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Methods in
Molecular Biology 1264

Volkmar Weissig
Marvin Edeas Editors

Mitochondrial
Medicine
Volume I,
Probing Mitochondrial Function
METHODS IN M O L E C U L A R B I O LO G Y

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
Mitochondrial Medicine

Volume I, Probing Mitochondrial Function

Edited by

Volkmar Weissig
Midwestern University, Glendale, AZ, USA

Marvin Edeas
ISANH, Paris, France
Editors
Volkmar Weissig Marvin Edeas
Midwestern University ISANH
Glendale, AZ, USA Paris, France

ISSN 1064-3745 ISSN 1940-6029 (electronic)

ISBN 978-1-4939-2256-7 ISBN 978-1-4939-2257-4 (eBook)
DOI 10.1007/978-1-4939-2257-4
Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2014960347

© Springer Science+Business Media New York 2015

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Preface

Mitochondrial Medicine is an interdisciplinary and rapidly growing new area of biomedical

research comprising genetic, biochemical, pathological, and clinical studies aimed at the
diagnosis and therapy of human diseases which are either caused by or associated with mito-
chondrial dysfunction. The term “Mitochondrial Medicine” was probably used for the first
time by Rolf Luft [1] who is widely accepted as the father of Mitochondrial Medicine. Over
50 years ago, it was he who described for the very first time a patient with clinical symptoms
caused by malfunctioning mitochondria [2].
The beginning of mitochondria-related research dates back to the end of the nine-
teenth century. During the 1890s, early cytological studies revealed the existence of
bacteria-resembling subcellular particles in the cytosol of mammalian cells. Robert Altman
termed them bioblasts, and he hypothesized that these particles were the basic unit of cel-
lular activity. The name mitochondrion, which means thread-like particles, was coined in
1898 by Carl Benda. During the 1940s, progress was made in the development of cell
fractionation techniques which ultimately allowed the isolation of intact mitochondria from
cell homogenates, thereby making them more accessible to biochemical studies.
Subsequently, by the end of the 1940s, activities of a variety of enzymes needed for fatty
acid oxidation, the Krebs cycle, and other metabolic pathways were found to be associated
with mitochondrial fractions.
Human mitochondrial DNA was discovered in 1963 [3], and Mitchell’s disputed
chemiosmotic theory [4] of ATP synthesis became generally accepted in the early 1970s. In
1972, Harman proposed the Mitochondrial Theory of Aging, according to which aging is
the result of the cumulative effects of mitochondrial DNA damage caused by free radicals
[5, 6]. In 1986, Miquel and Fleming published their hypothesis about the involvement of
mitochondria-originated free radicals in the process of ageing [7]. By 1981, mitochondrial
DNA was completely sequenced [8], and, 5 years later, its entire genetic content had been
described [9, 10]. Obviously, research on and with mitochondria has been conducted for
over 120 years continuously and with steady success. Nevertheless, the last decade of the
twentieth century saw another significant boost of interest in studying mitochondrial func-
tions. First, in 1988, two papers, one published in Science and the other in Nature [11, 12],
revealed for the very first time deletions and point mutations of mitochondria DNA to be
the cause for human diseases. Second, by around 1995, mitochondria well known as the
“powerhouse of the cell” have also been accepted as the “motor of cell death” [13] reflect-
ing the organelle’s key role in apoptosis. It is nowadays recognized that mitochondrial
dysfunction is either the cause of or at least associated with a large number and variety of
human disorders, ranging from neurodegenerative and neuromuscular diseases, obesity,
cardiovascular disorders, migraine, liver and kidney disease to ischemia-reperfusion injury
and cancer. Subsequently, increased pharmacological and pharmaceutical efforts have led to
the emergence of mitochondrial medicine as a new field of biomedical research [1, 14].
Future developments of techniques for probing and manipulating mitochondrial functions
will eventually lead to the treatment and prevention of a wide variety of pathologies and
chronic diseases, “the future of medicine will come through mitochondria” [15].

v
vi Preface

Our book is dedicated to showcasing the tremendous efforts and the progress that has
been made over the last decades in developing techniques and protocols for probing, imag-
ing, and manipulating mitochondrial functions. All chapters were written by leading experts
in their particular fields. The book is divided into two volumes. Volume I (Probing
Mitochondrial Function) is focused on methods being used for the assessment of mitochon-
drial function under physiological conditions as well as in healthy isolated mitochondria.
Volume II (Manipulating Mitochondrial Function) describes techniques developed for
manipulating and assessing mitochondrial function under general pathological conditions
and specific disease states.

Volume I

Stefan Lehr and coworkers critically evaluate in a review chapter a commonly used isolation
procedure for mitochondria utilizing differential (gradient) centrifugation and depict major
challenges to achieve “functional” mitochondria as basis for comprehensive physiological
studies. The same authors provide in a protocol chapter an isopycnic density gradient cen-
trifugation strategy for the isolation of mitochondria with a special focus on quality control
of prepared intact, functional mitochondria. The isolation of interorganellar membrane
contact sites is described by Alessandra d’Azzo and colleagues. They outline a protocol
tailored for the isolation of mitochondria, mitochondria-associated ER membranes, and
glycosphingolipid-enriched microdomains from the adult mouse brain, primary neuro-
spheres, and murine embryonic fibroblasts. The analysis of single mitochondria helps
uncovering a new level of biological heterogeneity and holds promises for a better under-
standing of mitochondria-related diseases. Peter Burke and colleagues describe a nanoscale
approach for trapping single mitochondria in fluidic channels for fluorescence microscopy.
Their method reduces background fluorescence, enhances focus, and allows simple experi-
mental buffer exchanges. Stephane Arbault and colleagues describe the preparation and use
of microwell arrays for the entrapment and fluorescence microscopy of single isolated mito-
chondria. Measuring variations of NADH of each mitochondrion in the array, this method
allows the analysis of the metabolic status of the single organelle at different energetic-
respiratory stages.
Deep resequencing allows the detection and quantification of low-level variants in
mitochondrial DNA (mtDNA). This massively parallel (“next-generation”) sequencing is
characterized by great depth and breadth of coverage. Brendan Payne and colleagues
describe a method for whole mtDNA genome deep sequencing as well as short amplicon
deep sequencing. In another chapter, the same group provides a method for characterizing
mtDNA within single skeletal muscle fibers. This approach allows the detection of somatic
mtDNA mutations existing within individual cells which may be missed by techniques
applied to the whole tissue DNA extract. The authors also apply single-cell mtDNA
sequencing for analyzing differential segregation of mtDNA during embryogenesis. They
demonstrate how to study this phenomenon by single-cell analysis of embryonic primordial
germ cells. Next-generation sequencing (NGS) as an effective method for mitochondrial
genome sequencing is also the subject of Shale Dames’ chapter. He and his group describe
an mtDNA enrichment method including library preparation and sequencing on “Illumina
NGS platforms” and provide also a short command line alignment script for downloading
via FTP. Conventional methods for mitochondrial DNA (mtDNA) extraction do not yield
the level of mtDNA enrichment needed for direct sequencing, and the necessary subsequent
 Preface vii

long-range PCR amplification may introduce bias into the sequence results. Alexander
Maslov and colleagues provide a protocol involving a paramagnetic bead-based purification
step for the preparation of mtDNA-enriched samples ready for direct sequencing. Lars Eide
and coworkers give a detailed protocol for the use of real-time qPCR to analyze the integ-
rity of mitochondrial DNA and RNA quantitatively. Their method has low material require-
ment, is low cost, and can detect modifications with high resolution.
Mitochondria in species ranging from yeast to human have been found to import a
small number of nucleus-encoded RNAs. With the advent of high-throughput RNA
sequencing, additional nucleus-encoded mitochondrial RNAs are being identified. Michael
Teitell and his group describe both an in vitro and in vivo import system for studying mito-
chondrial RNA import, processing, and functions.
In the last decade an increasing number of studies have been conducted aimed at quan-
tifying acquired changes in the concentration of circulating mitochondrial DNA (mtDNA)
as an indicator of mitochondrial function. Afshan Malik and colleagues provide a protocol
for accurately measuring the amount of human mtDNA in peripheral blood samples which
is based on the use of real-time quantitative PCR (qPCR) to quantify the amount of mtDNA
relative to nuclear DNA. Their protocol is suitable for high-throughput use and can be
modified for application to other body fluids, human cells, and tissues. The characterization
of mtDNA processing at the single-cell level is poorly defined. Laurent Chatre and Miria
Ricchetti describe a mitochondrial transcription and replication imaging protocol which is
based on modified fluorescence in situ hybridization and which allows the detection of
qualitative and quantitative alterations of the dynamics of mtDNA processing in human
cells undergoing physiological changes.
William Sivitz and colleagues describe a highly sensitive and specific nuclear magnetic
resonance-based assay which allows the simultaneous quantification of ATP and reactive
oxygen species using small amounts of mitochondrial isolates or permeabilized cells. Their
novel assay also avoids the problem of changing mitochondrial membrane potential while
ADP is converted to ATP, as occurs in conventional assays. Accurate detection of mito-
chondrial superoxide especially in living cells remains a difficult task. Werner Koopman and
coworkers describe a live-cell microscopy-based method for detecting superoxide in both
mitochondria and the entire cell using dihydroethidium. Boronate-based probes were
developed over the last decade for detection of hydrogen peroxide and peroxynitrite in
biological systems. However, most boronates lack specificity needed to distinguish between
hydrogen peroxide and peroxynitrite within a complex biological system. Jacek Zielonka
and colleagues describe how a newly developed mitochondria-targeted phenylboronic acid
can be used to detect and differentiate peroxynitrite-dependent and independent probe
oxidation. Time-resolved fluorescence spectrometry can be used to detect and characterize
mitochondrial metabolic oxidative changes by means of endogenous fluorescence. Alzbeta
Marcek Chorvatova and coworkers describe the detection and measurement of endogenous
mitochondrial NAD(P)H fluorescence in living cells in vitro using fluorescence lifetime
spectrometry imaging after excitation with a 405 nm picoseconds laser. Quantifying the
mitochondrial membrane potential is essential for understanding mitochondrial function.
Most of the current methodologies are based on the accumulation of cation indicators.
Roger Springett describes a new methodology which allows calculating the membrane
potential from the measured oxidation states of the b-hemes. To better understand the
impact of oxygen on cellular function, James Hynes and Conn Carey outline the procedure
for measuring in situ oxygenation of cells in 2D and 3D cultures. These authors also
illustrate how the impact of drug treatment on cell oxygenation can be assessed and how
viii Preface

the link between oxygenation and glycolytic metabolism can be examined. Egbert Mik and
Floor Harms have developed a method called Protoporphyrin IX—Triplet State Lifetime
Technique as a potential tool for noninvasive monitoring of mitochondrial function in the
clinic. In their chapter they describe the application of mitochondrial respirometry for
monitoring mitochondrial oxygen tension and mitochondrial oxygen consumption in the
skin of experimental animals. The selective monitoring of mitochondria-produced hydro-
gen peroxide inside living systems can be challenging. Alexander Lippert and colleagues
describe the synthesis of the small molecular probe MitoPY1 and its application for measur-
ing hydrogen peroxide in vitro and in live cells. The authors also provide an example pro-
cedure for measuring mitochondrial hydrogen peroxide in a cell culture model of Parkinson's
disease. Erich Gnaiger and colleagues describe how the Amplex Red assay can be used to
detect hydrogen peroxide production in combination with the simultaneous assessment of
mitochondrial bioenergetics by high-resolution respirometry. They have optimized instru-
mental and methodological parameters to analyze the effects of various substrate, uncou-
pler, and inhibitor titrations (SUIT) on respiration versus hydrogen peroxide production.
The authors also show an application example using isolated mouse brain mitochondria as
an experimental model for the simultaneous measurement of mitochondrial respiration and
hydrogen peroxide production in SUIT protocols. Andrey Abramov and Fernando
Bartolome describe a strategy for assessing NADH/NAD(P)H and FAD autofluorescence
in a time course-dependent manner. Their method provides information about NADH and
FAD redox indexes both reflecting the activity of the mitochondrial electron transport
chain. Their analysis of NADH autofluorescence after induction of maximal respiration can
also offer information about the pentose phosphate pathway activity where glucose can be
alternatively oxidized instead of pyruvate. Coenzyme Q10 (CoQ10) is an essential part of
the mitochondrial respiratory chain. Outi Itkonen and Ursula Turpeinen describe an accu-
rate and sensitive liquid chromatography tandem mass spectrometry method for the deter-
mination of mitochondrial CoQ10 in isolated mitochondria.
Assessing bioenergetic parameters of human pluripotent stem cells (hPSCs), including
embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provides consid-
erable insight into their mitochondrial functions and cellular properties, which allows
exposing potential energetic defects caused by mitochondrial diseases. Alessandro Prigione
and Vanessa Pfiffer describe a method that facilitates the assessment of the bioenergetic
profiles of hPSCs in a noninvasive fashion, while requiring only small sample sizes and
allowing for several replicates.
Due to the complexity of the interactions involved at the different levels of integration
in organ physiology, current molecular analyses of pathologies should be combined with
integrative approaches of whole organ function. By combining the principles of control
analysis with noninvasive 31P NMR measurement of the energetic intermediates and simul-
taneous measurement of heart contractile activity, Philippe Diolez and colleagues have
developed MoCA (Modular Control and Regulation Analysis), which is an integrative
approach designed to study in situ control and regulation of cardiac energetics during con-
traction in intact beating perfused isolated heart. In their review chapter the authors pres-
ent selected examples of the applications of MoCA to isolated intact beating heart, and they
also discuss wider application to cardiac energetics under clinical conditions with the direct
study of heart pathologies.
Mitochondrial proteins encoded on the cytosolic ribosomes carry specific patterns in
the precursor sequence needed for mitochondrial import. Rita Casadio and colleagues
discuss the feasibility of utilizing computational methods for detecting such mitochondrial
 Preface ix

targeting peptides in polypeptide sequences. These authors also introduce their newly
implemented web server and demonstrate its application to the whole human proteome for
detecting mitochondrial targeting peptides. Fabiana Perocchi and Yiming Cheng describe
evolutionary biology approaches for studying mitochondrial physiology. One strategy,
which they refer to as “comparative physiology,” allows the de novo identification of mito-
chondrial proteins involved in a physiological function. Another approach known as “phy-
logenetic profiling” allows predicting the function of uncharacterized proteins as well as
functional interactions by comparing phylogenetic profiles of uncharacterized and known
components. Besides DNA mutations, faulty posttranslational modifications can also cause
malfunction of mitochondrial proteins. Suresh Mishra and colleagues describe procedures
for the isolation of mitochondria from cells and for separating the mitochondrial proteins
by two-dimensional gel electrophoresis. The employment of antibodies specific to each
posttranslational modification allows them to assess posttranslational modifications of mito-
chondrial proteins. Posttranslational protein glutathionylation regulates protein function in
response to cellular redox changes and is involved in carbon monoxide-induced cellular
pathways. Helena Viera and Ana S. Almeida describe a technique for the assessment of
mitochondrial protein glutathionylation in response to carbon monoxide exposure.
High-resolution melting (HRM) allows detecting homozygous or heterozygous point
sequence variants and small deletions within specific PCR products. Marketa Tesarova and
colleagues provide an updated HRM-based protocol for routine variant screening of nuclear
genes encoding assembly factors and structural subunits of cytochrome c oxidase (COX).
Their general recommendations given for HRM analysis are applicable for examining any
genetic region of interest. Anton Vila-Sanjurjo and colleagues have designed a computa-
tional approach named Heterologous Inferential Analysis or HIA for making predictions
on the disruptive potential of a large subset of mt-rRNA variants. The authors demonstrate
that in the case of certain mitochondrial variants for which sufficient information regarding
their genetic and pathological manifestation is available, HIA data alone can be used to
predict their pathogenicity.
Mitochondria play a key role in apoptosis. Vladimir Gogvadze and coworkers describe
how to evaluate the release of intermembrane space proteins during apoptosis, alterations
in the mitochondrial membrane potential, and oxygen consumption in apoptotic cells.
Fluorescent lifetime imaging microscopy-Förster resonant energy transfer (FLIM-FRET) is
a high-resolution technique for the detection of protein interactions in live cells. David
Andrews and colleagues provide a detailed protocol for applying this technique to assess the
interaction between BclXL and Bad at the mitochondrial outer membrane in live MCF7
breast cancer cells. Mitochondrial Ca2+ uptake is essential for regulating mitochondrial
function. Markus Waldeck-Weiermair and colleagues analyze the benefits and drawbacks of
various established old and new techniques to assess dynamic changes of mitochondrial
Ca2+ concentrations in a wide range of applications.
Untargeted lipidomics profiling by liquid chromatography-mass spectrometry (LC-
MS) allows the examination of lipids without any bias towards specific classes of lipids.
Bruce Kristal and group describe a workflow including the isolation of mitochondria from
liver tissue, followed by mitochondrial lipid extraction and the LC-MS conditions used for
data acquisition. The authors also highlight how, in this method, all ion fragmentation can
be used for the identification of species of lower abundances, which are often missed by
data-dependent fragmentation techniques.
Mitochondrial dynamics, i.e., mitochondrial location, number, and morphology, has an
essential function in numerous physiological and pathophysiological phenomena in the
x Preface

developing and adult human heart. Elizabeth Lipke and colleagues describe the application
of a computer-based tool (MATLAB, MQM) to quantify mitochondrial changes, in par-
ticular number, area, and location of mitochondria, during human pluripotent stem cell
differentiation into spontaneously contracting cardiomyocytes. Helena Bros and coworkers
present an ex vivo method for monitoring the movement of mitochondria within myelin-
ated sensory and motor axons from spinal nerve roots.

Volume II

The development of mitochondria-targeted pharmaceutical nanocarriers began at the end

of the 1990s with an accidental discovery of the vesicle-forming capacity of dequalinium
chloride. Volkmar Weissig describes a detailed protocol for the preparation, characteriza-
tion, and application of dequalinium-based nano vesicles called DQAsomes. Whether small
molecule xenobiotics (biocides, drugs, probes, toxins) will target mitochondria in living
cells without the assistance of any mitochondria-targeted delivery system can be predicted
using an algorithm derived from QSAR modeling and is described in detail by Richard
Horobin. Small molecules can be physicochemically targeted to mitochondria via conjuga-
tion to mitochondriotropic triphenylphosphonium cations. Utilizing this strategy, Richard
Hartley describes the preparation of MitoB and MitoP as exomarkers of mitochondrial
hydrogen peroxide. Gerard D’Souza and his group describe the use of triphenylphospho-
nium cations for the preparation of phospholipid conjugates which in turn are the basis for
preparing mitochondria-targeted liposomes. Triphenylphosphonium cations are used by
Jung-Joon Min and Dong-Yeon Kim for the synthesis of 18F-labeled fluoroalkyl triphe-
nylphosphonium conjugates as mitochondrial voltage sensors for PET myocardial imaging.
Fernanda Borges and her group describe the utilization of triphenylphosphonium cations
for the development and application of a new antioxidant based on dietary cinnamic acid.
Tamer Elbayoumi and colleagues have utilized the intrinsic mitochondriotropism of
Genistein to design mitochondria-targeted cationic lipid-based nanocarrier systems includ-
ing micelles and nanoemulsions. Since Genistein, a major soy isoflavone, exhibits extensive
proapoptotic anticancer effects which are mediated predominantly via induction of mito-
chondrial damage, this delivery system is potentially suited to enhance anticancer efficacy of
different coformulated chemotherapeutic agents. Shanta Dhara and her group outline in
one chapter the synthesis and characterization of a functionalized polymer for building
mitochondria-targeted nanoparticles (NPs), and in a second chapter she describes the
application of such mitochondria-specific nanoparticles for the delivery of a photosensitizer
to mitochondria for photodynamic therapy. Hideyoshi Harashima and Yuma Yamada
describe the construction and application of a mitochondria-targeted dual function
liposome-based nanocarrier termed DF-MITO-Porter.
The utilization of α-aminophosphonates as external probes in combination with 31P-NMR
allows the simultaneous pH measurements of cytosolic and acidic compartments in normal
and stressed cultured cells. Sylvia Pietria’s group has developed this strategy further by
using triphenylphosphonium derivatives of aminophosphonates as mitochondria-targeted
pH probes. The authors describe the synthesis and 31P-NMR pH titrating properties of
such mitochondria-targeted pH probes as well as their application in green alga cultures.
The formation of reactive oxygen species (ROS) in the inner mitochondrial membrane can
cause mitochondrial dysfunction eventually followed by induction of apoptosis. Antioxidants
conjugated with mitochondria-targeted, membrane-penetrating cations can be used to
 Preface xi

scavenge ROS inside mitochondria. Vladimir Skulachev and coworkers describe some
essential methodological aspects of the application of mitochondria-targeted cations
belonging to the MitoQ and SkQ groups which have shown promise for treating oxidative
stress-related pathologies. Lucia Biasutto and colleagues describe a step-by-step procedure
for synthesizing mitochondria-targeted derivatives of resveratrol and quercetin, two plant
polyphenols exhibiting potential health-promoting properties, as well as a method for
assessing their mitochondrial accumulation.
The mitochondrial respiratory chain is stress-responsive and responds to mitochondri-
ally targeted anticancer agent by destabilization and induction of massive ROS production
eventually leading to apoptosis. Jiri Neuzil’s group has developed mitochondrially targeted
anticancer agents epitomized by the mitochondrially targeted analogue of the redox-silent
compound vitamin E succinate, which belongs to the group of agents that kill cancer cells
via their mitochondria-destabilizing activity. The authors describe the use of native blue gel
electrophoresis and clear native electrophoresis coupled with in-gel activity assays as meth-
ods of choice for trying to understand the molecular mechanism of the effect of such
mitochondria-destabilizing agents. Many low-molecular-weight agents that may be of
potential clinical relevance act by targeting mitochondria, where they may suppress mito-
chondrial respiration. Jiri Neuzil and coworkers describe the methodology for assessing
respiration in cultured cells as well as in tumor tissue exposed to mitochondria-targeted
anticancer agents.
Nina Entelis, Ivan Tarassov, and colleagues have developed mitochondria-targeting
RNA vectors for the delivery of therapeutic oligoribonucleotides into human mitochondria.
Their group provides a detailed protocol for the transfection of cultured human cells with
small recombinant RNA molecules as well as methods for characterizing the mitochondrial
transfection efficiency. Genetic transformation of mitochondria in multicellular eukaryotes is
of fundamental importance for basic investigations and for applications to gene therapy or
biotechnology. Andre Dietrich’s group has developed a strategy to target nuclear transgene-
encoded RNAs into mitochondria in plants. In their chapter they give a detailed protocol for
mitochondrial targeting of trans-cleaving ribozymes destined to knockdown organelle RNAs
for regulation studies, inverse genetics, and biotechnological purposes.
Allotopic expression (AE) of mitochondrial proteins, i.e., nuclear localization and tran-
scription of mtDNA genes followed by cytoplasmic translation and transport into mito-
chondria, has been suggested as a strategy for gene replacement therapy in patients
harboring mitochondrial DNA mutations. Carl Pinkert and David Dunn describe the use
of AE for transgenic mouse modeling of the pathogenic human T8993G mutation in
mtATP6 as a case study for designing AE animal models.
There is increasing evidence that exposure to air pollutants is associated with human
disease and may act through epigenetic modification of the nuclear genome, but there have
been few publications describing their impact upon the mitochondrial genome. Hyang-
Min Byun and Timothy M. Barrow describe a protocol for the isolation of mitochondrial
DNA from peripheral blood samples and the analysis of 5-methlycytosine content by
bisulfite-pyrosequencing. Stanislaw Pshenichnyuk and Alberto Modelli describe the appli-
cation of two complementary experimental techniques, Electron Transmission Spectroscopy
(ETS) and Dissociative Electron Attachment Spectroscopy (DEAS), for studying the trans-
fer of electrons unto xenobiotics in the intermembrane space of mitochondria. Additional
support of experimental procedures by suitable quantum-chemical calculations is described
in detail and illustrated by an example of ETS/DEAS study of rhodamine which shows rich
fragmentation under gas-phase resonance electron attachment.
xii Preface

The link between mitochondrial dynamics and human pathologies has spawned signifi-
cant interest in developing methods for screening proteins involved in mitochondrial
dynamics as well as small molecules that modulate mitochondrial dynamics. Antonio
Zorzano and Juan Pablo Munoz describe in their chapter functional screening protocols for
the in vitro examination of mitochondrial parameters such as mitochondrial morphology,
reactive oxygen species (ROS) levels, mitochondrial calcium, and oxygen consumption
rate. Dysfunctional mitochondria communicate via retrograde signaling with the nucleus
leading to cell stress adaptation by changes in nuclear gene expression. Mitochondria to
nucleus signaling pathways have been widely studied in Saccharomyces cerevisiae, where
retrograde-target gene expression is regulated by RTG genes. Sergio Giannattasio and
coworkers describe a method for the assessment of the mitochondrial retrograde pathway
activation in yeast cells based on monitoring the mRNA levels of a variety of RTG-target
genes. Adaptations to energy stress or altered physiological condition can be assessed by
measuring changes of multiple bioenergetic parameters. Dmitri Papkovsky and Alexander
Zhdanov describe a simple methodology for high-throughput multiparametric assessment
of cell bioenergetics, called Cell Energy Budget (CEB) platform, and demonstrate its prac-
tical use with cell models.
Viable disease models for mitochondrial DNA diseases are much needed for elucidating
genotype/phenotype relationships and for improving disease management. Alessandro
Prigione discusses the potential advantages and critical challenges for the utilization of
induced pluripotent stem cells (iPSCs) from patients affected by mtDNA disorders for
modeling debilitating mtDNA diseases.
Heteroplasmic mice can be used for studying the segregation of different mtDNA hap-
lotypes in vivo against a defined nuclear background. Thomas Kolbe and colleagues describe
two methods involving either the transfer of ooplasm or the fusion of two blastomeres for
the creation of such mice models.
H. van der Spek and coworkers describe a robust and efficient method for visualizing
and quantifying mitochondrial morphology in Caenorhabditis elegans, which is a preferred
model for studying mitochondrial deficiencies caused by disease or drug toxicity. Their
method allows for a comprehensive analysis of mitochondrial morphology. Mitochondrial
DNA (mtDNA) is a useful and reliable biomarker of UV-induced genetic damage in both
animal and human skin. Mark Birch-Machin and Amy Bowman describe in their protocol
chapter the assessment of UV-induced mtDNA damage, including the extraction of cellular
DNA, qPCR to determine the relative amount of mtDNA, and qPCR to determine
UV-induced damage within a long strand of mtDNA.
Mitochondrial dysfunction is associated with the pathogenesis of septic disorders, even-
tually leading to a decline in energy supply. Matthias Hecker and colleagues give a protocol
for assessing the influence of short- and medium-chain fatty acids on mitochondrial respira-
tion using high-resolution respirometry under inflammatory and baseline conditions. Hun-
Kuk Park and Gi-Ja Lee describe the application of Atomic Force Microscopy (AFM)-based
shape analysis for the characterization of nanostructural changes of mitochondria. The
authors use AFM to study mitochondrial swelling in heart mitochondria during myocardial
ischemia-reperfusion injury employing a rat model. In some tissues such as the heart,
abnormal mitochondrial fusion and fission can go along with mitochondrial apoptosis, but
its contribution as cause vs. a consequence remains to be defined. Catherine Brenner and
her group give a protocol for the isolation of fresh mitochondria from rat heart by a
procedure adapted to the myofibrillar structure of the tissue, and they describe several min-
iaturized enzymatic assays for probing mitochondria-mediated apoptosis. The pathogenesis
 Preface xiii

of Parkinson’s disease (PD) is poorly understood and under intensive investigation.

Mitochondrial dysfunction has been linked to the sporadic form of PD. Daniella Arduino’s
group describes a method for the generation of cytoplasmic hybrid cells as a cellular model
of sporadic PD which is based upon the fusion of platelets harboring mtDNA from PD
patients with cells in which the endogenous mtDNA has been depleted. JC-1, a commer-
cially available fluorescence dye, is widely used for measuring changes in the mitochondrial
membrane potential. Dorit Ben-Shachar and coworkers show that JC-1 can also be used to
follow alterations in mitochondrial distribution and mitochondrial network connectivity.
The authors describe various applications of JC-1 staining to study mitochondrial abnor-
malities in different cell types derived from schizophrenia patients and healthy subjects.
We are extremely grateful to all authors for having spent significant parts of their valu-
able time to contribute to this book. It is our hope that together we have succeeded in
providing an essential source of know-how and a source of inspiration to all researchers
who are as fascinated as we are about this tiny organelle which so much seems to control
life and death of a single cell and the whole organism alike. Last but not least we would like
to thank John Walker, the series editor of Methods in Molecular Biology, for having accepted
our book proposal, which originated from our efforts in organizing a series of annual con-
ferences on Targeting Mitochondria, the fifth one of which has taken place in October
2014 in Berlin, Germany (www.targeting-mitochondria.com). We are also grateful to John
Walker for his unlimited guidance and help throughout the whole process.
I (V.W.) would like to thank my wife, Angelina Lynn Weikel, for her understanding and
strong support throughout the duration of this project.

Glendale, AZ, USA Volkmar Weissig

Paris, France Marvin Edeas

References

1. Luft R (1994) The development of mitochon- 7. Miquel J, Fleming J (1986) Oxygen radical-
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2. Luft R, Ikkos D, Palmieri G, Ernster L, Afzelius degenerative diseases. Alan R. Liss, New York,
B (1962) A case of severe hypermetabolism of NY, pp 51–74
nonthyroid origin with a defect in the mainte- 8. Anderson S, Bankier AT, Barrell BG et al. (1981)
nance of mitochondrial respiratory control: a Sequence and organization of the human mito-
correlated clinical, biochemical, and morpho- chondrial genome. Nature 290:457–465
logical study. J Clin Invest 41:1776–1804 9. Chomyn A, Cleeter MW, Ragan CI, Riley M,
3. Nass S, Nass MM (1963) Intramitochondrial Doolittle RF, Attardi G (1986) URF6, last
fibers with DNA characteristics. II. Enzymatic unidentified reading frame of human mtDNA,
and other hydrolytic treatments. J Cell Biol codes for an NADH dehydrogenase subunit.
19:613–629 Science 234:614–618
4. Mitchell P (1961) Coupling of phosphoryla- 10. Chomyn A, Mariottini P, Cleeter MW et al.
tion to electron and hydrogen transfer by a (1985) Six unidentified reading frames of
chemi-osmotic type of mechanism. Nature human mitochondrial DNA encode compo-
191:144–148 nents of the respiratory-chain NADH dehy-
5. Harman D (1972) Free radical theory of aging: drogenase. Nature 314:592–597
dietary implications. Am J Clin Nutr 11. Holt IJ, Harding AE, Morgan-Hughes JA
25:839–843 (1988) Deletions of muscle mitochondrial
6. Harman D (1972) The biologic clock: the DNA in patients with mitochondrial myopa-
mitochondria? J Am Geriatr Soc 20:145–147 thies. Nature 331:717–719
xiv Preface

12. Wallace DC, Singh G, Lott MT et al. (1988) 14. Weissig V (2003) Mitochondrial-targeted drug
Mitochondrial DNA mutation associated with and DNA delivery. Crit Rev Ther Drug Carrier
Leber’s hereditary optic neuropathy. Science Syst 20:1–62
242:1427–1430 15. Edeas M, Weissig V (2013) Targeting mitochon-
13. Brown GC, Nicholls DG, Cooper CE (1999) dria: strategies, innovations and challenges: the
Mitochondria and cell death. Princeton future of medicine will come through mitochon-
University Press, Princeton, NJ, pp vii–viii dria. Mitochondrion 13:389–390
Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix

1 Preparation of “Functional” Mitochondria: A Challenging Business . . . . . . . . 1

Stefan Lehr, Sonja Hartwig, and Jorg Kotzka
2 Isolation and Quality Control of Functional Mitochondria . . . . . . . . . . . . . . . 9
Sonja Hartwig, Jorg Kotzka, and Stefan Lehr
3 Isolation of Mitochondria-Associated ER Membranes (MAMs)
and Glycosphingolipid-Enriched Microdomains (GEMs)
from Brain Tissues and Neuronal Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Ida Annunziata, Annette Patterson, and Alessandra d’Azzo
4 Fluorescence Analysis of Single Mitochondria with Nanofluidic Channels . . . . 35
Ted Pham, Katayoun Zand, Douglas Wallace, and Peter Burke
5 Optical Microwell Arrays for Large-Scale Studies of Single Mitochondria
Metabolic Responses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Venkata Suresh Vajrala, Emmanuel Suraniti, Bertrand Goudeau,
Neso Sojic, and Stéphane Arbault
6 Deep Resequencing of Mitochondrial DNA . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Brendan A.I. Payne, Kristian Gardner, Jonathan Coxhead,
and Patrick F. Chinnery
7 Single-Cell Analysis of Mitochondrial DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Brendan A.I. Payne, Lynsey Cree, and Patrick F. Chinnery
8 A High-Throughput Next-Generation Sequencing Assay
for the Mitochondrial Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Shale Dames, Karen Eilbeck, and Rong Mao
9 Rapid Mitochondrial DNA Isolation Method for Direct Sequencing . . . . . . . . 89
Wilber Quispe-Tintaya, Ryan R. White, Vasily N. Popov, Jan Vijg,
and Alexander Y. Maslov
10 Analysis of Mitochondrial DNA and RNA Integrity
by a Real-Time qPCR-Based Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Wei Wang, Ying Esbensen, Katja Scheffler, and Lars Eide
11 Mitochondria-Targeted RNA Import . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Geng Wang, Eriko Shimada, Mahta Nili, Carla M. Koehler,
and Michael A. Teitell
12 Accurate Measurement of Circulating Mitochondrial DNA Content
from Human Blood Samples Using Real-Time Quantitative PCR . . . . . . . . . . 117
Saima Ajaz, Anna Czajka, and Afshan Malik
13 mTRIP: An Imaging Tool to Investigate Mitochondrial DNA
Dynamics in Physiology and Disease at the Single-Cell Resolution. . . . . . . . . . 133
Laurent Chatre and Miria Ricchetti

xv
xvi Contents

14 Simultaneous Quantification of Mitochondrial ATP and ROS Production . . . . 149

Liping Yu, Brian D. Fink, and William I. Sivitz
15 Live-Cell Assessment of Mitochondrial Reactive Oxygen Species
Using Dihydroethidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Marleen Forkink, Peter H.G.M. Willems, Werner J.H. Koopman,
and Sander Grefte
16 Detection and Differentiation Between Peroxynitrite
and Hydroperoxides Using Mitochondria-Targeted Arylboronic Acid . . . . . . . 171
Jacek Zielonka, Adam Sikora, Jan Adamus, and B. Kalyanaraman
17 Time-Resolved Spectrometry of Mitochondrial NAD(P)H Fluorescence
and Its Applications for Evaluating the Oxidative State in Living Cells . . . . . . . 183
Julia Horilova, Hauke Studier, Zuzana Nadova, Pavol Miskovsky,
Dusan Chorvat Jr, and Alzbeta Marcek Chorvatova
18 Novel Methods for Measuring the Mitochondrial Membrane Potential . . . . . . 195
Roger Springett
19 High-Throughput Real-Time Analysis of Cell Oxygenation
Using Intracellular Oxygen-Sensitive Probes . . . . . . . . . . . . . . . . . . . . . . . . . . 203
James Hynes and Conn Carey
20 In Vivo Assessment of Mitochondrial Oxygen Consumption . . . . . . . . . . . . . . 219
Floor A. Harms and Egbert G. Mik
21 Imaging Mitochondrial Hydrogen Peroxide in Living Cells . . . . . . . . . . . . . . . 231
Alexander R. Lippert, Bryan C. Dickinson, and Elizabeth J. New
22 Simultaneous High-Resolution Measurement of Mitochondrial Respiration
and Hydrogen Peroxide Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Gerhard Krumschnabel, Mona Fontana-Ayoub, Zuzana Sumbalova,
Juliana Heidler, Kathrin Gauper, Mario Fasching, and Erich Gnaiger
23 Measurement of Mitochondrial NADH and FAD Autofluorescence
in Live Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Fernando Bartolomé and Andrey Y. Abramov
24 Mitochondrial Coenzyme Q10 Determination via Isotope Dilution
Liquid Chromatography Tandem Mass Spectrometry . . . . . . . . . . . . . . . . . . . 271
Outi Itkonen and Ursula Turpeinen
25 Assessing the Bioenergetic Profile of Human Pluripotent Stem Cells . . . . . . . . 279
Vanessa Pfiffer and Alessandro Prigione
26 Integrative Methods for Studying Cardiac Energetics . . . . . . . . . . . . . . . . . . . 289
Philippe Diolez, Véronique Deschodt-Arsac, Guillaume Calmettes,
Gilles Gouspillou, Laurent Arsac, Pierre dos Santos, Pierre Jais,
and Michel Haissaguerre
27 Computer-Based Prediction of Mitochondria-Targeting Peptides . . . . . . . . . . 305
Pier Luigi Martelli, Castrense Savojardo, Piero Fariselli, Gianluca Tasco,
and Rita Casadio
28 Prediction of Mitochondrial Protein Function
by Comparative Physiology and Phylogenetic Profiling . . . . . . . . . . . . . . . . . . 321
Yiming Cheng and Fabiana Perocchi
 Contents xvii

29 Assessment of Posttranslational Modification of Mitochondrial Proteins . . . . . 331

Sudharsana R. Ande, G. Pauline Padilla-Meier, and Suresh Mishra
30 Assessment of Mitochondrial Protein Glutathionylation
as Signaling for CO Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Ana S. Almeida and Helena L.A. Vieira
31 High-Resolution Melting Analysis for Identifying Sequence Variations
in Nuclear Genes for Assembly Factors and Structural Subunits
of Cytochrome C Oxidase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Alžběta Vondráčková, Kateřina Veselá, Jiří Zeman, and Markéta Tesařová
32 Heterologous Inferential Analysis (HIA) as a Method to Understand
the Role of Mitochondrial rRNA Mutations in Pathogenesis . . . . . . . . . . . . . . 369
Joanna L. Elson, Paul M. Smith, and Antón Vila-Sanjurjo
33 Analysis of Mitochondrial Dysfunction During Cell Death. . . . . . . . . . . . . . . . 385
Vladimir Gogvadze, Sten Orrenius, and Boris Zhivotovsky
34 The Use of FLIM-FRET for the Detection of Mitochondria-Associated
Protein Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
Elizabeth J. Osterlund, Qian Liu, and David W. Andrews
35 Assessment of Mitochondrial Ca2+ Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421
András T. Deak, Claire Jean-Quartier, Alexander I. Bondarenko,
Lukas N. Groschner, Roland Malli, Wolfgang F. Graier,
and Markus Waldeck-Weiermair
36 Qualitative Characterization of the Rat Liver Mitochondrial Lipidome
Using All Ion Fragmentation on an Exactive Benchtop Orbitrap MS . . . . . . . . 441
Susan S. Bird, Irina G. Stavrovskaya, Rose M. Gathungu, Fateme Tousi,
and Bruce S. Kristal
37 Characterization of Mitochondrial Populations
During Stem Cell Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
Petra Kerscher, Blakely S. Bussie, Katherine M. DeSimone,
David A. Dunn, and Elizabeth A. Lipke
38 An Ex Vivo Model for Studying Mitochondrial Trafficking in Neurons . . . . . . 465
Helena Bros, Raluca Niesner, and Carmen Infante-Duarte

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473
Contributors

ANDREY Y. ABRAMOV • Neurosciences group, Instituto de Investigacion Hospital 12 de

Octubre (i+12), Av Cordoba, Madrid, Spain
JAN ADAMUS • Institute of Applied Radiation Chemistry, Lodz University of Technology,
Lodz, Poland
SAIMA AJAZ • Diabetes Research Group, School of Medicine, King’s College London, London, UK
ANA S. ALMEIDA • Chronic Diseases Research Center (CEDOC), Faculdade de Ciências
Médicas, Universidade Nova de Lisboa, Lisbon, Portugal; Instituto de Biologia
Experimental e Tecnológica (IBET), Oeiras, Portugal; Instituto de Tecnologia Química
e Biológica (ITQB), Universidade Nova de Lisboa, Oeiras, Portugal
SUDHARSANA R. ANDE • Department of Internal Medicine, University of Manitoba,
Winnipeg, MB, Canada
DAVID W. ANDREWS • Department of Biochemistry, University of Toronto, Toronto, ON,
Canada; Odette Cancer Research Program, Sunnybrook Research Institute, Toronto,
ON, Canada
IDA ANNUNZIATA • Department of Genetics, St. Jude Children Research Hospital, Memphis,
TN, USA
STÉPHANE ARBAULT • ISM, CNRS, University of Bordeaux, Pessac, France
LAURENT ARSAC • INSERM U1045, Centre de Recherche Cardio-Thoracique de Bordeaux
& LIRYC, Institut de Rythmologie et Modélisation Cardiaque, Université de Bordeaux,
Lormont, France
FERNANDO BARTOLOMÉ • Neurosciences group, Instituto de Investigacion Hospital 12 de
Octubre (i+12), Av Cordoba, Madrid, Spain
SUSAN S. BIRD • Thermo Fisher Scientific, Cambridge, MA, USA
ALEXANDER I. BONDARENKO • Institute of Molecular Biology and Biochemistry, Center
of Molecular Medicine, Medical University of Graz, Graz, Austria
HELENA BROS • NeuroCure Clinical Research Center, Institute for Medical Immunology,
Charité-Universitätsmedizin Berlin, Berlin, Germany; Experimental and Clinical
Research Center, a joint cooperation between the Charité-Universitätsmedizin Berlin
and the Max-Delbrück Center for Molecular Medicine, Berlin, Germany
PETER BURKE • Integrated Nanosystem Research Facility, Electrical Engineering
and Computer Science, University of California Irvine, Irvine, CA, USA
BLAKELY S. BUSSIE • Department of Chemical Engineering, Auburn University, Auburn,
AL, USA
GUILLAUME CALMETTES • Department of Medicine (Cardiology), David Geffen School of
Medicine, University of California, Los Angeles, CA, USA
CONN CAREY • Luxcel Biosciences Ltd., BioInnovation Centre, UCC, Cork, Ireland
RITA CASADIO • Biocomputing Group, CIRI Health Sciences & Technologies (HST),
University of Bologna, Bologna, Italy
LAURENT CHATRE • Team “Stability of nuclear and mitochondrial DNA”, Unité de
Génétique Moléculaire des Levures, CNRS UMR3525, Institut Pasteur, Paris, France
YIMING CHENG • Gene Center, Ludwig-Maximilians-Universität, Munich, Germany;
Institute of Human Genetics, Helmholtz Zentrum Munich, Munich, Germany

xix
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