Automated Quantification and Network Analysis of Redox Dynamics in Neuronal Mitochondria

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Computational Systems Biology in Medicine and Biotechnology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2399))

Abstract

Mitochondria are complex organelles with multifaceted roles in cell biology, acting as signaling hubs that implicate them in cellular physiology and pathology. Mitochondria are both the target and the origin of multiple signaling events, including redox processes and calcium signaling which are important for organellar function and homeostasis. One way to interrogate mitochondrial function is by live cell imaging. Elaborated approaches perform imaging of single mitochondrial dynamics in living cells and animals. Imaging mitochondrial signaling and function can be challenging due to the sheer number of mitochondria, and the speed, propagation, and potential short half-life of signals. Moreover, mitochondria are organized in functionally coupled interorganellar networks. Therefore, advanced analysis and postprocessing tools are needed to enable automated analysis to fully quantitate mitochondrial signaling events and decipher their complex spatiotemporal connectedness. Herein, we present a protocol for recording and automating analyses of signaling in neuronal mitochondrial networks.

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References

  1. Choi HB, Gordon GRJ, Zhou N, Tai C, Rungta RL, Martinez J et al (2012) Metabolic communication between astrocytes and neurons via bicarbonate-responsive soluble adenylyl cyclase. Neuron 75:1094–1104

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Herrero-Mendez A, Almeida A, Fernández E, Maestre C, Moncada S, Bolaños JP (2009) The bioenergetic and antioxidant status of neurons is controlled by continuous degradation of a key glycolytic enzyme by APC/C–Cdh1. Nat Cell Biol 11:747–752

    CAS  PubMed  Google Scholar 

  3. Hoppins S, Nunnari J (2012) Mitochondrial dynamics and apoptosis-the ER connection. Science 337:1052–1054

    CAS  PubMed  Google Scholar 

  4. Vaseva AV, Marchenko ND, Ji K, Tsirka SE, Holzmann S, Moll UM (2012) p53 opens the mitochondrial permeability transition pore to trigger necrosis. Cell 149:1536–1548

    CAS  PubMed  PubMed Central  Google Scholar 

  5. MacAskill AF, Kittler JT (2010) Control of mitochondrial transport and localization in neurons. Trends Cell Biol 20(2):102–112

    CAS  PubMed  Google Scholar 

  6. Youle RJ, Narendra DP (2011) Mechanisms of mitophagy. Nat Rev Mol Cell Biol 12:9–14

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Wallace DC (2012) Mitochondria and cancer. Nat Rev Cancer 12:685–698

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Lin MT, Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443:787–795

    CAS  PubMed  Google Scholar 

  9. Nunnari J, Suomalainen A (2012) Mitochondria: in sickness and in health. Cell 148:1145–1159

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Corrado M, Scorrano L, Campello S (2012) Mitochondrial dynamics in cancer and neurodegenerative and neuroinflammatory diseases. Int J Cell Biol 2012:729290

    PubMed  PubMed Central  Google Scholar 

  11. Kurz FT, Kembro JM, Flesia AG, Armoundas AA, Cortassa S, Aon MA et al (2017) Network dynamics: quantitative analysis of complex behavior in metabolism, organelles, and cells, from experiments to models and back. Wiley Interdiscip Rev Syst Biol Med 9(1)

    Google Scholar 

  12. Hamanaka RB, Chandel NS (2010) Mitochondrial reactive oxygen species regulate cellular signaling and dictate biological outcomes. Trends Biochem Sci 35:505–513

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Al-Mehdi AB, Pastukh VM, Swiger BM, Reed DJ, Patel MR, Bardwell GC et al (2012) Perinuclear mitochondrial clustering creates an oxidant-rich nuclear domain required for hypoxia-induced transcription. Sci Sign 5:ra47

    Google Scholar 

  14. Kurz FT, Aon MA, O'Rourke B, Armoundas AA (2018) Assessing spatiotemporal and functional Organization of Mitochondrial Networks. In: 1st (ed) Mitochondrial Bioenergetics. Humana Press, NY, New York, NY, pp 383–402

    Google Scholar 

  15. Murphy MP (2008) How mitochondria produce reactive oxygen species. Biochem J 417(1):1–13

    Google Scholar 

  16. Hirst J (2013) Mitochondrial complex I. Annu Rev Biochem 82:551–575

    CAS  PubMed  Google Scholar 

  17. Ibrahim W, Lee US, Yen HC, St Clair DK, Chow CK (2000) Antioxidant and oxidative status in tissues of manganese superoxide dismutase transgenic mice. Free Radic Biol Med 28:397–402. https://doi.org/10.1016/S0891-5849(99)00253-1

    Article  CAS  PubMed  Google Scholar 

  18. Niethammer P, Grabher C, Look AT, Mitchison TJ (2009) A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. Nature 459:996–999

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Weismann D, Hartvigsen K, Lauer N, Bennett KL, Scholl HPN, Issa PC et al (2011) Complement factor H binds malondialdehyde epitopes and protects from oxidative stress. Nature 478:76–81

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Schwarzländer M, Dick TP, Meyer AJ, Morgan B (2016) Dissecting redox biology using fluorescent protein sensors. Antioxid Redox Signal 24(13):680–712

    PubMed  Google Scholar 

  21. Breckwoldt MO, Wittmann C, Misgeld T, Kerschensteiner M, Grabher C (2015) Redox imaging using genetically encoded redox indicators in zebrafish and mice. Biol Chem 396:511–522.0294

    CAS  PubMed  Google Scholar 

  22. Kurz CT, Aon MA, O'Rourke B, Armoundas AA (2017) Functional implications of cardiac mitochondria clustering, in: mitochondrial dynamics in cardiovascular medicine. Springer, Cham, Cham, pp 1–24

    Google Scholar 

  23. Hanson GT, Aggeler R, Oglesbee D, Cannon M, Capaldi RA, Tsien RY et al (2004) Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem 279:13044–13053

    CAS  PubMed  Google Scholar 

  24. Dooley CT, Dore TM, Hanson GT, Jackson WC, Remington SJ, Tsien RY (2004) Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J Biol Chem 279:22284–22293

    CAS  PubMed  Google Scholar 

  25. Schwarzländer M, Fricker MD, Sweetlove LJ (2009) Monitoring the in vivo redox state of plant mitochondria: effect of respiratory inhibitors, abiotic stress and assessment of recovery from oxidative challenge. Biochim Biophys Acta 1787:468–475

    PubMed  Google Scholar 

  26. Guzman JN, Sanchez-Padilla J, Wokosin D, Kondapalli J, Ilijic E, Schumacker PT et al (2010) Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1. Nature 468:696–700

    CAS  PubMed  PubMed Central  Google Scholar 

  27. van Lith M, Tiwari S, Pediani J, Milligan G, Bulleid NJ (2011) Real-time monitoring of redox changes in the mammalian endoplasmic reticulum. J Cell Sci 124:2349–2356

    PubMed  PubMed Central  Google Scholar 

  28. Gutscher M, Pauleau AL, Marty L, Brach T, Wabnitz GH, Samstag Y et al (2008) Real-time imaging of the intracellular glutathione redox potential. Nat Methods 5:553–559

    CAS  PubMed  Google Scholar 

  29. Albrecht SC, Barata AG, Großhans J, Teleman AA, Dick TP (2011) In vivo map** of hydrogen peroxide and oxidized glutathione reveals chemical and regional specificity of redox homeostasis. Cell Metab 14(6):819–829

    CAS  PubMed  Google Scholar 

  30. Singh S, Kerndt CC, Davis D, Ringer's Lactate (2020) StatPearls. StatPearls Publishing, Treasure Island (FL)

    Google Scholar 

  31. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682

    CAS  PubMed  Google Scholar 

  32. Breckwoldt MO, Pfister FMJ, Bradley PM, Marinković P, Williams PR, Brill MS et al (2014) Multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo. Nat Med 20:555–560

    CAS  PubMed  Google Scholar 

  33. Misgeld T, Nikić I, Kerschensteiner M (2007) In vivo imaging of single axons in the mouse spinal cord. Nat Protoc 2:263–268

    CAS  PubMed  Google Scholar 

  34. Drew PJ, Shih AY, Driscoll JD, Knutsen PM, Blinder P, Davalos D et al (2010) Chronic optical access through a polished and reinforced thinned skull. Nat Methods 7:981–984

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Kerschensteiner M, Reuter MS, Lichtman JW, Misgeld T (2008) Ex vivo imaging of motor axon dynamics in murine triangularis sterni explants. Nat Protoc 3:1645–1653

    PubMed  PubMed Central  Google Scholar 

  36. Li K (2008) The image stabilizer plugin for ImageJ. www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html (02/17/2022)

    Google Scholar 

  37. Kurz FT, Derungs T, Aon MA, O'Rourke B, Armoundas AA (2015) Mitochondrial networks in cardiac myocytes reveal dynamic coupling behavior. Biophys J 108:1922–1933

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Kurz FT, Aon MA, O'Rourke B, Armoundas AA (2010) Spatio-temporal oscillations of individual mitochondria in cardiac myocytes reveal modulation of synchronized mitochondrial clusters. Proc Natl Acad Sci 107:14315–14320

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Kurz FT, Aon MA, O'Rourke B, Armoundas AA (2010) Wavelet analysis reveals heterogeneous time-dependent oscillations of individual mitochondria. Am J Physiol Heart Circ Physiol 299(5):H1736–H1740

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Vetter L, Cortassa S, O'Rourke B, Armoundas AA, Bedja D, Jende JME et al (2020) Diabetes increases the vulnerability of the cardiac mitochondrial network to criticality. Front Physiol 11:175

    PubMed  PubMed Central  Google Scholar 

  41. Kurz FT, Aon MA, O'Rourke B, Armoundas AA (2014) Cardiac mitochondria exhibit dynamic functional clustering. Front Physiol 5:599

    Google Scholar 

  42. Breckwoldt MO, Armoundas AA, Aon MA, Bendszus M, O'Rourke B, Schwarzländer M et al (2016) Mitochondrial redox and pH signaling occurs in axonal and synaptic organelle clusters. Sci Rep 6:23251–23212

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Aon MA, Cortassa S, Marbán E, O'Rourke B (2003) Synchronized whole cell oscillations in mitochondrial metabolism triggered by a local release of reactive oxygen species in cardiac myocytes. J Biol Chem 278:44735–44744

    CAS  PubMed  Google Scholar 

  44. Chazotte B (2011) Labeling mitochondria with TMRM or TMRE. Cold Spring Harb Protoc:895–897

    Google Scholar 

  45. Poburko D, Santo-Domingo J, Demaurex N (2011) Dynamic regulation of the mitochondrial proton gradient during cytosolic calcium elevations. J Biol Chem 286:11672–11684

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Akerboom J, Carreras Calderón N, Tian L, Wabnig S, Prigge M, Tolö J et al (2013) Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front. Mol. Neurosci 6:2

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Schwarzländer M, Logan DC, Johnston IG, Jones NS, Meyer AJ, Fricker MD et al (2012) Pulsing of membrane potential in individual mitochondria: a stress-induced mechanism to regulate respiratory Bioenergetics in Arabidopsis. Plant Cell 24:1188–1201

    PubMed  PubMed Central  Google Scholar 

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Acknowledgments

Experiments that form the basis of this protocol were performed in the laboratory of T. Misgeld (TU Munich) and Martin Kerschensteiner (LMU Munich). M.O.B. acknowledges helpful discussions with T. Dick (DKFZ Heidelberg) and M. Schwarzländer (University of Münster). M.O.B. and F.T.K. were supported by a physician-scientist fellowship of the Medical Faculty, University of Heidelberg and by the Hoffmann-Klose Foundation (University of Heidelberg). F.T.K. was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, KU 3555/1-1) and a research grant from Heidelberg University Hospital.

Author Contributions: F.T.K. and M.O.B. conceived the study. M.O.B. performed microscopy experiments. F.T.K. provided analytical tools. M.O.B. and F.T.K. performed image analysis of the data. F.T.K. and M.O.B. wrote the manuscript.

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Correspondence to Felix T. Kurz or Michael O. Breckwoldt .

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Kurz, F.T., Breckwoldt, M.O. (2022). Automated Quantification and Network Analysis of Redox Dynamics in Neuronal Mitochondria. In: Cortassa, S., Aon, M.A. (eds) Computational Systems Biology in Medicine and Biotechnology. Methods in Molecular Biology, vol 2399. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1831-8_12

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  • DOI: https://doi.org/10.1007/978-1-0716-1831-8_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1830-1

  • Online ISBN: 978-1-0716-1831-8

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