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Synaptosome Bioenergetics and Calcium Handling: Aging Response

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Synaptosomes

Part of the book series: Neuromethods ((NM,volume 141))

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Abstract

Synaptic function and role of mitochondria inside nerve terminals can be studied by the isolation of an enriched fraction of synaptosomes, which consist in nerve-ending particles that are formed during homogenization of brain tissue. Different procedures have been described for the isolation of an enriched synaptosomal fraction, most of them based on the use of gradients.

Neuronal function seems to be critically dependent on the energy provided by mitochondrial respiration. The determination of bioenergetic parameters such as mitochondrial membrane potential, respiratory rates, ATP content, and mitochondrial Ca2+ uptake in synaptosomal preparations can provide useful information for the understanding of the importance of mitochondrial function in neurotransmission.

Synaptic nerve terminals are constantly exposed to complex Ca2+ fluxes. At the presynaptic terminal, the recovery from calcium oscillations critically depends on the proper mitochondrial function to generate ATP and modulate Ca2+ homeostasis together with an efficient endoplasmic reticulum function.

The differential characteristics of synaptic and non-synaptic mitochondria in terms of bioenergetics and free radical production as well as the response to aging are discussed.

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Abbreviations

DAF-2:

4,5-Diaminofluorescein

DAF-2-DA:

4,5-Diaminofluorescein diacetate

EDTA:

Ethylenediaminetetraacetic acid

ER:

Endoplasmic reticulum

FCCP:

Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone

GSNO:

S-nitrosoglutathione

H2O2:

Hydrogen peroxide

l-NNA:

Nω-nitro-l-arginine

MPT:

Mitochondrial permeability transition

NAO:

10-N-nonylacridine orange

NMDA:

N-methyl-d-aspartate

NMDAR2B:

NMDA receptor NR2B subunit

nNOS:

Neuronal nitric oxide synthase

NO:

Nitric oxide

NOS:

Nitric oxide synthase

PSD-95:

Postsynaptic density protein 95 kDa

RNS:

Reactive nitrogen species

ROS:

Reactive oxygen species

SYTs:

Synaptotagmins

TMRE:

Tetramethylrhodamine, ethyl ester

VDAC:

Voltage-dependent anion channel

References

  1. Whittaker VP, Michaelson IA, Kirkland RJ (1964) The separation of synaptic vesicles from nerve-ending particles (‘synaptosomes’). Biochem J 90:293–303

    Article  CAS  Google Scholar 

  2. Rodríguez de Lores Arnaiz G, De Robertis E (1972) Properties of the isolated nerve endings. In: Bronner F, Kleinzeller A (eds) Current topics in membranes and transport, vol III. Academic Press, New York, pp 237–272

    Google Scholar 

  3. Nicholls DG (2010) Stochastic aspects of transmitter release and bioenergetic dysfunction in isolated nerve terminals. Biochem Soc Trans 38:457–459

    Article  CAS  Google Scholar 

  4. Ly CV, Verstreken P (2006) Mitochondria at the synapse. Neuroscientist 12:291–299

    Article  CAS  Google Scholar 

  5. Attwell D, Laughlin SB (2001) An energy budget for signaling in the grey matter of the brain. J Cereb Blood Flow Metab 21:1133–1145

    Article  CAS  Google Scholar 

  6. De Robertis E, Pellegrino de Iraldi A, Rodríguez de Lores Arnaiz G et al (1961) On the isolation of nerve endings and synaptic vesicles. J Biophys Biochem Cytol 9:229–235

    Article  Google Scholar 

  7. Whittaker VP (1959) The isolation and characterization of acetylcholine-containing particles from brain. Biochem J 72:694–706

    Article  CAS  Google Scholar 

  8. Bellamy D (1959) The distribution of bound acetylcholine and choline acetylase in rat and pigeon brain. Biochem J 72:165–168

    Article  CAS  Google Scholar 

  9. De Robertis E, Pellegrino de Iraldi A, Rodríguez de LoresArnaiz G et al (1962) Cholinergic and non-cholinergic nerve endings in rat brain. I. Isolation and subcellular distribution of acetylcholine and acetylcholinesterase. J Neurochem 9:23–35

    Article  Google Scholar 

  10. Gray EG, Whittaker VP (1962) The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation. J Anat 96:79–88

    Google Scholar 

  11. Rodríguez de Lores Arnaiz G, Alberici M, De Robertis E (1967) Ultrastructural and enzymic studies of cholinergic and noncholinergic synaptic membranes isolated from brain cortex. J Neurochem 14:215–225

    Article  Google Scholar 

  12. Lai JC, Clark JB (1976) Preparation and properties of mitochondria derived from synaptosomes. Biochem J 154:423–432

    Article  CAS  Google Scholar 

  13. Cotman CW, Matthews DA (1971) Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization. Biochim Biophys Acta 249:380–394

    Article  CAS  Google Scholar 

  14. Rodríguez de Lores Arnaiz G, Girardi E (1977) The increase in respiratory capacity of brain subcellular fractions after the administration of the convulsant 3-mercaptopropionic acid. Life Sci 21:637–646

    Article  Google Scholar 

  15. Lores-Arnaiz S, Lombardi P, Karadayian AG et al (2016) Brain cortex mitochondrial bioenergetics in synaptosomes and non-synaptic mitochondria during aging. Neurochem Res 41:353–363

    Article  CAS  Google Scholar 

  16. Lores-Arnaiz S, Bustamante J (2011) Age-related alterations in mitochondrial physiological parameters and nitric oxide production in synaptic and non-synaptic brain cortex mitochondria. Neuroscience 188:117–124

    Article  CAS  Google Scholar 

  17. Anderson MF, Sims NR (2000) Improved recovery of highly enriched mitochondrial fractions from small brain tissue samples. Brain Res Protocol 5:95–101

    Article  CAS  Google Scholar 

  18. Dunkley PR, Jarvie PE, Robinson PJ (2008) A rapid Percoll gradient procedure for preparation of synaptosomes. Nat Protoc 3:1718–1728

    Article  CAS  Google Scholar 

  19. Tenreiro P, Rebelo S, Martins F et al (2017) Comparison of simple sucrose and percoll based methodologies for synaptosome enrichment. Anal Biochem 517:1–8

    Article  CAS  Google Scholar 

  20. Nicholls DG (1993) The glutamatergic nerve terminal. Eur J Biochem 212:613–631

    Article  CAS  Google Scholar 

  21. Dodd PR, Hardy JA, Oakley AE et al (1981) A rapid method for preparing synaptosomes: comparison, with alternative procedures. Brain Res 226:107–118

    Article  CAS  Google Scholar 

  22. Dunkley PR, Jarvie PE, Heath JW et al (1986) A rapid method for isolation of synaptosomes on Percoll gradients. Brain Res 372:115–129

    Article  CAS  Google Scholar 

  23. Gylys KH, Fein JA, Cole GM (2000) Quantitative characterization of crude synaptosomal fraction (P-2) components by flow cytometry. J Neurosci Res 61:186–192

    Article  CAS  Google Scholar 

  24. Sudhof TC (1995) The synaptic vesicle cycle: a cascade of protein-proteininteractions. Nature 375:645–653

    Article  CAS  Google Scholar 

  25. Kornau HC, Schenker LT, Kennedy MB et al (1995) Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 269:1737–1740

    Article  CAS  Google Scholar 

  26. Blanco Garcia J, Aldinucci C, Maiorca SM et al (2009) Physiopathological effects of the NO donor 3-Morpholinosydnonimine on rat cortical synaptosomes. Neurochem Res 34:931–941

    Article  CAS  Google Scholar 

  27. Gylys KH, Fein JA, Yang F et al (2004) Enrichment of presynaptic and postsynaptic markers by size-based gating analysis of synaptosome preparations from rat and human cortex. Cytometry A 60:90–96

    Article  Google Scholar 

  28. Hollenbeck PJ, Saxton WM (2005) The axonal transport of mitochondria. J Cell Sci 118:5411–5419

    Article  CAS  Google Scholar 

  29. Guo X, Macleod GT, Wellington A et al (2005) GTPase dMiro is required for axonal transport of mitochondria to Drosophila synapses. Neuron 47:379–393

    Article  CAS  Google Scholar 

  30. Ma H, Cai Q, Lu W et al (2009) KIF5B motor adaptor syntabulin maintains synaptic transmission in sympathetic neurons. J Neurosci 29:13019–13029

    Article  CAS  Google Scholar 

  31. Stowers RS, Megeath LJ, Górska-Andrzejak J et al (2002) Axonal transport of mitochondria to synapses depends on Milton, a novel Drosophila protein. Neuron 36:1063–1077

    Article  CAS  Google Scholar 

  32. Lin MY, Sheng ZH (2015) Regulation of mitochondrial transport in neurons. Exp Cell Res 334:35–44

    Article  CAS  Google Scholar 

  33. Sheng ZH (2014) Mitochondrial trafficking and anchoring in neurons: new insight and implications. J Cell Biol 204:1087–1098

    Article  CAS  Google Scholar 

  34. Brown MR, Sullivan PG, Geddes JW (2006) Synaptic mitochondria are more susceptible to Ca2+ overload than non-synaptic mitochondria. J Biol Chem 281:11658–11668

    Article  CAS  Google Scholar 

  35. Naga KK, Sullivan PG, Geddes JW (2007) High cyclophilin D content of synaptic mitochondria results in increased vulnerability to permeability transition. J Neurosci 27:7469–7475

    Article  CAS  Google Scholar 

  36. Yarana C, Sanit J, Chattipakorn N et al (2012) Synaptic and non synaptic mitochondria demonstrate a different degree of calcium-induced mitochondrial dysfunction. Life Sci 90:808–814

    Article  CAS  Google Scholar 

  37. Choi SW, Gerencser AA, Lee DW et al (2011) Intrinsic bioenergetic properties and stress sensitivity of dopaminergic synaptosomes. J Neurosci 31:4524–4534

    Article  CAS  Google Scholar 

  38. Brand MD, Nicholls DG (2011) Assessing mitochondrial dysfunction in cells. Biochem J 435:297–312

    Article  CAS  Google Scholar 

  39. Choi SW, Gerencser AA, Nicholls DG (2009) Bioenergetic analysis of isolated cerebrocortical nerve terminals on a microgram scale: spare respiratory capacity and stochastic mitochondrial failure. J Neurochem 109:1179–1191

    Article  CAS  Google Scholar 

  40. Chavan V, Willis J, Walker SK et al (2015) Central presynaptic terminals are enriched in ATP but the majority lack mitochondria. PLoS One 10(4):e0125185. https://doi.org/10.1371/journal.pone.0125185

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Bustamante J, Di Libero E, Fernandez-Cobo M et al (2004) Kinetic analysis of thapsigargin-induced thymocyte apoptosis. Free Radic Bio Med 37:1490–1498

    Article  CAS  Google Scholar 

  42. Alekseenko AV, Lemeshchenko VV, Pekun TG et al (2012) Glutamate-induced free radical formation in rat brain synaptosomes is not dependent on intrasynaptosomal mitochondria membrane potential. Neurosci Lett 513:238–242

    Article  CAS  Google Scholar 

  43. Hrynevich SV, Pekun TG, Waseem TV et al (2015) Influence of glucose deprivation on membrane potentials of plasma membranes, mitochondria and synaptic vesicles in rat brain synaptosomes. Neurochem Res 40:1188–1196

    Article  CAS  Google Scholar 

  44. Berridge MJ (1997) Elementary and global aspects of calcium signalling. J Physiol 499:291–306

    Article  CAS  Google Scholar 

  45. Bootman MD, Collins TJ, Peppiatt CM et al (2001) Calcium signalling-an overview. Semin Cell Dev Biol 12:3–10

    Article  CAS  Google Scholar 

  46. Bucurenciu I, Bischofberger J, Jonas P (2010) A small number of open Ca2+ channels trigger transmitter release at a central GABAergic synapse. Nat Neurosci 13:19–21

    Article  CAS  Google Scholar 

  47. Bacci A, Verderio C, Pravettoni E et al (1999) Synaptic and intrinsic mechanisms shape synchronous oscillations in hippocampal neurons in culture. Eur J Neurosci 11:389–397

    Article  CAS  Google Scholar 

  48. Berridge MJ (1998) Neuronal calcium signaling. Neuron 21:13–26

    Article  CAS  Google Scholar 

  49. Berridge MJ, Lipp P, Bootman MD (2000) The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1:11–21

    Article  CAS  Google Scholar 

  50. Ishii K, Hirose K, Iino M (2006) Ca2+ shuttling between endoplasmic reticulum and mitochondria underlying Ca2+ oscillations. EMBO Rep 7:390–396

    Article  CAS  Google Scholar 

  51. Bannai H, Inonie H, Nakayama T et al (2004) Kinesin dependent, rapid, bi-directional transport of ER sub-compartment in dendrites of hippocampal neurons. J Cell Sci 117:163–175

    Article  CAS  Google Scholar 

  52. Mironov SL, Symonchuk N (2006) ER vesicles and mitochondria move and communicate at synapses. J Cell Sci 119:4926–4934

    Article  CAS  Google Scholar 

  53. Markram H, Helm PJ, Sakmann B (1995) Dendritic calcium transients evoked by single back-propagating action potentials in rat neocortical pyramidal neurons. J Physiol 485:1–20

    Article  CAS  Google Scholar 

  54. Billups B, Forsythe ID (2002) Presynaptic mitochondrial calcium sequestration influences transmission at mammalian central synapses. J Neurosci 22:5840–5847

    Article  CAS  Google Scholar 

  55. Medler K, Gleason EL (2002) Mitochondrial Ca(2+) buffering regulates synaptic transmission between retinal amacrine cells. J Neurophysiol 87:1426–1439

    Article  CAS  Google Scholar 

  56. Belair EL, Vallee J, Robitaille R (2005) Long-term in vivo modulation of synaptic efficacy at the neuromuscular junction of Rana pipiens frogs. J Physiol 569:163–178

    Article  CAS  Google Scholar 

  57. Mattson MP (2008) Glutamate and neurotrophic factors in neuronal plasticity and disease. Ann N Y Acad Sci 1144:97–112

    Article  CAS  Google Scholar 

  58. Nicholls DG (2008) Oxidative stress and energy crises in neuronal dysfunction. Ann N Y Acad Sci 1147:53–60 o

    Article  CAS  Google Scholar 

  59. Bustamante J, Czerniczyniec A, Lores-Arnaiz S (2016) Ketamine effect on intracellular and mitochondrial calcium mobilization. Biocell 40:11–14

    CAS  Google Scholar 

  60. Logan CV, Szabadkai G, Sharpe JA et al (2014) Loss-of-function mutations in MICU1 cause a brain and muscle disorder linked to primary alterations in mitochondrial calcium signaling. Nat Genet 46:188–193

    Article  CAS  Google Scholar 

  61. Rangaraju V, Calloway N, Ryan TA (2014) Activity-driven local ATP synthesis is required for synaptic function. Cell 156:825–835

    Article  CAS  Google Scholar 

  62. Katz B, Miledi R (1967) Ionic requirements of synaptic transmitter release. Nature 215:651

    Article  CAS  Google Scholar 

  63. Sabatini BL, Regehr WG (1996) Timing of neurotransmission at fast synapses in the mammalian brain. Nature 384:170–172

    Article  CAS  Google Scholar 

  64. Perin MS, Fried VA, Mignery GA et al (1990) Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature 345:260–263

    Article  CAS  Google Scholar 

  65. Südhof TC (2012) Calcium control of neurotransmitter release. Cold Spring Harb Perspect Biol 4:a011353

    Article  Google Scholar 

  66. Kavalali ET (2015) The mechanisms and functions of spontaneous neurotransmitter release. Nat Rev Neurosci 16(1):5–16

    Article  CAS  Google Scholar 

  67. Kilbride SM, Telford JE, Davey GP (2008) Age-related changes in H2O2 production and bioenergetics in rat brain synaptosomes. Biochem Biophys Acta 1777:783–788

    CAS  PubMed  Google Scholar 

  68. Tarasenko A, Krupko O, Himmelreich N (2011) Presynaptic kainate and NMDA receptors are implicated in the modulation of GABA release from cortical and hippocampal nerve terminals. Neurochem Int 59:81–89

    Article  CAS  Google Scholar 

  69. Tarasenko A, Krupko O, Himmelreich N (2012) Reactive oxygen species induced by presynaptic glutamate receptor activation is involved in [3H] GABA release from rat brain cortical nerve terminals. Neurochem Int 61:1044–1051

    Article  CAS  Google Scholar 

  70. Abdel-Rahman EA, Mahmoud AM, Aaliya A et al (2016) Resolving contributions of oxygen-consuming and ROS-generating enzymes at the 2016 synapse. Oxidative Med Cell Longev 2016:1. https://doi.org/10.1155/2016/1089364

    Article  CAS  Google Scholar 

  71. Pubill D, Chipana C, Camins A et al (2005) Free radical production induced by methamphetamine in rat striatal synaptosomes. Toxicol Appl Pharmacol 204:57–68

    Article  CAS  Google Scholar 

  72. Sipos I, Tretter L, Adam-Vizi V (2003) Quantitative relationship between inhibition of respiratory complexes and formation of reactive oxygen species in isolated nerve terminals. J Neurochem 84:112–118

    Article  CAS  Google Scholar 

  73. Sipos I, Tretter L, Adam-Vizi V (2003) The production of reactive oxygen species in intact isolated nerve terminals is independent of the mitochondrial membrane potential. Neurochem Res 28:1575–1581

    Article  CAS  Google Scholar 

  74. Faber-Zuschratter H, Seidenbecher T, Reymann K et al (1996) Ultrastructural distribution of NADPH-diaphorase in the normal hippocampus and after long-term potentiation. J Neural Transm 103:807–817

    Article  CAS  Google Scholar 

  75. Batista CM, de Paula KC, Cavalcante LA et al (2001) Subcellular localization of neuronal nitric oxide synthase in the superficial gray layer of the rat superior colliculus. Neurosci Res 41:67–70

    Article  CAS  Google Scholar 

  76. Czerniczyniec A, Bustamante J, Lores-Arnaiz S (2006) Modulation of brain mitochondrial function by deprenyl. Neurochem Int 48:235–241

    Article  CAS  Google Scholar 

  77. Brown GC, Cooper CE (1994) Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase. FEBS Lett 356:295–298

    Article  CAS  Google Scholar 

  78. López-Figueroa MO, Caamaño C, Morano MI et al (2000) Direct evidence of nitric oxide presence within mitochondria. Biochem Biophys Res Commun 272:129–133

    Article  Google Scholar 

  79. Genova ML, Bovina C, Marchetti M et al (1997) Decrease of rotenone inhibition is a sensitive parameter of complex I damage in brain non-synaptic mitochondria of aged rats. FEBS Lett 410:467–469

    Article  CAS  Google Scholar 

  80. Lenaz G, D’Aurelio M, Merlo Pich M et al (2000) Mitochondrial bioenergetics in aging. Biochem Biophys Acta 1459:397–404

    CAS  PubMed  Google Scholar 

  81. Navarro A, Boveris A (2007) Brain mitochondrial dysfunction in aging: conditions that improve survival, neurological performance and mitochondrial function. Front Biosci 12:1154–1163

    Article  CAS  Google Scholar 

  82. Davey GP, Peuchen S, Clark JB (1998) Energy thresholds in brain mitochondria. Potentialinvolvement in neurodegeneration. J Biol Chem 273:12753–12757

    Article  CAS  Google Scholar 

  83. Nicoletti VG, Tendi EA, Lalicata C et al (1995) Changes of mitochondrial cytochrome c oxidase and FoF1 ATP synthase subunits in rat cerebral cortex during aging. Neurochem Res 20:1465–1470

    Article  CAS  Google Scholar 

  84. Manczak M, Jung Y, Park BS et al (2005) Time course of mitochondrial gene expressions in mice brains: implications for mitochondrial dysfunction, oxidative damage, and cytochrome c in aging. J Neurochem 92:494–504

    Article  CAS  Google Scholar 

  85. Barazzoni R, Nair KS (2001) Changes in uncoupling protein-2 and -3 expression in aging rat skeletal muscle, liver, and heart. Am J Physiol Endocrinol Metab 280:E413–E419

    Article  CAS  Google Scholar 

  86. Stauch KL, Purnell PR, Fox HS (2014) Aging synaptic mitochondria exhibit dynamic proteomic changes while maintaining bioenergetic function. Aging (Albany NY) 6:320–334

    Article  CAS  Google Scholar 

  87. Kokoszka JE, Coskun P, Esposito LA et al (2001) Increased mitochondrial oxidative stress in the Sod2 (+/−) mouse results in the age-related decline of mitochondrial function culminating in increased apoptosis. Proc Natl Acad Sci USA 98:2278–2283

    Article  CAS  Google Scholar 

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Acknowledgments

This research was supported by grants from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PIP 112-20110100271) and Universidad de Buenos Aires (UBA, 20020130100255).

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Authors and Affiliations

  1. Facultad de Farmacia y Bioquímica, Fisicoquímica, Universidad de Buenos Aires, Buenos Aires, Argentina

    Silvia Lores-Arnaiz & Analía G. Karadayian

  2. Instituto de Bioquímica y Medicina Molecular (IBIMOL), CONICET-Universidad de Buenos Aires, Buenos Aires, Argentina

    Silvia Lores-Arnaiz & Analía G. Karadayian

  3. Instituto de Biología Celular y Neurociencias “Prof. E. De Robertis” (IBCN), CONICET-Universidad de Buenos Aires, Buenos Aires, Argentina

    Georgina Rodríguez de Lores Arnaiz

  4. Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina

    Georgina Rodríguez de Lores Arnaiz

  5. Centro de Altos Estudios en Ciencias de la Salud (CAECIHS), Universidad Abierta Interamericana, Buenos Aires, Argentina

    Juanita Bustamante

Authors
  1. Silvia Lores-Arnaiz
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  2. Georgina Rodríguez de Lores Arnaiz
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  3. Analía G. Karadayian
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  4. Juanita Bustamante
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Corresponding author

Correspondence to Silvia Lores-Arnaiz .

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Editors and Affiliations

  1. Department of Psychology, Neuroscience & Behavior, McMaster University, Hamilton, ON, Canada

    Kathryn M. Murphy

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Lores-Arnaiz, S., Rodríguez de Lores Arnaiz, G., Karadayian, A.G., Bustamante, J. (2018). Synaptosome Bioenergetics and Calcium Handling: Aging Response. In: Murphy, K. (eds) Synaptosomes. Neuromethods, vol 141. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8739-9_8

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