF1 show increased basal synaptic transmission ............................................................................................................................................ 111 Figure 33. IF1-KO mice show a deficit in exploratory activity ............................................ 114 Figure 34. Cognitive abilities increase in mice with higher IF1 dose ................................. 115 Figure 35. mtROS guide the enhanced long term memory upon IF1 overexpression ...... 116 Figure 36. MitoQ treatment quenches the induction of the antioxidant system in TG mice ............................................................................................................................................ 117 Figure 37. mtROS scavenging reverses the enhanced synaptic transmission of IF1overexpressing mice ........................................................................................................... 119 Figure 38. Overexpression of IF1 in neurons preserves exploratory activity and memory in aged mice ........................................................................................................................... 120 Figure 39. Tissue-specific content and regulation of IF1 expression in human and mouse tissues ................................................................................................................................. 124 Figure 40. The availability of active IF1 regulates the fraction of inhibited ATP synthase 127 Figure 41. IF1 promotes mitochondrial organization and neuronal function in mice ......... 130 Supplementary Figure S1. No off-target mutations were detected for single guide RNA 1 .......................................................................................................................................... 169 Supplementary Figure S2. No off-target mutations were detected for single guide RNA 5 .......................................................................................................................................... 170 Supplementary Figure S1. IF1 expression is compensated in heterozygous mice and confirmation of the transcriptome results by real-time PCR ............................................... 171. 8.

(17) Abbreviations 2D ACSF AMPA AMPK ANT BN CA1 CamKIIα Cas CI CII CIII CIV CoQ CRISPR CRL DAPIT DIV DMEM DNP DRP1 ER ERK 1/2 ETC FCCP fEPSP FVB/NJ GAPDH GluA1 GluN1 GPX GSEA GSH GSR HIF-1 IBM IEF IF1/ATPIF1 IMM iTRAQ KO LC3B LDHA LRPPRC LTP MAMs MEFs. 2-dimensional Artificial cerebrospinal fluid α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic AMP-activated protein kinase Antimycin A Blue native Cornu Ammonis 1 Calcium/calmodulin-dependent protein kinase II alpha (CAMK2A) CRISPR-associated Complex I Complex II Complex III Complex IV Ubiquinone Clustered regularly interspaced short palindromic repeats Control Diabetes-associated protein in insulin-sensitive tissues (ATP5MD) Days in vitro Dulbecco's modified Eagle medium 2,4-dinitrophenol Dynamin 1-like protein (DNM1L) Endoplasmic reticulum Extracellular signal-regulated kinases 1/2 Electron transport chain Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone Field excitatory postsynaptic potential Friend Virus B NIH Jackson Glyceraldehyde-3-phosphate dehydrogenase Glutamate receptor AMPA type subunit 1 (GRIA1) Glutamate receptor NMDA type subunit 1 (GRIN1) Glutathione peroxidase Gene set enrichment analysis Glutathione Glutathione-disulfide reductase Hypoxia inducible factor 1 Inner boundary membrane Isoelectrofocusing ATP synthase inhibitory factor subunit 1 (ATP5IF1) Inner mitochondrial membrane Isobaric tags for relative and absolute quantitation Knockout Microtubule-associated protein 1 light chain 3 beta (MAP1LC3B) Lactate dehydrogenase A Leucine-rich pentatricopeptide repeat containing protein Long-term potentiation Mitochondrial associated membranes Mouse embryonic fibroblasts. 9.

(18) MFN1 MFN2 MICOS mtDNA mtROS NFκB NMDA NRF2 NRK OCR OL OMM OPA1 OSCP OSR OXPHOS PCA PFA pI pIF1 PRDX PTP RET ROS ROT SAMP8 SC sgRNA SOD1 SOD2 TB stimulation TBS TCA cycle TFAM TG TMRM TOR UPRmt VDAC WB α-F1 β-F1 Δp ΔΨm. 10. Mitofusin 1 Mitofusin 2 Mitochondrial contact site and cristae organizing system mitochondrial DNA Mitochondrial reactive oxygen species Nuclear factor κ light-chain-enhancer of activated B cells N-methyl-D-aspartate Nuclear factor erythroid 2-like 2 (NFE2L2) Normal rat kidney cells Oxygen consumption rate Oligomycin Outer mitochondrial membrane Optic atrophy 1 Oligomycin sensitivity-conferring protein (ATP5PO) Oligomycin-sensitive respiration Oxidative phosphorylation Principal component analysis Paraformaldehyde Isoelectric point Precursor IF1 protein Peroxiredoxin Permeability transition pore Reverse electron transport Reactive oxygen species Rotenone Senescence accelerated mouse-prone 8 mice Supercomplex Single guide RNA Superoxide dismutase 1 Superoxide dismutase 2 Theta-burst stimulation Tris-buffered saline Tricarboxylic acid cycle Transcription factor A, mitochondrial Transgenic mice overexpressing IF1 Tetramethylrhodamine methyl ester Target of rapamycin Mitochondrial unfolded protein response Voltage-dependent anion-selective channel Western blot ATP synthase F1 subunit alpha (ATP5F1A) ATP synthase F1 subunit beta (ATP5F1B) Proton-motive force Mitochondrial membrane potential.

(19) INTRODUCTION.

(20) Introduction 1. Mitochondria are central organelles in cellular physiology Mitochondria are pivotal organelles for the cells because they provide most of the ATP to sustain cellular activities and are critical hubs in intermediary metabolism, cell death execution and intracellular signaling (Pagliarini and Rutter, 2013). Mitochondria are dynamic organelles by nature because they can change their number, structure, protein composition and function to adapt to different cellular needs and situations (Bahat and Gross, 2019). Mitochondria have been traditionally regarded as the “powerhouses” of the cell since they produce most of cellular energy in a process known as oxidative phosphorylation (OXPHOS) (Mitchell, 1961). However, this notion has been expanded in recent years after the demonstration of the role played by mitochondria in the execution of programmed cell death (Liu et al., 1996, Susin et al., 1998) and in establishing pathways to communicate their status to the nucleus (Chandel, 2015, Quiros et al., 2016) and to other compartments (LopezCrisosto et al., 2015, Fernandez-Mosquera et al., 2019) for the execution of integrated cellular responses. Mitochondrial communication is exerted by signaling molecules that modulate the activity of different proteins resulting in the activation/inactivation of particular genetic and/or epigenetic programs controlling proliferation, differentiation and stress responses (Shadel and Horvath, 2015, Chandel, 2015). The list of cellular programs affected by the mitochondrial signalosome is just being glimpse, and remarkably, some of them are responsible for promoting adaptive mechanisms that allow cells to withstand subsequent stresses, a concept coined as “mitohormesis” (Ristow, 2014, Yun and Finkel, 2014).. 2. Mitochondrial structure and dynamics 2.1. Mitochondrial compartments Mitochondria have a bacterial symbiotic origin (Gray, 2012), explaining their “semiautonomous” nature in the eukaryotic cells. Mitochondria are bounded by two membranes, the outer and inner mitochondrial membranes, that enclose two compartments within, the intermembrane space and the matrix, respectively (Fig. 1A). The outer mitochondrial membrane (OMM) defines the outmost border of the organelle and is permeable to small solutes, allowing their free exchange with the cytosol (Palade, 1953). A major protein in the OMM is the voltage-dependent anion-selective channel (VDAC), which forms pores in the membrane and represents the main transporter of ions, metabolites, ATP and other nucleotides, and also interacts with enzymes, coupling different metabolic pathways (Colombini, 2016) (Fig. 1A). The OMM also contains key proteins involved in mitochondrial dynamics, in motility along the tracks paced by the cytoskeleton (Schwarz, 2013) and in stablishing contacts with other mitochondria, through inter-mitochondrial junctions (Picard et al., 2015), with the endoplasmic reticulum (ER), forming mitochondrial associated membranes (MAMs) (Lopez-Crisosto et al., 2015). Moreover, the OMM serves as a platform 13.

(21) for the assembly of signaling complexes involved in innate immune responses (Gurung et al., 2015). In contrast to the OMM, the inner mitochondrial membrane (IMM) represent a highly selective barrier with very low permeability to molecules and ions. This is a necessary condition for the IMM to be the insulating membrane across which the proton electrochemical gradient is built to drive ATP production by OXPHOS (Mitchell, 1961). The IMM can be divided into two differentiated compartments: the inner boundary membrane (IBM), which is close to the OMM, and the cristae membrane, which forms the characteristic invaginations of the IMM delimiting mitochondrial cristae (Zick et al., 2009) (Fig. 1A). Both IMM compartments show an asymmetric distribution of lipids and proteins. The IBM homes proteins involved in IMM fusion (which is the last step of mitochondrial fusion) and the machinery responsible for the regulated import of proteins into mitochondria, while the proteins responsible for OXPHOS and for the biogenesis of iron/sulfur (Fe/S) clusters are preferentially located in the cristae membrane. The IBM and the cristae membrane are connected by crista junctions, which stablish a dynamic boundary between both compartments and insulate the cristae lumen from the IMS, allowing the functional compartmentalization of mitochondria (Cogliati et al., 2016). The mitochondrial contact site and cristae organizing system (MICOS) is a protein complex organized in clusters mainly in cristae junctions that plays a crucial role in the formation and maintenance of cristae architecture (Friedman et al., 2015, Kozjak-Pavlovic, 2017) (Fig. 1A). Crista architecture depends on the coordinated action of MICOS with complexes of the OXPHOS system (Paumard et al., 2002, Friedman et al., 2015) and other factors such as prohibitins (Merkwirth et al., 2008) and the pro-fusion protein optic atrophy 1 (OPA1) (Frezza et al., 2006). Interestingly, crista structure is dynamic and change in different cellular conditions, for instance cristae remodeling is a key process in programmed cell death (Frezza et al., 2006). The matrix is the innermost mitochondrial compartment and hosts the molecules involved in key metabolic pathways including tricarboxylic acid (TCA) cycle, b-oxidation of fatty acids, urea cycle and pyrimidine biosynthesis, as well as mitochondrial DNA (mtDNA) and the machinery for its replication and expression (Logan, 2007). Mitochondria have their own genetic material, reflecting their symbiotic origin. Human mtDNA contains 37 genes: 22 transfer RNA, 2 ribosomal RNA and 13 protein-coding genes (Chinnery and Hudson, 2013). Each mitochondrion harbors multiple mtDNA copies, with the number varying upon tissue and cellular energy demand, and are packaged into nucleoids by different proteins, for instance mitochondrial transcription factor A (TFAM) (Spelbrink, 2010). The 13 mtDNAencoded proteins constitute central subunits of the OXPHOS complexes I, III, IV and the ATP synthase (Chinnery and Hudson, 2013). However, the list of mtDNA-encoded proteins has been recently expanded after the identification of two peptides involved in signaling from mitochondria: humanin (Guo et al., 2003) and MOTS-c (Lee et al., 2015). The remainder of. 14.

(22) the mitochondrial proteome, which consists of about 1500 proteins (Calvo et al., 2006), is encoded in the nuclear genome and is translated in cytosolic ribosomes. Nuclear-encoded proteins are imported into mitochondria through the action of a dedicated machinery consisting of protein complexes in the OMM and IMM that recognize and sort the proteins to the different mitochondrial compartments according to specific targeting sequences (Pfanner and Wiedemann, 2002, Neupert and Brunner, 2002).. Figure 1. Mitochondrial organization and dynamics. A) The electron micrograph shows the ultrastructure of a typical mitochondrion (scale bar, 200 nm). Taken from (Alberts et al., 2002). Schematic of the mitochondrial compartments: outer mitochondrial membrane (OMM), intermembrane space (IMS), inner mitochondrial membrane (IMM), which is subdivided into inner boundary membrane (IBM) and cristae membrane, and matrix. The mitochondrial protein import machinery (yellow) is located in the OMM and IBM. The mitochondrial contact site and cristae organizing system (MICOS, green) is located in cristae junctions and contributes to the functional division between IMS and cristae lumen, and between IBM and cristae membrane. OXPHOS complexes (blue) are located in the cristae membrane. B) Cycle illustrating mitochondrial dynamics. Two mitochondria fuse in a process mainly geared by mitofusins 1 and 2 (MFN1/2) and optic atrophy 1 (OPA1). Fission is mediated by the recruitment of dynamin 1-like protein (DRP1) to the OMM and constriction triggered by endoplasmic reticulum (ER) tubules. VDAC, voltage-dependent anion-selective channel; TOM, translocase of the OMM; SAM, sorting and assembly machinery; TIM, translocase of the IMM; CV, ATP synthase. 15.

(23) 2.2. Mitochondrial dynamics Mitochondria are not static organelles, rather dynamical ones moving through the cytoplasm and experiencing fusion and fission events, forming a heterogeneous network or fragmenting according to the metabolic needs of the cell (Mishra and Chan, 2014). Mitochondrial morphology depends on the relative balance of both processes: reduced fission (or increased fusion) favors the formation of elongated networks, while reduced fusion (or increased fission) promotes mitochondrial fragmentation (Fig. 1B). Both processes depend on the activity of GTP-hydrolyzing enzymes of the dynamin superfamily. Mitochondrial fusion involves the sequential fusion of the OMM of two nearby mitochondria, which is mediated by mitofusin 1 (MFN1) and MFN2, followed by fusion of the IMM, by the action of OPA1 (Fig. 1B). On the other hand, fission is mediated by the recruitment of dynamin 1 like protein (DRP1) from the cytosol to the OMM, by binding to fission receptors, including mitochondrial fission 1 (FIS1) and mitochondrial fission factor (MFF), among others (Mishra and Chan, 2014). Final scission of the mitochondrial membranes requires their constriction in a process mediated by wrapping of ER tubules around the OMM (Friedman et al., 2011) and polymerization of actin filaments, which provide the necessary force for the constriction (Korobova et al., 2013) (Fig. 1B). Mitochondrial dynamics is crucial for the segregation of mitochondria throughout the cell, which is very relevant is specialized cells such as neurons (Yu and Pekkurnaz, 2018), and in cell division (Mishra and Chan, 2014). Moreover, it represents an important mechanism of mitochondrial quality control (Sebastian et al., 2017), by allowing the fission of “damaged” mitochondria and their subsequent elimination by autophagy. The primary sensor of mitochondrial status is the mitochondrial membrane potential (ΔΨm), which collapses in “damaged” or malfunctioning mitochondria. The PTEN-induced kinase 1 (PINK1) is stabilized in the OMM of de-energized mitochondria and recruits the ubiquitin ligase Parkin, which in turn ubiquitinates OMM proteins such as VDAC, being recognized by the autophagic machinery for their selective degradation (Pickrell and Youle, 2015). In addition, the regulated cycles of fusion and fission events play a key role in adapting energy production in mitochondria to the cellular energy demand (Liesa and Shirihai, 2013). Situations in which the energy demand exceeds the supply, mitochondria tend to form an elongated network to increase the efficiency of ATP production by OXPHOS (Liesa and Shirihai, 2013). On the other hand, when the energy demand is lower than the provision, mitochondria to tend to fragment, favoring uncoupling respiration from ATP production.. 3. Organization and regulation of the oxidative phosphorylation system The OXPHOS system is the set of protein complexes, diffusible carriers and transporters located in the IMM that produce most of the ATP under normal conditions (Fig. 2A). The complexes I, II, III and IV and two diffusible carriers form the basic electron transport. 16.

(24) chain (ETC), which generates a proton electrochemical gradient across the IMM. The ATP synthase produces ATP using the free energy stored in the gradient, and the adenine nucleotide translocase (ANT) and mitochondrial phosphate carrier provide the precursors for ATP synthesis (ADP and Pi) (Fig. 2A).. Figure 2. Schematic of the oxidative phosphorylation system. A) The mitochondrial OXPHOS system consists of the complexes I-IV (CI-IV) of the electron transport chain, the ATP synthase, two diffusible electron carriers –ubiquinone (CoQ) and cytochrome c (Cyt c)– and two transporters –the adenine nucleotide translocase (ANT) and phosphate carrier (PiC). Other dehydrogenases transfer electrons from additional substrates into the CoQ pool: the glycerol-3-phosphate dehydrogenase (G3PDH), the dihydroorotate dehydrogenase (DHODH) and the complex acyl-CoA dehydrogenase (ACDH), electron-transferring flavoprotein (ETF) and ETF-dehydrogenase (ETFDH). The energy obtained from electron transfer is used to generate the proton-motive force (Δp), which drives ATP production and solute transport. B) Three-dimensional reconstruction from Protein Data Bank of the bovine ATP synthase indicating the major parts and the different subunits. C) Cryo-EM map of the porcine ATP synthase tetramer, depicting the four protomers (I-IV) (obtained from (Gu et al., 2019)). Note the V-shape of each dimer. IMS, intermembrane space; G3P, glycerol-3phosphate; DHAP, dihydroxyacetone phosphate; DHO, 4,5-dihydroorotate.. 3.1. Components of the electron transport chain The oxidation of different metabolic substrates feeds electrons to the ETC, which couples the thermodynamically favorable transfer of electrons to reduce molecular oxygen to the generation of the proton electrochemical gradient across the IMM (Mitchell, 1961) (Fig. 2A). The potential energy stored in the gradient is measured as proton-motive force (Δp) and. 17.

(25) has two components: (i) a chemical component (ΔpH), due to the different concentration of protons in the IMS and the matrix, and (ii) an electrical one, arising from the asymmetric distribution of charges across the IMM that is known as mitochondrial membrane potential (ΔΨm). Besides driving OXPHOS, Δp is used for mitochondrial import of proteins translated in the cytosol (Neupert and Brunner, 2002), transport of small solutes across the IMM through dedicated transporters (LaNoue and Schoolwerth, 1979) and the biogenesis of Fe/S clusters (Veatch et al., 2009). Complex I (NADH:ubiquinone oxidoreductase) accepts the electrons from NADH and transfers them to ubiquinone (CoQ) pool, pumping protons into the IMS (Brandt, 2006) (Fig. 2A). Mammalian complex I is the largest ETC complex and is built from 44 different proteins, 7 of which are encoded in the mtDNA (Zhu et al., 2016). It has a L-shape structure composed of a hydrophilic and a hydrophobic domain. The former contains the functional modules responsible for NADH oxidation (module N), and electron transfer to the CoQ pool (module Q), whereas the latter contains the module that pumps the protons (module P) (Zhu et al., 2016). Complex II (succinate:ubiquinone oxidoreductase) is the smallest ETC complex and transfers the electrons derived from fumarate oxidation to the CoQ pool (Sun et al., 2005) (Fig. 2A). It is the succinate dehydrogenase enzyme of the TCA cycle and has FAD as a cofactor. It consists of 4 core subunits encoded in the nuclear DNA and does not transport protons into the IMS. Other FAD-dependent dehydrogenases transfer electrons from the oxidation of lipids and dihydroorotate to the CoQ pool: electron-transferring-flavoprotein dehydrogenase and dihydroorotate dehydrogenase, respectively (Alcazar-Fabra et al., 2016) (Fig. 2A). Moreover, electrons obtained in the cytosol, such as the ones derived from glycolysis in the form of NADH, also feed the mitochondrial ETC. Even though the IMM is impermeable to NADH, the reducing equivalents can be imported into mitochondria through two enzymatic systems: (i) the malate-aspartate shuttle, whose net effect is the recycling of cytosolic NAD+ and the production of mitochondrial NADH, and (ii) the glycerol phosphate shuttle, which transfers reducing equivalents from cytosolic NADH to the CoQ pool using FADH2 as a coenzyme (LaNoue and Schoolwerth, 1979). Complex III (ubiquinol:cytochrome c oxidoreductase) is central in the ETC because accepts electrons coming from NADH (through CI) and FADH2 (through CII), which converge in the CoQ pool (Fig. 2A). It transports protons into the intermembrane space by a Q-cycle (Crofts et al., 2003). Complex III is an homodimer with two protomers built from 11 different subunits, one of which is encoded in the mtDNA (Iwata et al., 1998). Complex IV (cytochrome c oxidase) is the terminal enzyme of the ETC and transfers electrons from cytochrome c to reduce O2 into the water of respiration, while pumping protons to the IMS (Fig. 2A). Complex IV consists of 13 subunits, 3 of which are encoded in the mtDNA and form the catalytic core involved in electron transfer and proton transport 18.

(26) (Tsukihara et al., 1996). The additional nuclear-encoded subunits surround the core, protecting it from oxidative damage and are important for the assembly, function and dimerization of the complex (Fontanesi et al., 2006).. 3.2. The mitochondrial ATP synthase: structure, assembly and expression The ATP synthase is the engine of OXPHOS catalyzing ATP synthesis in a process driven by Δp (Boyer, 1997). The mammalian ATP synthase is a protein complex consisting of 27 subunits (18 different proteins) that are arranged into two main functional domains: (i) the membrane-embedded Fo (consisting of subunits a and 8c) and (ii) the soluble catalytic F1 domain (built from 3a, 3b, g, d and e subunits) (Walker, 2013, Kuhlbrandt, 2019) (Fig. 2B). Both domains are connected by a central stalk, which contains g, d and e subunits, and a peripheral stalk, which is made up of subunits b, d, F6 and oligomycin sensitivity-conferring protein (OSCP) (Walker, 2013, Kuhlbrandt, 2019) (Fig. 2B). Additional subunits have been described in the membrane domain when the enzyme is purified in the presence of phospholipids: e, f, g, A6L, diabetes-associated protein in insulin-sensitive tissues (DAPIT) and the 6.8-kDa proteolipid (6.8PL), two of which (a and A6L) are encoded in the mtDNA (Kuhlbrandt, 2019). ATP synthesis is driven by the influx of protons into the matrix through subunit a that triggers the rotation of the c-ring in the membrane Fo domain. The central stalk transfers the torque to the barrel of α3β3 subunits of the F1 domain triggering the conformational changes that drive ATP synthesis from ADP and Pi by a rotatory catalytic mechanism (Srivastava et al., 2018, Murphy et al., 2019). The peripheral stalk acts as a stator, preventing the unproductive rotation of the α3β3 barrel (Hahn et al., 2018). The ATP synthase is a reversible engine because when the energy stored in the form of Δp drops below the free energy stored in the form of ATP (i.e. when mitochondria become de-energized), ATP hydrolysis triggers the rotation of the c-ring in the opposite direction generating a proton gradient across the IMM (Walker, 2013, Saita et al., 2015). The assembly of the human ATP synthase is a multi-step process that has been recently described after the identification of key assembly intermediates (He et al., 2018, Song et al., 2018). The pathway involves the independent assembly of the F1 domain and cring and their subsequent association with the peripheral stalk and the supernumerary subunits e, f and g building a key intermediate complex. This intermediate provides the template for integration of the mtDNA-encoded subunits a and A6L, thus forming the proton channel. DAPIT and 6.8PL are the last subunits to be integrated in the complex, stabilizing the mtDNA-encoded subunits and promoting the dimerization and oligomerization of ATP synthase complexes in higher order assemblies. Super-assemblies of the ATP synthase (Wittig and Schagger, 2009, Nesci and Pagliarani, 2019) play an important role in cristae architecture because generate the. 19.

(27) curvature of the IMM that promotes cristae formation (Strauss et al., 2008, Guo et al., 2017). In fact, liposome-reconstituted dimers of the ATP synthase from yeast and algae selfassemble into rows and bend the lipid bilayer locally without the need of additional proteins or lipids, generating membrane curvature resembling that of cristae (Blum et al., 2019). The recently determined structure of the complete porcine ATP synthase shows that the long rows of ATP synthase running along the cristae edges (Davies et al., 2012) are oligomers of the enzyme formed by associated tetramers (Gu et al., 2019) (Fig. 2B). Importantly, supernumerary subunits of the enzyme are important for its supramolecular association into dimers (Hahn et al., 2016) and oligomers, since their ablation causes loss of ATP synthase superassemblies and respiratory defects (Ohsakaya et al., 2011, Fujikawa et al., 2014). Additional players contribute to cristae architecture such as the pro-fusion protein OPA1, which promotes ATP synthase oligomerization and preserves mitochondrial function upon complex III inhibition (Quintana-Cabrera et al., 2018). Little is known about the expression of the ATP synthase in mammalian tissues. In this regard, expression of the catalytic subunit of the enzyme, b-F1-ATPase (b-F1), is primarily regulated at the translational level (Izquierdo et al., 1990). Translation of b-F1mRNA is boosted in rat liver in the first postnatal hour as a mechanism that contributes to mitochondrial differentiation in adaptation of newborns to the aerobic extrauterine environment (Izquierdo and Cuezva, 1997). Conversely, its translation is repressed in most prevalent human carcinomas, reducing mitochondrial bioenergetic function and reflecting the parallelism in the rewiring of metabolic pathways in cancer and development (Cuezva et al., 2002, Isidoro et al., 2004, Willers and Cuezva, 2011). Mechanistically, the RNA-binding protein Ras-GTPase-activating protein SH3-domain-binding protein 1 (G3BP1) (Ortega et al., 2010) and the microRNA miR-127-5p (Willers et al., 2012) are known to limit b-F1-mRNA translation and may explain the differential expression of the protein in cancer and in development.. 3.3. Regulation of oxidative phosphorylation In vivo, the activity of the OXPHOS system is primarily determined by the cellular ATP demand (Wilson, 2017). Therefore, OXPHOS activity is tightly coupled to the rate of ATP utilization, hence providing a steady state in which there is little variation in cellular ATP levels. Other regulatory inputs are the cellular energy state (ATP/ADP Pi ratio) and the availability of intramitochondrial substrates for the ETC (mainly the NAD+/NADH ratio) (Wilson, 2017). Moreover, different signaling pathways also modulate OXPHOS function (Huttemann et al., 2007). Cellular Ca2+ is an important signaling factor that, besides controlling processes related to excitability and exocytosis, controls mitochondrial respiration (Glancy and Balaban, 2012). Although ER is the main cellular Ca2+ store, mitochondria also take up Ca2+ from the. 20.

(28) cytosol through the mitochondrial calcium uniporter (Baughman et al., 2011, De Stefani et al., 2011) and release it in a transient manner (Drago et al., 2011). Moreover, mitochondria and the ER exchange Ca2+, phospholipids and other ions and metabolites through ERmitochondria encounter structure (ERMES), a protein complex tethering both organelles identified in yeast (Kornmann et al., 2009, Vance, 2014). Cellular Ca2+ promotes mitochondrial respiration by imposing an energy demand to restore its basal cytosolic concentration (Llorente-Folch et al., 2013) and by operating as a signaling molecule activating different mitochondrial enzymes. Extramitochondrial Ca2+ activates ARALAR, the brain isoform of the aspartate-glutamate carrier, which constitutes the malate-aspartate shuttle (Llorente-Folch et al., 2013), and also the mitochondrial ATPMg/Pi carrier (Rueda et al., 2015), thereby stimulating the entry of reducing equivalents and adenine nucleotides into mitochondria to feed OXPHOS. Moreover, matrix Ca2+ activates pyruvate dehydrogenase, the TCA cycle enzymes a-ketoglutarate dehydrogenase and isocitrate dehydrogenase (Denton and McCormack, 1986), and also the ATP synthase (Glancy and Balaban, 2012). Matrix Ca2+ also activates mitochondrial soluble adenylate kinase (Di Benedetto et al., 2014) and triggers signaling mediated by intramitochondrial cAMP and cAMP-dependent protein kinase activity, which can phosphorylate different proteins of the OXPHOS system to regulate its efficiency (Acin-Perez et al., 2009, Di Benedetto et al., 2013, Garcia-Bermudez et al., 2015). Different post-translational modifications have been identified in subunits of the ATP synthase with high-throughput proteomic techniques (Stram and Payne, 2016, Covian and Balaban, 2012, Garcia-Bermudez and Cuezva, 2016). For instance, OSCP K139 can be acetylated and is a target for the deacetylase SIRT3, fine tuning mitochondrial ATP production by OXPHOS with nutrient availability and energy demand (Vassilopoulos et al., 2014). However, the physiological relevance of many of the modifications identified remains to be fully understood. Small solutes such as the metabolites α-ketoglutarate (Chin et al., 2014) and 2hydroxyglutarate (Fu et al., 2015), which directly bind to the ATP synthase, and Ca2+ (Glancy and Balaban, 2012) modulate the activity of the enzyme. S100A1 is a Ca2+-sensing protein that is expressed predominantly in cardiac muscle and interacts with the F1 domain in a Ca2+dependent manner increasing its activity (Boerries et al., 2007). Hence, S100A1 may be involved in coupling Ca2+ cycling during muscle contraction to adjust energy production with demand (Boerries et al., 2007). Highlighting an intricate relationship between OXPHOS and cell death execution, the anti-apoptotic protein Bcl-xL interacts with the ATP synthase, increasing its activity and efficient energy conservation due to reduced ion leak (Alavian et al., 2011). Hence, Bcl-xL may play an additional role as a regulator of energy metabolism in neurons, enhancing synaptic function by promoting efficient ATP production by OXPHOS (Alavian et al., 2011).. 21.

(29) Moreover, dissociation of Bcl-xL from the ATP synthase upon excitotoxic damage may contribute to apoptotic neuronal death by triggering mitochondrial dysfunction (Veas-Perez de Tudela et al., 2015).. 3.4. Production of reactive oxygen species in mitochondria Mitochondria are thought to be the most prominent contributors to reactive oxygen species (ROS) production in most cell types (Holmstrom and Finkel, 2014). Eleven distinct sites have been identified in mitochondria that leak electrons to molecular oxygen producing superoxide or hydrogen peroxide (Brand, 2016). These sites are associated with substrate oxidation (dehydrogenases transferring electrons from NADH or FADH2) and OXPHOS (Brand, 2016). The major sites of superoxide production are generally accepted to be within ETC complexes I and III (Holmstrom and Finkel, 2014) (Fig. 3A). In contrast to the idea that ROS production and effects are nonspecific, which comes from the damaging effects of high ROS levels, physiological ROS production and modulation of signaling pathways is proposed to be localized and controlled (Holmstrom and Finkel, 2014, Scialo et al., 2017). Complex I produces superoxide in the mitochondrial matrix while complex III produces it in the matrix and IMS (Bleier et al., 2015) (Fig. 3A). Superoxide is rapidly dismutated into hydrogen peroxide either non-enzymatically or by the action of superoxide dismutases (SOD2 in the mitochondrial matrix and SOD1 in the IMS and cytosol) (Fig. 3A). Hydrogen peroxide is more stable than superoxide and is capable of crossing membranes, so it has a greater contribution to signaling (Holmstrom and Finkel, 2014) (Fig. 3A). Hydrogen peroxide is reduced into water by the action of glutathione peroxidase 1 (GPX1) and peroxiredoxins 3 and 5 (PRDX3 and PRDX5) in the mitochondrial matrix, or catalase and several GPX and PRDX in the cytoplasm (Martinez-Reyes and Cuezva, 2014) (Fig. 3A). Moreover, cells are endowed with the non-enzymatic antioxidant glutathione (GSH), which is kept in the reduced state by the generation of NADPH through the pentose phosphate pathway (Holmstrom and Finkel, 2014). Remarkably, the detrimental effects of ROS arise when their production exceed the capacity of the antioxidants defenses (Sies et al., 2017). Interestingly, ROS scavengers are starting to be considered ROS sensors that activate different pathways, thus integrating antioxidant and signaling functions (Rhee et al., 2018). It has been proposed that the production of mitochondrial ROS (mtROS) is enhanced when the proton-motive force is high (Murphy, 2009), i.e. when mitochondria become hyperpolarized. This condition is necessary for mtROS production by reverse electron transport (RET), a phenomenon in which electrons are transferred from the CoQ pool back to complex I, which produces superoxide (Scialo et al., 2017). RET occurs by over-reduction of the CoQ pool, which is mainly due to increased activity of complex II, but also by inhibition of complexes III and/or IV (Scialo et al., 2017). Moreover, RET is associated with the inhibition of the ATP synthase (Mills et al., 2016). RET highlights the relevance of site-specific. 22.

(30) ROS production in signaling, because it is important for macrophage activation upon bacterial infection (Murphy, 2019), for the re-organization of the ETC in response to the metabolic fuel and extends lifespan in Drosophila melanogaster (Scialo et al., 2017).. Figure 3. ROS production and supramolecular organization of the electron transport chain. A) The main sources of superoxide (O2–.) are complexes I and III, while complex II also produces it. Superoxide is dismutated into hydrogen peroxide (H2O2) by the action of superoxide dismutase 1 (SOD1) in the intermembrane space (IMS) and SOD2 in the matrix. Hydrogen peroxide can cross membranes, oxidize cellular biomolecules and is reduced into water by the action of catalase (CAT), glutathione peroxidases (GPX) and peroxiredoxins (PRDX), which exist both inside and outside mitochondria. B) Schematic of the main respiratory supercomplexes and organization of the electron transport. The respirasome (complexes I, III and IV) encloses a pool of ubiquinone (CoQ) and cytochrome c (Cyt c). Supercomplex containing complexes I and III transfers electrons to complex IV through a common pool of Cyt c. Electrons coming from complex II are transferred to a common pool of CoQ, which transfers the electrons to the common pool of Cyt c or to a superassembly containing complexes I and IV. For simplicity the main electron routes are indicated. OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane.. 23.

(31) 3.5. Supramolecular organization of the OXPHOS complexes The organization of the five complexes of the OXPHOS system has been debated for a long time. Firstly, solid model proposed that ETC complexes were assembled in a higher-order entity to favor fast channeling and catalysis of substrates (Keilin and Hartree, 1947, Slater, 1953). Later on, fluid model proposed the opposite view in which the complexes freely diffused along the IMM, randomly colliding between themselves allowing electron transfers to occur (Hackenbrock, 1977). Thanks to technical advances, mainly involving the development of blue native gel electrophoresis in which the OXPHOS complexes of the IMM are solubilized and resolved in native conditions, the organization of the OXPHOS system was analyzed. The findings indicated the existence of supramolecular associations of the complexes, known as supercomplexes (SCs) (Schagger and Pfeiffer, 2000). The doubts casted about the existence of supercomplexes were dissipated when their functionality was demonstrated (Acin-Perez et al., 2008) and high-resolution structures of the respirasome, which is a SC containing complexes I, III and IV, was determined (Melber and Winge, 2016) (Fig. 3B). This led to the proposal of the “plasticity” model as a refinement of the fluid model, in which the OXPHOS complexes except complex II can dynamic associate into SCs (Enriquez, 2016). Different SCs has been proposed: the respirasome, complexes I and III, and complexes III and IV (Fig. 3B). Moreover, the ATP synthase also organizes in higher-order superassemblies (Nesci and Pagliarani, 2019), as previously described. The dynamic association of the OXPHOS complexes adds a layer of structural and functional complexity to the system (Enriquez, 2016). The physiological relevance of SCs has been a matter of debate, with different functions proposed (Enriquez, 2016). The association of OXPHOS complexes may canalize electron flux, by enclosing the diffusible electron carriers (CoQ and cytochrome c) within the SCs, thus favoring efficient electron transfer (Schagger, 2001). Moreover, the existence of different SCs has been shown to optimize the efficiency of electron transfer in the ETC depending on the metabolic substrate being used (LapuenteBrun et al., 2013). The relative number of electrons feeding the ETC from NADH or FADH2 depends on the metabolic fuel. Full oxidation of glucose yields 10 electrons in the form of NADH and 2 in the form of FADH2 per glucose molecule, i.e. a NADH:FADH2 ratio of 5; whereas in the case of fatty acids such as palmitate the ratio falls to 2 (Speijer, 2011). Accordingly, when glucose is the main metabolic fuel, most CI is associated to CIII forming a SC with or without CIV that receives electrons from NADH (Schagger and Pfeiffer, 2000, Lapuente-Brun et al., 2013) (Fig. 3B). When electrons primarily come from FADH2, CIII is released from its preferential association with CI and receives the electrons coming through CII (Lapuente-Brun et al., 2013) (Fig. 3B). Efficient electron flux is accomplished by the partition of two functional CoQ pools: (ii) one enclosed in CI-containing SCs, that. 24.

(32) preferentially transfers electrons coming from NADH, and (ii) one free pool in the IMM that receives electrons from FADH2 (Fig. 3B). More recent evidences point that the redox status of the CoQ pool is the metabolic gauge controlling the organization of the ETC complexes into the different SCs (Guaras et al., 2016). When electrons mainly come from FADH2, the CoQ oxidation capacity saturates (i.e. the CoQ pool becomes overreduced) inducing RET and ROS production by CI, resulting in local oxidation of its subunits, its degradation and release from the SC (Guaras et al., 2016). In this regard, an additional role of SCs maybe limiting the production of mtROS. This is based on their capacity to favoring electron transfer (Enriquez, 2016) by promoting an oxidized state in the redox centers of the ETC (Panov et al., 2007). In particular, studies performed in isolated mitochondria, liposome preparations and in cultured cells indicate that ROS production is lower when complex I is associated to complex III (Maranzana et al., 2013, Lopez-Fabuel et al., 2016). Interestingly, in neurons, which highly depend on OXPHOS for energy provision, complex I is mainly associated to complex III forming SCs, while in astrocytes, which are more glycolytic and produce more mtROS, there is more free complex I (Lopez-Fabuel et al., 2016). The differential expression of NDUFS1, a core subunit of complex I present in the N module, appears to be the mechanism explaining the different assembly in both cell types, which contributes to their different bioenergetic and redox profiles (Lopez-Fabuel et al., 2016). Finally, SCs may also be necessary for structural stabilization of individual complexes (Fernandez-Vizarra et al., 2007, Enriquez, 2016). In particular, it has been reported that deficiency of complexes III or IV compromises complex I assembly and stability (Acin-Perez et al., 2004, Diaz et al., 2006). However, the functional relevance of SCs needs to be fully clarified and additional questions need to be addressed regarding the mechanisms of assembly of OXPHOS complexes.. 4. Mitochondrial functions 4.1. Mitochondria are biosynthetic hubs and the gate to cell death Mitochondria are largely known for their bioenergetic function (Mitchell, 1961, Boyer, 1997), hence they are crucial organelles for the correct functioning of energy demanding tissues such as brain (Magistretti, 1999). Moreover, mitochondria are also biosynthetic hubs because they provide anabolic precursors, such as the intermediates of the TCA cycle citrate and oxaloacetate (Boroughs and DeBerardinis, 2015). Importantly, an essential function of mitochondria is the biogenesis of Fe/S clusters, which are important cofactors of diverse cellular proteins involved in genome maintenance, protein translation and mitochondrial respiration (Veatch et al., 2009), and also heme groups (Ajioka et al., 2006). Besides providing energy and precursors necessary for living cells, mitochondria are the gate to of programmed cell death. Programmed cell death is a physiological and highly. 25.