Sources and triggers of oxidative damage in neurodegeneration

Neurodegeneration describes a group of more than 300 neurological diseases, characterised by neuronal loss and intra- or extracellular protein depositions, as key neuropathological features. Multiple factors play role in the pathogenesis of these group of disorders: mitochondrial dysfunction, membrane damage, calcium dyshomeostasis, metallostasis, defect clearance and renewal mechanisms, to name a few. All these factors, without exceptions, have in common the involvement of immensely increased generation of free radicals and occurrence of oxidative stress, and as a result - exhaustion of the scavenging potency of the cellular redox defence [1] mechanisms. Besides genetic predisposition and environmental exposure to toxins, the main risk factor for developing neurodegeneration is age. And although the “Free radical theory of ageing” was declared dead, it is undisputable that accumulation of damage occurs with age, especially in systems that are regulated by free radical messengers and those that oppose oxidative stress, protein oxidation and the accuracy in protein synthesis and degradation machinery has difficulties to be maintained. This brief review provides a comprehensive summary on the main sources of free radical damage, occurring in the setting of neurodegeneration

Functional role of mitochondrial reactive oxygen species in physiology

The major energy generator in the cell – mitochondria produce reactive oxygen species as a by-product of a number of enzymatic reactions and the production of ATP. Emerging evidence suggests that mitochondrial ROS regulate diverse physiological parameters and that dysregulated ROS signalling may contribute to a development of processes which lead to human diseases. ROS produced in mitochondrial enzymes are triggers of monoamine-induced calcium signal in astrocytes, playing important role in physiological and pathophysiological response to dopamine. Generation of ROS in mitochondria leads to peroxidation of lipids, which is considered to be one of the most important mechanisms of cell injury under condition of oxidative stress. However, it also can induce activation of mitochondrial and cellular phospholipases that can trigger a variety of the signals – from activation of ion channels to stimulation of calcium signal. Mitochondria are shown to be the oxygen sensor in astrocytes, therefore inhibition of respiration by hypoxia induces ROS production which leads to lipid peroxidation, activation of phospholipase C and induction of IP3-mediated calcium signal. Propagation of astrocytic calcium signal stimulates breathing activity in response to hypoxia. Thus, ROS produced by mitochondrial enzymes or electron transport chain can be used as a trigger for signalling cascades in central nervous system and deregulation of this process leads to pathology

Alpha-synuclein and beta-amyloid different targets, same players: calcium, free radicals and mitochondria in the mechanism of neurodegeneration

Two of the most devastating neurodegenerative diseases are consequences out of misfolding and aggregation of key proteins-alpha synuclein and beta-amyloid. Although the primary targets for the two proteins are different, they both share a common mechanism that involves formation of pore-like structure on the plasma membrane, consequent dysregulation of calcium homeostasis, mitochondrial dysfunction and oxidative damage. The combined effect of all this factors ultimately leads to neuronal cell death. Whereas beta amyloid acts on the astrocytic plasma membrane, exhibiting a tight dependence to the membrane cholesterol content, alpha–synuclein does not distinguish between type of membrane or cell. Additionally, oligomeric forms of both proteins produce reactive oxygen species through different mechanisms: beta-amyloid through activation of the NADPH oxidase and alpha-synuclein through non-enzymatic way. Finally, both peptides in oligomeric form induce mitochondrial depolarisation through calcium overload and free radical production that ultimately lead to opening of the mitochondrial permeability transition pore and trigger cell death

Mitochondrial dysfunction and energy deprivation in the mechanism of neurodegeneration

Energy-producing organelles mitochondria are involved in a number of cellular functions. Deregulation of mitochondrial function due to mutations or effects of mitochondrial toxins is proven to be a trigger for diverse pathologies, including neurodegenerative disorders. Despite the extensive research done in the last decades, the mechanisms by which mitochondrial dysfunction leads to neuronal deregulation and cell death have not yet been fully elucidated. Brain cells are specifically dependent on mitochondria due to their high energy demands to maintain neuronal ion gradients and signal transduction, and also, to mediate neuronal health through the processes of mitochondrial calcium homeostasis, mitophagy, mitochondrial reactive oxygen species production and mitochondrial dynamics. Some of these processes have been independently implicated in the mechanism of neuronal loss in neurodegeneration. Moreover, it is increasingly recognised that these processes are interdependent and interact within the mitochondria to ensure proper neuronal function and survival

Lipid peroxidation is essential for α-synuclein-induced cell death.

Parkinson's disease is the second most common neurodegenerative disease and its pathogenesis is closely associated with oxidative stress. Deposition of aggregated α-synuclein (α-Syn) occurs in familial and sporadic forms of Parkinson's disease. Here, we studied the effect of oligomeric α-Syn on one of the major markers of oxidative stress, lipid peroxidation, in primary co-cultures of neurons and astrocytes. We found that oligomeric but not monomeric α-Syn significantly increases the rate of production of reactive oxygen species, subsequently inducing lipid peroxidation in both neurons and astrocytes. Pre-incubation of cells with isotope-reinforced polyunsaturated fatty acids (D-PUFAs) completely prevented the effect of oligomeric α-Syn on lipid peroxidation. Inhibition of lipid peroxidation with D-PUFAs further protected cells from cell death induced by oligomeric α-Syn. Thus, lipid peroxidation induced by misfolding of α-Syn may play an important role in the cellular mechanism of neuronal cell loss in Parkinson's disease. We have found that aggregated α-synuclein-induced production of reactive oxygen species (ROS) that subsequently stimulates lipid peroxidation and cell death in neurons and astrocytes. Specific inhibition of lipid peroxidation by incubation with reinforced polyunsaturated fatty acids (D-PUFAs) completely prevented the effect of α-synuclein on lipid peroxidation and cell death

Viper toxins affect membrane characteristics of human erythrocytes

Elucidating electrokinetic stability by which surface charges regulate toxins interaction with erythrocytes is crucial for understanding the cell functionality. Electrokinetic properties of human erythrocytes upon treatment of Vipoxin, phospholipase A2 (PLA2) and Vipoxin acidic component (VAC), isolated from Vipera ammodytes meridionalis venom were studied using particle microelectrophoresis. PLA2 and Vipoxin treatments alter the osmotic fragility of erythrocyte membranes. The increased stability of cells upon viper toxins is presented by the increased zeta potential of erythrocytes before sedimentation of cells during electric field applied preventing the aggregation of cells. Lipid peroxidation of low dose toxin-treated erythrocytes shows reduced LP products compared to untreated cells. The apparent proton efflux and conductivity assays are performed and the effectiveness PLA2 > Vipoxin>VAC is discussed. The reported results open perspectives to a further investigation of the electrokinetic properties of the membrane after viper toxins treatment to shed light on the molecular mechanisms driving the mechanisms of inflammation and neurodegenerative diseases

Assessment of ROS Production in the Mitochondria of Live Cells

Production of reactive oxygen species (ROS) in the mitochondria plays multiple roles in physiology, and excessive production of ROS leads to the development of various pathologies. ROS in the mitochondria are generated by various enzymes, mainly in the electron transporvt chain, and it is important to identify not only the trigger but also the source of free radical production. It is important to measure mitochondrial ROS in live, intact cells, because activation of ROS production could be initiated by changes in extramitochondrial processes which could be overseen when using isolated mitochondria. Here we describe the approaches, which allow to measure production of ROS in the matrix of mitochondria in live cells. We also demonstrate how to measure kinetic changes in lipid peroxidation in mitochondria of live cells. These methods could be used for understanding the mechanisms of pathology in a variety of disease models and also for testing neuro- or cardioprotective chemicals

Assessment of ROS Production in the Mitochondria of Live Cells

Production of reactive oxygen species (ROS) in the mitochondria plays multiple roles in physiology, and excessive production of ROS leads to the development of various pathologies. ROS in the mitochondria are generated by various enzymes, mainly in the electron transporvt chain, and it is important to identify not only the trigger but also the source of free radical production. It is important to measure mitochondrial ROS in live, intact cells, because activation of ROS production could be initiated by changes in extramitochondrial processes which could be overseen when using isolated mitochondria. Here we describe the approaches, which allow to measure production of ROS in the matrix of mitochondria in live cells. We also demonstrate how to measure kinetic changes in lipid peroxidation in mitochondria of live cells. These methods could be used for understanding the mechanisms of pathology in a variety of disease models and also for testing neuro- or cardioprotective chemicals

Singlet oxygen stimulates mitochondrial bioenergetics in brain cells.

Oxygen, in form of reactive oxygen species (ROS), has been shown to participate in oxidative stress, one of the major triggers for pathology, but also is a main contributor to physiological processes. Recently, it was found that 1267 nm irradiation can produce singlet oxygen without photosensitizers. We used this phenomenon to study the effect of laser-generated singlet oxygen on one of the major oxygen-dependent processes, mitochondrial energy metabolism. We have found that laser-induced generation of 1O2 in neurons and astrocytes led to the increase of mitochondrial membrane potential, activation of NADH- and FADH-dependent respiration, and importantly, increased the rate of maximal respiration in isolated mitochondria. The activation of mitochondrial respiration stimulated production of ATP in these cells. Thus, we found that the singlet oxygen generated by 1267 nm laser pulse works as an activator of mitochondrial respiration and ATP production in the brain

Age-related changes in the energy of human mesenchymal stem cells

Aging is a physiological process that leads to a higher risk for the most devastating diseases. There are a number of theories of human aging proposed, and many of them are directly or indirectly linked to mitochondria. Here, we used mesenchymal stem cells (MSCs) from young and older donors to study age-related changes in mitochondrial metabolism. We have found that aging in MSCs is associated with a decrease in mitochondrial membrane potential and lower NADH levels in mitochondria. Mitochondrial DNA content is higher in aged MSCs, but the overall mitochondrial mass is decreased due to increased rates of mitophagy. Despite the higher level of ATP in aged cells, a higher rate of ATP consumption renders them more vulnerable to energy deprivation compared to younger cells. Changes in mitochondrial metabolism in aged MSCs activate the overproduction of reactive oxygen species in mitochondria which is compensated by a higher level of the endogenous antioxidant glutathione. Thus, energy metabolism and redox state are the drivers for the aging of MSCs/mesenchymal stromal cells