Docosahexaenoic acid accumulation in the prenatal brain: a purported role in combating oxidative stress

Mitochondria may be both the source and the target of oxidative stress in neurodegenerative disease. In models of excitotoxicity, neuronal injury is triggered by the influx of calcium into neurons and then into mitochondria. Our studies suggest that an important consequence of this calcium movement is the generation of ROS by mitochondria. Studies with isolated mitochondria suggest that calcium may enhance ROS generation by mitochondria, especially when complex I is impaired. However, these studies are complicated by a lack of specificity of detection methods like Amplex Red. One feature of mitochondria is their movement within neurons. We used fluorescent proteins targeted to mitochondria to follow trafficking in neurons. Neurotoxins like glutamate, zinc and peroxide, which feature oxidative stress in their mechanism of action, affect mitochondrial movement, morphology or both. We speculate that restricting the delivery of mitochondria to their targets within neurons could impair neuronal viability.     

Amyloid-β triggers the release of neuronal hexokinase 1 from mitochondria

Brain accumulation of the amyloid-β peptide (Aβ) and oxidative stress underlie neuronal dysfunction and memory loss in Alzheimer's disease (AD). Hexokinase (HK), a key glycolytic enzyme, plays important pro-survival roles, reducing mitochondrial reactive oxygen species (ROS) generation and preventing apoptosis in neurons and other cell types. Brain isozyme HKI is mainly associated with mitochondria and HK release from mitochondria causes a significant decrease in enzyme activity and triggers oxidative damage. We here investigated the relationship between Aβ-induced oxidative stress and HK activity. We found that Aβ triggered HKI detachment from mitochondria decreasing HKI activity in cortical neurons. Aβ oligomers further impair energy metabolism by decreasing neuronal ATP levels. Aβ-induced HKI cellular redistribution was accompanied by excessive ROS generation and neuronal death. 2-deoxyglucose blocked Aβ-induced oxidative stress and neuronal death. Results suggest that Aβ-induced cellular redistribution and inactivation of neuronal HKI play important roles in oxidative stress and neurodegeneration in AD.     

Amino-Nogo-A antagonizes reactive oxygen species generation and protects immature primary cortical neurons from oxidative toxicity

Nogo-A is originally identified as an inhibitor of axon regeneration from the CNS myelin. Nogo-A is mainly expressed by oligodendrocytes, and also by some neuronal subpopulations, particularly in the developing nervous system. Although extensive studies have uncovered regulatory roles of Nogo-A in neurite outgrowth inhibition, precursor migration, neuronal homeostasis, plasticity and neurodegeneration, its cell-autonomous functions in neurons are largely uncharacterized. Here, we show that HIV-1 trans-activating-mediated amino-Nogo-A protein transduction into cultured primary cortical neurons achieves an almost complete neuroprotection against oxidative stress induced by exogenous hydrogen peroxide (H(2)O(2)). Endogenously expressed neuronal Nogo-A is significantly downregulated upon H(2)O(2) treatment. Furthermore, knockdown of Nogo-A results in more susceptibility to acute oxidative insults and markedly increases neuronal death. Interacting with peroxiredoxin 2 (Prdx2), amino-Nogo-A reduces reactive oxygen species (ROS) generation and extracellular signal-regulated kinase phosphorylation to exert neuroprotective effects. Structure-function mapping experiments reveal that, out of NiG-Δ20, a novel region comprising residues 290-562 of amino-Nogo-A is indispensable for preventing oxidative neuronal death. Moreover, mutagenesis analysis confirms that cysteine residues 424, 464 and 559 are involved in the inhibition of ROS generation and neuroprotective role of amino-Nogo-A. Our data suggest that neuronal Nogo-A might play a cell-autonomous role in improving neuronal survival against oxidative insult through interacting with Prdx2 and scavenging of ROS.     

Dynamics of intracellular calcium and free radical production during ischemia in pyramidal neurons.

Biochemical cascades initiated by oxidative stress and excitotoxic intracellular calcium rises are thought to converge on mitochondrial dysfunction. We investigated the contribution of mitochondrial dysfunction to free radical (FR) overproduction in rat CA1 pyramidal neurons of organotypic slices subjected to a hypoxic-hypoglycemic insult. Ischemia-induced FR generation was decreased by the mitochondrial complex I blocker, rotenone, indicating that mitochondria are the principal source of ischemic FR production. Measurements of mitochondrial calcium with the mitochondrial calcium probe dihydroRhod-2, revealed that FR production during and after the anoxic episode correlates with the accumulation of mitochondrial calcium. However, the mitochondrial calcium uptake inhibitor Ru360 did not prevent FR generation during ischemia and attenuated it to some degree during reoxygenation. On the other hand, the mitochondrial permeability transition blocker cyclosporinA (CsA) completely arrested both ischemic FR generation and mitochondrial calcium overload, and prevented deterioration of neuronal intrinsic membrane properties. CsA had no effect on the accumulation of intracellular calcium during ischemia-reperfusion. Nicotinamide, a blocker of NAD+ hydrolysis, reproduced the CsA effects on FR generation, mitochondrial calcium accumulation and cytoplasmic calcium increases. These observations suggest that a major determinant of ischemic FR generation in pyramidal neurons is the uncoupling of the mitochondrial respiratory chain, which may be associated with the mitochondrial permeability transition.     

Mitochondrial uncoupling proteins: new perspectives for evolutionary ecologists

Reactive oxygen species (ROS)-induced damage on host cells and molecules has been considered the most likely proximal mechanism responsible for the age-related decline in organismal performance. Organisms have two possible ways to reduce the negative effect of ROS: disposing of effective antioxidant defenses and minimizing ROS production. The unbalance between the amount of ROS produced and the availability of antioxidant defenses determines the intensity of so-called oxidative stress. Interestingly, most studies that deal with the effect of oxidative stress on organismal performance have focused on the antioxidant defense compartment and, surprisingly, have neglected the mechanisms that control ROS production within mitochondria. Uncoupling proteins (UCPs), mitochondrial transporters of the inner membrane, are involved in the control of redox state of cells and in the production of mitochondrial ROS. Given their function, UCPs might therefore represent a major mechanistic link between metabolic activity and fitness. We suggest that by exploring the role of expression and function of UCPs both in experimental as well as in comparative studies, evolutionary biologists may gain better insight into this link.     

Calcium-induced generation of reactive oxygen species in brain mitochondria is mediated by permeability transition

Mitochondrial uptake of calcium in excitotoxicity is associated with subsequent increase in reactive oxygen species (ROS) generation and delayed cellular calcium deregulation in ischemic and neurodegenerative insults. The mechanisms linking mitochondrial calcium uptake and ROS production remain unknown but activation of the mitochondrial permeability transition (mPT) may be one such mechanism. In the present study, calcium increased ROS generation in isolated rodent brain and human liver mitochondria undergoing mPT despite an associated loss of membrane potential, NADH and respiration. Unspecific permeabilization of the inner mitochondrial membrane by alamethicin likewise increased ROS independently of calcium, and the ROS increase was further potentiated if NAD(H) was added to the system. Importantly, calcium per se did not induce a ROS increase unless mPT was triggered. Twenty-one cyclosporin A analogs were evaluated for inhibition of calcium-induced ROS and their efficacy clearly paralleled their potency of inhibiting mPT-mediated mitochondrial swelling. We conclude that while intact respiring mitochondria possess powerful antioxidant capability, mPT induces a dysregulated oxidative state with loss of GSH- and NADPH-dependent ROS detoxification. We propose that mPT is a significant cause of pathological ROS generation in excitotoxic cell death.     

Differential effects of thyroid status on regional H2O2 production in slow- and fast-twitch muscle of ducklings

Birds seem to employ powerful physiological strategies to curb the harmful effects of reactive oxygen species (ROS) because they generally live longer than predicted by the free radical theory of aging. However, little is known about the physiological mechanisms that confer protection to birds against excessive ROS generation. Hence, we investigated the ability of birds to control mitochondrial ROS generation during physiologically stressful periods. In our study, we analyzed the relationship between the thyroid status and the function of intermyofibrillar and subsarcolemmal mitochondria located in glycolytic and oxidative muscles of ducklings. We found that the intermyofibrillar mitochondria of both glycolytic and oxidative muscles down regulate ROS production when plasma T₃ levels rise. The intermyofibrillar mitochondria of the gastrocnemius muscle (an oxidative muscle) produced less ROS and were more sensitive than the pectoralis muscle (a glycolytic muscle) to changes in plasma T₃. Such differences in the ROS production by glycolytic and oxidative muscles were associated with differences in the membrane proton permeability and in the rate of free radical leakage within the respiratory chain. This is the first evidence which shows that in birds, the amount of ROS that the mitochondria release is dependent on: (1) their location within the muscle; (2) the type of muscle (glycolytic or oxidative) and (3) on the thyroid status. Reducing muscle mitochondrial ROS generation might be an important mechanism in birds to limit oxidative damage during periods of physiological stress.     

Rethinking the excitotoxic ionic milieu: the emerging role of Zn(2+) in ischemic neuronal injury

Zn(2+) plays an important role in diverse physiological processes, but when released in excess amounts it is potently neurotoxic. In vivo trans-synaptic movement and subsequent post-synaptic accumulation of intracellular Zn(2+) contributes to the neuronal injury observed in some forms of cerebral ischemia. Zn(2+) may enter neurons through NMDA channels, voltage-sensitive calcium channels, Ca(2+)-permeable AMPA/kainate (Ca-A/K) channels, or Zn(2+)-sensitive membrane transporters. Furthermore, Zn(2+) is also released from intracellular sites such as metallothioneins and mitochondria. The mechanisms by which Zn(2+) exerts its potent neurotoxic effects involve many signaling pathways, including mitochondrial and extra-mitochondrial generation of reactive oxygen species (ROS) and disruption of metabolic enzyme activity, ultimately leading to activation of apoptotic and/or necrotic processes. As is the case with Ca(2+), neuronal mitochondria take up Zn(2+) as a way of modulating cellular Zn(2+) homeostasis. However, excessive mitochondrial Zn(2+) sequestration leads to a marked dysfunction of these organelles, characterized by prolonged ROS generation. Intriguingly, in direct comparison to Ca(2+), Zn(2+) appears to induce these changes with a considerably greater degree of potency. These effects are particularly evident upon large (i.e., micromolar) rises in intracellular Zn(2+) concentration ([Zn(2+)](i)), and likely hasten necrotic neuronal death. In contrast, sub-micromolar [Zn(2+)](i) increases promote release of pro-apoptotic factors, suggesting that different intensities of [Zn(2+)](i) load may activate distinct pathways of injury. Finally, Zn(2+) homeostasis seems particularly sensitive to the environmental changes observed in ischemia, such as acidosis and oxidative stress, indicating that alterations in [Zn(2+)](i) may play a very significant role in the development of ischemic neuronal damage.     

Geniposide Protects Primary Cortical Neurons against Oligomeric Aβ1-42-Induced Neurotoxicity through a Mitochondrial Pathway

Mitochondrial dysfunction plays a key role in the progression of Alzheimer’s disease (AD). The accumulation of amyloid-beta peptide (Aβ) in the brains of AD patients is thought to be closely related to neuronal mitochondrial dysfunction and oxidative stress. Therefore, protecting mitochondria from Aβ-induced neurotoxicity is an effective strategy for AD therapeutics. In a previous study, we found that geniposide, a pharmacologically active compound purified from gardenia fruit, has protective effects on oxidative stress and mitochondrial dysfunction in AD transgenic mouse models. However, whether geniposide has a protective effect on Aβ-induced neuronal dysfunction remains unknown. In the present study, we demonstrate that geniposide protects cultured primary cortical neurons from Aβ-mediated mitochondrial dysfunction by recovering ATP generation, mitochondrial membrane potential (MMP), and cytochrome c oxidase (CcO) and caspase 3/9 activity; by reducing ROS production and cytochrome c leakage; as well as by inhibiting apoptosis. These findings suggest that geniposide may attenuate Aβ-induced neuronal injury by inhibiting mitochondrial dysfunction and oxidative stress.     

Cdc42-dependent activation of NADPH oxidase is involved in ethanol-induced neuronal oxidative stress

It has been suggested that excessive reactive oxygen species (ROS) and oxidative stress play an important role in ethanol-induced damage to both the developing and mature central nervous system (CNS). The mechanisms underlying ethanol-induced neuronal ROS, however, remain unclear. In this study, we investigated the role of NADPH oxidase (NOX) in ethanol-induced ROS generation. We demonstrated that ethanol activated NOX and inhibition of NOX reduced ethanol-promoted ROS generation. Ethanol significantly increased the expression of p47(phox) and p67(phox), the essential subunits for NOX activation in cultured neuronal cells and the cerebral cortex of infant mice. Ethanol caused serine phosphorylation and membrane translocation of p47(phox) and p67(phox), which were prerequisites for NOX assembly and activation. Knocking down p47(phox) with the small interfering RNA was sufficient to attenuate ethanol-induced ROS production and ameliorate ethanol-mediated oxidative damage, which is indicated by a decrease in protein oxidation and lipid peroxidation. Ethanol activated cell division cycle 42 (Cdc42) and overexpression of a dominant negative (DN) Cdc42 abrogate ethanol-induced NOX activation and ROS generation. These results suggest that Cdc42-dependent NOX activation mediates ethanol-induced oxidative damages to neurons.     

Rat MYH,a glycosylase for repair of oxidatively damaged DNA,has brain-specific isoforms that localize to neuronal mitochondria

Mitochondrial genomes are exposed to a heavy load of reactive oxygen species (ROS) that damage DNA. Since in neurons, mitochondrial DNA integrity must be maintained over the entire mammalian life span, neuronal mitochondria most likely repair oxidatively damaged DNA. We show that the Escherichia coli MutY DNA glycosylase homolog (MYH) in rat (rMYH) involved in repair of oxidative damage is abundantly expressed in the rat brain, with isoforms that are exclusive to brain tissue. Confocal microscopy and western analyses reveal localization of rMYH in neuronal mitochondria. To assess involvement of MYH in the neuronal response to oxidative DNA damage, we used a rat model of respiratory hypoxia, in which acutely reduced blood oxygenation leads to generation of superoxide, and formation and subsequent removal of 8-hydroxy-2'-deoxyguanosine (8OHdG). Removal of 8OHdG is accompanied by a spatial increase in rMYH immunoreactivity in the brain and an increase in levels of one of the three mitochondrial MYH isoforms, suggesting that inducible and non-inducible MYH isoforms exist in the brain. The mitochondrial localization of oxidative DNA damage repair enzymes in neurons may represent a specialized neuronal mechanism that safeguards mitochondrial genomes in the face of routine and accidental exposures to heavy loads of injurious ROS.     

Possible plant mitochondria involvement in cell adaptation to drought stress. A case study: durum wheat mitochondria

Although plant cell bioenergetics is strongly affected by abiotic stresses, mitochondrial metabolism under stress is still largely unknown. Interestingly, plant mitochondria may control reactive oxygen species (ROS) generation by means of energy-dissipating systems. Therefore, mitochondria may play a central role in cell adaptation to abiotic stresses, which are known to induce oxidative stress at cellular level. With this in mind, in recent years, studies have been focused on mitochondria from durum wheat, a species well adapted to drought stress. Durum wheat mitochondria possess three energy-dissipating systems: the ATP-sensitive plant mitochondrial potassium channel (PmitoK(ATP)); the plant uncoupling protein (PUCP); and the alternative oxidase (AOX). It has been shown that these systems are able to dampen mitochondrial ROS production; surprisingly, PmitoK(ATP) and PUCP (but not AOX) are activated by ROS. This was found to occur in mitochondria from both control and hyperosmotic-stressed seedlings. Therefore, the hypothesis of a 'feed-back' mechanism operating under hyperosmotic/oxidative stress conditions was validated: stress conditions induce an increase in mitochondrial ROS production; ROS activate PmitoK(ATP) and PUCP that, in turn, dissipate the mitochondrial membrane potential, thus inhibiting further large-scale ROS production. Another important aspect is the chloroplast/cytosol/mitochondrion co-operation in green tissues under stress conditions aimed at modulating cell redox homeostasis. Durum wheat mitochondria may act against chloroplast/cytosol over-reduction: the malate/oxaloacetate antiporter and the rotenone-insensitive external NAD(P)H dehydrogenases allow cytosolic NAD(P)H oxidation; under stress this may occur without high ROS production due to co-operation with AOX, which is activated by intermediates of the photorespiratory cycle.     

Mechanism, measurement, and prevention of oxidative stress in male reproductive physiology

Numerous factors influence male fertility. Among these factors is oxidative stress (OS), which has elicited an enormous interest in researchers in recent period. Reactive oxygen species (ROS) are continuously produced by various metabolic and physiologic processes. OS occurs when the delicate balance between the production of ROS and the inherent antioxidant capacity of the organism is distorted. Spermatozoa are particularly sensitive to ROS as their plasma membrane contains polyunsaturated fatty acids (PUFA), which oxidizes easily. They also lack cytoplasm to generate a robust preventive and repair mechanism against ROS. The transition metal ions that are found in the body have a catalytic effect in the generation of ROS. Lifestyle behaviours such as smoking and alcohol use and environmental pollution further enhance the generation of ROS and thus, cause destructive effects on various cellular organelles like mitochondria, sperm DNA etc. This article analyzes the detrimental effects of OS on male fertility, measurement of OS and effective ways to decrease or eliminate them completely. We have also provided information on oxidative stress in other systems of the body, which may be applied to future research in the field of reproductive biology.     

4-hydroxynonenal and neurodegenerative diseases

The development of oxidative stress, in which production of highly reactive oxygen species (ROS) overwhelms antioxidant defenses, is a feature of many neurological diseases: ischemic, inflammatory, metabolic and degenerative. Oxidative stress is increasingly implicated in a number of neurodegenerative disorders characterized by abnormal filament accumulation or deposition of abnormal forms of specific proteins in affected neurons, like Alzheimer's disease (AD), Pick's disease, Lewy bodies related diseases, amyotrophic lateral sclerosis (ALS), and Huntington disease. Causes of neuronal death in neurodegenerative diseases are multifactorial. In some familiar cases of ALS mutation in the gene for Cu/Zn superoxide dismutase (SOD1) can be identified. In other neurodegenerative diseases ROS have some, usually not clear, role in early pathogenesis or implications on neuronal death in advanced stages of illness. The effects of oxidative stress on "post-mitotic cells", such as neurons may be cumulative, hence, it is often unclear whether oxidative damage is a cause or consequence of neurodegeneration. Peroxidation of cellular membrane lipids, or circulating lipoprotein molecules generates highly reactive aldehydes among which one of most important is 4-hydroxynonenal (HNE). The presence of HNE is increased in brain tissue and cerebrospinal fluid of AD patients, and in spinal cord of ALS patients. Immunohistochemical studies show presence of HNE in neurofibrilary tangles and in senile plaques in AD, in the cytoplasm of the residual motor neurons in sporadic ALS, in Lewy bodies in neocortical and brain stem neurons in Parkinson's disease (PD) and in diffuse Lewy bodies disease (DLBD). Thus, increased levels of HNE in neurodegenerative disorders and immunohistochemical distribution of HNE in brain tissue indicate pathophysiological role of oxidative stress in these diseases, and especially HNE in formation of abnormal filament deposites.     

Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism

The recent knowledge on mitochondria as the substantial source of reactive oxygen species, namely superoxide and hydrogen peroxide efflux from mitochondria, is reviewed, as well as nitric oxide and subsequent peroxynitrite generation in mitochondria and their effects. The reactive oxygen species formation in extramitochondrial locations, in peroxisomes, by cytochrome P450, and NADPH oxidase reaction, is also briefly discussed. Conditions are pointed out under which mitochondria represent the major ROS source for the cell: higher percentage of non-phosphorylating and coupled mitochondria, in vivo oxygen levels leading to increased intensity of the reverse electron transport in the respiratory chain, and nitric oxide effects on the redox state of cytochromes. We formulate hypotheses on the crucial role of ROS generated in mitochondria for the whole cell and organism, in concert with extramitochondrial ROS and antioxidant defense. We hypothesize that a sudden decline of mitochondrial ROS production converts cells or their microenvironment into a “ROS sink” represented by the instantly released excessive capacity of ROS-detoxification mechanisms. A partial but immediate decline of mitochondrial ROS production may be triggered by activation of mitochondrial uncoupling, specifically by activation of recruited or constitutively present uncoupling proteins such as UCP2, which may counterbalance the mild oxidative stress.     

Mechanisms of Rapid Reactive Oxygen Species Generation in Response to Cytosolic Ca2+ or Zn2+ Loads in Cortical Neurons

Excessive “excitotoxic” accumulation of Ca²⁺ and Zn²⁺ within neurons contributes to neurodegeneration in pathological conditions including ischemia. Putative early targets of these ions, both of which are linked to increased reactive oxygen species (ROS) generation, are mitochondria and the cytosolic enzyme, NADPH oxidase (NOX). The present study uses primary cortical neuronal cultures to examine respective contributions of mitochondria and NOX to ROS generation in response to Ca²⁺ or Zn²⁺ loading. Induction of rapid cytosolic accumulation of either Ca²⁺ (via NMDA exposure) or Zn²⁺ (via Zn²⁺/Pyrithione exposure in 0 Ca²⁺) caused sharp cytosolic rises in these ions, as well as a strong and rapid increase in ROS generation. Inhibition of NOX activation significantly reduced the Ca²⁺-induced ROS production with little effect on the Zn²⁺- triggered ROS generation. Conversely, dissipation of the mitochondrial electrochemical gradient increased the cytosolic Ca²⁺ or Zn²⁺ rises caused by these exposures, consistent with inhibition of mitochondrial uptake of these ions. However, such disruption of mitochondrial function markedly suppressed the Zn²⁺-triggered ROS, while partially attenuating the Ca²⁺-triggered ROS. Furthermore, block of the mitochondrial Ca²⁺ uniporter (MCU), through which Zn²⁺ as well as Ca²⁺ can enter the mitochondrial matrix, substantially diminished Zn²⁺ triggered ROS production, suggesting that the ROS generation occurs specifically in response to Zn²⁺ entry into mitochondria. Finally, in the presence of the sulfhydryl-oxidizing agent 2,2''-dithiodipyridine, which impairs Zn²⁺ binding to cytosolic metalloproteins, far lower Zn²⁺ exposures were able to induce mitochondrial Zn²⁺ uptake and consequent ROS generation. Thus, whereas rapid acute accumulation of Zn²⁺ and Ca²⁺ each can trigger injurious ROS generation, Zn²⁺ entry into mitochondria via the MCU may do so with particular potency. This may be of particular relevance to conditions like ischemia in which cytosolic Zn²⁺ buffering is impaired due to acidosis and oxidative stress.     

Homocysteine potentiates β-amyloid neurotoxicity: role of oxidative stress

The cause of neuronal degeneration in Alzheimer's disease (AD) has not been completely clarified, but has been variously attributed to increases in cytosolic calcium and increased generation of reactive oxygen species (ROS). The beta-amyloid fragment (Abeta) of the amyloid precursor protein induces calcium influx, ROS and apoptosis. Homocysteine (HC), a neurotoxic amino acid that accumulates in neurological disorders including AD, also induces calcium influx and oxidative stress, which has been shown to enhance neuronal excitotoxicity, leading to apoptosis. We examined the possibility that HC may augment Abeta neurotoxicity. HC potentiated the Abeta-induced increase in cytosolic calcium and apoptosis in differentiated SH-SY-5Y human neuroblastoma cells. The antioxidant vitamin E and the glutathione precursor N-acetyl-L-cysteine blocked apoptosis following cotreatment with HC and Abeta, indicating that apoptosis is associated with oxidative stress. These findings underscore that moderate accumulation of excitotoxins at concentrations that alone do not appear to initiate adverse events may enhance the effects of other factors known to cause neurodegeneration such as Abeta.     

The role of an astrocytic NADPH oxidase in the neurotoxicity of amyloid beta peptides

Amyloid beta peptide (Abeta) accumulates in the CNS in Alzheimer's disease. Both the full peptide (1-42) or the 25-35 fragment are toxic to neurons in culture. We have used fluorescence imaging technology to explore the mechanism of neurotoxicity in mixed asytrocyte/neuronal cultures prepared from rat or mouse cortex or hippocampus, and have found that Abeta acts preferentially on astrocytes but causes neuronal death. Abeta causes sporadic transient increases in [Ca2+]c in astrocytes, associated with a calcium dependent increased generation of reactive oxygen species (ROS) and glutathione depletion. This caused a slow dissipation of mitochondrial potential on which abrupt calcium dependent transient depolarizations were superimposed. The mitochondrial depolarization was reversed by mitochondrial substrates glutamate, pyruvate or methyl succinate, and by NADPH oxidase (NOX) inhibitors, suggesting that it reflects oxidative damage to metabolic pathways upstream of mitochondrial complex I. The Abeta induced increase in ROS and the mitochondrial depolarization were absent in cells cultured from transgenic mice lacking the NOX component, gp91phox. Neuronal death after 24 h of Abeta exposure was dramatically reduced both by NOX inhibitors and in gp91phox knockout mice. Thus, by raising [Ca2+]c in astrocytes, Abeta activates NOX, generating oxidative stress that is transmitted to neurons, causing neuronal death.     

Yeast apurinic/apyrimidinic endonuclease Apn1 protects mammalian neuronal cell line from oxidative stress

Reactive oxygen species (ROS) have been implicated as one of the agents responsible for many neurodegenerative diseases. A critical target for ROS is DNA. Most oxidative stress-induced DNA damage in the nucleus and mitochondria is removed by the base excision repair pathway. Apn1 is a yeast enzyme in this pathway which possesses a wider substrate specificity and greater enzyme activity than its mammalian counterpart for removing DNA damage, making it a good therapeutic candidate. For this study we targeted Apn1 to mitochondria in a neuronal cell line derived from the substantia nigra by using a mitochondrial targeting signal (MTS) in an effort to hasten the removal of DNA damage and thereby protect these cells. We found that following oxidative stress, mitochondrial DNA (mtDNA) was repaired more efficiently in cells containing Apn1 with the MTS than controls. There was no difference in nuclear repair. However, cells that expressed Apn1 without the MTS showed enhanced repair of both nuclear and mtDNA. Both Apn1-expressing cells were more resistant to cell death following oxidative stress compared with controls. Therefore, these results reveal that the expression of Apn1 in neurons may be of potential therapeutic benefit for treating patients with specific neurodegenerative diseases.     

Implication of PTEN in production of reactive oxygen species and neuronal death in in vitro models of stroke and Parkinson's disease

Oxidative stress plays crucial role in the pathogenesis of neurodegenerative diseases. However, the precise mechanism for an increased production of reactive oxygen species (ROS) under pathological conditions is not yet fully understood. We have recently demonstrated an implication of phosphatase and tensin homologue deleted on chromosome 10 (PTEN), a tumor suppressor, in ROS generation and neuronal apoptosis induced by staurosporine. These findings raised further interest whether PTEN functions as a common mediator of oxidative stress in neurodegenerative processes. To address this issue, neural cells were exposed to oxygen-glucose deprivation (OGD) and to the neurotoxin 1-methyl-4-phenylpyridinium iodide (MPP(+)), which mimic cerebral ischemia and Parkinson's disease, respectively. OGD for 4 h followed by 16 h of reoxygenation or incubation with MPP(+) (250 microM) for 48 h induced 33% and 45% neuronal death in rat hippocampal and in human dopaminergic SH-SY5Y neurons, respectively, accompanied by a gradual increase in the intracellular level of ROS. The increase in ROS by OGD and by MPP(+) did not cause oxidative inactivation of PTEN and thus, PTEN remains constitutively active. In support, the protein level of PTEN was not reduced in both cell cultures after challenging with OGD or MPP(+). Importantly, the elevated intracellular ROS levels and the neuronal death caused by OGD or by MPP(+) toxicity were significantly inhibited when PTEN was downregulated by a specific antisense oligonucleotide or by siRNA. Because SOD2 protein level is not altered either by knockdown of PTEN nor by an inhibition of the PI3K/Akt signalling, we suggest that SOD2 do not contribute to the pathomechanism of oxidative stress induced by PTEN or by inhibiting the related Akt signalling. The present study highlights PTEN as a crucial and common mediator of ROS generation and neuronal death and suggests that PTEN could become a potential therapeutic target for interfering with neurodegeneration.