Mechanism of Oxidative Stress in Neurodegeneration

Biological tissues require oxygen to meet their energetic demands. However, the consumption of oxygen also results in the generation of free radicals that may have damaging effects on cells. The brain is particularly vulnerable to the effects of reactive oxygen species due to its high demand for oxygen, and its abundance of highly peroxidisable substrates. Oxidative stress is caused by an imbalance in the redox state of the cell, either by overproduction of reactive oxygen species, or by dysfunction of the antioxidant systems. Oxidative stress has been detected in a range of neurodegenerative disease, and emerging evidence from in vitro and in vivo disease models suggests that oxidative stress may play a role in disease pathogenesis. However, the promise of antioxidants as novel therapies for neurodegenerative diseases has not been borne out in clinical studies. In this review, we critically assess the hypothesis that oxidative stress is a crucial player in common neurodegenerative disease and discuss the source of free radicals in such diseases. Furthermore, we examine the issues surrounding the failure to translate this hypothesis into an effective clinical treatment

Carbon monoxide neurotoxicity is triggered by oxidative stress induced by ROS production from three distinct cellular sources

Carbon monoxide (CO) poisoning is one of the leading causes of toxic mortality and morbidity. We have studied the generation of reactive oxygen species in cortical neurons in culture in response to toxic doses of CO exposure. Fluorescence microscopy was used to measure the rate of free radical generation, lipid peroxidation, GSH level and also mitochondrial metabolism. We have found that toxic concentrations of CO released from CORM-401 induced mitochondrial depolarisation and inhibition of NADH dependent respiration to a lesser degree than when compared to ischaemia. Energy collapse was not observed within 40 min of CO exposure. We have found that CO induces the generation of reactive oxygen species resulting in lipid peroxidation and a decrease in GSH via three different mechanisms: from mitochondria during the first minutes of CO exposure, from xanthine oxidase at around 20 min exposure due to energy deprivation, and considerable ROS production from NADPH oxidase in the post CO exposure period (re-oxygenation). Inhibition of these different phases with mitochondrial antioxidants, inhibitors of xanthine oxidase, or NADPH oxidase, protected neurons and astrocytes against CO-induced oxidative stress and cell death. The most profound effect was seen during NADPH oxidase inhibition. Thus, oxidative stress has a remarkably significant role in CO-induced neuronal cell death and preventing its occurrence during reoxygenation is of great importance in the consideration of a positive, neurologically protective therapeutic outcome for CO exposed patients

Interaction of oxidative stress and misfolded proteins in the mechanism of neurodegeneration

Aggregation of the misfolded proteins β-amyloid, tau, huntingtin, and α-synuclein is one of the most important steps in the pathology underlying a wide spectrum of neurodegenerative disorders, including the two most common ones—Alzheimer's and Parkinson's disease. Activity and toxicity of these proteins depends on the stage and form of aggregates. Excessive production of free radicals, including reactive oxygen species which lead to oxidative stress, is proven to be involved in the mechanism of pathology in most of neurodegenerative disorders. Both reactive oxygen species and misfolded proteins play a physiological role in the brain, and only deregulation in redox state and aggregation of the proteins leads to pathology. Here, we review the role of misfolded proteins in the activation of ROS production from various sources in neurons and glia. We discuss if free radicals can influence structural changes of the key toxic intermediates and describe the putative mechanisms by which oxidative stress and oligomers may cause neuronal death

Hyperammonemia induces mitochondrial dysfunction and neuronal cell death

BACKGROUND & AIMS: In liver cirrhosis, astrocytic swelling is believed to be the principal mechanism of ammonia neurotoxicity leading to hepatic encephalopathy (HE). The role of neuronal dysfunction in HE is not clear. We aimed to explore the impact of hyperammonemia on mitochondrial function in primary co-cultures of neurons and astrocytes and in acute brain slices of cirrhotic rats using live cell imaging. METHODS: To primary co-cultures of astrocytes and neurons, low concentrations (1 and 5μM) of NH4Cl were applied. In rats with bile-duct ligation (BDL)-induced cirrhosis, a model known to induce hyperammonemia and minimal HE, acute brain slices were studied. One group of BDL rats were treated twice daily with the ammonia scavenger ornithine phenylacetate (OP, 0.3g/kg). Fluorescence measurements of changes in mitochondrial membrane potential (ΔΨm), cytosolic and mitochondrial reactive oxygen species (ROS) production, lipid peroxidation (LP) rates, and cell viability were performed using confocal microscopy. RESULTS: Neuronal cultures treated with NH4Cl exhibited mitochondrial dysfunction, ROS overproduction and reduced cell viability (27.8±2.3% and 41.5±3.7%, respectively) compared to untreated cultures (15.7±1.0%, both p<0.0001). BDL led to increased cerebral LP (p=0.0003) and cytosolic ROS generation (p<0.0001), which was restored by OP (both p<0.0001). Mitochondrial function was severely compromised in BDL resulting in hyperpolarization of ΔΨm with consequent overconsumption of ATP and augmentation of mitochondrial ROS production. Administration of OP restored ΔΨm. In BDL animals, neuronal loss was observed in hippocampal areas, which was partially prevented by OP. CONCLUSIONS: Our results elucidate that low-grade hyperammonemia in cirrhosis can severely impact on brain mitochondrial function. Profound neuronal injury was observed in hyperammonemic conditions, which was partially reversible by OP. This points towards a novel mechanism of HE development. LAY SUMMARY: The impact of hyperammonemia, a common finding in patients with liver cirrhosis, on brain mitochondrial function was investigated in this study. The results show that ammonia in concentrations commonly seen in patients induces severe mitochondrial dysfunction, overproduction of damaging oxygen molecules and profound injury and death of neurons in rat brain cells. These findings point towards a novel mechanism of ammonia-induced brain injury in liver failure and potential novel therapeutic targets

Inorganic polyphosphate regulates AMPA and NMDA receptors and protects against glutamate excitotoxicity via activation of P2Y receptors

Glutamate is one of the most important neurotransmitters in the process of signal transduction in the central nervous system. Excessive amounts of this neurotransmitter lead to glutamate excitotoxicity which is accountable for neuronal death in acute neurological disorders including stroke, trauma, and in neurodegenerative diseases. Inorganic polyphosphate (PolyP) plays multiple roles in the mammalian brain, including function as a calcium-dependent gliotransmitter mediating communication between astrocytes, while its role in the regulation of neuronal activity is unknown. Here we studied the effect of polyP on glutamate-induced calcium signal in primary rat neurons in both physiological and pathological conditions. We found that pre-incubation of primary neurons with polyP reduced glutamate- and AMPA- but not the NMDA-induced calcium signal. However, in rat hippocampal acute slices polyP reduced ion flux through NMDA and AMPA receptors in native neurons. The effect of polyP on glutamate and specifically on the AMPA receptors was dependent on the presence of P2Y1 but not of P2X receptor inhibitors and also could be mimicked by P2Y1 agonist 2MeSADP. Pre-incubation of cortical neurons with polyP significantly reduced the initial calcium peak as well as the number of neurons with delayed calcium deregulation in response to high concentrations of glutamate and resulted in protection of neurons against glutamate-induced cell death. As a result, activation of P2Y1 receptors by polyP reduced calcium signal acting through AMPA receptors, thus protecting neurons against glutamate excitotoxicity by reduction of the calcium overload and restoration of mitochondrial function.Significance StatementOne of the oldest polymers in the evolution of living matter is the inorganic polyphosphate. It is shown to play a role of gliotransmitter in the brain; however, the role of polyphosphate in neuronal signalling is not clear. Here we demonstrate that inorganic polyphosphate is able to reduce calcium signal, induced by physiological or high concentrations of glutamate. The effect of polyphosphate on glutamate-induced calcium signal in neurons is due to the effect of this polymer on the AMPA receptors.The effect of polyP on glutamate- and AMPA-induced calcium signal is dependent on P2Y receptor antagonist. The ability of polyphosphate to restrict glutamate-induced calcium signal lies in the basis of its protection of neurons against glutamate excitotoxicity

Interaction of Mitochondrial Calcium and ROS in Neurodegeneration

Neurodegenerative disorders are currently incurable devastating diseases which are characterized by the slow and progressive loss of neurons in specific brain regions. Progress in the investigation of the mechanisms of these disorders helped to identify a number of genes associated with familial forms of these diseases and a number of toxins and risk factors which trigger sporadic and toxic forms of these diseases. Recently, some similarities in the mechanisms of neurodegenerative diseases were identified, including the involvement of mitochondria, oxidative stress, and the abnormality of Ca signaling in neurons and astrocytes. Thus, mitochondria produce reactive oxygen species during metabolism which play a further role in redox signaling, but this may also act as an additional trigger for abnormal mitochondrial calcium handling, resulting in mitochondrial calcium overload. Combinations of these factors can be the trigger of neuronal cell death in some pathologies. Here, we review the latest literature on the crosstalk of reactive oxygen species and Ca in brain mitochondria in physiology and beyond, considering how changes in mitochondrial metabolism or redox signaling can convert this interaction into a pathological event

Lipid peroxidation is involved in calcium dependent upregulation of mitochondrial metabolism in skeletal muscle

BACKGROUND: Skeletal muscle cells continuously generate reactive oxygen species (ROS). Excessive ROS can affect lipids resulting in lipid peroxidation (LPO). Here we investigated the effects of myotube intracellular calcium-induced signaling eliciting contractions on the LPO induction and the impact of LPO-product 4-hydroxynonenal (4-HNE) on physiology/pathology of myotubes using C2C12 myoblasts. METHODS: C2C12 myoblasts were differentiated into myotubes, stimulated with caffeine and analyzed for the induction of LPO and formation of 4-HNE protein adducts. Further effects of 4-HNE on mitochondrial bioenergetics, NADH level, mitochondrial density and expression of mitochondrial metabolism genes were determined. RESULTS: Short and long-term caffeine stimulation of myotubes promoted superoxide production, LPO and formation of 4-HNE protein adducts. Furthermore, low 4-HNE concentrations had no effect on myotube viability and cellular redox homeostasis, while concentrations from 10 μM and above reduced myotube viability and significantly disrupted homeostasis. A time and dose-dependent 4-HNE effect on superoxide production and mitochondrial NADH-autofluorescence was observed. Finally, 4-HNE had strong impact on maximal respiration, spare respiratory capacity, ATP production, coupling efficiency of mitochondria and mitochondrial density. CONCLUSION: Data presented in this work make evident for the first time that pathological 4-HNE levels elicit damaging effects on skeletal muscle cells while acute exposure to physiological 4-HNE induces transient adaptation. GENERAL SIGNIFICANCE: This work suggests an important role of 4-HNE on the regulation of myotube's mitochondrial metabolism and cellular energy production. It further signifies the importance of skeletal muscle cells hormesis in response to acute stress in order to maintain essential biological functions

KEAP₁ inhibition is neuroprotective and suppresses the development of epilepsy

Hippocampal sclerosis is a common acquired disease that is a major cause of drug-resistant epilepsy. A mechanism that has been proposed to lead from brain insult to hippocampal sclerosis is the excessive generation of reactive oxygen species, and consequent mitochondrial failure. Here we use a novel strategy to increase endogenous antioxidant defences using RTA 408, which we show activates nuclear factor erythroid 2-related factor 2 (Nrf2, encoded by NFE2L2) through inhibition of kelch like ECH associated protein 1 (KEAP1) through its primary sensor C151. Activation of Nrf2 with RTA 408 inhibited reactive oxygen species production, mitochondrial depolarization and cell death in an in vitro model of seizure-like activity. RTA 408 given after status epilepticus in vivo increased ATP, prevented neuronal death, and dramatically reduced (by 94%) the frequency of late spontaneous seizures for at least 4 months following status epilepticus. Thus, acute KEAP1 inhibition following status epilepticus exerts a neuroprotective and disease-modifying effect, supporting the hypothesis that reactive oxygen species generation is a key event in the development of epilepsy

Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons

Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP. Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies