Christian A. Combs

The Mammalian Longevity-associated Gene Product p66shc Regulates Mitochondrial Metabolism

Previous studies have determined that mice with a homozygous deletion in the adapter protein p66shc have an extended life span and that cells derived from these mice exhibit lower levels of reactive oxygen species. Here we demonstrate that a fraction of p66shc localizes to the mitochondria and that p66shc-/- fibroblasts have altered mitochondrial energetics. In particular, despite similar cytochrome content, under basal conditions, the oxygen consumption of spontaneously immortalized p66shc-/- mouse embryonic fibroblasts were lower than similarly maintained wild type cells. Differences in oxygen consumption were particularly evident under chemically uncoupled conditions, demonstrating that p66shc-/- cells have a reduction in both their resting and maximal oxidative capacity. We further demonstrate that reconstitution of p66shc expression in p66shc-/- cells increases oxygen consumption. The observed defect in oxidative capacity seen in p66shc-/- cells is partially offset by augmented levels of aerobic glycolysis. This metabolic switch is manifested by p66shc-/- cells exhibiting an increase in lactate production and a stricter requirement for extracellular glucose in order to maintain intracellular ATP levels. In addition, using an in vivo NADH photobleaching technique, we demonstrate that mitochondrial NADH metabolism is reduced in p66shc-/- cells. These results demonstrate that p66shc regulates mitochondrial oxidative capacity and suggest that p66shc may extend life span by repartitioning metabolic energy conversion away from oxidative and toward glycolytic pathways.

Measuring In Vivo Mitophagy

Alterations in mitophagy have been increasingly linked to aging and age-related diseases. There are, however, no convenient methods to analyze mitophagy in vivo. Here, we describe a transgenic mouse model in which we expressed a mitochondrial-targeted form of the fluorescent reporter Keima (mt-Keima). Keima is a coral-derived protein that exhibits both pH-dependent excitation and resistance to lysosomal proteases. Comparison of a wide range of primary cells and tissues generated from the mt-Keima mouse revealed significant variations in basal mitophagy. In addition, we have employed the mt-Keima mice to analyze how mitophagy is altered by conditions including diet, oxygen availability, Huntingtin transgene expression, the absence of macroautophagy (ATG5 or ATG7 expression), an increase in mitochondrial mutational load, the presence of metastatic tumors, and normal aging. The ability to assess mitophagy under a host of varying environmental and genetic perturbations suggests that the mt-Keima mouse should be a valuable resource.

Phospholipases D1 and D2 Regulate Different Phases of Exocytosis in Mast Cells

The rat mast cell line RBL-2H3 contains both phospholipase D (PLD)1 and PLD2. Previous studies with this cell line indicated that expressed PLD1 and PLD2 are both strongly activated by stimulants of secretion. We now show by use of PLDs tagged with enhanced green fluorescent protein that PLD1, which is largely associated with secretory granules, redistributes to the plasma membrane in stimulated cells by processes reminiscent of exocytosis and fusion of granules with the plasma membrane. These processes and secretion of granules are suppressed by expression of a catalytically inactive mutant of PLD1 or by the presence of 50 mM 1-butanol but not tert-butanol, an indication that these events are dependent on the catalytic activity of PLD1. Of note, cholera toxin induces translocation of PLD1-labeled granules to the plasma membrane but not fusion of granules with plasma membrane or secretion. Subsequent stimulation of calcium influx with Ag or thapsigargin leads to rapid redistribution of PLD1 to the plasma membrane and accelerated secretion. Also of note, PLD1 is recycled from plasma membrane back to granules within 4 h of stimulation. PLD2, in contrast, is largely confined to the plasma membrane, but it too participates in the secretory process, because expression of catalytically inactive PLD2 also blocks secretion. These data indicate a two-step process: translocation of granules to the cell periphery, regulated by granule-associated PLD1, and a calcium-dependent fusion of granules with the plasma membrane, regulated by plasma membrane-associated PLD2 and possibly PLD1.

Autophagy regulates endothelial cell processing, maturation and secretion of von Willebrand factor

Endothelial secretion of von Willebrand factor (VWF) from intracellular organelles known as Weibel-Palade bodies (WPBs) is required for platelet adhesion to the injured vessel wall. Here we demonstrate that WPBs are often found near or within autophagosomes and that endothelial autophagosomes contain abundant VWF protein. Pharmacological inhibitors of autophagy or knockdown of the essential autophagy genes Atg5 or Atg7 inhibits the in vitro secretion of VWF. Furthermore, although mice with endothelial-specific deletion of Atg7 have normal vessel architecture and capillary density, they exhibit impaired epinephrine-stimulated VWF release, reduced levels of high–molecular weight VWF multimers and a corresponding prolongation of bleeding times. Endothelial-specific deletion of Atg5 or pharmacological inhibition of autophagic flux results in a similar in vivo alteration of hemostasis. Thus, autophagy regulates endothelial VWF secretion, and transient pharmacological inhibition of autophagic flux may be a useful strategy to prevent thrombotic events.

Mitochondrial reticulum for cellular energy distribution in muscle

Intracellular energy distribution has attracted much interest and has been proposed to occur in skeletal muscle via metabolite-facilitated diffusion; however, genetic evidence suggests that facilitated diffusion is not critical for normal function. We hypothesized that mitochondrial structure minimizes metabolite diffusion distances in skeletal muscle. Here we demonstrate a mitochondrial reticulum providing a conductive pathway for energy distribution, in the form of the proton-motive force, throughout the mouse skeletal muscle cell. Within this reticulum, we find proteins associated with mitochondrial proton-motive force production preferentially in the cell periphery and proteins that use the proton-motive force for ATP production in the cell interior near contractile and transport ATPases. Furthermore, we show a rapid, coordinated depolarization of the membrane potential component of the proton-motive force throughout the cell in response to spatially controlled uncoupling of the cell interior. We propose that membrane potential conduction via the mitochondrial reticulum is the dominant pathway for skeletal muscle energy distribution.

Mitochondrial respiration protects against oxygen-associated DNA damage

Oxygen is not only required for oxidative phosphorylation but also serves as the essential substrate for the formation of reactive oxygen species (ROS), which is implicated in ageing and tumorigenesis. Although the mitochondrion is known for its bioenergetic function, the symbiotic theory originally proposed that it provided protection against the toxicity of increasing oxygen in the primordial atmosphere. Using human cells lacking Synthesis of Cytochrome c Oxidase 2 (SCO2-/-), we have tested the oxygen toxicity hypothesis. These cells are oxidative phosphorylation defective and glycolysis dependent; they exhibit increased viability under hypoxia and feature an inverted growth response to oxygen compared with wild-type cells. SCO2-/- cells have increased intracellular oxygen and nicotinamide adenine dinucleotide (NADH) levels, which result in increased ROS and oxidative DNA damage. Using this isogenic cell line, we have revealed the genotoxicity of ambient oxygen. Our study highlights the importance of mitochondrial respiration both for bioenergetic benefits and for maintaining genomic stability in an oxygen-rich environment.

The ABCA1 transporter functions on the basolateral surface of hepatocytes

ABCA1 on the cell surface and in endosomes plays an essential role in the cell-mediated lipidation of apoA-I to form nascent HDL. Our previous studies of transgenic mice overexpressing ABCA1 suggested that ABCA1 in the liver plays a major role in regulating plasma HDL levels. The site of function of ABCA1 in the polarized hepatocyte was currently assessed by expression of an adenoviral construct encoding a human ABCA1-GFP fusion protein in the polarized hepatocyte-like WIF-B cell line. Consistent with localization of ABCA1 at the basolateral (vascular) cell surface, expression of ABCA1-GFP stimulated apoA-I mediated efflux of WIF-B cell cholesterol into the culture medium. Confocal fluorescence microscopy revealed that ABCA1-GFP was expressed solely on the basolateral surface and associated endocytic vesicles. These findings suggest an important role for hepatocyte basolateral membrane ABCA1 in the regulation of the levels of intracellular hepatic cholesterol, as well as plasma HDL.

Restricted mitochondrial protein acetylation initiates mitochondrial autophagy

Summary Because nutrient-sensing nuclear and cytosolic acetylation mediates cellular autophagy, we investigated whether mitochondrial acetylation modulates mitochondrial autophagy (mitophagy). Knockdown of GCN5L1, a component of the mitochondrial acetyltransferase machinery, diminished mitochondrial protein acetylation and augmented mitochondrial enrichment of autophagy mediators. This program was disrupted by SIRT3 knockdown. Chronic GCN5L1 depletion increased mitochondrial turnover and reduced mitochondrial protein content and/or mass. In parallel, mitochondria showed blunted respiration and enhanced ‘stress-resilience’. Genetic disruption of autophagy mediators Atg5 and p62 (also known as SQSTM1), as well as GCN5L1 reconstitution, abolished deacetylation-induced mitochondrial autophagy. Interestingly, this program is independent of the mitophagy E3-ligase Parkin (also known as PARK2). Taken together, these data suggest that deacetylation of mitochondrial proteins initiates mitochondrial autophagy in a canonical autophagy-mediator-dependent program and shows that modulation of this regulatory program has ameliorative mitochondrial homeostatic effects.

Direct Imaging of Dehydrogenase Activity within Living Cells Using Enzyme-Dependent Fluorescence Recovery after Photobleaching (ED-FRAP)

Reduced nicotine adenine dinucleotide (NADH) is a key metabolite involved in cellular energy conversion and many redox reactions. We describe the use of confocal microscopy in conjunction with enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH as a topological assay of NADH generation capacity within living cardiac myocytes. Quantitative validation of this approach was performed using a dehydrogenase system, in vitro. In intact cells the NADH ED-FRAP was sensitive to temperature (Q(10) of 2.5) and to dehydrogenase activation by dichloroacetate or cAMP (twofold increase for each). In addition, NADH ED-FRAP was correlated with flavin adenine dinucleotide (FAD(+)) fluorescence. These data, coupled with the cellular patterns of NADH ED-FRAP changes with dehydrogenase stimulation, suggest that NADH ED-FRAP is localized to the mitochondria. These results suggest that ED-FRAP enables measurement of regional dynamics of mitochondrial NADH production in intact cells, thus providing information regarding region-specific intracellular redox reactions and energy metabolism.

NADH enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP): Applications to enzyme and mitochondrial reaction kinetics, in vitro

NADH enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) was evaluated for studying enzyme kinetics in vitro and in isolated mitochondria. Mass, optical, and nuclear magnetic resonance spectroscopy data were consistent with the UV NADH photolysis reaction being NADH --> NAD* + H+ + e-. The overall net reaction was O2 + 2NADH + 2H+ --> 2NAD+ + 2H2O, or in the presence of other competing electron acceptors such as cytochrome c, NADH + 2Cyt(ox) --> NAD+ + H+ + 2Cyt(red). Solution pH could differentiate between these free-radical scavenging pathways. These net reactions represent the photooxidation of NADH to NAD+. Kinetic models and acquisition schemes were developed, varying [NADH] and [NAD] by altering NADH photolysis levels, for extracting kinetic parameters. UV irradiation levels used did not damage mitochondrial function or enzymatic activity. In mitochondria, [NADH] is a high affinity product inhibitor that significantly reduced the NADH regeneration rate. Matrix NADH regeneration only slightly exceeded the net rate of NADH consumption, suggesting that the NADH regeneration process is far from equilibrium. Evaluation of NADH regeneration in active mitochondria, in comparison to rotenone-treated preparations, revealed other regulatory elements in addition to matrix [NADH] and [NAD] that have yet to be fully characterized. These studies demonstrate that the rapid UV photolysis of NADH to NAD is an effective tool in evaluating the steady-state kinetic properties of enzyme systems. Initial data support the notion that the NADH regeneration process is far from equilibrium in mitochondria and is potentially controlled by NADH levels as well as several other matrix factors.