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

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

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

Nrf2 depletion in the context of loss-of-function Keap1 leads to mitolysosome accumulation

Transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2) is the principal determinant of the cellular redox homeostasis, contributing to mitochondrial function, integrity and bioenergetics. The main negative regulator of Nrf2 is Kelch-like ECH associated protein 1 (Keap1), a substrate adaptor for Cul3/Rbx1 ubiquitin ligase, which continuously targets Nrf2 for ubiquitination and proteasomal degradation. Loss-of-function mutations in Keap1 occur frequently in lung cancer, leading to constitutive Nrf2 activation. We used the human lung cancer cell line A549 and its CRISPR/Cas9-generated homozygous Nrf2-knockout (Nrf2-KO) counterpart to assess the role of Nrf2 on mitochondrial health. To confirm that the observed effects of Nrf2 deficiency are not due to clonal selection or long-term adaptation to the absence of Nrf2, we also depleted Nrf2 by siRNA (siNFE2L2), thus creating populations of Nrf2-knockdown (Nrf2-KD) A549 cells. Nrf2 deficiency decreased mitochondrial respiration, but increased the mitochondrial membrane potential, mass, DNA content, and the number of mitolysosomes. The proportion of ATG7 and ATG3 within their respective LC3B conjugates was increased in Nrf2-deficient cells with mutant Keap1, whereas the formation of new autophagosomes was not affected. Thus, in lung cancer cells with loss-of-function Keap1, Nrf2 facilitates mitolysosome degradation thereby ensuring timely clearance of damaged mitochondria

Alpha-Synuclein Oligomers Interact with Metal Ions to Induce Oxidative Stress and Neuronal Death in Parkinson's Disease

Protein aggregation and oxidative stress are both key pathogenic processes in Parkinson's disease, although the mechanism by which misfolded proteins induce oxidative stress and neuronal death remains unknown. In this study, we describe how aggregation of alpha-synuclein (α-S) from its monomeric form to its soluble oligomeric state results in aberrant free radical production and neuronal toxicity

Pharmacological sequestration of mitochondrial calcium uptake protects against dementia and β-amyloid neurotoxicity

All forms of dementia including Alzheimer's disease are currently incurable. Mitochondrial dysfunction and calcium alterations are shown to be involved in the mechanism of neurodegeneration in Alzheimer's disease. Previously we have described the ability of compound Tg-2112x to protect neurons via sequestration of mitochondrial calcium uptake and we suggest that it can also be protective against neurodegeneration and development of dementia. Using primary co-culture neurons and astrocytes we studied the effect of Tg-2112x and its derivative Tg-2113x on β-amyloid-induced changes in calcium signal, mitochondrial membrane potential, mitochondrial calcium, and cell death. We have found that both compounds had no effect on β-amyloid or acetylcholine-induced calcium changes in the cytosol although Tg2113x, but not Tg2112x reduced glutamate-induced calcium signal. Both compounds were able to reduce mitochondrial calcium uptake and protected cells against β-amyloid-induced mitochondrial depolarization and cell death. Behavioral effects of Tg-2113x on learning and memory in fear conditioning were also studied in 3 mouse models of neurodegeneration: aged (16-month-old) C57Bl/6j mice, scopolamine-induced amnesia (3-month-old mice), and 9-month-old 5xFAD mice. It was found that Tg-2113x prevented age-, scopolamine- and cerebral amyloidosis-induced decrease in fear conditioning. In addition, Tg-2113x restored fear extinction of aged mice. Thus, reduction of the mitochondrial calcium uptake protects neurons and astrocytes against β-amyloid-induced cell death and contributes to protection against dementia of different ethology. These compounds could be used as background for the developing of a novel generation of disease-modifying neuroprotective agents

α-synuclein oligomers interact with ATP synthase and open the permeability transition pore in Parkinson's disease.

Protein aggregation causes α-synuclein to switch from its physiological role to a pathological toxic gain of function. Under physiological conditions, monomeric α-synuclein improves ATP synthase efficiency. Here, we report that aggregation of monomers generates beta sheet-rich oligomers that localise to the mitochondria in close proximity to several mitochondrial proteins including ATP synthase. Oligomeric α-synuclein impairs complex I-dependent respiration. Oligomers induce selective oxidation of the ATP synthase beta subunit and mitochondrial lipid peroxidation. These oxidation events increase the probability of permeability transition pore (PTP) opening, triggering mitochondrial swelling, and ultimately cell death. Notably, inhibition of oligomer-induced oxidation prevents the pathological induction of PTP. Inducible pluripotent stem cells (iPSC)-derived neurons bearing SNCA triplication, generate α-synuclein aggregates that interact with the ATP synthase and induce PTP opening, leading to neuronal death. This study shows how the transition of α-synuclein from its monomeric to oligomeric structure alters its functional consequences in Parkinson's disease