Samantha Giordano

Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling

Reactive oxygen and nitrogen species change cellular responses through diverse mechanisms that are now being defined. At low levels, they are signalling molecules, and at high levels, they damage organelles, particularly the mitochondria. Oxidative damage and the associated mitochondrial dysfunction may result in energy depletion, accumulation of cytotoxic mediators and cell death. Understanding the interface between stress adaptation and cell death then is important for understanding redox biology and disease pathogenesis. Recent studies have found that one major sensor of redox signalling at this switch in cellular responses is autophagy. Autophagic activities are mediated by a complex molecular machinery including more than 30 Atg (AuTophaGy-related) proteins and 50 lysosomal hydrolases. Autophagosomes form membrane structures, sequester damaged, oxidized or dysfunctional intracellular components and organelles, and direct them to the lysosomes for degradation. This autophagic process is the sole known mechanism for mitochondrial turnover. It has been speculated that dysfunction of autophagy may result in abnormal mitochondrial function and oxidative or nitrative stress. Emerging investigations have provided new understanding of how autophagy of mitochondria (also known as mitophagy) is controlled, and the impact of autophagic dysfunction on cellular oxidative stress. The present review highlights recent studies on redox signalling in the regulation of autophagy, in the context of the basic mechanisms of mitophagy. Furthermore, we discuss the impact of autophagy on mitochondrial function and accumulation of reactive species. This is particularly relevant to degenerative diseases in which oxidative stress occurs over time, and dysfunction in both the mitochondrial and autophagic pathways play a role.

Assessing bioenergetic function in response to oxidative stress by metabolic profiling

It is now clear that mitochondria are an important target for oxidative stress in a broad range of pathologies, including cardiovascular disease, diabetes, neurodegeneration, and cancer. Methods for assessing the impact of reactive species on isolated mitochondria are well established but constrained by the need for large amounts of material to prepare intact mitochondria for polarographic measurements. With the availability of high-resolution polarography and fluorescence techniques for the measurement of oxygen concentration in solution, measurements of mitochondrial function in intact cells can be made. Recently, the development of extracellular flux methods to monitor changes in oxygen concentration and pH in cultures of adherent cells in multiple-sample wells simultaneously has greatly enhanced the ability to measure bioenergetic function in response to oxidative stress. Here we describe these methods in detail using representative cell types from renal, cardiovascular, nervous, and tumorigenic model systems while illustrating the application of three protocols to analyze the bioenergetic response of cells to oxidative stress.

Autophagy as an essential cellular antioxidant pathway in neurodegenerative disease

Oxidative stress including DNA damage, increased lipid and protein oxidation, are important features of aging and neurodegeneration suggesting that endogenous antioxidant protective pathways are inadequate or overwhelmed. Importantly, oxidative protein damage contributes to age-dependent accumulation of dysfunctional mitochondria or protein aggregates. In addition, environmental toxins such as rotenone and paraquat, which are risk factors for the pathogenesis of neurodegenerative diseases, also promote protein oxidation. The obvious approach of supplementing the primary antioxidant systems designed to suppress the initiation of oxidative stress has been tested in animal models and positive results were obtained. However, these findings have not been effectively translated to treating human patients, and clinical trials for antioxidant therapies using radical scavenging molecules such as α-tocopherol, ascorbate and coenzyme Q have met with limited success, highlighting several limitations to this approach. These could include: (1) radical scavenging antioxidants cannot reverse established damage to proteins and organelles; (2) radical scavenging antioxidants are oxidant specific, and can only be effective if the specific mechanism for neurodegeneration involves the reactive species to which they are targeted and (3) since reactive species play an important role in physiological signaling, suppression of endogenous oxidants maybe deleterious. Therefore, alternative approaches that can circumvent these limitations are needed. While not previously considered an antioxidant system we propose that the autophagy-lysosomal activities, may serve this essential function in neurodegenerative diseases by removing damaged or dysfunctional proteins and organelles.

Differentiation of SH-SY5Y cells to a neuronal phenotype changes cellular bioenergetics and the response to oxidative stress

Cell differentiation is associated with changes in metabolism and function. Understanding these changes during differentiation is important in the context of stem cell research, cancer, and neurodegenerative diseases. An early event in neurodegenerative diseases is the alteration of mitochondrial function and increased oxidative stress. Studies using both undifferentiated and differentiated SH-SY5Y neuroblastoma cells have shown distinct responses to cellular stressors; however, the mechanisms remain unclear. We hypothesized that because the regulation of glycolysis and oxidative phosphorylation is modulated during cellular differentiation, this would change bioenergetic function and the response to oxidative stress. To test this, we used retinoic acid (RA) to induce differentiation of SH-SY5Y cells and assessed changes in cellular bioenergetics using extracellular flux analysis. After exposure to RA, the SH-SY5Y cells had an increased mitochondrial membrane potential, without changing mitochondrial number. Differentiated cells exhibited greater stimulation of mitochondrial respiration with uncoupling and an increased bioenergetic reserve capacity. The increased reserve capacity in the differentiated cells was suppressed by the inhibitor of glycolysis 2-deoxy-d-glucose. Furthermore, we found that differentiated cells were substantially more resistant to cytotoxicity and mitochondrial dysfunction induced by the reactive lipid species 4-hydroxynonenal or the reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone. We then analyzed the levels of selected mitochondrial proteins and found an increase in complex IV subunits, which we propose contributes to the increase in reserve capacity in the differentiated cells. Furthermore, we found an increase in MnSOD that could, at least in part, account for the increased resistance to oxidative stress. Our findings suggest that profound changes in mitochondrial metabolism and antioxidant defenses occur upon differentiation of neuroblastoma cells to a neuron-like phenotype.

Distinct effects of rotenone, 1-methyl-4-phenylpyridinium and 6-hydroxydopamine on cellular bioenergetics and cell death.

Parkinson’s disease is characterized by dopaminergic neurodegeneration and is associated with mitochondrial dysfunction. The bioenergetic susceptibility of dopaminergic neurons to toxins which induce Parkinson’s like syndromes in animal models is then of particular interest. For example, rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP+), and 6-hydroxydopamine (6-OHDA), have been shown to induce dopaminergic cell death in vivo and in vitro. Exposure of animals to these compounds induce a range of responses characteristics of Parkinson’s disease, including dopaminergic cell death, and Reactive Oxygen Species (ROS) production. Here we test the hypothesis that cellular bioenergetic dysfunction caused by these compounds correlates with induction of cell death in differentiated dopaminergic neuroblastoma SH-SY5Y cells. At increasing doses, rotenone induced significant cell death accompanied with caspase 3 activation. At these concentrations, rotenone had an immediate inhibition of mitochondrial basal oxygen consumption rate (OCR) concomitant with a decrease of ATP-linked OCR and reserve capacity, as well as a stimulation of glycolysis. MPP+ exhibited a different behavior with less pronounced cell death at doses that nearly eliminated basal and ATP-linked OCR. Interestingly, MPP+, unlike rotenone, stimulated bioenergetic reserve capacity. The effects of 6-OHDA on bioenergetic function was markedly less than the effects of rotenone or MPP+ at cytotoxic doses, suggesting a mechanism largely independent of bioenergetic dysfunction. These studies suggest that these dopaminergic neurotoxins induce cell death through distinct mechanisms and differential effects on cellular bioenergetics.

Bioenergetic adaptation in response to autophagy regulators during rotenone exposure.

Parkinsons disease is the second most common neurodegenerative disorder with both mitochondrial dysfunction and insufficient autophagy playing a key role in its pathogenesis. Among the risk factors, exposure to the environmental neurotoxin rotenone increases the probability of developing Parkinsons disease. We previously reported that in differentiated SH‐SY5Y cells, rotenone‐induced cell death is directly related to inhibition of mitochondrial function. How rotenone at nM concentrations inhibits mitochondrial function, and whether it can engage the autophagy pathway necessary to remove damaged proteins and organelles, is unknown. We tested the hypothesis that autophagy plays a protective role against rotenone toxicity in primary neurons. We found that rotenone (10–100 nM) immediately inhibited cellular bioenergetics. Concentrations that decreased mitochondrial function at 2 h, caused cell death at 24 h with an LD50 of 10 nM. Overall, autophagic flux was decreased by 10 nM rotenone at both 2 and 24 h, but surprisingly mitophagy, or autophagy of the mitochondria, was increased at 24 h, suggesting that a mitochondrial‐specific lysosomal degradation pathway may be activated. Up‐regulation of autophagy by rapamycin protected against cell death while inhibition of autophagy by 3‐methyladenine exacerbated cell death. Interestingly, while 3‐methyladenine exacerbated the rotenone‐dependent effects on bioenergetics, rapamycin did not prevent rotenone‐induced mitochondrial dysfunction, but caused reprogramming of mitochondrial substrate usage associated with both complex I and complex II activities. Taken together, these data demonstrate that autophagy can play a protective role in primary neuron survival in response to rotenone; moreover, surviving neurons exhibit bioenergetic adaptations to this metabolic stressor.

Estrogen and Cardiovascular Disease: Is Timing Everything?

Cardiovascular disease (CVD) is the leading cause of death for both women and men, accounting for 1 in every 3 deaths in the United States (US) [1]. Since the mid-1980s, CVD has killed more women than men each year. In 2011 alone, CVD caused about 10,000 more deaths in women than men [1]. CVD in women is a disease of aging, rarely occurring before the 6th decade of life [2]. It has been proposed that deprivation of ovarian hormones, specifically estrogen, in menopause is causally related to increased CVD risk in aging women [3]. Observational and randomized controlled trials showed differential effects of menopausal hormone therapy (MHT), which included estrogen, on CVD risk: observational studies almost uniformly suggested benefit, while randomized trials showed harm, particularly in elderly women who were many years post-menopausal [4,5]. Multiple hypotheses have been proposed to explain the differences between the unfavorable effects of MHT in randomized studies and the body of observational evidence supporting the beneficial effects of MHT. Prominent among these is the “timing hypothesis” which proposes that MHT started in the perimenopausal or early postmenopausal period is cardioprotective, whereas MHT begun late after menopause increases the risk of CVD [6]. In this review we discuss observational studies and randomized controlled trials of MHT in women and examine the age-dependent effects of estrogen in animal models of acute vascular injury, as well as the effects of estrogen on cellular (macrophage and vascular smooth muscle cell (VSMC)) responses to inflammatory stimuli in vitro. STUDIES OF MENOPAUSAL HORMONES IN WOMEN Observational Studies A meta-analysis of 25 observational studies showed a decreased relative risk of CVD and coronary heart disease (CHD) in postmenopausal women taking MHT compared to those who had never taken hormones (RR=0.70; CI, 0.65-0.75) [7]. The largest and most frequently cited of these, the Nurses’ Health Study (NHS), was a prospective observational study that enrolled 121,700 female nurses 30-55 years of age (Table 1) [8]. The 20 year follow up study of the 70,533 postmenopausal participants (accruing 808,825 person-years of follow up) demonstrated significantly fewer CVD events, non-fatal myocardial infarctions (MIs) or fatal CHD in women on MHT compared to MHT never-users after adjustment for age, body mass index (BMI), weight, diabetes history, hypertension, increased cholesterol, age of menopause, smoking, and family history (RR=0.61; 95% CI 0.52-0.71). Table 1 Studies of Menopausal Hormones The major limitation of the NHS and other observational studies are its non-randomized design [9]. Observational studies are inherently unable to control for selection bias and for confounding differences between treatment groups. Although all of the participants in the NHS were female nurses, there may have been significant differences in unknown or unmeasured variables between the groups due to the non-randomized design of the study. Further studies demonstrated that women who chose to use MHT were more often Caucasian, healthier, wealthier and had more access to healthcare than non-users [10, 11, 12]. To control for these confounding factors, randomized placebo controlled trials were needed to determine the efficacy of MHT as a preventive strategy for CVD.

High-Density Lipoprotein Regulation of Mitochondrial Function

Lipoproteins play a key role in regulating plasma and tissue levels of cholesterol. Apolipoprotein B (apoB)-containing lipoproteins, including chylomicrons, very-low density lipoprotein (VLDL) and low-density lipoprotein (LDL), serve as carriers of triglycerides and cholesterol and deliver these metabolites to peripheral tissues. In contrast, high-density lipoprotein (HDL) mediates Reverse Cholesterol Transport (RCT), a process by which excess cholesterol is removed from the periphery and taken up by hepatocytes where it is metabolized and excreted. Anti-atherogenic properties of HDL have been largely ascribed to apoA-I, the major protein component of the lipoprotein particle. The inflammatory response associated with atherosclerosis and ischemia-reperfusion (I-R) injury has been linked to the development of mitochondrial dysfunction. Under these conditions, an increase in reactive oxygen species (ROS) formation induces damage to mitochondrial structural elements, leading to a reduction in ATP synthesis and initiation of the apoptotic program. Recent studies suggest that HDL-associated apoA-I and lysosphingolipids attenuate mitochondrial injury by multiple mechanisms, including the suppression of ROS formation and induction of autophagy. Other apolipoproteins, however, present in lower abundance in HDL particles may exert opposing effects on mitochondrial function. This chapter examines the role of HDL-associated apolipoproteins and lipids in the regulation of mitochondrial function and bioenergetics.

Targeted delivery of human iPS-ECs overexpressing IL-8 receptors inhibits neointimal and inflammatory responses to vascular injury in the rat

Interleukin-8 (IL8) is highly expressed by injured arteries in a variety of diseases and is a chemoattractant for neutrophils which express IL8 receptors IL8RA and RB (IL8RA/B) on their membranes. Neutrophils interact with the damaged endothelium and initiate an inflammatory cascade at the site of injury. We have generated a novel translational targeted cell therapy for acute vascular injury using adenoviral vectors to overexpress IL8RA/B and green fluorescent protein (GFP) on the surface of endothelial cells (ECs) derived from human induced pluripotent stem cells (HiPS-IL8RA/B-ECs). We hypothesize that HiPS-IL8RA/B-ECs transfused intravenously into rats with balloon injury of the carotid artery will target to the injured site and compete with neutrophils, thus inhibiting inflammation and neointima formation. Young adult male Sprague-Dawley rats underwent balloon injury of the right carotid artery and received intravenous transfusion of saline vehicle, 1.5 × 10(6) HiPS-ECs, 1.5 × 10(6) HiPS-Null-ECs, or 1.5 × 10(6) HiPS-IL8RA/B-ECs immediately after endoluminal injury. Tissue distribution of HiPS-IL8RA/B-ECs was analyzed by a novel GFP DNA qPCR method. Cytokine and chemokine expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 h postinjury by ELISA and immunohistochemistry, respectively. Neointimal, medial areas, and reendothelialization were measured 14 days postinjury. HiPS-IL8RA/B-ECs homed to injured arteries, inhibited inflammatory mediator expression and inflammatory cell infiltration, accelerated reendothelialization, and attenuated neointima formation after endoluminal injury while control HiPS-ECs and HiPS-Null-ECs did not. HiPS-IL8RA/B-ECs transfused into rats with endoluminal carotid artery injury target to the injured artery and provide a novel strategy to treat vascular injury.

High-density lipoprotein, mitochondrial dysfunction and cell survival mechanisms

Ischemic injury is associated with acute myocardial infarction, percutaneous coronary intervention, coronary artery bypass grafting and open heart surgery. The timely re-establishment of blood flow is critical in order to minimize cardiac complications. Reperfusion after a prolonged ischemic period, however, can induce severe cardiomyocyte dysfunction with mitochondria serving as a major target of ischemia/reperfusion (I/R) injury. An increase in the formation of reactive oxygen species (ROS) induces damage to mitochondrial respiratory complexes leading to uncoupling of oxidative phosphorylation. Mitochondrial membrane perturbations also contribute to calcium overload, opening of the mitochondrial permeability transition pore (mPTP) and the release of apoptotic mediators into the cytoplasm. Clinical and experimental studies show that ischemic preconditioning (ICPRE) and postconditioning (ICPOST) attenuate mitochondrial injury and improve cardiac function in the context of I/R injury. This is achieved by the activation of two principal cell survival cascades: 1) the Reperfusion Injury Salvage Kinase (RISK) pathway; and 2) the Survivor Activating Factor Enhancement (SAFE) pathway. Recent data suggest that high density lipoprotein (HDL) mimics the effects of conditioning protocols and attenuates myocardial I/R injury via activation of the RISK and SAFE signaling cascades. In this review, we discuss the roles of apolipoproteinA-I (apoA-I), the major protein constituent of HDL, and sphingosine 1-phosphate (S1P), a lysosphingolipid associated with small, dense HDL particles as mediators of cardiomyocyte survival. Both apoA-I and S1P exert an infarct-sparing effect by preventing ROS-dependent injury and inhibiting the opening of the mPTP.