Source:
http://info.med.yale.edu/labinvest/abstracts/98months/9812dec/1298_013.html

Tumor Necrosis Factor Is Delivered to Mitochondria Where a Tumor Necrosis Factor-Binding Protein Is Localized

Elizabeth C. Ledgerwood, Johannes B. Prins,Nicholas A. Bright,David R. Johnson, Karen Wolfreys, Jordan S. Pober,Steven O'Rahilly, John R. Bradley

Departments of Medicine (ECL, JBP, KW, SO'R, JRB) and Clinical Biochemistry (ECL, JBP, NAB, SO'R), University of Cambridge, Addenbrooke's Hospital, Cambridge, United Kingdom; and Boyer Center for Molecular Medicine (DRJ, JSP), Yale University School of Medicine, New Haven, Connecticut

The roles of the known tumor necrosis factor (TNF) receptors (TNFR-I and TNFR-II) and their associated signaling pathways in mediating the diverse actions of TNF remain incompletely defined. We have found that a proportion of exogenous TNF is delivered to mitochondria as well as to lysosomes. Using confocal and immunoelectron microscopy and Western blotting of subcellular fractions, we have identified a 60-kd protein in the inner mitochondrial membrane that is recognized by a monoclonal antibody to TNFR-II. In isolated mitochondria, this protein binds [125I\]-TNF. This provides evidence of a mitochondrial binding protein for an extracellular ligand and demonstrates the presence of a pathway capable of delivering TNF from the cell surface to mitochondria. These findings suggest that TNF effects on cells may be due in part to a direct effect on mitochondria. (Lab Invest 1998, 78:1583-1589).

Localization of HIV RNA in mitochondria of infected cells: potential role in cytopathogenicity.

J Cell Biol 1994 Sep;126(6):1353-1360

Somasundaran M, Zapp ML, Beattie LK, Pang L, Byron KS, Bassell GJ, Sullivan JL, Singer RH

The intracellular distribution of HIV-1 RNA transcripts in infected cells was studied using in situ hybridization detected by electron microscopy and cellular fractionation. Although viral RNA and core protein could be detected throughout the cytoplasm and nucleus, viral RNA was found in significantly increased amounts in mitochondria relative to the cytoplasm and nucleus. In contrast, cellular poly(A) RNA or viral gag proteins were not increased in the mitochondria. A cell line containing an integrated latent genome that could be induced to express viral RNA after phorbol ester stimulation showed an increase in viral RNA accumulation in mitochondria parallel with the increase in HIV expression levels. Concomitant with HIV expression, there was a decrease in mitochondrial viability. Using immunofluorescent markers to detect probes to HIV RNA transcripts and antibodies to mitochondrial proteins simultaneously in single cells, there was an inverse relationship between the amount of viral RNA and mitochondrial integrity. High levels of viral RNA in mitochondria were found in acutely (but not chronically) infected cells. We propose that HIV RNA import into mitochondria can compromise mitochondrial function.

Mitochondria as a Critical Target for Toxicity
Gurmit Singh and Wilhelmina C.M. Duivenvoorden
Hamilton Regional Cancer Centre
699 Concession St, Hamilton, Ontario, Canada

Mitochondria perform a variety of important cellular functions. In addition to synthesizing ATP by oxidative phosphorylation, they synthesize lipids, heme, amino acids, pyrimidines and are involved in regulating intracellular pH and ion homeostasis. During the last two decades, mitochondrial DNA (mtDNA) from a number of species has been sequenced. The organelle DNA has been used to exploit the evolutionary aspects of the origin of mitochondria. The endosymbiotic bacterial ancestry suggests that some of the functions fundamental to bacterial life may be retained in mitochondria. Thus, an understanding of both bacterial genes and bacterial function form a basis for identifying novel mitochondrial functions.

There is direct evidence that mtDNA is 5- to 500-fold more sensitive than nuclear DNA to damage induced by several chemicals, with the highest differential relating to polycyclic aromatic hydrocarbons. Damage to mtDNA contributes to the cytotoxic, mutagenic and carcinogenic potential of several drugs and environmental chemicals into reactive electrophilic metabolites, especially since metabolic activation of xenobiotic compounds can occur within or at the surface of mitochondria. Furthermore, the lack of protective histones or non-histone proteins, the limited DNA repair capacity and the attachment of mtDNA to the inner membrane make the mtDNA more susceptible to damage by electrophilic compounds such as peroxides, epoxides, N-nitroso compounds, nitroxides, semiquinones, etc. Some of these electrophiles may be a result of increased metabolic activity in cell induced by hormones, neurotransmitters, etc. Damage to organelle DNA could result in a change in mitochondrial function resulting in alterations in cellular functions, which may manifest as a toxic event in the tissue.

The electrochemical gradient in mitochondria consists of two components, namely a pH gradient and a membrane potential. The two mechanisms that generate an electrochemical gradient are: (1) active pumping of protons across the membrane and (2) the movement of the protons coupled to electron transfer. The magnitude of the mitochondrial membrane potential differs depending on the cell type. The mitochondrial membrane potential in cells from different tissues from highest to lowest is as follows; cardiac muscle cells > skeletal muscle cells > smooth muscle cells > macrophages > hepatocytes > fibroblasts > neuronal cells > keratinocytes > bladder epithelial cells > resting T and B lymphocytes. Significance of the cell-specific characteristics of mitochondria are poorly understood. However, tissue-specific toxicities could be related to the electrochemical gradient within mitochondria of various tissues. In certain instances, the dissipation of the gradient could result in altered cellular function or could initiate damage of the vulnerable genome resulting in toxic cellular effects. Damage to mitochondria by various drugs and toxicants has been well established and good morphological data has been available for a very long time.

In conclusion, various chemicals can disrupt mitochondrial functions via disruption of the electrochemical gradient or damage to the mtDNA resulting in cellular toxicity.

(This work is supported by the Medical Research Council of Canada to G. Singh)

Environmental Health Perspectives 106, Supplement 5, October 1998

Nitric Oxide and Its Congeners in Mitochondria: Implications for Apoptosis
Christoph Richter

Laboratory of Biochemistry, Swiss Federal Institute of Technology Zürich, Switzerland

Abstract
Apoptosis is an evolutionarily conserved form of physiologic cell death important for tissue development and homeostasis. The causes and execution mechanisms of apoptosis are not completely understood. Nitric oxide (NO) and its congeners, oxidative stress, Ca2+, proteases, nucleases, and mitochondria are considered mediators of apoptosis. Recent findings strongly suggest that mitochondria contain a factor or factors that upon release from the destabilized organelles, induce apoptosis. We have found that oxidative stress-induced release of Ca2+ from mitochondria followed by Ca2+ reuptake (Ca2+ cycling) causes destabilization of mitochondria and apoptosis. The protein product of the protooncogene bcl-2 protects mitochondria and thereby prevents apoptosis. We have also found that NO and its congeners can induce Ca2+ release from mitochondria. Thus, nitrogen monoxide (·NO) binds to cytochrome oxidase, blocks respiration, and thereby causes mitochondrial deenergization and Ca2+ release. Peroxynitrite (ONOO-), on the other hand, causes Ca2+ release from mitochondria by stimulating a specific Ca2+ release pathway. This pathway requires oxidized nicotinamide adenine dinucleotide (NAD+) hydrolysis to adenosine diphosphate ribose and nicotinamide. NAD+ hydrolysis is only possible when some vicinal thiols are cross-linked. ONOO- is able to oxidize them. Our findings suggest that NO and its congeners can induce apoptosis by destabilizing mitochondria via deenergization and/or by inducing a specific Ca2+ release followed by Ca2+ cycling. -- Environ Health Perspect 106 (Suppl 5):1125-1130 (1998).

http://ehpnet1.niehs.nih.gov/docs/1998/Suppl-5/1125-1130richter/abstract.html

Key words: nitrogen monoxide, nitric oxide, peroxynitrite, calcium, bcl-2, nicotinamide adenine dinucleotide, respiration, membrane potential

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DIABETES, VOLUME 45, FEBRUARY 1996 PAGES 113 - 126

Perspectives in Diabetes
Mitochondria and Diabetes
Genetic, Biochemical, and Clinical Implications of the Cellular Energy Circuit
Klaus-Dieter Gerbitz, Klaus Gempel, and Dieter Brdiczka

Physiologically, a postprandial glucose rise induces metabolic signal sequences that use several steps in common in both the pancreas and peripheral tissues but result in different events due to specialized tissue functions. Glucose transport performed by tissue-specific glucose transporters is, in general, not rate limiting. The next step is phosphorylation of glucose by cell-specific hexoki-nases. In the ß-cell, glucokinase (or hexokinase IV) is activated upon binding to a pore protein in the outer mitochondrial membrane at contact sites between outer and inner membranes. The same mechanism applies for hexokinase II in skeletal muscle and adipose tissue. The activation of hexokinases depends on a contact site-specific structure of the pore, which is voltage-dependent and influenced by the electric potential of the inner mitochondrial membrane.

Mitochondria lacking a membrane potential because of defects in the respiratory chain would thus not be able to increase the glucose-phosphorylating enzyme activity over basal state. Binding and activation of hexokinases to mitochondrial contact sites lead to an acceleration of the formation of both ADP and glucose-6-phosphate (G-6-P). ADP directly enters the mitochondrion and stimulates mitochondrial oxidative phosphorylation. G-6-P is an important intermediate of energy metabolism at the switch position between glycolysis, glycogen synthesis, and the pentose-phosphate shunt. Initiated by blood glucose elevation, mitochondrial oxidative phosphorylation is accelerated in a concerted action coupling glycolysis to mitochondrial metabolism at three different points: first, through NADH transfer to the respiratory chain complex I via the malate/aspartate shuttle; second, by providing FADH2 to complex II through the glycerol-phosphate/dihydroxy-acetone-phosphate cycle; and third, by the action of hexo(gluco)kinases providing ADP for complex V, the ATP synthetase.

As cytosolic and mitochondrial isozymes of creatine kinase (CK) are observed in insulinoma cells, the phospho-creatine (CrP) shuttle, working in brain and muscle, may also be involved in signaling glucose-induced insulin secretion in ß-cells. An interplay between the plasma membrane-bound CK and the mitochondrial CK could provide a mechanism to increase ATP locally at the KATP channels, coordinated to the activity of mitochondrial CrP production. Closure of the KATP channels by ATP would lead to an increase of cytosolic and, even more, mitochondrial calcium and finally to insulin secretion. Thus in ß-cells, glucose, via bound glucokinase, stimulates mitochondrial CrP synthesis. The same signaling sequence is used in the opposite direction in muscle during exercise when high ATP turnover increases the creatine level that stimulates mitochondrial ATP synthesis and glucose phosphorylation via hexokinase. Furthermore, this cytosolic/mitochondrial cross-talk is also involved in activation of muscle glycogen synthesis by glucose. The activity of mitochondrially bound hexokinase provides G-6-P and stimulates UTP production through mitochondrial nucleoside diphosphate kinase.

Pathophysiologically, there are at least two genetically different forms of diabetes linked to energy metabolism: the first example is one form of maturity-onset diabetes of the young (MODY2), an autosomal dominant disorder caused by point mutations of the glucokinase gene; the second example is several forms of mitochondrial diabetes caused by point and length mutations of the mitochondrial DNA (mtDNA) that encodes several subunits of the respiratory chain complexes. Because the mtDNA is vulnerable and accumulates point and length mutations during aging, it is likely to contribute to the manifestation of some forms of NIDDM. Furthermore, point mutations in the muscle hexokinase II have recently been described in some NIDDM families, and decreased glycogen synthesis due to low G-6-P levels has been found as the first hint of peripheral insulin resistance in apparently healthy offspring of diabetic parents. Thus, we postulate that genetic defects at quite different sites of energy metabolism can lead to diabetes and that, in some forms of NIDDM, a common defect in the cytosolic-mitochondrial interplay of energy production can result in both impaired insulin secretion in the ß-cell and peripheral resistance against the hormone in the muscle and adipose tissue. Diabetes 45:113-126,1996

J. Timothy Greenamyre, MD, PhD
Emory University
Atlanta, GA, USA

Mitochondria, Excitotoxicity and Neurodegeneration

Excitotoxicity and mitochondrial impairment have been implicated in the pathogenesis of both Huntington's disease (HD) and Parkinson's disease (PD). These two pathogenic processes are intertwined mechanistically.

Mitochondrial dysfunction with ATP depletion impairs the activity of the Na+/K+-ATPase that maintains neuronal membrane potential. As a result, the voltage-dependent blockade by Mg++ of the N-methyl-D-aspartate (NMDA) class of glutamate receptor is reduced. In this setting, low concentrations of extracellular glutamate may become lethal. Additionally, mitochondrial impairment severely disrupts intracellular calcium homeostasis. Thus, mitochondrial dysfunction can produce secondary excitotoxicity. Mitochondria are also targets of excitotoxicity, such that NMDA receptor activation leads to mitochondrial swelling and generation of reactive oxygen species. Mitochondria isolated from HD patients have a reduced capacity to accumulate calcium; with graded calcium loads, they depolarize more readily and recover less well than control mitochondria. Normal mitochondria can be induced to behave like HD mitochondria by incubating them with polyglutamine-containing proteins similar to the mutant huntingtin protein that causes HD. In PD, a selective defect in mitochondrial complex I appears to be important in many cases. As the nigral dopamine neurons die, another group of neurons in the subthalamic nucleus become overactive. These subthalamic neurons are glutamatergic and project back to the dysfunctional dopamine neurons. It has been hypothesized that this increased glutamatergic drive upon already impaired neurons contributes to disease progression. Using a rat model of PD, we have found that chronic infusion of an NMDA antagonist into the subthalamic nucleus reduces degeneration of nigral dopamine neurons by more than 50%. Thus, mitochondrial defects and excitotoxicity appear to play important roles in HD and PD. Therapeutic strategies targeting these processes are likely to provide neuroprotection and slow disease progression.