Synaptic Mitochondria Are More Susceptible to Ca2+Overload than Nonsynaptic Mitochondria

Mitochondria in nerve terminals are subjected to extensive Ca2+fluxes and high energy demands, but the extent to which the synaptic mitochondria buffer Ca2+ is unclear. In this study, we identified a difference in the Ca2+ clearance ability of nonsynaptic versus synaptic mitochondrial populations enriched from rat cerebral cortex. Mitochondria were isolated using Percoll discontinuous gradients in combination with high pressure nitrogen cell disruption. Mitochondria in the nonsynaptic fraction originate from neurons and other cell types including glia, whereas mitochondria enriched from a synaptosomal fraction are predominantly neuronal and presynaptic in origin. There were no differences in respiration or initial Ca2+ loads between nonsynaptic and synaptic mitochondrial populations. Following both bolus and infusion Ca2+ addition, nonsynaptic mitochondria were able to accumulate significantly more exogenously added Ca 2+ than the synaptic mitochondria before undergoing mitochondrial permeability transition, observed as a loss in mitochondrial membrane potential and decreased Ca2+ uptake. The limited ability of synaptic mitochondria to accumulate Ca2+ could result from several factors including a primary function of ATP production to support the high energy demand of presynaptic terminals, their relative isolation in comparison with the threads or clusters of mitochondria found in the soma of neurons and glia, or the older age and increased exposure to oxidative damage of synaptic versus nonsynaptic mitochondria. By more readily undergoing permeability transition, synaptic mitochondria may initiate neuron death in response to insults that elevate synaptic levels of intracellular Ca2+, consistent with the early degeneration of distal axon segments in neurodegenerative disorders. Mitochondria in nerve terminals are subjected to extensive Ca2+fluxes and high energy demands, but the extent to which the synaptic mitochondria buffer Ca2+ is unclear. In this study, we identified a difference in the Ca2+ clearance ability of nonsynaptic versus synaptic mitochondrial populations enriched from rat cerebral cortex. Mitochondria were isolated using Percoll discontinuous gradients in combination with high pressure nitrogen cell disruption. Mitochondria in the nonsynaptic fraction originate from neurons and other cell types including glia, whereas mitochondria enriched from a synaptosomal fraction are predominantly neuronal and presynaptic in origin. There were no differences in respiration or initial Ca2+ loads between nonsynaptic and synaptic mitochondrial populations. Following both bolus and infusion Ca2+ addition, nonsynaptic mitochondria were able to accumulate significantly more exogenously added Ca 2+ than the synaptic mitochondria before undergoing mitochondrial permeability transition, observed as a loss in mitochondrial membrane potential and decreased Ca2+ uptake. The limited ability of synaptic mitochondria to accumulate Ca2+ could result from several factors including a primary function of ATP production to support the high energy demand of presynaptic terminals, their relative isolation in comparison with the threads or clusters of mitochondria found in the soma of neurons and glia, or the older age and increased exposure to oxidative damage of synaptic versus nonsynaptic mitochondria. By more readily undergoing permeability transition, synaptic mitochondria may initiate neuron death in response to insults that elevate synaptic levels of intracellular Ca2+, consistent with the early degeneration of distal axon segments in neurodegenerative disorders. Mitochondria are important regulators of cellular Ca2+ homeostasis, producers of ATP via oxidative phosphorylation, and regulators of cell death pathways (for reviews see Refs. 1Nicholls D.G. Budd S.L. Physiol. Rev. 2000; 80: 315-360Crossref PubMed Scopus (1068) Google Scholar and 2Sullivan P.G. Rabchevsky A.G. Waldmeier P.C. Springer J.E. J. Neurosci. Res. 2005; 79: 231-239Crossref PubMed Scopus (321) Google Scholar). Mitochondria assist in maintaining Ca2+ homeostasis by sequestering and releasing Ca2+ (2Sullivan P.G. Rabchevsky A.G. Waldmeier P.C. Springer J.E. J. Neurosci. Res. 2005; 79: 231-239Crossref PubMed Scopus (321) Google Scholar, 3Bernardi P. Physiol. 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When the mitochondria become overloaded with Ca2+, they undergo the cataclysmic mitochondrial permeability transition (mPT) 3The abbreviations used are: mPT, mitochondrial permeability transition; BSA, bovine serum albumin; CaG5N, Ca2+ Green-5N hexapotassium salt; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; CNS, central nervous system; COXIV, cytochrome oxidase subunit IV; CsA, cyclosporin A; DCF, 2′-7′-dichlorodihydro-fluorescein diacetate; F344, Fisher 344 rats; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; PSD-95, post-synaptic density 95 protein; ROS, reactive oxygen species; RuRed, Ruthenium Red; SD, Sprague-Dawley rats; TMRE, tetramethylrhodamine, ethyl ester perchlorate; TTBS, Tris-buffered saline containing 0.05% Tween 20; VDAC, voltage-dependent anion channel. via formation of a nonselective pore that allows solutes of 1500 daltons or smaller to pass through the usually impermeable inner mitochondrial membrane with a resultant rupture of the outer mitochondrial membrane caused by osmotic swelling (2Sullivan P.G. 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PubMed Scopus Google Scholar). of their morphology, or presynaptic mitochondria may Ca2+ than mitochondria in other of neurons and other cell Previous studies have compared isolated nonsynaptic and synaptic mitochondria with regard to and (19Lai J.C. Clark J.B. and Scholar, J.W. Res. PubMed Scopus Google Scholar, J.C. Clark J.B. 1979; PubMed Scopus Google Scholar, J.C. J.M. S.C. Clark J.B. J. Neurochem. PubMed Scopus Google Scholar, J.C. Clark J.B. J. Neurochem. PubMed Scopus Google Scholar, A. A. J. Neurosci. PubMed Scopus Google Scholar, M. G. S. A. G. J. Bioenerg. Biomembr. 23: PubMed Scopus Google Scholar, M. G. J. A. Res. 36: PubMed Scopus Google Scholar, M. A. J. Bioenerg. Biomembr. 2000; PubMed Scopus Google Scholar). in Ca2+ between isolated synaptic and nonsynaptic mitochondria have In studies using density mitochondria were isolated from and synaptic The synaptic a and levels and of and were to mitochondria M. G. J. A. Res. 36: PubMed Scopus Google Scholar, M. A. J. Bioenerg. Biomembr. 2000; PubMed Scopus Google Scholar). In synaptic and nonsynaptic mitochondria were in of and Percoll density gradients result in of the mitochondrial and synaptosomal and mitochondria isolated from synaptic and nonsynaptic populations and A. A. J. Neurosci. PubMed Scopus Google Scholar, J. Neurochem. PubMed Scopus Google Scholar). The of the to the ability of isolated synaptic versus nonsynaptic brain mitochondria to accumulate exogenously added bovine serum phosphate carbonyl cyanide 4-(trifluoromethoxy) carbonyl cyanide Ca 2+ and Ruthenium were from and the were from Percoll from and were from ethyl ester Ca 2+ Green-5N hexapotassium and 2′-7′-dichlorodihydro-fluorescein were from were by the of and Sprague-Dawley of were used in studies with the of the studies the with in Fisher 344 of the were from M.R. Sullivan P.G. Geddes J.W. O. J. Neurosci. 2004; PubMed Scopus Google the were and the were The were and in a containing the of isolation buffer BSA, to with The and of Percoll in isolation buffer added The resultant a discontinuous Percoll with the containing Percoll in isolation by a Percoll and the in a Percoll The density gradients were in a in a fixed of Percoll density gradients from the of nonsynaptic mitochondria the Percoll density Following and J. Neurochem. PubMed Scopus Google were from the density fraction in and of isolation buffer The were by The and the in the of isolation nitrogen cell to used to the within this fraction M.R. Sullivan P.G. Geddes J.W. O. J. Neurosci. 2004; PubMed Scopus Google Scholar, P.G. J. Biol. Chem. 2004; Scholar). the nonsynaptic mitochondria and were in the nitrogen we demonstrated that the nitrogen mitochondrial function M.R. Sullivan P.G. Geddes J.W. O. J. Neurosci. 2004; PubMed Scopus Google Scholar). The nonsynaptic mitochondrial and the synaptosomal mitochondrial were in of Percoll added to and discontinuous Percoll density as from of the and of isolation buffer BSA, is to with The were and The resultant in of isolation buffer and The mitochondrial in isolation buffer to a of and using the of isolated mitochondria using a oxygen as P.G. Exp. Neurol. 1999; PubMed Scopus Google Scholar). of isolated nonsynaptic or synaptic mitochondria were in a and in respiration buffer BSA, The of oxygen the of the response of isolated mitochondria to the of oxidative and added in and P.G. C. K. O. Neurol. 2003; PubMed Scopus Google Scholar). The by the of oxygen the presence of by the of and presence of isolated mitochondrial with of were used in the The of mitochondrial respiration by and J. Biol. Chem. Full Text PDF PubMed Google were also of oxygen of in respiration enriched in nonsynaptic and synaptic mitochondria were in of respiration buffer in a with and in the used to Ca and used to in with a by a and and Ca2+ added by a via infusion (5Chalmers S. Nicholls D.G. J. Biol. Chem. 2003; 278: 19062-19070Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, Nicholls D. J. Biochem. 1982; PubMed Scopus Google of of or through bolus of of or Ca the mitochondria were no able to buffer the added Ca The added the of The are of The were by the to the of the Ca 2+ infusion or before the bolus using the and The which the the to the which the mitochondria were overloaded and no of Ca 2+ from the Ca 2+ as the of Ca2+ added or to the which the the production using in the as M.R. Geddes J.W. Sullivan P.G. J. Bioenerg. Biomembr. 2004; 36: 401-406Crossref PubMed Scopus (75) Google Scholar, P.G. Rabchevsky A.G. M. A. J. Neurol. 2004; PubMed Scopus Google Scholar, P.G. 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Isolated mitochondria of were added to of respiration buffer with and as oxidative production as the of in production in the presence of or also to that were within the of the nonsynaptic and synaptic mitochondria in isolation buffer a were The resultant mitochondrial in of isolation buffer with and The used buffer added to the relative from the and of the were with the of The were by using or with Following the were The were in in containing 0.05% Tween The were in the primary in The primary used in cytochrome oxidase subunit post-synaptic density 95 and voltage-dependent anion in primary the were in and in in and or in The were in and were in the the were using a and the were using or a of with when The are as the from and is in the of synaptic the isolation discontinuous Percoll gradients and a nitrogen the synaptic and nonsynaptic mitochondria the nitrogen and through discontinuous Percoll density The nitrogen cell the damage to mitochondria caused by N. Dubinsky J.M. Neurosci. PubMed Scopus Google and demonstrated to mitochondria M.R. Sullivan P.G. Geddes J.W. O. J. Neurosci. 2004; PubMed Scopus Google Scholar). The mitochondrial from from were the nonsynaptic fraction and the synaptic were using to the outer mitochondrial membrane the inner mitochondrial membrane and the synaptosomal The nonsynaptic mitochondrial fraction the mitochondrial membrane and but The were the Percoll and the nitrogen disruption. Following the Percoll the synaptic fraction demonstrated both mitochondrial but and synaptosomal of the isolation The mitochondrial and enriched the Percoll density and The the Percoll density and the nitrogen cell were the synaptosomal the were through the Percoll the mitochondrial increased in of mitochondria in both the synaptic and nonsynaptic mitochondrial in Nonsynaptic and mitochondrial the of oxygen in presence of and as the oxidative respiration were observed in the nonsynaptic and synaptic mitochondria There no difference in oxygen in of the of respiration in the the that both populations of isolated mitochondria were and the isolation Ca 2+ in Nonsynaptic versus Mitochondria in nonsynaptic and synaptic mitochondria were with Ca2+ CaG5N, and a TMRE, and in a in a of the the mitochondria were in a respiration buffer containing and The used in that high mitochondrial is than of within the R.A. J. Physiol. 2000; PubMed Scopus Google Scholar). a oxidative and were the mitochondria to a high by the of the which caused the mitochondria to and their to the added to the ATP and the high early as in to that both the nonsynaptic and the synaptic mitochondrial were and the ability of isolated nonsynaptic and synaptic mitochondria to buffer Ca 2+ using in rat Ca2+ of of using infusion into the The infusion the mitochondria were no able to accumulate the added Ca2+ as demonstrated by in added the of and a of Ca2+ within the mitochondria. the were to the and of were also using and In both rat the nonsynaptic mitochondrial populations were able to buffer significantly more of the Ca2+ than the synaptic mitochondria. in both of the the of Ca2+ The we bolus of of of to isolated mitochondria populations to loads of Ca2+ Following the of Ca2+, the increased by a loss in which to the of Ca2+ via the Ca2+ the and as the mitochondria were able to take up the Ca2+ with a the synaptic mitochondrial populations were to buffer as Ca2+ before undergoing mPT as mitochondria. The of Ca2+ in the populations were with the bolus addition, but with the bolus the of Ca 2+ decreased in synaptic versus nonsynaptic and this with of The loss of caused by Ca2+, the of the Ca2+ able to the there a in the of Ca2+ of Ca2+ of up to mPT by nonsynaptic mitochondrial populations as compared with synaptic mitochondrial populations isolated from and brain When the mPT cyclosporin there in the of Ca2+ to mPT by both nonsynaptic and synaptic mitochondria and the of Ca2+ in nonsynaptic mitochondrial but in synaptic mitochondria. In the presence of CsA, Ca2+ to mPT by nonsynaptic mitochondria than in synaptic mitochondria The of nonsynaptic mitochondria also using the Ca2+ bolus the that the initial Ca2+ within the mitochondria between the nonsynaptic and synaptic mitochondria the isolation CCCP, a added the of to efflux of the Ca2+ There no difference in the of the addition, no substantial differences in the of Ca2+ with the Ca2+ in synaptic versus nonsynaptic mitochondria as a of the in Ca2+ observed in the mitochondrial populations. Ca2+ using both the infusion and the bolus the Ca2+ as demonstrated by the of RuRed, which Ca2+ In the presence of RuRed, there no loss of the of Ca between the Nonsynaptic and that the of nonsynaptic and synaptic mitochondria to accumulate Ca2+ is production and oxidative damage in the synaptic There no difference in using DCF, in isolated nonsynaptic and synaptic mitochondria The of this that nonsynaptic mitochondria isolated from rat accumulate significantly more Ca2+ than the synaptic mitochondria before undergoing difference in Ca2+ observed in of both bolus Ca2+ and Ca 2+ infusion CsA, of Ca2+ mPT increased the that could in nonsynaptic mitochondria but have a in synaptic mitochondria. The of Ca2+ both populations mitochondrial Ca 2+ the in uniporter and by the of The differences were to initial Ca2+ loads in the isolated mitochondria or the production of mitochondrial There were no differences between nonsynaptic and synaptic mitochondria in the of oxygen the of that the to oxidative the of in both populations of mitochondria. Previous synaptic mitochondria (19Lai J.C. Clark J.B. and Scholar, J.C. Clark J.B. 1979; PubMed Scopus Google Scholar, P.G. C. K. O. Neurol. 2003; PubMed Scopus Google Scholar, P.G. Neurol. 2000; PubMed Scopus Google or Percoll J. Neurochem. PubMed Scopus Google discontinuous density gradients and as to synaptosomal of synaptic mitochondria were in the of the isolation J.C. Clark J.B. 1979; PubMed Scopus Google and to the synaptic mitochondria from N. Brustovetsky T. Purl K.J. Capano M. Crompton M. Dubinsky J.M. J. Neurosci. 2003; 23: 4858-4867Crossref PubMed Google Scholar). In the study, rat brain mitochondria were isolated from nonsynaptic and synaptic using discontinuous Percoll gradients and high pressure nitrogen cell disruption. The nonsynaptic and synaptic mitochondria their both populations were to the nitrogen and both through discontinuous Percoll of the nitrogen M.R. Sullivan P.G. Geddes J.W. O. J. Neurosci. 2004; PubMed Scopus Google the of mitochondrial N. Brustovetsky T. Purl K.J. Capano M. Crompton M. Dubinsky J.M. J. Neurosci. 2003; 23: 4858-4867Crossref PubMed Google Scholar). mitochondria are the presynaptic terminals the of the and mitochondria are in the J. Google Scholar, D.G. J. Biochem. PubMed Scopus Google Scholar). The mitochondria within the synaptic fraction are from both and neurons from Within the presynaptic mitochondria a of In to ATP and Ca2+ they to and The in the presynaptic are with ATP and in to of mitochondria are to high Ca2+ with Cell 2000; PubMed Scopus Google Scholar). Nonsynaptic mitochondria originate from both neurons and In rat cell density is the neuronal and mitochondria a smaller of the in as compared with neurons A. M. J.M. PubMed Scopus Google Scholar). a to that used in the to nonsynaptic and T. Fiskum G. J. Neurosci. PubMed Scopus Google found a of mitochondria A. Res. Res. PubMed Scopus Google in the nonsynaptic mitochondrial fraction in to in mitochondria isolated from The is to decreased in A. Res. Res. PubMed Scopus Google that the nonsynaptic mitochondrial fraction may predominantly is to mitochondria from neurons versus in the CNS. mitochondria isolated from primary of neurons and T. Fiskum G. J. Neurosci. PubMed Scopus Google the are and the Ca2+ of the mitochondrial populations have The differences in mitochondrial Ca2+ were both bolus Ca2+ and Ca2+ to the isolated synaptic and nonsynaptic mitochondria. both a loss of or the in mitochondrial Ca2+ to in brain and liver mitochondria (5Chalmers S. Nicholls D.G. J. Biol. Chem. 2003; 278: 19062-19070Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). the bolus there are high of the (5Chalmers S. Nicholls D.G. J. Biol. Chem. 2003; 278: 19062-19070Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). In addition, the phosphate and and in response to the bolus may from from a slow of Ca2+ (5Chalmers S. Nicholls D.G. J. Biol. Chem. 2003; 278: 19062-19070Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). of a infusion allows mitochondria to accumulate the Ca2+ with and allows a more of the Ca2+ of the mitochondria (5Chalmers S. Nicholls D.G. J. Biol. Chem. 2003; 278: 19062-19070Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, D.G. S. S. Cell 2003; PubMed Scopus Google Scholar). with slow the of Ca2+ added are than Ca 2+ levels in presynaptic terminals, which are and to in the of a J. Neurosci. PubMed Google Scholar). 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In the of the that mitochondria isolated from rat have increased to undergo mPT in response to added Ca2+ as compared with mitochondria isolated from a difference is the result of differences in mitochondrial or initial Ca2+ but may the neuronal of synaptic mitochondria versus the cellular of nonsynaptic mitochondria or the of synaptic versus nonsynaptic mitochondria. is also that the isolated of synaptic mitochondria in comparison with the threads and clusters found in other cellular in or the age and oxidative damage to synaptic mitochondria may the differences in Ca2+ the mechanisms the Ca2+ differences to the the of distal axon segments and presynaptic terminals in several neurodegenerative and neuronal