Inhibition of autophagy with bafilomycin and chloroquine decreases mitochondrial quality and bioenergetic function in primary neurons.

Autophagy is an important cell recycling program responsible for the clearance of damaged or long-lived proteins and organelles. Pharmacological modulators of this pathway have been extensively utilized in a wide range of basic research and pre-clinical studies. Bafilomycin A1 and chloroquine are commonly used compounds that inhibit autophagy by targeting the lysosomes but through distinct mechanisms. Since it is now clear that mitochondrial quality control, particularly in neurons, is dependent on autophagy, it is important to determine whether these compounds modify cellular bioenergetics. To address this, we cultured primary rat cortical neurons from E18 embryos and used the Seahorse XF96 analyzer and a targeted metabolomics approach to measure the effects of bafilomycin A1 and chloroquine on bioenergetics and metabolism. We found that both bafilomycin and chloroquine could significantly increase the autophagosome marker LC3-II and inhibit key parameters of mitochondrial function, and increase mtDNA damage. Furthermore, we observed significant alterations in TCA cycle intermediates, particularly those downstream of citrate synthase and those linked to glutaminolysis. Taken together, these data demonstrate a significant impact of bafilomycin and chloroquine on cellular bioenergetics and metabolism consistent with decreased mitochondrial quality associated with inhibition of autophagy.

Mapping the Human Platelet Lipidome Reveals Cytosolic Phospholipase A2 as a Regulator of Mitochondrial Bioenergetics during Activation.

Human platelets acutely increase mitochondrial energy generation following stimulation. Herein, a lipidomic circuit was uncovered whereby the substrates for this are exclusively provided by cPLA2, including multiple fatty acids and oxidized species that support energy generation via ß-oxidation. This indicates that acute lipid membrane remodeling is required to support energetic demands during platelet activation. Phospholipase activity is linked to energy metabolism, revealing cPLA2 as a central regulator of both lipidomics and energy flux. Using a lipidomic approach (LipidArrays), we also estimated the total number of lipids in resting, thrombin-activated, and aspirinized platelets. Significant diversity between genetically unrelated individuals and a wealth of species was revealed. Resting platelets demonstrated ∼5,600 unique species, with only ∼50% being putatively identified. Thrombin elevated ∼900 lipids >2-fold with 86% newly appearing and 45% inhibited by aspirin supplementation, indicating COX-1 is required for major activation-dependent lipidomic fluxes. Many lipids were structurally identified. With ∼50% of the lipids being absent from databases, a major opportunity for mining lipids relevant to human health and disease is presented.

Bioenergetic adaptation in response to autophagy regulators during rotenone exposure.

Parkinson's 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 Parkinson's 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.

The Bioenergetic Health Index: a new concept in mitochondrial translational research.

Bioenergetics has become central to our understanding of pathological mechanisms, the development of new therapeutic strategies and as a biomarker for disease progression in neurodegeneration, diabetes, cancer and cardiovascular disease. A key concept is that the mitochondrion can act as the 'canary in the coal mine' by serving as an early warning of bioenergetic crisis in patient populations. We propose that new clinical tests to monitor changes in bioenergetics in patient populations are needed to take advantage of the early and sensitive ability of bioenergetics to determine severity and progression in complex and multifactorial diseases. With the recent development of high-throughput assays to measure cellular energetic function in the small number of cells that can be isolated from human blood these clinical tests are now feasible. We have shown that the sequential addition of well-characterized inhibitors of oxidative phosphorylation allows a bioenergetic profile to be measured in cells isolated from normal or pathological samples. From these data we propose that a single value-the Bioenergetic Health Index (BHI)-can be calculated to represent the patient's composite mitochondrial profile for a selected cell type. In the present Hypothesis paper, we discuss how BHI could serve as a dynamic index of bioenergetic health and how it can be measured in platelets and leucocytes. We propose that, ultimately, BHI has the potential to be a new biomarker for assessing patient health with both prognostic and diagnostic value.

Bioenergetics and the oxidative burst: protocols for the isolation and evaluation of human leukocytes and platelets.

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging. Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis. This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry. An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

A review of the mitochondrial and glycolytic metabolism in human platelets and leukocytes: implications for their use as bioenergetic biomarkers.

The assessment of metabolic function in cells isolated from human blood for treatment and diagnosis of disease is a new and important area of translational research. It is now becoming clear that a broad range of pathologies which present clinically with symptoms predominantly in one organ, such as the brain or kidney, also modulate mitochondrial energetics in platelets and leukocytes allowing these cells to serve as "the canary in the coal mine" for bioenergetic dysfunction. This opens up the possibility that circulating platelets and leukocytes can sense metabolic stress in patients and serve as biomarkers of mitochondrial dysfunction in human pathologies such as diabetes, neurodegeneration and cardiovascular disease. In this overview we will describe how the utilization of glycolysis and oxidative phosphorylation differs in platelets and leukocytes and discuss how they can be used in patient populations. Since it is clear that the metabolic programs between leukocytes and platelets are fundamentally distinct the measurement of mitochondrial function in distinct cell populations is necessary for translational research.

Functional genetic polymorphisms in the aromatase gene CYP19 vary the response of breast cancer patients to neoadjuvant therapy with aromatase inhibitors.

Aromatase (CYP19) is a critical enzyme in estrogen biosynthesis and aromatase inhibitors (AI) are employed widely for endocrine therapy in postmenopausal women with breast cancer. We hypothesized that single nucleotide polymorphisms (SNPs) in the CYP19 gene may alter the effectiveness of AI therapy in the neoadjuvant setting. Genomic DNA was obtained for sequencing from 52 women pre-AI and post-AI treatment in this setting. Additionally, genomic DNA obtained from 82 samples of breast cancer and 19 samples of normal breast tissue was subjected to resequencing. No differences in CYP19 sequence were observed between tumor and germ-line DNA in the same patient. A total of 48 SNPs were identified including 4 novel SNPs when compared with previous resequencing data. For genotype-phenotype association studies, we determined the levels of aromatase activity, estrone, estradiol, and tumor size in patients pre-AI and post-AI treatment. We defined two tightly linked SNPs (rs6493497 and rs7176005 in the 5'-flanking region of CYP19 exon 1.1) that were significantly associated with a greater change in aromatase activity after AI treatment. In a follow-up study of 200 women with early-stage breast cancer who were treated with adjuvant anastrozole, these same two SNPs were also associated with higher plasma estradiol levels in patients pre-AI and post-AI treatment. Electrophoretic mobility shift and reporter gene assays confirmed likely functional effects of these two SNPs on transcription of CYP19. Our findings indicate that two common genetic polymorphisms in the aromatase gene CYP19 vary the response of breast cancer patients to aromatase inhibitors.