Jyothisri Kondapalli

Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1

Parkinson’s disease is a pervasive, ageing-related neurodegenerative disease the cardinal motor symptoms of which reflect the loss of a small group of neurons, the dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial oxidant stress is widely viewed as being responsible for this loss, but why these particular neurons should be stressed is a mystery. Here we show, using transgenic mice that expressed a redox-sensitive variant of green fluorescent protein targeted to the mitochondrial matrix, that the engagement of plasma membrane L-type calcium channels during normal autonomous pacemaking created an oxidant stress that was specific to vulnerable SNc dopaminergic neurons. The oxidant stress engaged defences that induced transient, mild mitochondrial depolarization or uncoupling. The mild uncoupling was not affected by deletion of cyclophilin D, which is a component of the permeability transition pore, but was attenuated by genipin and purine nucleotides, which are antagonists of cloned uncoupling proteins. Knocking out DJ-1 (also known as PARK7 in humans and Park7 in mice), which is a gene associated with an early-onset form of Parkinson’s disease, downregulated the expression of two uncoupling proteins (UCP4 (SLC25A27) and UCP5 (SLC25A14)), compromised calcium-induced uncoupling and increased oxidation of matrix proteins specifically in SNc dopaminergic neurons. Because drugs approved for human use can antagonize calcium entry through L-type channels, these results point to a novel neuroprotective strategy for both idiopathic and familial forms of Parkinson’s disease.

Mitochondrial oxidant stress triggers cell death in simulated ischemia-reperfusion.

To clarify the relationship between reactive oxygen species (ROS) and cell death during ischemia-reperfusion (I/R), we studied cell death mechanisms in a cellular model of I/R. Oxidant stress during simulated ischemia was detected in the mitochondrial matrix using mito-roGFP, a ratiometric redox sensor, and by Mito-Sox Red oxidation. Reperfusion-induced death was attenuated by over-expression of Mn-superoxide dismutase (Mn-SOD) or mitochondrial phospholipid hydroperoxide glutathione peroxidase (mito-PHGPx), but not by catalase, mitochondria-targeted catalase, or Cu,Zn-SOD. Protection was also conferred by chemically distinct antioxidant compounds, and mito-roGFP oxidation was attenuated by NAC, or by scavenging of residual O(2) during the ischemia (anoxic ischemia). Mitochondrial permeability transition pore (mPTP) oscillation/opening was monitored by real-time imaging of mitochondrial calcein fluorescence. Oxidant stress caused release of calcein to the cytosol during ischemia, a response that was inhibited by chemically diverse antioxidants, anoxia, or over-expression of Mn-SOD or mito-PHGPx. These findings suggest that mitochondrial oxidant stress causes oscillation of the mPTP prior to reperfusion. Cytochrome c release from mitochondria to the cytosol was not detected until after reperfusion, and was inhibited by anoxic ischemia or antioxidant administration during ischemia. Although DNA fragmentation was detected after I/R, no evidence of Bax activation was detected. Over-expression of the anti-apoptotic protein Bcl-X(L) in cardiomyocytes did not confer protection against I/R-induced cell death. Moreover, murine embryonic fibroblasts with genetic depletion of Bax and Bak, or over-expression of Bcl-X(L), failed to show protection against I/R. These findings indicate that mitochondrial ROS during ischemia triggers mPTP activation, mitochondrial depolarization, and cell death during reperfusion through a Bax/Bak-independent cell death pathway. Therefore, mitochondrial apoptosis appears to represent a redundant death pathway in this model of simulated I/R. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.

Menadione triggers cell death through ROS-dependent mechanisms involving PARP activation without requiring apoptosis.

Low levels of reactive oxygen species (ROS) can function as redox-active signaling messengers, whereas high levels of ROS induce cellular damage. Menadione generates ROS through redox cycling, and high concentrations trigger cell death. Previous work suggests that menadione triggers cytochrome c release from mitochondria, whereas other studies implicate the activation of the mitochondrial permeability transition pore as the mediator of cell death. We investigated menadione-induced cell death in genetically modified cells lacking specific death-associated proteins. In cardiomyocytes, oxidant stress was assessed using the redox sensor RoGFP, expressed in the cytosol or the mitochondrial matrix. Menadione elicited rapid oxidation in both compartments, whereas it decreased mitochondrial potential and triggered cytochrome c redistribution to the cytosol. Cell death was attenuated by N-acetylcysteine and exogenous glutathione or by overexpression of cytosolic or mitochondria-targeted catalase. By contrast, no protection was observed in cells overexpressing Cu,Zn-SOD or Mn-SOD. Overexpression of antiapoptotic Bcl-X(L) protected against staurosporine-induced cell death, but it failed to confer protection against menadione. Genetic deletion of Bax and Bak, cytochrome c, cyclophilin D, or caspase-9 conferred no protection against menadione-induced cell death. However, cells lacking PARP-1 showed a significant decrease in menadione-induced cell death. Thus, menadione induces cell death through the generation of oxidant stress in multiple subcellular compartments, yet cytochrome c, Bax/Bak, caspase-9, and cyclophilin D are dispensable for cell death in this model. These studies suggest that multiple redundant cell death pathways are activated by menadione, but that PARP plays an essential role in mediating each of them.

Calcium entry induces mitochondrial oxidant stress in vagal neurons at risk in Parkinson's disease

Mitochondrial oxidant stress is widely viewed as being critical to pathogenesis in Parkinsons disease. But the origins of this stress are poorly defined. One possibility is that it arises from the metabolic demands associated with regenerative activity. To test this hypothesis, we characterized neurons in the dorsal motor nucleus of the vagus (DMV), a population of cholinergic neurons that show signs of pathology in the early stages of Parkinsons disease, in mouse brain slices. DMV neurons were slow, autonomous pacemakers with broad spikes, leading to calcium entry that was weakly buffered. Using a transgenic mouse expressing a redox-sensitive optical probe targeted to the mitochondrial matrix, we found that calcium entry during pacemaking created a basal mitochondrial oxidant stress. Knocking out DJ-1 (also known as PARK7), a gene associated with early-onset Parkinsons disease, exacerbated this stress. These results point to a common mechanism underlying mitochondrial oxidant stress in Parkinsons disease and a therapeutic strategy to ameliorate it.

Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis

8-Oxoguanine DNA glycosylase (Ogg1) repairs 8-oxo-7,8-dihydroxyguanine (8-oxoG), one of the most abundant DNA adducts caused by oxidative stress. In the mitochondria, Ogg1 is thought to prevent activation of the intrinsic apoptotic pathway in response to oxidative stress by augmenting DNA repair. However, the predominance of the beta-Ogg1 isoform, which lacks 8-oxoG DNA glycosylase activity, suggests that mitochondrial Ogg1 functions in a role independent of DNA repair. We report here that overexpression of mitochondria-targeted human alpha-hOgg1 (mt-hOgg1) in human lung adenocarcinoma cells with some alveolar epithelial cell characteristics (A549 cells) prevents oxidant-induced mitochondrial dysfunction and apoptosis by preserving mitochondrial aconitase. Importantly, mitochondrial alpha-hOgg1 mutants lacking 8-oxoG DNA repair activity were as effective as wild-type mt-hOgg1 in preventing oxidant-induced caspase-9 activation, reductions in mitochondrial aconitase, and apoptosis, suggesting that the protective effects of mt-hOgg1 occur independent of DNA repair. Notably, wild-type and mutant mt-hOgg1 coprecipitate with mitochondrial aconitase. Furthermore, overexpression of mitochondrial aconitase abolishes oxidant-induced apoptosis whereas hOgg1 silencing using shRNA reduces mitochondrial aconitase and augments apoptosis. These findings suggest a novel mechanism that mt-hOgg1 acts as a mitochondrial aconitase chaperone protein to prevent oxidant-mediated mitochondrial dysfunction and apoptosis that might be important in the molecular events underlying oxidant-induced toxicity.

Mitochondrial oxidant stress in locus coeruleus is regulated by activity and nitric oxide synthase

Loss of noradrenergic locus coeruleus (LC) neurons is a prominent feature of aging-related neurodegenerative diseases, such as Parkinsons disease (PD). The basis of this vulnerability is not understood. To explore possible physiological determinants, we studied LC neurons using electrophysiological and optical approaches in ex vivo mouse brain slices. We found that autonomous activity in LC neurons was accompanied by oscillations in dendritic Ca2+ concentration that were attributable to the opening of L-type Ca2+ channels. This oscillation elevated mitochondrial oxidant stress and was attenuated by inhibition of nitric oxide synthase. The relationship between activity and stress was malleable, as arousal and carbon dioxide increased the spike rate but differentially affected mitochondrial oxidant stress. Oxidant stress was also increased in an animal model of PD. Thus, our results point to activity-dependent Ca2+ entry and a resulting mitochondrial oxidant stress as factors contributing to the vulnerability of LC neurons.

The indirect pathway of the nucleus accumbens shell amplifies neuropathic pain

We examined adaptations in nucleus accumbens (NAc) neurons in mouse and rat peripheral nerve injury models of neuropathic pain. Injury selectively increased excitability of NAc shell indirect pathway spiny projection neurons (iSPNs) and altered their synaptic connectivity. Moreover, injury-induced tactile allodynia was reversed by inhibiting and exacerbated by exciting iSPNs, indicating that they not only participated in the central representation of pain, but gated activity in ascending nociceptive pathways.

Sirtuin 3 deficiency does not augment hypoxia-induced pulmonary hypertension.

Alveolar hypoxia elicits increases in mitochondrial reactive oxygen species (ROS) signaling in pulmonary arterial (PA) smooth muscle cells (PASMCs), triggering hypoxic pulmonary vasoconstriction. Mice deficient in sirtuin (Sirt) 3, a nicotinamide adenine dinucleotide-dependent mitochondrial deacetylase, demonstrate enhanced left ventricular hypertrophy after aortic banding, whereas cells from these mice reportedly exhibit augmented hypoxia-induced ROS signaling and hypoxia-inducible factor (HIF)-1 activation. We therefore tested whether deletion of Sirt3 would augment hypoxia-induced ROS signaling in PASMCs, thereby exacerbating the development of pulmonary hypertension (PH) and right ventricular hypertrophy. In PASMCs from Sirt3 knockout (Sirt3(-/-)) mice in the C57BL/6 background, we observed that acute hypoxia (1.5% O2; 30 min)-induced changes in ROS signaling, detected using targeted redox-sensitive, ratiometric fluorescent protein sensors (roGFP) in the mitochondrial matrix, intermembrane space, and the cytosol, were indistinguishable from Sirt3(+/+) cells. Acute hypoxia-induced cytosolic calcium signaling in Sirt3(-/-) PASMCs was also indistinguishable from Sirt3(+/+) cells. During sustained hypoxia (1.5% O2; 16 h), Sirt3 deletion augmented mitochondrial matrix oxidant stress, but this did not correspond to an augmentation of intermembrane space or cytosolic oxidant signaling. Sirt3 deletion did not affect HIF-1α stabilization under normoxia, nor did it augment HIF-1α stabilization during sustained hypoxia (1.5% O2; 4 h). Sirt3(-/-) mice housed in chronic hypoxia (10% O2; 30 d) developed PH, PA wall remodeling, and right ventricular hypertrophy that was indistinguishable from Sirt3(+/+) littermates. Thus, Sirt3 deletion does not augment hypoxia-induced ROS signaling or its consequences in the cytosol of PASMCs, or the development of PH. These findings suggest that Sirt3 responses may be cell type specific, or restricted to certain genetic backgrounds.

α-Synuclein-Dependent Calcium Entry Underlies Differential Sensitivity of Cultured SN and VTA Dopaminergic Neurons to a Parkinsonian Neurotoxin

Abstract Parkinson’s disease (PD) is a debilitating neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra (SN). Although mitochondrial dysfunction and dysregulated α-synuclein (aSyn) expression are postulated to play a role in PD pathogenesis, it is still debated why neurons of the SN are targeted while neighboring dopaminergic neurons of the ventral tegmental area (VTA) are spared. Using electrochemical and imaging approaches, we investigated metabolic changes in cultured primary mouse midbrain dopaminergic neurons exposed to a parkinsonian neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). We demonstrate that the higher level of neurotoxicity in SN than VTA neurons was due to SN neuron-specific toxin-induced increase in cytosolic dopamine (DA) and Ca2+, followed by an elevation of mitochondrial Ca2+, activation of nitric oxide synthase (NOS), and mitochondrial oxidation. The increase in cytosolic Ca2+ was not caused by MPP+-induced oxidative stress, but rather depended on the activity of both L-type calcium channels and aSyn expression, suggesting that these two established pathogenic factors in PD act in concert.

Systemic isradipine treatment diminishes calcium-dependent mitochondrial oxidant stress

The ability of the Cav1 channel inhibitor isradipine to slow the loss of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons and the progression of Parkinson’s disease (PD) is being tested in a phase 3 human clinical trial. But it is unclear whether and how chronic isradipine treatment will benefit SNc DA neurons in vivo. To pursue this question, isradipine was given systemically to mice at doses that achieved low nanomolar concentrations in plasma, near those achieved in patients. This treatment diminished cytosolic Ca2+ oscillations in SNc DA neurons without altering autonomous spiking or expression of Ca2+ channels, an effect mimicked by selectively knocking down expression of Cav1.3 channel subunits. Treatment also lowered mitochondrial oxidant stress, reduced a high basal rate of mitophagy, and normalized mitochondrial mass — demonstrating that Cav1 channels drive mitochondrial oxidant stress and turnover in vivo. Thus, chronic isradipine treatment remodeled SNc DA neurons in a way that should not only diminish their vulnerability to mitochondrial challenges, but to autophagic stress as well.