Mitochondrial N-methyl-d-aspartate receptor activation enhances bioenergetics by calcium-dependent and -Independent mechanisms.

Our laboratory has demonstrated that functional N-methyl-d-aspartate-like receptors are present on neuronal mitochondria (NMDAm). This novel site gates the influx of Ca2+ and causes a several-fold increase in ATP levels. Although elevations in ATP in other cell types have been linked to increases in mitochondrial Ca2+, it has not been established whether the same holds true for calcium uptake via NMDAm. In this study, we have investigated the effect of NMDAm activation on a variety of bioenergetic parameters. Our findings suggest that mitochondrial bioenergetics are not only modulated by NMDAm activation in a Ca2+-dependent but also in a Ca2+-independent manner.

Selective Vulnerability of Striatal D2 versus D1 Dopamine Receptor-Expressing Medium Spiny Neurons in HIV-1 Tat Transgenic Male Mice.

Despite marked regional differences in HIV susceptibility within the CNS, there has been surprisingly little exploration into the differential vulnerability among neuron types and the circuits they underlie. The dorsal striatum is especially susceptible, harboring high viral loads and displaying marked neuropathology, with motor impairment a frequent manifestation of chronic infection. However, little is known about the response of individual striatal neuron types to HIV or how this disrupts function. Therefore, we investigated the morphological and electrophysiological effects of HIV-1 trans-activator of transcription (Tat) in dopamine subtype 1 (D1) and dopamine subtype 2 (D2) receptor-expressing striatal medium spiny neurons (MSNs) by breeding transgenic Tat-expressing mice to Drd1a-tdTomato- or Drd2-eGFP-reporter mice. An additional goal was to examine neuronal vulnerability early during the degenerative process to gain insight into key events underlying the neuropathogenesis. In D2 MSNs, exposure to HIV-1 Tat reduced dendritic spine density significantly, increased dendritic damage (characterized by swellings/varicosities), and dysregulated neuronal excitability (decreased firing at 200-300 pA and increased firing rates at 450 pA), whereas insignificant morphologic and electrophysiological consequences were observed in Tat-exposed D1 MSNs. These changes were concomitant with an increased anxiety-like behavioral profile (lower latencies to enter a dark chamber in a light-dark transition task, a greater frequency of light-dark transitions, and reduced rearing time in an open field), whereas locomotor behavior was unaffected by 2 weeks of Tat induction. Our findings suggest that D2 MSNs and a specific subset of neural circuits within the dorsal striatum are preferentially vulnerable to HIV-1.SIGNIFICANCE STATEMENT Despite combination antiretroviral therapy (cART), neurocognitive disorders afflict 30-50% of HIV-infected individuals and synaptodendritic injury remains evident in specific brain regions such as the dorsal striatum. A possible explanation for the sustained neuronal injury is that the neurotoxic HIV-1 regulatory protein trans-activator of transcription (Tat) continues to be expressed in virally suppressed patients on cART. Using inducible Tat-expressing transgenic mice, we found that dopamine subtype 2 (D2) receptor-expressing medium spiny neurons (MSNs) are selectively vulnerable to Tat exposure compared with D1 receptor-expressing MSNs. This includes Tat-induced reductions in D2 MSN dendritic spine density, increased dendritic damage, and disruptions in neuronal excitability, which coincide with elevated anxiety-like behavior. These data suggest that D2 MSNs and specific circuits within the basal ganglia are preferentially vulnerable to HIV-1.

Direct exposure to N-methyl-d-aspartate alters mitochondrial function.

N-methyl-d-aspartate (NMDA) receptors have long been known to be associated with the plasma membrane, providing a channel for the passage of extracellular Ca(2+) into the cytosol during synaptic transmission. Recent results from our laboratory indicate that in addition to this classic location, an NMDA-sensitive site (NMDAm) may also exist within the inner mitochondrial membrane. We report direct exposure of mitochondrial to NMDA enhances the production of reactive oxygen species and attenuate ROS-induced cytochrome c release, all the while slowing the rate of Ca(2+)-induced mitochondrial swelling. Treatment with NMDA did not alter the mitochondrial membrane potential. The findings of this study lend further support for the existence of NMDAm and suggest that this site may serve to stabilize mitochondrial function.

6-Hydroxydopamine as a tool to understand adaptive immune system-induced dopamine neurodegeneration in Parkinson's disease.

CONTEXT: Neuroimmunological response is associated with neurodegeneration in the human substantia nigra (SN) in Parkinson's disease (PD). OBJECTIVE: To explore the possibility that the neurotoxin, 6-hydroxydopamine (6-OHDA), could be used as a tool in mice to understand the immune response in PD. MATERIALS AND METHODS: We employed unilateral administration of 6-OHDA into the mouse SN. At 1 week, 2 weeks and 4 weeks post-injection, we used immunohistochemistry for the markers Iba-1 and gp91PHOX to investigate activated microglia in the SN. To examine the adaptive immune response, we used immunohistochemistry for CD3-positive T-lymphocytes, CD45R-positive B-lymphocytes and anti-mouse immunoglobulin-G (IgG). Dopamine neuron loss was examined using immunohistochemistry for the dopamine neuron marker, tyrosine hydroxylase. RESULTS: Compared to vehicle, 6-OHDA administration induced an intense IgG deposition in the SN as well as increased infiltration of both T- and B- lymphocytes into the injected side of the midbrain. The adaptive immune response was associated with extensive destruction of dopamine neurons and extensive microglial activation at every time point in the 6-OHDA groups. CONCLUSION: Our results suggest that 6-OHDA administration in mice can a potential tool for understanding mechanisms underlying adaptive immune activation-induced neurodegeneration in PD.

Identification of an N-methyl-D-aspartate receptor in isolated nervous system mitochondria.

NMDA ionotropic glutamate receptors gate the cytoplasmic influx of calcium, which may, depending on the intensity of the stimulus, subserve either normal synaptic communication or cell death. We demonstrate that when isolated mitochondria are exposed to calcium and NMDA agonists, there is a significant increase in mitochondrial calcium levels. The agonist/antagonist response studies on purified mitochondria suggest the presence of a receptor on mitochondria with features similar to plasma membrane NMDA receptors. Immunogold electron microscopy of hippocampal tissue sections revealed extensive localization of NR2a subunit immunoreactivity on mitochondria. Transient transfection of neuronal GT1-7 cells with an NR1-NR2a NMDA receptor subunit cassette specifically targeting mitochondria resulted in a significant increase in mitochondrial calcium and neuroprotection against glutamate-induced cell death. Mitochondria prepared from GT1-7 cells in which the NR1 subunit of NMDA receptors was silenced demonstrated a decrease in calcium uptake. Our findings are the first to demonstrate that mitochondria express a calcium transport protein that shares characteristics with the NMDA receptor and may play a neuroprotective role.

Progress in understanding basal ganglia dysfunction as a common target for methamphetamine abuse and HIV-1 neurodegeneration.

HIV-1 infection with concurrent methamphetamine (MA) abuse results in exacerbated neurodegenerative changes and rapid progression of a form of sub-cortical dementia termed HIV-1 associated dementia (HAD). A notable feature of HAD is the involvement of the dopaminergic system manifested as parkinsonian like movement abnormalities. The HIV-1 transactivator of transcription (Tat) protein is very often used in experimental studies trying to understand neurotoxic consequences of HIV-1 infection, since the pathophysiological changes induced by Tat mirrors, in part, the means by which HIV-1 infection of the nervous system results in neuronal damage. Understanding the interaction of Tat and MA in the basal ganglia and the resultant injury to the dopaminergic system in rodent models as well as cell culture will shed light on the dopaminergic pathology occurring in HIV-1 infected-MA abusers. The aim of this review is to update the reader on the current knowledge of MA and HIV-1 neurotoxicity, specifically Tat, and discuss the progress in understanding how MA synergizes with the HIV-1 transactivator protein Tat to damage the basal ganglia.

Post-Injury Administration of Mitochondrial Uncouplers Increases Tissue Sparing and Improves Behavioral Outcome following Traumatic Brain Injury in Rodents.

Following experimental traumatic brain injury (TBI), a rapid and significant necrosis occurs at the site of injury which coincides with significant mitochondrial dysfunction. The present study is driven by the hypothesis that TBI-induced glutamate release increases mitochondrial Ca(2+)cycling/overload, ultimately leading to mitochondrial dysfunction. Based on this premise, mitochondrial uncoupling during the acute phases of TBI-induced excitotoxicity should reduce mitochondrial Ca(2+) uptake (cycling) and reactive oxygen species (ROS) production since both are mitochondrial membrane potential dependent. In the present study, we utilized a cortical impact model of TBI to assess the potential use of mitochondrial uncouplers (2,4-DNP, FCCP) as a neuroprotective therapy. Young adult male rats were intraperitoneally administered vehicle (DMSO), 2,4-DNP (5 mg/kg), or FCCP (2.5 mg/kg) at 5 min post-injury. All animals treated with the uncouplers demonstrated a significant reduction in the amount of cortical damage and behavioral improvement following TBI. In addition, mitochondria isolated from the injured cortex at 3 or 6 h post-injury demonstrated that treatment with the uncouplers significantly improved several parameters of mitochondrial bioenergetics. These results demonstrate that post-injury treatment with mitochondrial uncouplers significantly (p < 0.01) increases cortical tissue sparing ( approximately 12%) and significantly (p< 0.01) improves behavioral outcome following TBI. The mechanism of neuroprotection most likely involves the maintenance of mitochondrial homeostasis by reducing mitochondrial Ca(2+) loading and subsequent mitochondrial dysfunction. These results further implicate mitochondrial dysfunction as an early event in the pathophysiology of TBI and that targeting acute mitochondrial events can result in long-term neuroprotection and improve behavioral outcome following brain injury.

Protective effects of NIM811 in transient focal cerebral ischemia suggest involvement of the mitochondrial permeability transition.

Cerebral ischemia followed by reperfusion activates numerous pathways that lead to cell death. One such pathway involves the release of large quantities of the excitatory amino acid glutamate into the synapse and activation of N-methyl-D-aspartate receptors. This causes an increase in mitochondrial calcium levels ([Ca(2+)](m)) and a production of reactive oxygen species (ROS), both of which may induce the mitochondrial permeability transition (MPT). As a consequence, there is eventual mitochondrial failure culminating in either apoptotic or necrotic cell death. Thus, agents that inhibit MPT might prove useful as therapeutic interventions in cerebral ischemia. In this study, we have investigated the neuroprotective efficacy of the novel compound NIM811. Similar in structure of its parent compound cyclosporin A, NIM811 is a potent inhibitor of the MPT. Unlike cyclosporin A, however, it is essentially void of immunosuppressive actions, allowing the role of MPT to be clarified in ischemia/reperfusion injury. The results of these studies demonstrate that NIM811 provides almost 40% protection in a model of transient focal cerebral ischemia. This was associated with a nearly 10% reduction in mitochondrial reactive species formation and 34% and 38% reduction of cytochrome c release in core and penumbra, respectively. Treatment with NIM811 also increased calcium retention capacity by approximately 20%. Interestingly, NIM811 failed to improve ischemia-induced impairment of bioenergetics. The neuroprotective effects of NIM811 were not due to drug-induced alterations in cerebral perfusion after ischemia. Activation of MPT appears to be an important process in ischemia/reperfusion injury and may be a therapeutic target.

HIV-1 neuropathogenesis: glial mechanisms revealed through substance abuse.

Neuronal dysfunction and degeneration are ultimately responsible for the neurocognitive impairment and dementia manifest in neuroAIDS. Despite overt neuronal pathology, HIV-1 does not directly infect neurons; rather, neuronal dysfunction or death is largely an indirect consequence of disrupted glial function and the cellular and viral toxins released by infected glia. A role for glia in HIV-1 neuropathogenesis is revealed in experimental and clinical studies examining substance abuse-HIV-1 interactions. Current evidence suggests that glia are direct targets of substance abuse and that glia contribute markedly to the accelerated neurodegeneration seen with substance abuse in HIV-1 infected individuals. Moreover, maladaptive neuroplastic responses to chronic drug abuse might create a latent susceptibility to CNS disorders such as HIV-1. In this review, we consider astroglial and microglial interactions and dysfunction in the pathogenesis of HIV-1 infection and examine how drug actions in glia contribute to neuroAIDS.

Human immunodeficiency virus-1 protein tat and methamphetamine interactions.

The human immunodeficiency virus-1 (HIV-1) affects the central nervous system (CNS) in approximately 30% of infected individuals and basal ganglia structures seem to be most affected. The HIV-1-transactivating protein, Tat, has been suggested to be pathogenically relevant in HIV-1-induced neuronal injury. The abuse of methamphetamine (METH), which is great among this patient population, also affects the basal ganglia, causing degeneration of dopaminergic terminals. In previous studies, we demonstrated that coexposure to these two toxins caused a synergistic loss of striatal dopamine and binding to the dopamine transporter (DAT), suggesting a loss of dopamine terminals. Because the loss of dopamine and DAT, however, do not necessarily reflect dopamine terminal degeneration, we have used silver staining and TH immunohistochemistry to further examine this issue. We have also examined the glial reaction using GFAP as a marker of astrocyte activation and OX-42 as a marker of activated microglia. Lastly, we have begun to explore the mechanism of synergy by investigating the role that the cytokine TNF-alpha might play in Tat + METH synergy. Our data indicate that the synergistic loss of dopamine is likely the result of dopamine terminal degeneration. This injury is not a direct result of the number of activated glia but does involve TNF-alpha.

Inhibition of tumor necrosis factor-alpha signaling prevents human immunodeficiency virus-1 protein Tat and methamphetamine interaction.

Our previous studies demonstrated that the psychostimulant methamphetamine (MA) and the human immunodeficiency virus-1 (HIV-1) protein Tat interacted to cause enhanced dopaminergic neurotoxicity. The present study examined whether tumor necrosis factor-alpha (TNF-alpha) mediates the interaction between Tat and MA. In Sprague-Dawley rats, injections of Tat caused a small but significant increase in striatal TNF-alpha level, whereas MA resulted in no change. The increase in TNF-alpha induced by Tat + MA was not significantly different from that induced by Tat alone. Temporal analysis of TNF-alpha levels revealed a 50-fold increase 4 h after Tat administration. In C57BL/6 mice, Tat + MA induced a 50% decline in striatal dopamine levels, which was significantly attenuated in mice lacking both receptors for TNF-alpha. TNF-alpha synthesis inhibitors significantly attenuated Tat + MA neurotoxicity in hippocampal neuronal culture. The results suggest that Tat-induced elevation of TNF-alpha may predispose the dopaminergic terminals to subsequent damage by MA.

Involvement of cytokines in human immunodeficiency virus-1 protein Tat and methamphetamine interactions in the striatum.

Human immunodeficiency virus-1 (HIV-1) infection of the brain causes elevation in pro-inflammatory cytokines and inflammatory changes in the striatum. HIV-1-infected individuals who also abuse drugs including the psychostimulant methamphetamine (MA) develop more severe encephalitis and neuronal damage compared to HIV-1-infected patients who do not abuse drugs. In previous studies, we demonstrated that the HIV-1 protein Tat and MA interacted to cause enhanced loss of dopamine in the rat striatum via the destruction of dopaminergic terminals. Since both Tat and MA activate glia and induce cytokine production, we investigated the role of cytokines in the synergistic neurotoxicity induced by Tat and MA using cytokine arrays. Significant increases in monocyte chemotactic protein (MCP-1), interleukin-1 alpha (IL-1alpha) and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels were noted 4 h following Tat + MA treatment compared to saline, Tat or MA. MCP-1 and TIMP-1 levels remained elevated 16 h after Tat + MA compared to saline or MA but were not different from the Tat-treated group at this time point. Weak, but significant elevations in cytokine-induced neutrophil chemoattractant-3 (CINC-3), ciliary neurotrophic factor (CNTF) and macrophage inflammatory protein-3 alpha (MIP-3alpha) were also noted with Tat + MA. The interaction of Tat and MA was prevented in mice genetically deficient in MCP-1 with a consequent attenuation of Tat + MA neurotoxicity. Our findings suggest that HIV-1 infection with concurrent drug abuse might profoundly increase chemokine levels in the striatum resulting in enhanced damage to the dopaminergic system.

NeuroAIDS, drug abuse, and inflammation: building collaborative research activities.

Neurological complications of human immunodeficiency virus (HIV) infection are a public health problem despite the availability of active antiretroviral therapies. The neuropathogenesis of HIV infection revolves around a complex cascade of events that include viral infection and glial immune activation, monocyte-macrophage brain infiltration, and secretion of a host of viral and cellular inflammatory and neurotoxic molecules. Although there is evidence that HIV-infected drug abusers experience more severe neurological disease, the biological basis for this finding is unknown. A scientific workshop organized by the National Institute on Drug Abuse (NIDA) was held on March 23-24, 2006 to address this question. The goal of the meeting was to bring together basic science and clinical researchers who are experts in NeuroAIDS, glial immunity, drugs of abuse, and/or pharmacology in order to find new approaches to understanding interactions between drug abuse and neuroAIDS. The format of the meeting was designed to stimulate open discussion and forge new multidisciplinary research collaborations. This report includes transcripts of active discussions and short presentations from invited participants. The presentations were separated into sections that included: Glial Biology, Inflammation, and HIV; Pharmacology, Neurotoxicology, and Neuroprotection; NeuroAIDS and Virology; and Virus-Drug and Immune-Drug Interactions. Research priorities were identified. Additional information about this meeting is available through links from the NIDA AIDS Research Program website ( http://www.nida.nih.gov/about/organization/arp/arp-websites.htm ).

The uncoupling agent 2,4-dinitrophenol improves mitochondrial homeostasis following striatal quinolinic acid injections.

It is now generally accepted that excitotoxic cell death involves bioenergetic failure resulting from the cycling of Ca2+ and the generation of reactive oxygen species (ROS) by mitochondria. Both Ca2+ cycling and ROS formation by mitochondria are dependent on the mitochondrial membrane potential (Deltapsi(m)) that results from the proton gradient that is generated across the inner membrane. Mitochondrial uncoupling refers to a condition in which protons cross the inner membrane back into the matrix while bypassing the ATP synthase. As a consequence of this "short-circuit," there is a reduction in Deltapsi(m). We have previously demonstrated that animals treated with the classic uncoupling agent 2,4-dinitrophenol (DNP) show significant protection against brain damage following striatal injections of the NMDA agonist quinolinic acid (QA). In an effort to elucidate the mechanism of neuroprotection, we have assessed the effects of DNP on several parameters of mitochondrial function caused by QA. The results presented herein demonstrate that treatment with DNP attenuates QA-induced increases in mitochondrial Ca2+ levels and ROS formation and also improves mitochondrial respiration. Our findings indicate that DNP may confer protection against acute brain injury involving excitotoxic pathways by mechanisms that maintain mitochondrial function.

Enhanced toxicity to the catecholamine tyramine in polyglutamine transfected SH-SY5Y cells.

Huntington's disease (HD) is a progressive neurodegenerative disorder, of which the pathogenesis is not completely understood. In patients with Huntington's disease, there is a mutation in the gene encoding the protein huntingtin, which results in an expanded polyglutamine sequence leading to degeneration of the basal ganglia. There is mounting evidence that metabolism of the transmitter dopamine by the enzyme monoamine oxidase may contribute to striatal damage in mitochondrial toxin-induced models of HD. In this study, we have examined the role of the catecholamine tyramine in neural SH-SY5Y cells transfected with normal and expanded polyglutamine repeat numbers. Our findings demonstrate that cells containing a pathological number of polyglutamines are more sensitive to tyramine than cells with a non-pathological number. Tyramine-induced cell death was attenuated by MAO inhibitors as well as with catalase and the iron chelator deferoxamine, suggesting that H202 might mediate the observed toxicity. These observations support the notion that the metabolism of dopamine plays a role in neuron death in Huntington's disease.

The mitochondrial uncoupler 2,4-dinitrophenol attenuates tissue damage and improves mitochondrial homeostasis following transient focal cerebral ischemia.

Ischemic stroke is caused by acute neuronal degeneration provoked by interruption of cerebral blood flow. Although the mechanisms contributing to ischemic neuronal degeneration are myriad, mitochondrial dysfunction is now recognized as a pivotal event that can lead to either necrotic or apoptotic neuronal death. Lack of suitable 'upstream' targets to prevent loss of mitochondrial homeostasis has, so far, restricted the development of mechanistically based interventions to promote neuronal survival. Here, we show that the uncoupling agent 2,4 dinitrophenol (DNP) reduces infarct volume approximately 40% in a model of focal ischemia-reperfusion injury in the rat brain. The mechanism of protection involves an early decrease in mitochondrial reactive oxygen species formation and calcium uptake leading to improved mitochondrial function and a reduction in the release of cytochrome c into the cytoplasm. The observed effects of DNP were not associated with enhanced cerebral perfusion. These findings indicate that compounds with uncoupling properties may confer neuroprotection through a mechanism involving stabilization of mitochondrial function.

Proteomic analysis of protein expression and oxidative modification in r6/2 transgenic mice: a model of Huntington disease.

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by motor, psychiatric, and cognitive symptoms. The genetic defect responsible for the onset of the disease, expansion of CAG repeats in exon 1 of the gene that codes for huntingtin on chromosome 4, has been unambiguously identified. On the other hand, the mechanisms by which the mutation causes the disease are not completely understood yet. However, defects in energy metabolism of affected cells may cause oxidative damage, which has been proposed as one of the underlying molecular mechanisms that participate in the etiology of the disease. In our effort to investigate the extent of oxidative damage occurring at the protein level, we used a parallel proteomic approach to identify proteins potentially involved in processes upstream or downstream of the disease-causing huntingtin in a well established HD mouse model (R6/2 transgenic mice). We have demonstrated that the expression levels of dihydrolipoamide S-succinyltransferase and aspartate aminotransferase increase consistently over the course of disease (10-week-old mice). In contrast, pyruvate dehydrogenase expression levels were found to be decreased in 10-week-old HD transgenic mice compared with young (4-week-old) mice. Our experimental approach also led to the identification of oxidatively modified proteins. Six proteins were found to be significantly oxidized in old R6/2 transgenic mice compared with either young transgenic mice or non-transgenic mice. These proteins are alpha-enolase, gamma-enolase (neuron-specific enolase), aconitase, the voltage-dependent anion channel 1, heat shock protein 90, and creatine kinase. Because oxidative damage has proved to play an important role in the pathogenesis and the progression of Huntington disease, our results for the first time identify specific oxidatively modified proteins that potentially contribute to the pathogenesis of Huntington disease.

Mitochondrial uncoupling as a potential therapeutic target in acute central nervous system injury.

Mitochondrial dysfunction, resulting from the disruption of calcium homeostasis and the generation of toxic reactive oxygen species, is a central process leading to neuronal injury and death following acute CNS insults. Interventions aimed at preventing disturbances in mitochondrial function have therefore become targets of intense investigation. Mitochondrial uncoupling is a condition in which electron transport is disconnected from the production of ATP. As a consequence, there is a decrease in the mitochondrial membrane potential, which can temporarily decrease calcium influx and attenuate free radical formation. The potential use of pharmacological agents with uncoupling properties may provide a novel therapeutic approach for the treatment of acute neuronal injury.

Striatal damage and oxidative stress induced by the mitochondrial toxin malonate are reduced in clorgyline-treated rats and MAO-A deficient mice.

Intrastriatal administration of the succinate dehydrogenase (SDH) inhibitor malonate produces neuronal injury by a "secondary excitotoxic" mechanism involving the generation of reactive oxygen species (ROS). Recent evidence indicates dopamine may contribute to malonate-induced striatal neurodegeneration; infusion of malonate causes a pronounced increase in extracellular dopamine and dopamine deafferentation attenuates malonate toxicity. Inhibition of the catabolic enzyme monoamine oxidase (MAO) also attenuates striatal lesions induced by malonate. In addition to forming 3,4-dihydroxyphenylacetic acid, metabolism of dopamine by MAO generates H2O2, suggesting that dopamine metabolism may be a source of ROS in malonate toxicity. There are two isoforms of MAO, MAO-A and MAO-B. In this study, we have investigated the role of each isozyme in malonate-induced striatal injury using both pharmacological and genetic approaches. In rats treated with either of the specific MAO-A or -B inhibitors, clorgyline or deprenyl, respectively, malonate lesion volumes were reduced by 30% compared to controls. In knock-out mice lacking the MAO-A isoform, malonate-induced lesions were reduced by 50% and protein carbonyls, an index ROS formation, were reduced by 11%, compared to wild-type animals. In contrast, mice deficient in MAO-B showed highly variable susceptibility to malonate toxicity precluding us from determining the precise role of MAO-B in this form of brain damage. These findings indicate that normal levels of MAO-A participate in expression of malonate toxicity by a mechanism involving oxidative stress.