Isolation of Mitochondria from the CNS

Tibor Kristian1

1 Department of Anesthesiology, Organized Research Center, School of Medicine, University of Maryland, Baltimore, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.22
DOI:  10.1002/0471142301.ns0722s52
Online Posting Date:  July, 2010
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This unit contains a protocol describing the isolation of brain mitochondria by using discontinuous Percoll gradient centrifugation. The Percoll density gradient centrifugation separates synaptosomes, myelin, and free nonsynaptic mitochondria released from cells during tissue homogenization into individual fractions. Mitochondria entrapped in synaptosomes (synaptic mitochondria) can be liberated using nitrogen cavitation and then further purified by Percoll gradient centrifugation. These methods yield mitochondria that exhibit good respiratory coupling and high respiratory rates. Curr. Protoc. Neurosci. 52:7.22.1‐7.22.12. © 2010 by John Wiley & Sons, Inc.

Keywords: brain; synaptosomes; nonsynaptic; mitochondria; Percoll

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Isolation of Nonsynaptic and Synaptic Mitochondria from Brain Using Percoll Gradient Centrifugation
  • Support Protocol 1: Isolation of Synaptic Mitochondria from Synaptosomes Using Nitrogen Cavitation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Isolation of Nonsynaptic and Synaptic Mitochondria from Brain Using Percoll Gradient Centrifugation

  • Isolation medium (IM; see recipe)
  • Rodents: one rat, usually an adult male Sprague‐Dawley or Fisher 344 (200‐300 g); or two mice, wild type or transgenic (strain C57Bl/6; 20‐25 g).
  • Percoll (GE Healthcare, cat. no. 17089101) gradient solutions (see recipe)
  • Bovine serum albumin (BSA), fatty acid‐free (10 mg/ml; see recipe)
  • Isolation medium with no EGTA (IM‐EGTA; see recipe)
  • Protease inhibitor cocktail (Sigma, cat. no. P8340)
  • 50‐ml disposable centrifuge tubes, polypropylene (Fisher Scientific, cat. no. 055396)
  • 50‐ml glass beakers
  • Glass vessel Teflon pestle Potter‐Elvehjem Homogenizer (30 ml; Colonial Scientific, cat. no. 358049)
  • Decapicones (Braintree Scientific, Model DC‐200)
  • Small animal guillotine (Harvard Apparatus, cat. no. PY8 73‐1918)
  • Beebee bone scissors (FST, cat. no. 16044‐10)
  • 12‐in. long nickel stainless spatula
  • Medium straight‐edged scissors (FST, cat. no. 14002‐14)
  • 10‐ml polycarbonate centrifuge tubes for the JA‐21 rotor (16 mm × 76 mm; Beckman Coulter, cat. no. 355630)
  • Plastic transparent pipets
  • Single‐use disposable Pasteur pipets
  • Beckman Coulter (J2‐MC) high‐speed refrigerated centrifuge with fixed‐angle rotor JA 21 or equivalent high‐speed centrifuge and rotor that holds 10‐ml centrifuge tubes
  • 5‐ and 1‐ml volumetric pipets
  • Glass stirring rod
  • 1.5‐ml microcentrifuge tubes
NOTE: All protocols that use live animals must be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) prior to initiation of the study.NOTE: Ice‐cold IM is used in all steps.

Support Protocol 1: Isolation of Synaptic Mitochondria from Synaptosomes Using Nitrogen Cavitation

  • Cell disruption vessel (45 ml; Parr Instrument, cat. no. 4639)
  • Stir bar
  • Nitrogen tank
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Literature Cited

   Beal, M.F. 2005. Mitochondria take center stage in aging and neurodegeneration. Ann. Neurol. 58:495‐505.
   Booth, R.F. and Clark, J.B. 1979. A method for the rapid separation of soluble and particulate components of rat brain synaptosomes. FEBS Lett. 107:387‐392.
   Brown, M.R., Sullivan, P.G., Dorenbos, K.A., Modafferi, E.A., Geddes, J.W., and Steward, O. 2004. Nitrogen disruption of synaptoneurosomes: An alternative method to isolate brain mitochondria. J. Neurosci. Methods 137:299‐303.
   Brown, M.R., Sullivan, P.G., and Geddes, J.W. 2006. Synaptic mitochondria are more susceptible to Ca2+ overload than nonsynaptic mitochondria. J. Biol. Chem. 281:11658‐11668.
   Brustovetsky, N. and Dubinsky, J.M. 2000. Limitations of cyclosporin A inhibition of the permeability transition in CNS mitochondria. J. Neurosci. 20:8229‐8237.
   Brustovetsky, N., Jemmerson, R., and Dubinsky, J.M. 2002. Calcium‐induced Cytochrome c release from rat brain mitochondria is altered by digitonin. Neurosci. Lett. 332:91‐94.
   Brustovetsky, N., Brustovetsky, T., Purl, K.J., Capano, M., Crompton, M., and Dubinsky, J.M. 2003. Increased susceptibility of striatal mitochondria to calcium‐induced permeability transition. J. Neurosci. 23:4858‐4867.
   Chinopoulos, C., Starkov, A.A., and Fiskum, G. 2003. Cyclosporin A‐insensitive permeability transition in brain mitochondria: inhibition by 2‐aminoethoxydiphenyl borate. J. Biol. Chem. 278:27382‐27389.
   Clark, J.B. and Nicklas, W.J. 1970. The metabolism of rat brain mitochondria. Preparation and characterization. J. Biol. Chem. 245:4724‐4731.
   Colbeau, A., Nachbaur, J., and Vignais, P.M. 1971. Enzymic characterization and lipid composition of rat liver subcellular membranes. Biochim. Biophys. Acta 249:462‐492.
   Dunkley, P.R., Jarvie, P.E., Heath, J.W., Kidd, G.J., and Rostas, J.A. 1986. A rapid method for isolation of synaptosomes on Percoll gradients. Brain Res. 372:115‐129.
   Elias, P.M., Goerke, J., Friend, D.S., and Brown, B.E. 1978. Freeze‐fracture identification of sterol‐digitonin complexes in cell and liposome membranes. J. Cell Biol. 78,577‐596.
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   Friberg, H., Connern, C., Halestrap, A.P., and Wieloch, T. 1999. Differences in the activation of the mitochondrial permeability transition among brain regions in the rat correlate with selective vulnerability. J. Neurochem. 72:2488‐2497.
   Graham, J.M. 1999. Purification of a crude mitochondrial fraction by density‐gradient centrifugation. Curr. Protoc. Cell Biol. 4:3.4.1‐3.4.22.
   Harrison, S.M., Jarvie, P.E., and Dunkley, P.R. 1988. A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: viability of subcellular fractions. Brain Res. 441:72‐80.
   Hazelton, J.L., Petrasheuskaya, M., Fiskum, G., and Kristian, T. 2009. Cyclophilin D is expressed predominantly in mitochondria of gamma‐aminobutyric acidergic interneurons. J. Neurosci. Res. 87:1250‐1259.
   Kristal, B.S., Stavrovskaya, I.G., Narayanan, M.V., Krasnikov, B.F., Brown, A.M., Beal, M.F., and Friedlander, R.M. 2004. The mitochondrial permeability transition as a target for neuroprotection. J. Bioenerg. Biomembr. 36:309‐312.
   Kristian, T. 2004. Metabolic stages, mitochondria and calcium in hypoxic/ischemic brain damage. Cell Calcium 36:221‐233.
   Kristian, T., Gertsch, J., Bates, T.E., and Siesjo, B.K. 2000. Characteristics of the calcium‐triggered mitochondrial permeability transition in nonsynaptic brain mitochondria: Effect of cyclosporin A and ubiquinone O. J. Neurochem. 74:1999‐2009.
   Kristian, T., Hopkins, I.B., McKenna, M.C., and Fiskum, G. 2006. Isolation of mitochondria with high respiratory control from primary cultures of neurons and astrocytes using nitrogen cavitation. J. Neurosci. Methods 152:136‐143.
   Lai, J.C. and Clark, J.B. 1976. Preparation and properties of mitochondria derived from synaptosomes. Biochem. J. 154:423‐432.
   Lai, J.C., Walsh, J.M., Dennis, S.C., and Clark, J.B. 1977. Synaptic and non‐synaptic mitochondria from rat brain: Isolation and characterization. J. Neurochem. 28:625‐631.
   Morota, S., Hansson, M.J., Ishii, N., Kudo, Y., Elmer, E., and Uchino, H. 2007. Spinal cord mitochondria display lower calcium retention capacity compared with brain mitochondria without inherent differences in sensitivity to cyclophilin D inhibition. J. Neurochem. 103:2066‐2076.
   Naga, K.K., Sullivan, P.G., and Geddes, J.W. 2007. High cyclophilin D content of synaptic mitochondria results in increased vulnerability to permeability transition. J. Neurosci. 27:7469‐7475.
   Panov, A.V., Gutekunst, C.A., Leavitt, B.R., Hayden, M.R., Burke, J.R., Strittmatter, W.J., and Greenamyre, J.T. 2002. Early mitochondrial calcium defects in Huntington's disease are a direct effect of polyglutamines. Nat. Neurosci. 5:731‐736.
   Rosenthal, R.E., Hamud, F., Fiskum, G., Varghese, P.J., and Sharpe, S. 1987. Cerebral ischemia and reperfusion: prevention of brain mitochondrial injury by lidoflazine. J. Cereb. Blood Flow Metab. 7:752‐758.
   Sims, N.R. 1990. Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation. J. Neurochem. 55:698‐707.
   Sims, N.R. and Anderson, M.F. 2008. Isolation of mitochondria from rat brain using Percoll density gradient centrifugation. Nat. Protoc. 3:1228‐1239.
   Stavrovskaya, I.G. and Kristal, B.S. 2005. The powerhouse takes control of the cell: Is the mitochondrial permeability transition a viable therapeutic target against neuronal dysfunction and death? Free Radic. Biol. Med. 38:687‐697.
   Sullivan, P.G., Rabchevsky, A.G., Waldmeier, P.C., and Springer, J.E. 2005. Mitochondrial permeability transition in CNS trauma: Cause or effect of neuronal cell death? J. Neurosci. Res. 79:231‐239.
   Taupin, P., Ben‐Ari, Y., and Roisin, M.P. 1994. Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: Evoked release of glutamate, GABA, aspartate and glutamate decarboxylase activity in control and degranulated rat hippocampus. Brain Res. 644:313‐321.
   Zaidan, E. and Sims, N.R. 1994. The calcium content of mitochondria from brain subregions following short‐term forebrain ischemia and recirculation in the rat. J. Neurochem. 63:1812‐1819.
Key References
   Sims and Anderson, 2008. See above.
  This paper offers detailed protocols for isolating mitochondria from brain tissues of different sample sizes by using a modification of Percoll gradient centrifugation.
   Graham, 1999. See above.
  Describes the principles of gradient centrifugation and use of variety of density media that can be used to prepare pure fractions of mitochondria.
   Brown et al., 2004. See above.
  This paper describes the protocol that uses the nitrogen cavitation to isolate the whole mitochondrial population (nonsynaptic and synaptic mitochondria) from brain homogenate.
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