Oxoglutarate dehydrogenase complex controls glutamate-mediated neuronal death.

Brain injury is accompanied by neuroinflammation, accumulation of extracellular glutamate and mitochondrial dysfunction, all of which cause neuronal death. The aim of this study was to investigate the impact of these mechanisms on neuronal death. Patients from the neurosurgical intensive care unit suffering aneurysmal subarachnoid hemorrhage (SAH) were recruited retrospectively from a respective database. In vitro experiments were performed in rat cortex homogenate, primary dissociated neuronal cultures, B35 and NG108-15 cell lines. We employed methods including high resolution respirometry, electron spin resonance, fluorescent microscopy, kinetic determination of enzymatic activities and immunocytochemistry. We found that elevated levels of extracellular glutamate and nitric oxide (NO) metabolites correlated with poor clinical outcome in patients with SAH. In experiments using neuronal cultures we showed that the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme of the glutamate-dependent segment of the tricarboxylic acid (TCA) cycle, is more susceptible to the inhibition by NO than mitochondrial respiration. Inhibition of OGDHC by NO or by succinyl phosphonate (SP), a highly specific OGDHC inhibitor, caused accumulation of extracellular glutamate and neuronal death. Extracellular nitrite did not substantially contribute to this NO action. Reactivation of OGDHC by its cofactor thiamine (TH) reduced extracellular glutamate levels, Ca2+ influx into neurons and cell death rate. Salutary effect of TH against glutamate toxicity was confirmed in three different cell lines. Our data suggest that the loss of control over extracellular glutamate, as described here, rather than commonly assumed impaired energy metabolism, is the critical pathological manifestation of insufficient OGDHC activity, leading to neuronal death.

The effect of DS16570511, a new inhibitor of mitochondrial calcium uniporter, on calcium homeostasis, metabolism, and functional state of cultured cortical neurons and isolated brain mitochondria.

BACKGROUND: Disorders of mitochondrial Ca2+ homeostasis play a key role in the glutamate excitotoxicity of brain neurons. DS16570511 (DS) is a new penetrating inhibitor of mitochondrial Ca2+ uniporter complex (MCUC). The paper examines the effects of DS on the cultivated cortical neurons and isolated mitochondria of the rat brain. METHODS: The functions of neurons and mitochondria were examined using fluorescence microscopy, XF24 microplate-based сell respirometry, ion-selective microelectrodes, spectrophotometry, and polarographic technique. RESULTS: At the doses of 30 and 45 µM, DS reliably slowed down the onset of glutamate-induced delayed calcium deregulation of neurons and suppressed their death. 30 µM DS caused hyperpolarization of mitochondria of resting neurons, and 45 µM DS temporarily depolarized neuronal mitochondria. It was also demonstrated that 30-60 µM DS stimulated cellular respiration. DS was shown to suppress Ca2+ uptake by isolated brain mitochondria. In addition, DS inhibited ADP-stimulated mitochondrial respiration and ADP-induced decrease in the mitochondrial membrane potential. It was found that DS inhibited the activity of complex II of the respiratory chain. In the presence of Ca2+, high DS concentrations caused a collapse of the mitochondrial membrane potential. CONCLUSIONS: The data obtained indicate that, in addition to the inhibition of MCUC, DS affects the main energy-transducing functions of mitochondria. GENERAL SIGNIFICANCE: The using DS as a tool for studying MCUC and its functional role in neuronal cells should be done with care, bearing in mind multiple effects of DS, a proper evaluation of which would require multivariate analysis.

Viscoelasticity and Volume of Cortical Neurons under Glutamate Excitotoxicity and Osmotic Challenges.

Neural activity depends on the maintenance of ionic and osmotic homeostasis. Under these conditions, the cell volume must be regulated to maintain optimal neural function. A disturbance in the neuronal volume regulation often occurs in pathological conditions such as glutamate excitotoxicity. The cell volume, mechanical properties, and actin cytoskeleton structure are tightly connected to achieve the cell homeostasis. Here, we studied the effects of glutamate-induced excitotoxicity, external osmotic pressure, and inhibition of actin polymerization on the viscoelastic properties and volume of neurons. Atomic force microscopy was used to map the viscoelastic properties of neurons in time-series experiments to observe the dynamical changes and a possible recovery. The data obtained on cultured rat cortical neurons were compared with the data obtained on rat fibroblasts. The neurons were found to be more responsive to the osmotic challenges but less sensitive to the inhibition of actin polymerization than fibroblasts. The alterations of the viscoelastic properties caused by glutamate excitotoxicity were similar to those induced by the hypoosmotic stress, but, in contrast to the latter, they did not recover after the glutamate removal. These data were consistent with the dynamic volume changes estimated using ratiometric fluorescent dyes. The recovery after the glutamate-induced excitotoxicity was slow or absent because of a steady increase in intracellular calcium and sodium concentrations. The viscoelastic parameters and their changes were related to such parameters as the actin cortex stiffness, tension, and cytoplasmic viscosity.