ABSTRACT
KB-R7943, an inhibitor of a reversed Na(+)/Ca(2+) exchanger, exhibits neuroprotection against glutamate excitotoxicity. Taking into consideration that prolonged exposure of neurons to glutamate induces delayed calcium deregulation (DCD) and irreversible decrease of mitochondrial membrane potential (Deltapsi(mit)), we examined the effect of KB-R7943 on glutamate and kainate-induced [Ca(2+)](i) and on Deltapsi(mit) changes in rat cultured cerebellar granule neurons. 15 micromol/l KB-R7943 significantly delayed the onset of DCD in response to kainate but not in response to glutamate. In spite of [Ca(2+)](i) overload, KB-R7943 considerably improved the [Ca(2+)](i) recovery and restoration of Deltapsi(mit) after glutamate and kainate washout and increased cell viability after glutamate exposure. In resting neurons, KB-R7943 induced a statistically significant decrease in Deltapsi(mit). KB-R7943 also depolarized isolated brain mitochondria and slightly inhibited mitochondrial Ca(2+) uptake. These findings suggest that mild mitochondrial depolarization and diminution of Ca(2+) accumulation in the organelles might contribute to neuroprotective effect of KB-R7943.
Subject(s)
Calcium/metabolism , Glutamic Acid/toxicity , Neuroprotective Agents/pharmacology , Thiourea/analogs & derivatives , Animals , Cell Survival/drug effects , Glutamic Acid/pharmacology , Kainic Acid/pharmacology , Lithium/pharmacology , Membrane Potential, Mitochondrial/drug effects , Neurons/drug effects , Neurons/physiology , Rats , Rats, Wistar , Thiourea/pharmacologyABSTRACT
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 Huntington's 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 Huntington's 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.
Subject(s)
Aging/metabolism , Calcium Signaling/genetics , Corpus Striatum/metabolism , Huntington Disease/metabolism , Mitochondria/metabolism , Nerve Degeneration/metabolism , Aging/genetics , Animals , Calcium/metabolism , Cell Death/genetics , Cell Membrane Permeability/genetics , Cerebral Cortex/metabolism , Cerebral Cortex/physiopathology , Corpus Striatum/physiopathology , Disease Models, Animal , Female , Huntingtin Protein , Huntington Disease/genetics , Huntington Disease/physiopathology , Male , Membrane Potentials/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitochondria/genetics , Nerve Degeneration/genetics , Nerve Degeneration/physiopathology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Peptides/metabolism , Trinucleotide Repeat Expansion/geneticsABSTRACT
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.
