Janet M. Dubinsky

Mitochondrial Permeability Transition in the Central Nervous System: Induction by Calcium Cycling‐Dependent and ‐Independent Pathways

Abstract: Isolated rat CNS mitochondria and cultured cortical astrocytes were examined for behavior indicative of a mitochondrial permeability transition (mPT). Exposure of isolated CNS mitochondria to elevated calcium or phosphate or both produced loss of absorbance indicative of mitochondrial swelling. The absorbance decreases were prevented by ADP and Mg2+ and reduced by cyclosporin A, dithiothreitol, and N‐ethylmaleimide. Ruthenium red prevented calcium cycling‐induced, but only attenuated phosphate‐induced losses of absorbance. In cultured astrocytes permeabilized with digitonin or treated with the calcium ionophore, 4‐bromo‐A23187, elevations of external calcium altered mitochondrial morphology visualized with the dye, JC‐1, from rod‐like to rounded, swollen structures. Similar changes were observed in digitonin‐permeabilized astrocytes exposed to phosphate. The incidence of calcium‐induced changes in astrocyte mitochondria was prevented by Mg2+ and pretreatment with dithiothreitol and N‐ethylmaleimide, and was reduced by cyclosporin A, ADP, and butacaine alone or in combinations. Ruthenium red and the Na+/Ca2+ exchange inhibitor CGP 37157 blocked calcium cycling and prevented mitochondrial shape changes in digitonin‐treated, but not ionophore‐treated astrocytes. Thus, the demonstrated induction conditions and pharmacological profile indicated the existence of an mPT in brain mitochondria. The mPT occurred consequent to activation of calcium cycling‐dependent and ‐independent pathways. Induction of an mPT could contribute to neuronal injury following ischemia and reperfusion.

Calcium-induced cytochrome c release from CNS mitochondria is associated with the permeability transition and rupture of the outer membrane.

The mechanisms of Ca2+‐induced release of Cytochrome c (Cyt c) from rat brain mitochondria were examined quantitatively using a capture ELISA. In 75 or 125 mm KCl‐based media 1.4 µmol Ca2+/mg protein caused depolarization and mitochondrial swelling. However, this resulted in partial Cyt c release only in 75 mm KCl. The release was inhibited by Ru360, an inhibitor of the Ca2+ uniporter, and by cyclosporin A plus ADP, a combination of mitochondrial permeability transition inhibitors. Transmission electron microscopy (TEM) revealed that Ca2+‐induced swelling caused rupture of the outer membrane only in 75 mm KCl. Koenigs polyanion, an inhibitor of mitochondrial porin (VDAC), enhanced swelling and amplified Cyt c release. Dextran T70 that is known to enhance mitochondrial contact site formation did not prevent Cyt c release. Exposure of cultured cortical neurons to 500 µm glutamate for 5 min caused Cyt c release into the cytosol 30 min after glutamate removal. MK‐801 or CsA inhibited this release. Thus, the release of Cyt c from CNS mitochondria induced by Ca2+in vitro as well as in situ involved the mPT and appeared to require the rupture of the outer membrane.

Calcium-induced activation of the mitochondrial permeability transition in hippocampal neurons

The mitochondrial permeability transition (mPT) has been implicated in both excitotoxic and apoptotic neuronal cell death, despite the fact that it has not been previously identified in neurons. To study the mPT in hippocampal neurons, cultures were loaded with the mitochondrial dye JC‐1 and observed with confocal and conventional microscopy. After pretreatment with 4Br‐A23187 and subsequent calcium addition, the initially rodlike mitochondria increased in diameter until mitochondria became rounded in appearance. Morphological changes reversed when calcium was removed by EGTA. When neurons were loaded with both fura‐2‐AM and rhodamine 123, calcium loading produced an increase in cytosolic calcium, mitochondrial depolarization, and similar alterations in mitochondrial morphology. Smaller calcium challenges produced calcium cycling, delaying morphological changes until after secondary depolarization and calcium release to the cytosol. In neurons exposed to glutamate, confocal observation of JC‐1 fluorescence revealed comparable changes in mitochondrial morphology that were prevented when barium was substituted for calcium, or following pretreatment with the mPT inhibitor, cyclosporin A. These experiments establish conditions in which the mPT could be observed in situ in neurons in response to calcium loading. In addition, the timing of changes suggested that induction of the permeability transition in situ represents a sequence of multiple events that may reflect the multiple open conformations of the mPT pore. J. Neurosci. Res. 53:728–741, 1998.

On the mechanisms of neuroprotection by creatine and phosphocreatine

Creatine and phosphocreatine were evaluated for their ability to prevent death of cultured striatal and hippocampal neurons exposed to either glutamate or 3‐nitropropionic acid (3NP) and to inhibit the mitochondrial permeability transition in CNS mitochondria. Phosphocreatine (PCr), and to a lesser extent creatine (Cr), but not (5R,10S)‐(+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine hydrogen maleate (MK801), dose‐dependently ameliorated 3NP toxicity when applied simultaneously with the 3NP in Mg2+‐free media. Pre‐treatment of PCr for 2 or 5 days and Cr for 5 days protected against glutamate excitotoxicity equivalent to that achieved by MK801 post‐treatment. The combination of PCr or Cr pre‐treatment and MK801 post‐treatment did not provide additional protection, indicating that both prevented the toxicity attributable to secondary glutamate release. To determine if Cr or PCr directly inhibited the permeability transition, mitochondrial swelling and depolarization were assayed in isolated, purified brain mitochondria. PCr reduced the amount of swelling induced by calcium by 20%. Cr decreased mitochondrial swelling when inhibitors of creatine kinase octamer–dimer transition were present. However, in brain mitochondria prepared from rats fed a diet supplemented with 2% creatine for 2 weeks, the extent of calcium‐induced mitochondrial swelling was not altered. Thus, the neuroprotective properties of PCr and Cr may reflect enhancement of cytoplasmic high‐energy phosphates but not permeability transition inhibition.

Age-Dependent Changes in the Calcium Sensitivity of Striatal Mitochondria in Mouse Models of Huntington's Disease

Striatal and cortical mitochondria from knock‐in and transgenic mutant huntingtin mice were examined for their sensitivity to calcium induction of the permeability transition, a cause of mitochondrial depolarization and ATP loss. The permeability transition has been suggested to contribute to cell death in Huntingtons Disease. Mitochondria were examined from slowly progressing knock‐in mouse models with different length polyglutarnine expansions (Q20, Q50, Q92, Q111) and from the rapidly progressing transgenic R6/2 mice overexpressing exon I of human huntingtin with more than 110 polyglutamines. As previously observed in rats, striatal mitochondria from background strain CD1 and C57BL/6 control mice were more sensitive to calcium than cortical mitochondria. Between 5 and 12 months in knock‐in Q92 mice and between 8 and 12 weeks in knock‐in Q111 mice, striatal mitochondria developed resistance, becoming equally sensitive to calcium as cortical mitochondria, while those from Q50 mice were unchanged. Cortical mitochondrial calcium sensitivity did not change. In R6/2 mice striatal and cortical mitochondria were equally resistant to Ca2+ while striatal mitochondria from littermate controls were more susceptible. No increases in calcium sensitivity were observed in the mitochondria from Huntingtons Disease (HD) mice compared to controls. Neither motor abnormalities, nor expression of cyclophilin D corresponded to the changes in mitochondrial sensitivity. Polyglutamine expansions in huntingtin produced an early increased resistance to calcium in striatal mitochondria suggesting mitochondria undergo compensatory changes in calcium sensitivity in response to the many cellular changes wrought by polyglutamine expansion.

Neurochemical changes in Huntington R6/2 mouse striatum detected by in vivo 1H NMR spectroscopy

The neurochemical profile of the striatum of R6/2 Huntington’s disease mice was examined at different stages of pathogenesis using in vivo1H NMR spectroscopy at 9.4 T. Between 8 and 12 weeks, R6/2 mice exhibited distinct changes in a set of 17 quantifiable metabolites compared with littermate controls. Concentrations of creatine, glycerophosphorylcholine, glutamine and glutathione increased and N‐acetylaspartate decreased at 8 weeks. By 12 weeks, concentrations of phosphocreatine, taurine, ascorbate, glutamate, and myo‐inositol increased and phophorylethanolamine decreased. These metabolic changes probably reflected multiple processes, including compensatory processes to maintain homeostasis, active at different stages in the development of HD. The observed changes in concentrations suggested impairment of neurotransmission, neuronal integrity and energy demand, and increased membrane breakdown, gliosis, and osmotic and oxidative stress. Comparisons between metabolite concentrations from individual animals clearly distinguished HD transgenics from non‐diseased littermates and identified possible markers of disease progression. Metabolic changes in R6/2 striata were distinctly different from those observed previously in the quinolinic acid and 3NP models of HD. Longitudinal monitoring of changes in these metabolites may provide quantifiable measures of disease progression and treatment effects in both mouse models of HD and patients.

Two pathways for tBID‐induced cytochrome c release from rat brain mitochondria: BAK‐ versus BAX‐dependence

The mechanisms of truncated BID (tBID)‐induced Cyt c release from non‐synaptosomal brain mitochondria were examined. Addition of tBID to mitochondria induced partial Cyt c release which was inhibited by anti‐BAK antibodies, implicating BAK. Immunoblotting showed the presence of BAK, but not BAX, in brain mitochondria. tBID did not release Cyt c from rat liver mitochondria, which lacked both BAX and BAK. This indicated that tBID did not act independently of BAX and BAK. tBID plus monomeric BAX produced twice as much Cyt c release as did tBID or oligomeric BAX alone. Neither tBID alone nor in combination with BAX induced mitochondrial swelling. In both cases Cyt c release was insensitive to cyclosporin A plus ADP, inhibitors of the mitochondrial permeability transition (mPT). Recombinant Bcl‐xL inhibited Cyt c release induced by tBID alone or in combination with monomeric BAX. Koenigs polyanion, an inhibitor of VDAC, suppressed tBID‐induced Cyt c release from brain mitochondria mediated by BAK but not by BAX. Thus, tBID can induce mPT‐independent Cyt c release from brain mitochondria by interacting with exogenous BAX and/or with endogenous BAK that may involve VDAC. In contrast, neither adenylate kinase nor Smac/DIABLO was released from isolated rat brain mitochondria via BAK or BAX.

Activation of calcium‐independent phospholipase A2 (iPLA2) in brain mitochondria and release of apoptogenic factors by BAX and truncated BID

Cleaved or truncated BID (tBID) is known to oligomerize both BAK and BAX. Previously, BAK and BAX lacing the C‐terminal fragment (BAXΔC) were shown to induce modest cytochrome c (Cyt c) release from rat brain mitochondria when activated by tBID. We now show that tBID plus monomeric full‐length BAX induce extensive release of Cyt c, Smac/DIABLO, and Omi/HtrA2 (but not endonuclease G and the apoptosis inducing factor) comparable to the release induced by alamethicin. This occurs independently of the permeability transition without overt changes in mitochondrial morphology. The mechanism of the release may involve formation of reactive oxygen species (ROS) and activation of calcium‐independent phospholipase A2 (iPLA2). Indeed, increased ROS production and activated iPLA2 were observed prior to massive Cyt c release. Furthermore, the extent of inhibition of Cyt c release correlated with the degree of suppression of iPLA2 by the inhibitors propranolol, dibucaine, 4‐bromophenacyl bromide, and bromenol lactone. Consistent with a requirement for iPLA2 in Cyt c release from brain mitochondria, synthetic liposomes composed of lipids mimicking the outer mitochondrial membrane (OMM) but lacing iPLA2 failed to release 10 kDa fluorescent dextran (FD‐10) in response to tBID plus BAX. We propose that tBID plus BAX activate ROS generation, which subsequently augments iPLA2 activity leading to changes in the OMM that allow translocation of certain mitochondrial proteins from the intermembrane space.

Heterogeneity of nervous system mitochondria: location, location, location!

Mitochondrial impairments have been associated with many neurological disorders, from inborn errors of metabolism or genetic disorders to age and environmentally linked diseases of aging (DiMauro S., Schon E.A. 2008. Mitochondrial disorders in the nervous system. Annu. Rev., Neurosci. 31, 91-123.). In these disorders, specific nervous system components or brain regions appear to be initially more susceptible to the triggering event or pathological process. Such regional variation in susceptibility to multiple types of stressors raises the possibility that inherent differences in mitochondrial function may mediate some aspect of pathogenesis. Regional differences in the distribution or number of mitochondria, mitochondrial enzyme activities, enzyme expression levels, mitochondrial genes or availability of necessary metabolites become attractive explanations for selective vulnerability of a nervous system structure. While regionally selective mitochondrial vulnerability has been documented, regional variations in other cellular and tissue characteristics may also contribute to metabolic impairment. Such environmental variables include high tonic firing rates, neurotransmitter phenotype, location of mitochondria within a neuron, or the varied tissue perfusion pressure of different cerebral arterial branches. These contextual variables exert regionally distinct regulatory influences on mitochondria to tune their energy production to local demands. Thus to understand variations in mitochondrial functioning and consequent selective vulnerability to injury, the organelle must be placed within the context of its cellular, functional, developmental and neuroanatomical environment.

Age-related changes in regional brain mitochondria from Fischer 344 rats

Brain mitochondrial function has been posited to decline with aging. In order to test this hypothesis, cortical and striatal mitochondria were isolated from Fischer 344 rats at 2, 5, 11, 24 and 33 months of age. Mitochondrial membrane potential remained stable through 24 months, declining slightly in mitochondria from both brain regions at 33 months. The ability of calcium to induce mitochondrial swelling and depolarization, characteristics of the permeability transition, was remarkably stable through 24 months of age and increased at advanced ages only for cortical, but not striatal, mitochondria. Striatal mitochondria were more sensitive to calcium than were cortical mitochondria throughout the first 2 years of life. A two‐fold increased resistance to calcium was observed in striatal mitochondria between 5 and 11 months. Although these measurements do demonstrate changes in mitochondrial function with aging, the changes in polarization are relatively small and the increased cortical susceptibility to the permeability transition only occurred at very advanced ages. Thus mitochondrial decline with advanced age depends upon brain region.