Nickolay Brustovetsky

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.

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.

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.

Calcium-induced Cytochrome c release from rat brain mitochondria is altered by digitonin

To determine if calcium could release Cytochrome c (Cyt c) from brain mitochondria without activating the permeability transition (mPT), brain mitochondria were prepared in two different ways. Digitonin was used to lyse synaptosomes and release synaptosomal mitochondria or a Percoll gradient was used to separate non-synaptosomal mitochondria from the synaptosomes. In gradient-purified mitochondria, low levels of added digitonin produced swelling and Cyt c release. Digitonin augmented Ca(2+)-induced Cyt c release that was insensitive to the mPT inhibitors, cyclosporin A CsA and ADP. Similarly, in mitochondria prepared with digitonin, these inhibitors also failed to prevent Ca(2+)-induced Cyt c release. Thus the mPT-independent, Ca(2+)-induced Cyt c release pathway was attributable to alteration of the permeability properties of the outer mitochondrial membrane by digitonin.

EDTA-induced monovalent fluxes through the Ca2+ uniporter in brain mitochondria

The low ion permeability of the inner mitochondrial membrane is a crucial prerequisite for normal mitochondrial functioning. Considering the high, negative mitochondrial membrane potential (∆ψ), induction of a permeability pathway for protons and/or monovalent cations might have disastrous consequences, inhibiting oxidative phosphorylation and alterating mitochondrial Ca2+ buffering capacity. A rapid increase of cytosolic Ca2+, accompanying neurodegenerative disorders, may cause mitochondrial dysfunction due to various permeability mechanisms involving activation of Ca2+ cycling and/or induction of the mitochondrial permeability transition (mPT) pore. Induction of the mPT is a candidate intermediate step in both necrotic and apoptotic cell death pathways.1 Recently, we reported Ca2+-dependent induction of a novel low-conductance proton-permeable pathway linked to the Ca2+ uniporter.2,3 Under normal conditions with low cytosolic calcium the Ca2+ uniporter acts as a Ca2+-selective proton-impermeable channel. Under some circumstances the Ca2+ uniporter becomes permeable for monovalent cations as well. A rapid increase in extramitochondrial Ca2+ caused sustained mitochondrial depolarization, probably due to an induction of proton permeability.2,3 Repolarization by ruthenium red (RR), a potent inhibitor of the Ca2+ uniporter, linked this pathway to the unporter.2,3 An inverse correlation between Ca2+ fluxes and the sustained depolarization ruled out a substantial contribution of Ca2+ cycling to the sustained depolarization.2,3 Mg2+ also significantly influences operation of the Ca2+ uniporter. Bernardi et al.4 reported an electrogenic Na+ influx into liver mitochondria that could be induced by removal of external Mg2+. Because this Na+ influx was sensitive to inhibition by RR and La3+, Kapus et al.5 suggested that the Mg2+-dependent Na+ channel and the Ca2+ uniporter could be identical. In the present work we investigated whether Mg2+-regulated, monovalent cation permeability could be detected in brain mitochondria, similarly linked to the Ca2+ uniporter. Mitochondrial membrane permeability was evaluated by monitoring membrane potential with tetraphenylphosphonium (TPP+) and a TPP+-sensitive electrode in the mitochondrial suspension. Sequestration of external Mg2+ by 0.5 mM EDTA in NaCl medium partially depolarized mitochondria, suggesting induction of a proton or Na+ permeability (FIG. 1A). RR prevented the EDTA-induced depolarization and partially repolarized EDTA-treated mitochondria. Pretreatment with the divalent cation ionophore A23187 depleted intramitochondrial Mg2+ by providing a pathway for matrix Mg2+ efflux. In the presence of

Varied Responses of Central Nervous System Mitochondria to Calcium

Mitochondrial dysfunction and induction of the mitochondrial permeability transition (MPT) are candidate intermediate steps in both necrotic and apoptotic cell death pathways (Dubinsky and Levi, 1998; Hirsch et al., 1998; Schinder et al., 1996). In its classic definition (referred to here as the high-conductance MPT), the MPT is a nonselective, multiconductance pore in the inner mitochondrial membrane whose activation causes mitochondrial swelling and dysfunction (Nieminen et al., 1996; Schinder et al., 1996; Zoratti and Szabo, 1995). In liver and heart mitochondria, accumulation of excess matrix calcium combined with phosphate or with an oxidative event leads to opening of the high conductance MPT pore (Zoratti and Szabo, 1995). Induction of the MPT is modulated by mitochondrial membrane potential , matrix free fatty acids, redox status of mitochondrial protein thiols, and surface potential generated by the largely anionic phospholipids of the inner mitochondrial membrane (Zoratti and Szabo, 1995). Pharmacological inhibition of the MPT can be accomplished with the immunosuppressant cyclosporin and some of its analogs, adenine nucleotides, and the adenine nucleotide transporter inhibitor bongkrekic acid (Zoratti and Szabo, 1995). Mitochondrial swelling as measured by changes in absorbance has typically been studied in mitochondria after loading. High loads alone, or lower plus phosphate, uncoupler, or pro-oxidants, initiates transition, a process thought to propagate through the mitochondrial population (Bernardi, 1992; Broekemeier et al., 1989). When ruthenium red (RR) is added after to prevent its loss through reverse operation of the uniporter, application of