1. Metabolism of energy in Cells - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/2611/11/11_chapter 1.pdf · Chapter 1 Aijaz Ahmad Wani, PhD thesis, 2007 1 1. Metabolism of

Chapter 1

Aijaz Ahmad Wani, PhD thesis, 2007 1

1. Metabolism of energy in Cells To do mechanical work, organisms need energy. This energy is used for the

active transport of molecules, ions and synthesis of macromolecules and other

biomolecules. The free energy used in these processes comes from the

environment. However, energy from the nutrients cannot be utilized directly

by the cell. It has to be converted to chemical energy and stored in the form of

useful energy. This energy conversion occurs in mitochondria and is known

as oxidative phosphorylation (Figure 1.1). Under aerobic conditions, the

glucose metabolism starts with glycolysis in the cytosol and the overall

reaction results in conversion of glucose into two pyruvate molecules. A net

energy yield of two adenosine triphosphate (ATP) molecules is generated by

this reaction and also involves the reduction of two nicotinamide adenine

dinucleotide molecules (NADH). Under aerobic conditions, the NADH and

pyruvate are transferred to mitochondria. When pyruvate enters the

mitochondrial matrix, it reacts with coenzyme A to form acetyl-CoA, CO2 and

reduced NADH, the acetyl-CoA being further oxidized in the Krebs cycle. The

net energy yield of the overall oxidation of two pyruvate molecules from one

glucose molecule is conserved in ten NADH, two flavin adenine dinucleotide

(FADH2) and two ATP molecules. Under anaerobic conditions pyruvate is

converted into lactate by the enzyme lactate dehydrogenase in a reversible

reaction that enables glycolysis to proceed transiently in active tissues such

as contracting muscle. The lactate formed in these active tissues is secreted

into the bloodstream and then oxidized back to pyruvate in the liver (Berg et

al. 2002)

The acetyl-CoA can also be generated for Krebs cycle by the oxidation of fatty

acids in the mitochondrial matrix. Fatty acids are coupled to coenzyme A to

form acyl-CoA in the outer mitochondrial membrane. The long-chain acyl-

CoA esters are unable to traverse through mitochondrial membrane and are

carried across as carnitine esters. On the matrix side, the regenerated acyl-

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CoA molecules are shortened by two carbon atoms in a cyclic process called β-

oxidation. This continues until all the carbon atoms in the acyl-CoA have

been converted into two carbon acetyl-CoA molecules, which enter the Krebs

cycle. One molecule of NADH and one of FADH2 and two of acetyl-CoA are

produced in every cycle (Berg et al. 2002).

Figure 1.1: Energy metabolism in eukaryotic cells. Oxidation of nutrients in the cytosol and in the

mitochondria leads to adenosine triphosphate (ATP) synthesis by the oxidative phorphorylation system.

The ATP generated by this process is then used to drive biosynthetic reactions and other processes in

the cell that require energy.

2. Mitochondria Mitochondria are membrane-bound organelles present in almost all

eukaryotic cells except a few (Henze and Martin 2003). These organelles are

sometimes described as "cellular power plants", because they generate most

of the cell’s ATP supply that is used as a source of chemical energy. The

number of mitochondria in a cell varies by organism, tissue type or cell type.

Many cells possess only a single mitochondrion, while others can have several

million mitochondria (Alberts et al. 1994; Voet et al. 2006). A mitochondrion

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contains inner and outer membranes made up of phospholipid bilayers and

proteins. The two membranes are very much different in their properties.

Due to this double-membrane organization, mitochondrion is divided into five

compartments; the outer membrane, inner membrane, cristae (infoldings of

inner membrane) and the matrix. The size of mitochondrion ranges from 1 to 10 micrometers (μm).

The outer membrane encloses the entire organelle and has a protein-to-

phospholipid ratio similar to plasma membrane of eukaryotic cells (about 1:1

by weight). It contains many integral proteins called porins, which contain a

large internal channel (about 2-3 nm) that is permeable to all molecules of

about 5000 daltons or less (Henze and Martin 2003). Larger molecules can

only traverse the outer membrane by active transport through mitochondrial

membrane transport proteins. The outer membrane also contains enzymes

involved in activities such as elongation of fatty acids, oxidation of

epinephrine (adrenaline), and the degradation of tryptophan.

The space between outer and inner membrane is known as inter-membrane

space (IMS). The main function of IMS is nucleotide phosphorylation.

Channel proteins (porins) in the outer membrane allow free movement of ions

and small molecules into the IMS. The contents of the intermembrane space

are similar to that of the contents of cytoplasm. Enzymes destined for the

mitochondrial matrix can pass through this space via transport through

translocators. These are known as translocase of the outer membrane (TOM)

and transloacase of the inner mitochondrial membrane (TIM). The IMS tends

to have a low pH because of the proton gradient, which results when protons

are pumped from mitochondrial matrix into the intermembrane space during

the electron transport.

The inner mitochondrial membrane contains proteins with four types of

functions (Alberts et al. 1994): (a) Proteins carrying out the oxidation

reactions of the respiratory chain (b) ATP synthase, which makes ATP in the

matrix. (c) Specific transport proteins that regulate the passage of

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metabolites into and out of the matrix and (d) Protein import machinery. The

inner membrane contains more than 100 different polypeptides, and has a

very high protein to phospholipid ratio (more than 3:1 by weight, which is

about 1 protein for 15 phospholipids). It is rich in unusual phospholipids

known as cardiolipin, which was first isolated from beef hearts. Unlike the

outer membrane, the inner membrane does not contain porins, and is highly

impermeable. Most of the ions and molecules require special membrane

transporters to enter or exit the matrix. In addition, there is a membrane

potential across the inner membrane. The inner mitochondrial membrane is

compartmentalized into numerous cristae, which expand the surface area of

the inner mitochondrial membrane, enhancing its ability to generate ATP.

Mitochondria of cells, which have greater demand for ATP, such as muscle

and nerve cells, contain more cristae than typical liver mitochondria.

The matrix is the space enclosed by the inner membrane. The matrix is

important in the production of ATP with the help of the ATP synthase located

in the inner membrane. The matrix contains a mixture of hundreds of

enzymes, in addition to the special mitochondrial ribosomes, tRNAs, and

several copies of the mitochondrial genome. Of the enzymes, the major

functions include oxidation of pyruvate and fatty acids, and the citric acid

cycle (Alberts et al. 1994).

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Figure 1.2: Simplified structure of mitochondrion. Mitochondria are bounded by a double-membrane

system, consisting of inner and outer membranes. Folds of the inner membrane (cristae) extend into the

matrix.

2.1. The Respiratory Chain The respiratory chain in the inner mitochondrial membrane contains three

respiratory enzyme complexes through which electrons pass on their way

from NADH to O2. Although the respiratory chain usually proceeds in the

forward direction (towards the formation of water) due to the exergonic

nature of the reaction cascade, except for the final reaction, all of these steps

are fully reversible. In order to be reversed, sufficient energy must be

provided to drive the reaction in this direction. For example, the reducing

equivalents derived from succinate are usually carried by FAD (as FADH+H+).

These can be transferred to NAD (as NADH++H+) with the concomitant

hydrolysis of ATP. Electron transport across the other two phosphorylation

sites can also be reversed, again, only if sufficient energy is provided. The

respiratory chain consists of four major enzyme complexes (Figure 1.3)

located in the inner mitochondrial membrane. The enzymes of the respiratory

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chain are arranged so as to transport hydrogen ions from the matrix across

the inner membrane. When this occurs, a proton gradient develops in close

proximity to the F1F0 ATPase (ATP synthase) complex and provides sufficient

energy to drive ATP synthesis by causing a dehydration of ADP and Pi.

Reducing equivalents transported into the mitochondrial compartment by the

various substrate shuttle systems or generated in the compartment are

passed down the respiratory chain in carefully regulated steps. Reducing

equivalents enter the chain through the NAD-dehydrogenase complex

(complex I) via mitochondrial shuttles or via the FAD-ubiquinone complex

(complex II). With respect to the latter complex, reducing equivalents collect

via three pathways:

Succinate contributes its reducing equivalents to a flavoprotein with

an iron–sulfur center.

Glycerol 3-phosphate also uses FAD flavoprotein with an iron–sulfur

center, but it is a different protein.

The products of fatty acid oxidation (a mitochondrial process) are

picked up by a FAD-flavoprotein, transferred to an electron-

transferring flavoprotein, again with an iron–sulfur center, and then

transferred to ubiquinone.

In mammals, complex I consist of about 42 subunits (Weiss et al. 1981). Of

these, seven are encoded by the mitochondrial genome and the rest by the

nuclear genome synthesized on the ribosomes in the cytoplasm and imported

into the mitochondrial compartment. It is the largest of the four respiratory

chain complexes. Complex I is known as NADH-coenzyme Q reductase or

NADH dehydrogenase. As the name implies, this complex transfers a pair of

electrons from NADH to coenzyme Q, a lipid-soluble compound embedded in

the inner membrane. Complex I has a molecule of flavin mononucleotide

(FMN) and two binuclear iron-sulfur centers and four tetranuclear iron-

sulfur centers (Ohnishi 1993). Because of its FMN, it is called a flavoprotein.

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The complex catalyzes the transfer of electrons to complex III via ubiquinone

and this transfer is coupled with the vectorial transfer of protons across the

mitochondrial membrane. There are two distinct species of tightly bound

ubiquinones in complex I that differ in spin relaxation and redox properties. The transfer of electrons leads to the formation of a proton gradient (∆μH+)

that, in turn, drives ATP production. The stoichiometry of proton transfer for

complex I is 4H+/2e–. This distinguishes this complex from those that follow it

in the respiratory chain. The other two H+ translocating complexes (III and

IV) have a stoichiometry of 2 H+/2e–.

Complex II, succinate:quinone reductase (succinate dehydrogenase), is the

smallest of the respiratory chain complexes (Hederstedt and Ohnishi 1992).

None of the subunits of complex II are encoded by the mitochondrial genome.

The complex consists of four subunits with several different redox prosthetic

groups: a covalently bound FAD, three iron-sulfur clusters, and a cytochrome

b. The head of the complex protrudes out into the matrix where its FAD can

accept succinate-donated electrons from the citric acid cycle. Actually, this

enzyme is a component of the respiratory chain and the citric acid cycle. It too

is a flavoprotein because of its FAD content. The FAD is bound to a histidine

residue (Paudel 1994). When succinate is converted to fumarate in the citric

acid cycle, a concomitant reduction of FAD to FADH2 occurs. This FADH2

transfers its electrons to the iron-sulfur cluster, which, in turn, passes them

on to ubiquinone. Because of insufficient energy to elicit a proton gradient,

reducing equivalents and associated electrons entering the respiratory chain

via complex II yield only two ATPs via OXPHOS rather than the three ATPs

generated when entry occurs via complex I.

Once reducing equivalents enter the chain via site 1 or site 2 (the sites

correspond to entry via complex I or II), they are passed to complex III, the

ubiquinone-cytochrome bc1 reductase. This complex takes the electrons

passed to it from ubiquinone and then passes them to complex IV, cytochrome

c oxidase. This passage uses a unique redox pathway called the Q cycle

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(Slater 1985). Three different cytochromes (bc, b, c1) are involved as well as

an iron-sulfur protein. The iron is in the middle of a porphorin ring much like

that of hemoglobin and oscillates between the reduced and oxidized states

(ferrous to ferric).

The Q cycle begins when a molecule of reduced ubiquinone diffuses to the Qp

site on complex III near the outer face of the inner mitochondrial membrane.

An electron from the reduced ubiquinone is transferred to a mobile protein

called the Rieske protein. The electrons are then transferred to cytochrome c1.

This releases two H+ and leaves UQ–, a semiquinone anion form of

ubiquinone, at the Qp site. The second electron is then transferred to the bL

heme, converting UQ– to ubiquinone. The Rieske protein and cytochrome c1

are similar in structure; each has a globular domain and each is anchored to

the inner membrane by a hydophobic segment. The segments differ: the

Rieske protein has an N-terminal and the cytochrome c1 has a C-terminal.

The electron on the bL heme is passed to the bH heme against a membrane

potential of 0.15 V and is driven by the loss of redox potential as the electron

moves from bL to bH. The electron is then passed from bH to ubiquinone at the

second binding site, converting the ubiquinone to UQ–. The resulting UQ–

remains firmly bound to the Qn site. This completes the first half of the Q

cycle. The second half is similar in that a second molecule of reduced

ubiquinone is oxidized at the Qp site. One electron is passed to cytochrome c1

and the other is passed to heme bH. The bH electron is transferred to the

semiquinone anion UQ– at the Qn site. With the addition of two H+, this

produces UQH2. The UQH2 is released and returns to the coenzyme Q pool

and the Q cycle is complete. Actually, the Q cycle is an unbalanced proton

pump. Cytochrome c is a mobile electron carrier, as is ubiquinone. Electrons

travel from c to the water-soluble c1. The c1 associates loosely with the inner

mitochondrial membrane to acquire electrons from the iron-sulfur centers.

The c1 of complex III then migrates along the membrane in a reduced state so

as to give these electrons to complex IV.

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Complex IV, cytochrome-c oxidase, contains two heme centers (a and a3) as

well as two copper centers. The copper oscillates between the reduced

(cuprous) and oxidized (cupric) states. Complexes III and IV elicit a proton

gradient and thus ATP is formed at each of these sites. Complex IV accepts

the electrons from cytochrome c and directs them to molecular oxygen to form

water. The water thus formed quickly passes out of the compartment into the

cytoplasm.

2.2. Origin of mitochondria Mitochondria are only formed by the division of other mitochondria and

contain ribosomes and transfer RNAs that are similar to bacteria. They

contain their own DNA, which is circular as is true with bacteria, along with

their own transcriptional and translational machinery. Hence, it is generally

accepted that they were originally derived from endosymbiont prokaryotes.

Studies of mitochondrial DNA, which uses a variant genetic code, show that

the ancestor proto-mitochondrion was a member of the phylum

Proteobacteria (Futuyma and Douglas 2005). In particular, the pre-

mitochondrion was probably related to the rickettsias, group of eubacterial

obligate intracellular parasites (Gray et al. 1999). Infact, the most

mitochondrion-like bacterial genome found to date is that of Proteobacteria

Rickettsia Prowasekii (Andersson et al. 1998). The endosymbiotic hypothesis

suggests that mitochondria descended from specialized bacteria (probably

purple non-sulfur bacteria) that survived endocytosis by other cell types, and

became incorporated into the cytoplasm. The ability of symbiont bacteria to

conduct cellular respiration in host cells that had relied on glycolysis and

fermentation would have provided a considerable evolutionary advantage.

Similarly, host cells with symbiotic bacteria capable of photosynthesis would

also have an advantage. In both cases, the number of environments in which

the cells could survive would have been greatly expanded.

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The endosymbiotic relationship developed a few years ago and mitochondria

still show the signs of their ancient origin. Mitochondrial ribosomes in

mammals are the 70S type like bacteria, in contrast to the 80S ribosomes

found elsewhere in the cell (O’Brien 2003). One mitochondrion can contain 2-

10 copies of its genome (Wiesner et al. 1992). As in prokaryotes, there is a

very high proportion of coding DNA, and an absence of repeats, mitochondrial

genes are transcribed as multigenic transcripts, which are cleaved, and

polyadenylated to yield mature mRNAs. In humans, mitochondrial genes lack

introns (Anderson et al. 1981), conforming to the bacterial pattern. Further,

there are codon differences in mitochondria (Fernandez-Silva et al. 2003). In

the mitochondria, the UGA codon specifies tryptophan; AGA and AGG are

stop codons; and AUA, AUC, and AUU are each allowable start codons.

2.3. Functions of mitochondria Mitochondria not only produce most of the energy in eukaryotic cells but are

also involved in a number of other processes such as: lipid metabolism, Krebs

cycle, apoptosis (programmed cell death), cellular proliferation, regulation of

the cellular redox state, heme synthesis and steroid synthesis. Some

mitochondrial functions are performed only in specific types of cells. For

example, mitochondria in liver cells contain enzymes that allow them to

detoxify ammonia, a waste product of protein metabolism. A mutation in the

genes regulating any of these functions can result in mitochondrial diseases.

2.3.1. Heat production Under certain circumstances, protons re-enter the mitochondrial matrix

without contributing to ATP synthesis. This process is known as proton leak

or mitochondrial uncoupling and is due to the facilitated diffusion of protons

into the matrix. This process results in the unharnessed potential energy of

the proton electrochemical gradient being released as heat. The process is

mediated by a proton channel called thermogenin, or UCP1 (Mozo et al. 2005).

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Thermogenin is a 33kDa protein (Nicholls and Lindberg 1973) primarily

found in brown adipose tissue, or brown fat, and is responsible for non-

shivering thermogenesis. Brown adipose tissue is found in mammals and is

at its highest levels in early life and in hibernating animals. In humans,

brown adipose tissue is present at birth and decreases with age (Mozo et al.

2005).

2.3.2. Calcium storage The concentration of free calcium in the cell can regulate a number of

reactions and is important for signal transduction. Mitochondria store

calcium, thus maintains calcium homeostasis. The release of this calcium

back into the cells interior can initiate calcium spikes or waves. These events

coordinate processes such as neurotransmitter release in nerve cells and

release of hormones in endocrine cells.

2.3.3. Citric acid cycle Pyruvate molecules produced by glycolysis are actively transported across the

inner mitochondrial membrane, and into the matrix where these are oxidized

and combined with coenzyme A to form CO2, acetyl-CoA and NADH. The

acetyl-CoA is the primary substrate to enter the citric acid cycle. The

enzymes of the citric acid cycle are present in the mitochondrial matrix

except succinate dehydrogenase, which is bound to the inner mitochondrial

membrane. The citric acid cycle oxidizes the acetyl-CoA to carbon dioxide and

in the process produces reduced cofactors (three molecules of NADH and one

molecule of FADH2), which are a source of electrons for the electron transport

chain, and a molecule of GTP (that is converted to an ATP).

2.3.4. Oxidative phosphorylation Oxidative phosphorylation (OXPHOS) is the synthesis of ATP from ADP and

inorganic phosphate. It is carried out by five multi-subunit enzyme complexes

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(Figure 1.3). Electrons from NADH and FADH2 are transported to oxygen by

respiratory chain complexes; I, II, III and IV. The final step, the production of

ATP from ADP and inorganic phosphate is carried out by ATP synthase

(complex V). Rather than being isolated complexes, these enzymes seem to

occur together, forming super-complexes (Schagger and Pfeiffer 2000,

Schagger and Pfeiffer 2001), with varying stoichiometries depending on the

use of different solubilization detergents (Schagger 2002, Schafer et al. 2006).

The functional significance of this super-complex formation is thought to

involve catalytic enhancement by channeling of the substrates and

prevention of competition from other enzymes, prevention of the reactive

oxygen species (ROS) and stabilization of the individual OXPHOS enzyme

complexes (Schagger 2002, Acin-Perez et al. 2004). Two processes

characterize the operation of the respiratory chain, electron flow within the

enzyme complexes and the transport of protons across the inner

mitochondrial membrane. NADH is oxidized by the first and largest enzyme

of the respiratory chain, complex I. Electrons are passed through the enzyme

via prosthetic groups flavin mononucleotide (FMN) and seven iron-sulphur

clusters, to ubiquinone (UQ). FADH2 donates the electrons to complex II. The

enzyme operates both in the respiratory chain and in the Krebs cycle, where

it oxidizes succinate to fumarate, yielding FADH2. Electrons are carried

through complex II via flavin adenine dinucleotide (FAD) and three iron-

sulphur clusters to ubiquinone. Complex II also contains one b heme, the

functional role of which is currently unclear. The reaction of complex II is

reversible and the direction of electron flow through the enzyme is dictated

by the relative concentrations of the reactants and products. Thus, in

addition to being an entry point for electrons into the respiratory chain,

complex II also participates in regulation of the Krebs cycle. Electrons from

complexes I and II reduce ubiquinone (UQ) to ubiquinol (QH2), which is

hydrophobic and shuttles within the inner mitochondrial membrane,

transferring the electrons further to complex III. The electrons pass through

Chapter 1


the complex III dimer via cytochrome b, a binuclear iron-sulphur cluster and

cytochrome c1, which donates them to ferricytochrome c. Complex IV,

catalyzes the final step of the respiratory chain, the transfer of the electrons

from ferrocytochrome c to dioxygen, to produce water. The electron

transportation occurs via four redox centres, a binuclear copper centre (CuA),

a mononuclear copper centre (CuB) and two hemes (a & a3). In addition to

electron transport, complexes I, III and IV serve as proton pumps. The

enzymes use energy from the electron transfer to translocate protons from

the matrix side into the intermembrane space, producing an electrochemical

gradient across the inner mitochondrial membrane. The free energy released

from the flow of protons back across the membrane is then used by complex V

for chemical work to produce ATP from ADP and inorganic phosphate

(Schultz and Chan 2001, Berg et al. 2002).

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Figure 1.3: Schematic diagram of the OXPHOS system in mammalian mitochondria. Electrons (e-) from carbon oxidations (step 1, dotted lines) are transferred through NADH (step 2) into OXPHOS complex I (step 3), which is embedded in the lipid bilayer of the mitochondrial inner membrane (IMM), and are then transported to coenzyme Q (CoQ; step 4). Some electrons from organic acid oxidations are transferred through other flavin-containing enzyme complexes (step 5) directly to CoQ. CoQ delivers electrons through complex III (step 6) and cytochrome c (Cyt c; step 7) to the final electron acceptor complex IV (step 8), where oxygen is reduced to water. The electrons lose free energy at each transfer step, and in complexes I, III and IV the energy is harnessed and coupled to the movement of H+ (step 9, dashed lines) from the mitochondrial matrix to the intermembrane space (IMS). The proton gradient thus generated is used for the production of ATP by complex V (step 10). Except for complex II, all complexes contain some proteins encoded by the mitochondrial genome and others encoded by the nuclear genome. The number of subunits for each complex is indicated. CN: cyanide; FMN: flavin mononucleotide; mt: mitochondrial; Pi: phosphate. Adapted from Smeitink et al. 2003, Encyclopedia of life sciences.

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2.3.5. Generation of ROS by mitochondria The respiratory chain is also one of the main sources of ROS formation. It is

estimated that 1% to 3% of O2 reduced in mitochondria is in the form of

superoxide free radical (O2•–) (Turrens 2003). Superoxide (O2•–) is formed

when electrons passing through respiratory chain, leak and react with

molecular oxygen. O2•– is rapidly converted to hydrogen peroxide (H2O2) by

mitochondrial superoxide dismutase (MnSOD). In the presence of reduced

metals such as Fe2+, H2O2 can be converted to hydroxyl radical (OH•). These

three free radicals (O2•–, H2O2 and OH•) are collectively called as ROS and

can damage cellular macromolecules including DNA, proteins and lipids

(Andersen 2004). ROS production increases when respiratory flux is

depressed by a high ATP/ADP ratio, high electronegativity of auto oxidizable

redox carriers in complex I and complex III or a rise in oxygen tension (state

4 respiration). Defects in respiratory chain complexes and normal ageing also

lead to increased mitochondrial ROS production (Esposito et al. 1999;

Cadenas and Davies 2000). Although within a certain local concentration

range, ROS play important roles in regulating many cellular functions and

acting as a secondary messenger to activate specific transcription factors

such as NF-kB and AP-1 (Dalton et al. 1999), an excess production of ROS is

harmful to cells. ROS are extremely reactive molecules and oxidative damage

is believed to be involved in many neurodegenerative diseases,

mitochondriopathies and normal ageing (Shigenaga et al. 1994). A recent

study indicates that mitochondrial ROS homeostasis plays a key role in life

and death of eukaryotic cells (Fleury et al. 2002) as mitochondria not only

respond to ROS but also releases ROS in response to a number of pro-

apoptotic stimuli. However, mitochondria are not the sole source of ROS

within the cell; it is also formed in peroxisomes and cytosol.

Because of their potential harmful effects, excessive ROS must be promptly

eliminated from the cells by a variety of antioxidant defense mechanisms

including important enzymes such as superoxide dismutase (SOD), catalase

Chapter 1


and various peroxidases. The cytosolic copper/zinc-containing SOD

(Cu/ZnSOD, or SOD1) and the mitochondrial manganese-containing SOD

(MnSOD, or SOD2) are two essential enzymes responsible for catalyzing the

conversion of O2•– to H2O2, which is further eliminated by catalase and

peroxidases (Halliwell et al. 1999). Since mitochondrial respiratory chain is a

major site of O2•– generation in the cells, MnSOD plays an important role in

maintaining cellular ROS balance. Vicious cycle theories of aging and

oxidative stress propose that ROS produced by the electron transport chain

damages the mitochondrial DNA leading exponentially to more ROS

production and mitochondrial damage. Although this theory is widely

discussed in the field of research on aging and oxidative stress, there is little

supporting data available.

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Figure 1.4: A schematic representation of ROS generation through respiratory chain and other

sources, its consequences upon cell function. A fraction of oxygen is incompletely reduced by one

electron-transfer to generate ROS and organic free radicals, which are disposed by coordinated action of

antioxidant enzymes. If ROS escapes the antioxidant defense, they may cause oxidative damage and

mutate the mtDNA. This may finally result in defective electron transport chain (ETC). The defective

ETC not only works less efficiently in ATP synthesis but also generates more ROS, which will further

enhance the oxidative damage to various biomolecules in mitochondria or the whole cell.

2.3.6. Mitochondria and apoptosis Mitochondria play a central role in activating apoptotic cell death in response

to cellular dysfunction. Under certain conditions (e.g. high Ca2+, oxidative

stress and low ATP), mitochondrial permeability transition pore (PTP) opens

allowing diffusion of lower molecular weight solutes across the mitochondrial

inner membrane (Crompton 1999). This further results in mitochondrial

swelling and rupturing of outer membrane, leading to the release of proteins

ROS

Oxidative stress

Damage to lipids and proteins

Damage to mtDNA

Permeability transition pore (PTP) activation

Release of cytochrome and AIF

Apoptosis Energy deficit

Defective electron transport chain

mtDNA mutations

Defective mtDNA encoded subunits

ROS scavenging enzymes and antioxidants

Environmental insults, Peroxisomes

Vicious cycle

Cellular dysfunction/cell loss

Mitochondrial ETC

Damage to lipids and proteins

ROS scavenging enzymes and antioxidants

Environmental insults, Peroxisomes

Cellular dysfunction/cell loss

Redox imbalance

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present in the intermebrane space such as cytochrome c, SMAC/Diablo,

apoptosis inducing factor (AIF) and endonuclease G into the cytoplasm. These

proteins then activate the downstream pathways of cell death (Bernardi et al.

2001). The protein components of PTP are not known and it still unclear how

the PTP recognizes apoptotic signals and causes release of proapoptotic

proteins. The pore complex probably involves both inner membrane and outer

membrane proteins and its susceptibility to induction is believed to be

regulated by both pro and anti-apoptotic members of the Bcl-2 family

proteins (Harris and Thompson 2000; Adams and Cory 2001). The bcl-2 is a

family of proteins that are involved in the response to apoptosis. Some of

these proteins (bcl-2 and bcl-XL) are anti-apoptotic, while others (Bad, Bax or

Bid) are pro-apoptotic. The sensitivity of cells to apoptotic stimuli can depend

on the balance of pro and anti-apoptotic bcl-2 proteins. When there is an

excess of pro-apoptotic proteins the cells are more sensitive to apoptosis,

when there is an excess of anti-apoptotic proteins, the cells will tend to be

more resistant. An excess of pro-apoptotic bcl-2 proteins at the surface of the

mitochondria is thought to be important in the formation of the PTP.

The pro-apoptotic bcl-2 proteins are often found in the cytosol where they act

as sensors of cellular damage or stress. Following cellular stress they relocate

to the surface of the mitochondria where the anti-apoptotic proteins are

located. This interaction between pro- and anti-apoptotic proteins disrupts

the normal function of the anti-apoptotic bcl-2 proteins and can lead to the

formation of pores in the mitochondria and the release of cytochrome c and

other pro-apoptotic molecules from the intermembrane space. This in turn

leads to the formation of the apoptosome and the activation of the caspase

cascade. The release of cytochrome c from the mitochondria is particularly an

important event in the induction of apoptosis. Once cytochrome c has been

released into the cytosol it is able to interact with a protein called Apaf-1.

This leads to the recruitment of pro-caspase 9 into a multi-protein complex

with cytochrome c and Apaf-1 called the apoptosome. Formation of the

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apoptosome leads to activation of caspase 9 and the induction of apoptosis.

The role of mitochondria in the induction of apoptosis is summarized in figure

1.5. Mitochondria play a role in integrating many of the signaling pathways

that sense cellular dysfunction and decide whether to commit the cell to

apoptosis, it is tempting to speculate that mitochondrial dysfunction caused

by mitochondrial (mtDNA) or nuclear DNA (nDNA) mutations would promote

cell death. Supporting this hypothesis, progressive degeneration and loss of

neural tissue is a common feature of mitochondrial diseases, but it is unclear

whether the cell loss is apoptotic or necrotic and the degree of cell loss varies

considerably with brain region (Sparaco et al. 1993). Cell death may be

triggered by a range of distinct mechanisms including increases in ROS,

oxidation of the mitochondrial glutathione pool, elevation of free Ca2+, ATP

depletion or changes in intracellular pH, all of which can be affected by

mutations that cause mitochondrial dysfunction. ROS are an important

proapoptotic signal in a number of biological systems in response to cell

damage and in some cases may even be induced to signal cell death (Hwang

et al. 2001).

Understanding how and when cells die in response to mitochondrial

dysfunction is critical for understanding the pathophysiology of mitochondria

related diseases and to design therapies. Also it is important for

understanding what role mitochondrial dysfunction plays in ageing and other

neurodegenerative disorders.

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Figure 1.5: Illustration of the main apoptotic signaling pathways involving mitochondria

3. Mitochondrial DNA Mitochondria contain their own genome known as mitochondrial DNA

(mtDNA). Each cell contains hundreds to thousands of mtDNA molecules; the

phenomenon called polyplasmy and a single mitochondrion has 2-10 copies of

DNA. The mtDNA is maternally inherited, although a few mitochondria from

the sperm cell may enter the oozyte during fertilization, they are eliminated

by a ubiquitin-dependent mechanism (Sutovsky et al. 2000). Interestingly,

data related to human disease has shown that the paternal mtDNA can

escape this elimination and be transmitted to the muscle tissue of the

offspring (Schwartz and Vissing 2002). MtDNA is double-stranded circular

structure (Heavy and Light strand) and contains 16569 base pair nucleotide

sequence (Anderson et al. 1981; Andrews et al. 1999) (Figure 1.6). The two

strands of mtDNA have different base compositions; the heavy (H) strand is

Chapter 1


rich in guanine residues and light (L) strand is rich in thymine residues.

MtDNA encodes 37 genes, of which 28 are encoded by the H-strand and 9 by

the L-strand. Majority of the genes encode an RNA product. There are 22

mitochondrial tRNAs and two rRNAs (12S and 16S). The remaining 13 genes

encode proteins of the OXPHOS system. The mitochondrial genome is

compact, the genes lack introns and are tightly packed, overlapping each

other, or separated by only one or few non-coding bases. There are only two

non-coding regions in mtDNA, a displacement (D) loop of about 1 kilo baspair

(kb) between tRNA-phenyalanine and tRNA-proline. The other non-coding

region of approximately 30 nucleotides is located inside a tRNA cluster about

two-thirds of the way from the D-loop.

Figure 1.6: Structure of mitochondrial DNA. mtDNA encodes 37 genes, 2 rRNAs (12S and 16S), 22

tRNAs and 13 subunits of OXPHOS enzyme complexes: ND1-ND6 and ND4L for complex I, cytochrome

b gene for complex III, COX 1-3 genes for complex IV and ATPase 6 and 8 genes for complex V. D loop =

displacement loop, HSP and LSP = heavy and light strand promoters for transcription, OH and OL =

heavy and light strand replication origins.

Chapter 1


3.1. Replication and transcription of mtDNA The mitochondrial genome is replicated and transcribed within the organelle.

Its replication is under relaxed control and none of the mechanism assures

that each molecule is replicated once and only once during cell cycle. The cis

elements, which are located within the D-loop region of mtDNA, are

responsible for the regulation of both replication and transcription of mtDNA.

However, the trans-acting factors e.g. mtRNA polymerase, mtDNA

polymerase and other regulatory factors are encoded by nDNA (Garesse and

Vallejo 2001.)

The mechanism of mtDNA replication is currently under debate. For many

years the most accepted model was asymmetric (strand–asynchronous) model

of mtDNA replication (Clayton 1982). This model proposes two sites of

initiation of DNA synthesis, one for each strand, which lie far a part. The

synthesis of leading H-strand starts at a point in the major non-coding region

of mtDNA denoted as OH and OL. This model has been challenged by strand-

synchronous (symmetrical) model of mtDNA replication (Holt et al. 2000) in

which the origin of replication of both the leading and lagging strands is

located downstream from the original OH. The replication fork is thought to

move bidirectionally from the origin of replication along the parental mtDNA

strand until the OH is reached (Bowmaker et al. 2003). Differences between

mtDNA replication in cultured cells and in solid tissues have been observed

and it remains to be resolved whether or not both of these replication

mechanisms exist. The two mechanisms are perhaps regulated by different

physiological conditions in the cell.

The mtDNA contains 37 genes that are distributed on both H and L strand

and are expressed as three polycistronic transcription units. The replication

and transcription are linked, because the same RNA primer is used in both

transcription of the L-strand and replication of the mtDNA. The

mitochondrial heavy strand promoters (HSP) and light strand promoters

(LSP) are located about 150 bp apart on regulatory D-loop region.

Chapter 1


Transcription of the L-strand begins at the initiation point (L), located in the

LSP area of the D-loop (Figure 1.6). The L-strand is transcribed as a single

polycistronic precursor RNA containing eight tRNAs and the ND6 mRNA

(Attardi & Schatz 1988). Two models exist for the transcription of the H-

strand. Montoya et al. (1983) suggested that the RNA synthesis starts at two

transcription initiation points, H1 and H2, located in the HSP area of the D-

loop, while another model proposes the existence of only one major

transcription initiation point, H1 (Clayton 1992). The transcription of both

strands of mtDNA leads to a polycistronic primary RNA molecule in which

both of the rRNA genes and almost all the protein genes are flanked by tRNA

genes. This unique genetic organization has led to a proposal that the

cloverleaf secondary structures of the tRNA sequences may act as a signal for

the processing enzymes. Precise endonucleolytic excision of the tRNAs from

the polycistronic transcripts would then yield correctly processed rRNAs and

mRNAs (Ojala et al. 1981). In some cases, where there are no tRNAs flanking

the mRNA, there may be secondary structures resembling tRNA cloverleafs

that are recognized by the processing enzymes.

3.2. Mitochondrial DNA inheritance MtDNA is known to inherit maternally. No mtDNA from sperm enters the

fertilized ovum at the time of conception; the embryo is thus developing with

maternal mtDNA alone. However, a single report regarding the paternal

inheritance of a deletion mutation in mtDNA complex I gene indicated that

paternal inheritance of mtDNA is also possible (Schwartz et al. 2002).

Analysis of infants born after intracytoplasmic sperm injection failed to

identify paternal mtDNA with methods capable of detecting low levels as low

as 0.001% (Marchington et al. 2002; Danan et al. 1997; Houshmand et al.

1997). This implied that paternal mtDNA replication was either suppressed

or diluted beyond the limits of detection and its contribution may not be

necessary.

Chapter 1


Evidence for selective targeting of sperm mitochondria for degradation by the

ovum is also available (Sutovsky et al. 1999; Sutovsky et al. 2004). Failure of

ovum to eliminate paternal mtDNA could result in loss of the embryo at the

blastocyst stage (St. John et al. 2000). Investigators have also failed to detect

paternal mtDNA in patients with sporadic mitochondrial myopathies (Filosto

et al. 2000; Taylor et al. 2003). Thus, if paternal transmission does occur, it is

rare and might depend on the presence of particular paternal mutations that

allow the sperm’s mtDNA to escape destruction as well as to permit

replication. In any event, the dogma that human mtDNA is exclusively

maternally inherited remains as a basis for genetic counselling.

Figure 1.7: Mitochondrial Inheritance. As mitochondria are inherited almost exclusively from the

mother, defects in mtDNA will be passed on from the mother to her children.

3.3. Homoplasmy and heteroplasmy A single cell contains thousands of mtDNA molecules. In normal cases, the

sequence of all these DNA molecules will be identical. However, somatic

mtDNA mutations arise and accumulate with ageing, and could have a role

in the senescence of tissues. If the mutation is present in all the DNA

molecules of mitochondria, then it is known as homoplasmy. The most

common source of somatic mutation of mtDNA is the free radicals generated

by the respiratory chain itself. Most of the mitochondrial diseases are

Chapter 1


characterized by the coexistence of wild type and mutant mtDNA in various

proportions, called heteroplasmy. When the amount of wild-type DNA drops

below a certain level (40-20%), the functioning of the respiratory chain is

distributed and clinical symptoms emerge, a phenomenon called threshold

effect. The level of threshold effect is tissue specific, being lower in tissues

with high-energy demand e.g. heart, Brain, retina, kidney and skeletal

muscle. This means that these tissues are more vulnerable to energy defects.

Mitochondria are randomly segregated at cell division including oogenesis.

During oogenesis, wild-type and mutant mtDNA molecules are randomly

passed to oocytes creating a spectrum of heteroplasmy across the oocyte

population. Oocyte maturation is associated with the rapid replication of this

mtDNA population. This restricted-amplification event can lead to random

shift of mtDNA mutational load between generations and is responsible for

the mutated mtDNA observed in affected offsprings from mothers with

pathogenic mtDNA mutations.

3.4. Mitochondrial DNA mutations Defects in mtDNA can be either point mutations, deletions, duplications or

rearrangements. Point mutations are usually maternally inherited whereas

deletions or large-scale rearrangements are sporadic. As there are no introns,

no splice site mutations are found. MtDNA mutates 10-20 times faster than

nDNA, probably because mtDNA is less protected, especially from ROS

generated in its vicinity, and its repair mechanisms are less efficient

(Fernandez-Silva et al. 2003).

Deletions in mtDNA were the first described mutations to associate with

disease (Holt et al. 1988). The deletion can range from a single base or many

bases (about 6kb) and can be located on any part of mtDNA. The commonest

deletion is 5kb long spanning the region between cytochrome b and

cytochrome c oxidase subunit II (COX II), thus encompassing tRNA and

protein coding genes. The large-scale mtDNA deletions are commonly

Chapter 1


associated with diseases such as chronic progressive external

ophthalmoplegia (CPEO), Kearns-Sayre syndrome, and Pearson’s syndrome.

However, the pathological expression of deletion mutation is not only related

to these phenotypes but has also been described in association with all

mitochondrial syndromes. The prevalence of single deletion disorders is

estimated at 1.2 per 100 000 (Schapira 2006).

Deletions exist in heteroplasmic form, the proportion of deleted molecules

varies between tissues, and the degree of heteroplasmy can shift over time.

Single deletions arise as a primary mtDNA mutation, probably within the

oocyte, and are transmitted to offspring, which may then develop clinical

features. Some patients have duplications of mtDNA. Duplications might not

be pathogenic themselves, but could be an intermediate step in the

generation of deletions. Small size deletions have been described in

cytochrome oxidase, cytochrome b and complex I genes, which are associated

with a variety of clinical presentations.

Approximately, 100 point mutations have been described to be associated

with human disease (www.mitomap.org), but pathogenicity has not been

confirmed for all. These occur in protein coding, tRNA, and rRNA genes.

Evolution has produced related sets of mtDNA sequences, called haplogroups

that can be recognized by certain sequence changes. Since the mtDNA

haplogroups are associated with specific populations on different continents,

a great deal has been learned about the evolution of modern humans and

about human migrations by studying the haplogroup distributions in

different populations (Herrnstadt & Howell 2004). It has also been suggested

that haplogroup-specific polymorphisms may play a role in the pathogenesis

of mitochondrial diseases. The higher mutation rate of mtDNA makes it

difficult to distinguish pathogenic mutations from polymorphisms (Mitchell et

al. 2006). Furthermore, there is also no genotype-phenotype correlation, as

the same mutation can cause different phenotypes and the same clinical

features can be caused by different mutations. The G11778A mutation in

Chapter 1


ND4 was the first known mtDNA point mutation to be associated with

human disease (Wallace et al. 1988). Although, majority of pathogenic

mtDNA mutations are heteroplasmic, those causing Leber’s hereditary optic

neuropathy (LHON) are mostly homoplasmic. The mtDNA genes encoding

subunits of complex I are among the most frequently encountered carriers of

pathogenic mutations, and base pair changes causing variable clinical

features have been identified in all of them. The majority of the pathogenic

mutations reported to affect complex I seem to reside in the ND6 and ND1

genes, which have therefore been called hot spots for mutations causing

especially LHON (Chinnery et al. 2001, Valentino et al. 2004). In addition,

mutations in mitochondrial tRNA genes can lead to the absence or mis-

incorporation of certain amino acids during translation and can thereby cause

complex I deficiency (DiMauro and Hirano 2005). The effect of tRNA

mutation on the functioning of the OXPHOS enzymes varies depending on

codon usage, the subunits that are most dependent on the mutant tRNA for

protein elongation being more seriously affected (Triepels et al. 2001). In

addition, a mutation in a tRNA gene may affect the adjacent subunit gene

through changes in the processing of the polycistronic transcript (Bindoff et

al. 1993).

Criteria for a pathologically relevant mtDNA mutation are:

(i) Co-segregation of mutation with the particular clinical phenotype

(ii) Heteroplasmy

(iii) Absence of the particular mutation in more than 100 normal controls

(iv) Functional impairment in one or more respiratory chain complexes

(v) Haplogroup divergence of identical mutations in different index patients

(vi) Evolutionary conservation of each affected nucleotide

Chapter 1


Table 1.1: A list of confirmed pathogenic mtDNA point mutations associated with mitochondrial diseases (www.mitomap.org).

Mutation Gene Clinical Phenotype Amino acid change

Heteroplasmy

A8344G

tRNA Lys MERRF NA +

T8356C

tRNA Lys MERRF NA +

G12147A

tRNA His MERRF-MELAS / Cerebral edema NA +

A3243G

tRNA Leu (UUR) MELAS NA +

C3256T


T3271C


G4332A

tRNA Gln MELAS/Encephalopathy NA +

C7472insC

tRNA Ser (UCN) PEM/AMDF NA +/-

A3243G

tRNA Leu (UUR MM/CPEO NA +

A3302G

tRNA Leu (UUR) MM NA +

T14709C

tRNA Glu MM+DM NA +

A3260G

tRNA Leu (UUR) MM C NA +

T1095C

12S rRNA Sensory Neural Hearing Loss (SNHL) NA +/-

A1555G

12S rRNA Deafness NA -

A3243G

tRNA Leu (UUR) Diabetes Mellitus & Deafness NA +

T4336C tRNA Gln Alzheimer & Parkinson Disease/ Hearing loss and migraine

NA -

T14487C

ND6 Dystonia, Leigh Syndrome Met63Val +

G14459A

ND6 Dystonia, Leigh Syndrome Ala72Val +

T10158C

ND3 Leigh Syndrome Ser34Pro +

T10191C

ND3 LS-like/ESOC S45P +

C11777A

ND4 Leigh Syndrome Arg340Ser +

T12706C

ND5 Leigh Syndrome Phe124Leu +

T8993C

ATP6 LS/NARP Leu156Phe +

T8993G

ATP6 NARP Leu156Arg +

G13513A

ND5 MELAS/LS Asp393Asn +

A13514G

ND5 MELAS Asp393Gly +

A7445G

CO1 Deafness-Sensory Neural Hearing Loss Ter514Ter +/-

G3460A

ND1 LHON Ala52Thr +/-

G11778A

ND4 LHON Arg340His +/-

T14484C

ND6 LHON Met64Val +/-

G3733A

ND1 LHON Glu143Lys +/-

Chapter 1


Table 1.1 continued:

Besides this, a number of secondary or provisionally pathogenic variants are also reported, but their

pathogenicity has not yet been established. MM= mitochondrial myopathy, MICM= Maternally

Inherited Cardiomyopathy, NIDDM= Non-Insulin Dependent Diabetes Mellitus, MMC= mitochondrial

myopathy/cytopathy, MERRF= myoclonic epilepsy with ragged red fibresa, MELAS= mitochondrial

encephalopathy with stroke like episodes, NA= not applicable

4. Nuclear genes encoding respiratory chain proteins In contrast to the wealth of information gained on mtDNA and its disease-

related mutations, the number of nuclear OXPHOS-related genes that have

been proven to be associated with mitochondrial syndromes is still rather

small. However, a clinical genetic classification can now be proposed for these

defects (Zeviani et al. 2003) as follows:

4.1. Genes coding structural subunits of the respiratory chain

complexes Approximately 30% of the mitochondrial disorders involve deficiency of

complex I (Benit et al. 2001). The primary genetic defect may either be at the

mtDNA or at the nDNA level. Majority of complex I deficient cases seem to

follow an autosomal recessive mode of inheritance, suggesting a defect of

nuclear origin (Loeffen et al. 2000). In the past few years, several disease-

associated mutations have been discovered in some 36 nuclear-encoded

subunits of complex I (Triepels et al. 2001a; Petruzzella et al. 2002). These

include substitutions of functionally important amino acids, frame shifts and

C4171A

ND1 LHON L289M +/-

T10663C

ND4 LHON Val65Ala -

G14459A

ND6 LHON and dystonia, Leigh syndrome Ala72Val +

C14482G

ND6 LHON Met64Ile +

C14482A

ND6 LHON Met64Ile +/-

A14495G

ND6 LHON Leu60Ser +

C14568T

ND6 LHON Gly36Ser -

Chapter 1


premature stop codon mutations and also large-scale deletions (Triepels et al.

2000; Ogilvie et al. 2005). The NDUFV1 and NDUFS4 genes seem to be hot

spots for mutations in nuclear complex I genes. In most of these cases, the

clinical presentation is that of an early-onset progressive neurological

disorder with lactic acidosis, most often Leigh syndrome, occasionally

complicated by cardiomyopathy, or multisystem involvement. However, no

mutation in structural genes has been found in many cases of complex I

deficiency, suggesting that still unknown assembly factors for complex I

(Janssen et al. 2002) or other gene products involved in its formation and

activity may be responsible for these forms. Currently, in about 40% of

isolated complex I deficiencies, the OXPHOS defect can be explained by

mutations in structural nuclear genes (Table 1.2).

Complex II is a FAD-dependent enzyme at a cross-point between OXPHOS

and Krebs-cycle pathways. It comprises four protein subunits, all encoded by

nuclear genes (SDH-A, B, C, and D). Mutations in SDHA, the largest subunit

of complex II, are a rare cause of Leigh syndrome or late onset

neurodegenerative disease (Rustin & Rotig 2002). However, the most

interesting discovery concerning defects of complex II is their association

with inherited paragangliomas (Maher and Eng 2002; Ackrell 2002; Baysal

2002; Dannenberg et al. 2002). In 10-15% of the cases, these neuro-

ectodermal tumors are inherited in an autosomal dominant fashion with

incomplete penetrance. It now appears that mutations in SDHB, SDHC, and

SDHD are responsible for the majority of familial paragangliomas (Baysel et

al. 2002) and also for a significant fraction of non-familial tumors, including

phaeochromocytomas (tumors of the adrenal medulla) (Cascon et al. 2002,

Neumann et al. 2002).

Complex III catalyzes electron transfer from succinate and NADH-linked

dehydrogenases to cytochrome c. Complex III is made up of 11 subunits, of

which 10 are encoded by nDNA and one (cytochrome b) is coded by mtDNA.

Although pathogenic mutations in the gene encoding mitochondrial

Chapter 1


cytochrome b have been described, mutations in the nDNA encoded subunits

were never reported.

Coenzyme Q10 deficiency Coezyme Q10 (CoQ10), or ubiquinone, is a lipophilic component of the

electron-transport chain and transfers electrons from various dehydrogenases

to complex III and acts as a lipid-soluble antioxidant and as a membrane

stablizer. The first syndrome of CoQ10 deficiency was characterized by the

triad of recurrent myoglobinuria, brain involvement (seizures, ataxia, and

mental retardation) and ragged-red fibers/lipid storage in muscle. Coenzyme

Q10 is mainly synthesized intracellularly and requires many enzymatic steps.

The mevalonate pathway is a sequence of reactions that leads to farnesyl

pyrophosphate- the common substrate for synthesis of ubiquinone, cholesterol,

dolichol, dolicholphosphate, as well as for prenylation of proteins. Irrespective

of the genetic causes of the defect, which are presently unknown, early

recognition of CoQ deficiency is important, as its supplementation can lead to

clinical improvement.

4.2. Genes involved in the assembly of respiratory chain complexes This group comprises of genes encoding assembly factors for complex I, III

and IV. To date, only two complex I assembly factors (B17.2 and CIA30) have

been identified and how each functions is not clear. Mutations in B17.2L

chaperone are associated with progressive encephalopathy (Ogilvie et al.

2005) and that of CIA30 are reported to associate with

cardioencephalomyopathy (Dunning et al. 2007).

Human cytochrome oxidase (COX) is composed of 13 subunits: the three

largest ones are encoded by mtDNA genes, while the remaining subunits are

encoded by nuclear genes. The most frequent manifestation of isolated COX

deficiency in infancy is Leigh syndrome. Other phenotypes such as severe

cardiomyopathy or complex encephalocardiomyopathies are also known to

Chapter 1


associate with COX deficiency (Shoubridge 2001). No mutation in nDNA

encoded subunits of COX are reported, whereas all of the nuclear-gene

defects of COX identified till date are caused by mutations in assembly

factors of the enzyme, including SURF1, SCO1, SCO2, COX10 and COX15.

SURF1 is a 30kDa hydrophobic protein located in inner membrane of

mitochondria. Mutations in SURF1 are relatively frequent, accounting for the

majority of the Leigh syndrome cases.

Mutations in other COX assembly genes are rare and have been reported in

only a few families or singleton cases. Human SCO1 and SCO2 are nuclear-

encoded copper ion binding proteins, presumed to be responsible for the

insertion of copper into the COX holoenzyme. Whereas mutations in SCO1

were found in only one family, mutations in SCO2 are more frequent. Copper

supplementation can restore COX activity in cells harboring mutations in

genes involving copper transport, including SCO2 (Jaksch et al. 2001;

Salviati et al. 2002). Similar to COX10, COX15 (heme A farnesyl-transferase)

is involved in the synthesis of heme A, the prosthetic group for COX. The first

deleterious mutations in COX15 have been identified in a patient with fatal,

infantile hypertrophic cardiomyopathy (Antonicka et al. 2003a).

BCS1L, a mitochondrial inner-membrane protein, is a chaperone necessary

for the assembly of mitochondrial respiratory chain complex III. Mutations in

BCS1L have been shown in infantile cases of complex III deficiency

associated with neonatal proximal tubulopathy, hepatic involvement and

encephalopathy (de Lonlay et al. 2001) and in GRACILE (growth retardation,

aminoaciduria, cholestasis, iron overload, lactacidosis, and early death)

syndrome (Visapaa et al. 2002). More recently, BCS1L mutations were

associated with isolated encephalopathy (Fernandez-Vizarra et al. 2007) and

Bjornstad syndrome (Hinson et al. 2007)

Chapter 1


4.3. Genes involved in mtDNA maintenance MtDNA is dependent upon nDNA for the production of a number of proteins

involved in its replication, transcription, translation, repair, and

maintenance. Mutations of these genes can result in multiple mtDNA

deletions or depletion of mtDNA. Adenine nucleotide translocator-1 (ANT-1)

is an isoform specific to muscle, heart, and brain. It regulates the adenine

nucleotide pool within mitochondria. ANT1 mutations cause adult onset

autosomal dominant chronic progressive external opthalmoplegia (CPEO)

with ragged red fibers and multiple mtDNA deletions in skeletal muscle

(Kaukonen et al. 2000). Twinkle is a hexomeric 5′-3′ DNA helicase protein

encoded by the C10orf2 gene, which is responsible for unwinding the mtDNA

replication fork (Spelbrink et al. 2001; Korhonen et al. 2003). Inhibition of

twinkle in cultured cells results in rapid mtDNA depletion, whereas over-

expression of the gene leads to mtDNA accumulation, confirming its

importance in regulating copy number (Tyynismaa et al. 2004). Twinkle is

highly expressed in human skeletal muscle and in a specific splice variant in

testes, which is of interest since mtDNA replication is down regulated during

spermatogenesis. Twinkle co-localizes with mitochondrial transcription factor

A and mitochondrial single-stranded DNA-binding protein, and together they

are thought to stabilize mtDNA. Several mutations causing autosomal

dominant progressive external ophthalmoplegia (PEO) are located at or near

putative subunit interaction sites in the holoenzyme. The clinical

manifestations of C10orf2 (twinkle) mutations typically include PEO. In some

cases, this can be of late onset (above 50 years of age) and be associated with

myopathy and cardiomyopathy in addition to axonal neuropathy, diabetes,

deafness, and osteoporosis (Kiechl et al. 2004). MtDNA polymerase γ (POLG) is a heterodimer comprising a 140kDa alpha

subunit and a 41kDa beta subunit. It is located within the inner

mitochondrial membrane and is essential for mtDNA replication. The alpha

subunit is catalytic and contains both polymerase and exonuclease activities,

Chapter 1


the beta subunit facilitate DNA binding and promote DNA synthesis (Filosto

et al. 2003). Mutations of POLG have been associated with a range of clinical

phenotypes including PEO. The human POLG gene contains a 10-CAG repeat

length encoding a polyglutamine tract. A variation in this microsatellite has

been associated with male subfertility (Rovio et al. 2001; Jensen et al. 2004).

POLG mutations also cause Alpers syndrome, an autosomal recessive

disorder characterized by epilepsy, cortical blindness, micronodular hepatic

cirrhosis, and episodic psychomotor regression (Naviaux and Nguyen 2004;

Ferrari et al. 2005). POLG mutations have been identified in patients with

PEO and Parkinsonism.

Mitochondrial DNA depletion syndrome (MDS) is a clinically heterogeneous

group of disorders characterized by a reduction in mtDNA copy number. MDS

has been linked to mutations in two genes involved in deoxyribonucleotide

metabolism: thymidine kinase 2 (TK2) and deoxyguanosine kinase, which are

responsible for the myopathic form and the hepatoencephalopathic form of

MDS, respectively (Hirano et al. 2001). Both deoxyguanosine kinase and TK2

genes are involved in the formation of the mitochondrial nucleotide pool, as is

thymidine phosphorylase, whose deficiency causes a syndrome known as

mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). MNGIE

is associated with both depletion and multiple deletions of mtDNA.

Chapter 1


Table 1.2: Mutations in nuclear OXPHOS-related disease associated genes

Location Clinical phenotype Inheritance Accession no.

Respiratory-enzyme subunits

NDUFS1 (Complex I) 2q33-q34 Lactic acidosis, mitochondrial complex I defficiency

AR NM_005006

NDUFS2 (Complex I) 1q23 Cardiomyopathy and encephalomyopathy AR NM_004500

NDUFV1(Complex I) 11q13 Leigh syndrome

AR NM_007103

NDUFS4 (Complex I) 5q11.1 Fatal multisystem complex I deficiency AR NM_002495

NDUFS7 (Complex I) 19p13 Leigh syndrome

AR NM_024407

NDUFS8 (Complex I) 11q13 Leigh syndrome

AR NM_002496

SDHA (Complex II) 5p15/3q29* Leigh syndrome AR NM_004168

SDHB (Complex II) 1p36.1-p35 Phaeochromocytoma and cervical paraganglioma

AD NM_003000

SDHC (Complex II) 1q21 Familial paraganglioma - PGL3

AD NM_003001

SDHD (Complex II) 11q23 Familial paraganglioma - PGL1

AD NM_003002

Krebs cycle enzymes

FH (Fumarate hydratase) 1q42.3-q43 Multiple cutaneous and uterine leiomyomatosis

AD NM_000143

Assembly factors

B17.2L (Complex I) 5q12.1 Encephalopathy AD NM_174889

CIA30 (Complex I) 15q13.3 Cardioencephalomyopathy

AD NM_01601

SURF1 (COX assembly) 9q34 Leigh syndrome

AR NM_003172

SCO1 (COX assembly) 17p13-p12 Ketacidotic coma and hepatopathy

AR NM_004589

SCO2 (COX assembly) 22q13 Hypertrophic cardiomyopathy

AR NM_005138

COX10 (COX assembly) 17p13.1-q11.1 Tubulopathy and Leukodystrophy

AR NM_001303

COX15 (COX assembly) 10q24 Hypertrophic cardiomyopathy

AR NM_078470

BCS1L (Complex III assembly)

2q33-37

Tubulopathy, encephalopathy, and liver failure, complex III Deficiency, GRACILE

AD

NM_004328

MtDNA maintenance

TP (thymidine phosphorylase)

22q13.32-qter Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)

AR NM_001953

ANT1 (adenine nucleotide translocator 1)

4q34 Autosomal dominant progressive external ophthalmoplegia (adPEO)

AD J04982

C10 ORF2 (Twinkle) 10q24 Autosomal dominant progressive external ophthalmoplegia (adPEO)

AD AF292005

POLG1 (Polymerase gamma γ)

15q25 Autosomal dominant, autosomal recessive progressive external ophthalmoplegia (adPEO, arPEO)

AD, AR NM_002693

Chapter 1


Table 1.2 continued: DGUOK (deoxyguanosine Kinase)

2p13 MtDNA depletion syndrome, hepato-cerebral form

AR NM_080918

TK2 16q22 MtDNA depletion syndrome, myopathic form

AR NM_004614

DNC (deoxynucleotide carrier)

17q25.3 Congenital microcephaly of Amish (MCPHA) AR AJ251857

Mitochondrial-factors related to OXPHOS

SPG7 (paraplegin) 16q24.3 Spastic paraplegia-7

AR AH007611

FRDA (Frataxin) 9q13 Friedreich ataxia

AR U43747

ABC7

Xq13.1-q13.1 Sideroblastic anemia, spinocerebellar ataxia

X-linked AF133659

DDP1 Xp22 Deafness-dystonia syndrome (Mohr-Tranebjaerg)

X-linked NM_004085

OPA1 3q28-q29 Optic atrophy

AD NM_015560

TAZ (tafazzin) Xq28 Barth syndrome

X-linked NM_000116

AD= autosomal dominant, AR= autosomal recessive. 4.4 Genes encoding mitochondrial factors indirectly related to OXPHOS Neurodegenerative disorders linked to OXPHOS defects have also been

attributed to mutations in several nuclear genes indirectly related to

respiration and energy production (Zeviani et al. 2001) (Table 1.2). This

group includes paraplegin, a mitochondrial metalloprotease associated with

autosomal recessive (or dominant) spastic paraplegia; ABC7, an iron

mitochondrial exporter, which controls the generation of cytosolic iron sulfur

proteins and is responsible for X-linked sideroblastic anemia and ataxia;

frataxin, a mitochondrial iron-storage protein which is responsible for

Friedreich’s ataxia; and DDP1, a component of the import machinery for

mitochondrial carrier proteins, which is responsible for X-linked deafness-

dystonia syndrome (Mohr-Tranebjaerg syndrome).

Chapter 1


5. Mitochondrial encephalomyopathies Human diseases caused by impaired respiratory chain function have been

increasingly recognized in recent years. Although neurological diseases are

the commonest consequence of respiratory chain dysfunction, it is now

apparent that virtually any tissue in the body can be affected. The

neurological diseases associated with respiratory chain impairment are

collectively known as mitochondrial encephalomyopathies (MCPs), a term

which reflects the common involvement of both the central nervous system

and skeletal muscle in these patients. Organs such as the brain, heart and

skeletal muscle are highly energy dependent and thus vulnerable to defects

in energy metabolism. Skeletal muscle involvement presents with exercise

intolerance, weakness and myalgias, in association with involvement of other

organs, most commonly with encephalopathy or cardiomyopathy (Scaglia et

al. 2004). Lactic acidosis with an increased lactate/pyruvate ratio is

frequently observed. MCPs are caused by mutations in both nuclear and

mitocondrial DNA (DiMauro and Gurgel-Giannetti 2005).

Proteins most frequently affected by mutations are those of the respiratory

chain and oxidative phosphorylation. That is why mitochondrial

encephalomyopathies are sometimes also called as respiratory chain

disorders. However, MCPs may also be due to defects in pathways and

components of the mitochondrion other than respiratory chain. Proteins of

the respiratory chain that are encoded by nDNA; after being synthesized in

the cytoplasm, are imported into mitochondria, where they assemble together

with their mtDNA encoded counterparts to form holoenzymes in the

mitochondrial inner membrane. MCPs present with a wide spectrum of

disease and their clinical features overlap (Leonard and Schapira 2000a & b).

Concerning mtDNA, a single mutation or different mutations in the same

gene may present with different clinical manifestations, while as the same

clinical phenotype may be caused by different mutations, called genetic

Chapter 1


heterogeneity (Dimauro 1996). MtDNA mutations were first reported in 1988

(Holt et al. 1988; Wallace et al. 1988). Subsequently, the first nuclear

mutation leading to MCP was identified (Bourgeois et al. 1992).

5.1. Classical mitochondrial syndromes The onset of MCPs ranges from early embryogenesis to late adulthood. The

MCPs can present at any age (Wolf and Smeitink 2002). The frequency is

approximately 1 per 5000 individuals. The clinical phenotypes associated

with classical mitochondrial disorders are:

5.1.1. Chronic progressive external opthalmoplegia Chronic progressive external ophthalmoplegia (CPEO) is the commonest

manifestation of a mtDNA mutation and is characterized by ophthalmoplegia

and ptosis (Holt et al. 1989). Later on cataracts, retinitis pigmentosa,

deafness, fatigue, ataxia, limb weakness, neuropathy, cardiomyopathy and

renal insufficiency may develop (Schapira and Cock 1999; McFarland et al.

2002). The particular susceptibility of the extra-ocular muscles is explained

by three to four times greater mitochondrial volume compared with limb

muscles (Schapira and Cock 1999). The clinical course is usually benign in

that additional tissue or organ failure is rare with low risk of serious

disability. CPEO is due to mtDNA deletions (40% of the cases), nDNA

mutations and point mutations in mtDNA encoding tRNAs (Zeviani et al.

1989).

5.1.2. Kearns-sayre syndrome Kearns-Sayre syndrome (KSS) is a subtype of CPEO with pigmentary

retinopathy, cardiac conduction defects, cerebellar ataxia, raised CSF protein

and onset is above 20 years (Kearns and Sayre 1958). Its expression is

systemic, but most common expressions are in the eyes, with

ophthalmoplegia and retinal degeneration, specifically retinitis pigmentosa.

Chapter 1


Other characteristic features are dysphagia, proximal weakness, hearing loss,

cerebellar ataxia and cardiac conduction defects. The prognosis of KSS is

worse than that of CPEO and patients rarely survive beyond the age of 30

years. KSS is due to sporadic, single large deletions ranging from 1.3 to 8.8

kb (90% of the cases) or duplications. The origins of replication and

transcription within the mtDNA are usually spared (Schapira and Cock 1999).

5.1.3. Pearson syndrome Pearson syndrome is characterized by sideroblastic anemia and exocrine

pancreas dysfunction. Bone marrow biopsy is characterized by normal

cellularity, but vacuolization of the precursor cells (Pearson et al. 1979;

Schapira and Cock 1999). Additional features are failure to thrive, and

chronic diarrhoea with villous atrophy. With disease progression,

hepatomegaly, raised transaminases, hyperbilirubinaemia, coagulopathy and

tubular dysfunction with aminoaciduria and glucosuria (Fanconi’s syndrome)

may occur (Schapira and Cock 1999). The syndrome is usually fatal in

infancy. In patients who survive beyond infancy, the syndrome evolves into

KSS. It is caused by a deletion in mtDNA with a heteroplasmy rate of up to

90% in blood (McFarland et al. 2002).

5.1.4. Mitochondrial encephalomyopathy with lactic acidosis and

stroke-like episodes Mitochondrial encephalomyopathy with lactic acidosis and stroke-like

episodes (MELAS) syndrome is characterized by migraine-like headache,

recurrent vomiting, seizures, short stature, normal early development, lactic

acidosis and ragged-red fibers in muscle (Pavlakis et al. 1984). The classical

MELAS phenotype affects children at 5-15 years of age. However, stroke-like

episodes usually occur in early infancy whereas several atypical

manifestations, like delayed motor development and failure to thrive are

observed (Sue et al. 1999; Okhuijsen-Kroes et al. 2001). In adults, common

Chapter 1


clinical manifestations include sensorineural hearing impairment, diabetes,

myopathy, cardiomyopathy and cognitive decline. Complex I is frequently the

most affected respiratory chain enzyme detected in MELAS (Ciafaloni et al.

1992). About 80% of the MELAS patients have the heteroplasmic missense

mutation A3243G in the tRNA Leu(UUR) gene (Goto et al. 1990). The

genotype-phenotype correlation of A3243G mutation is rather loose, since the

observed clinical manifestations are not restricted solely on MELAS.

Identification of mutations in mitochondrial ND genes associated with

MELAS or with MELAS/LHON overlap syndrome further address the link

between complex I defect and MELAS phenotype (Corona et al. 2001).

5.1.5. Myoclonic epilepsy with ragged-red fibers Myoclonic epilepsy with ragged-red fibers (MERRF) is a neuromuscular

disorder characterized by myoclonus, epilepsy, muscle weakness, cerebellar

ataxia, deafness and dementia (Fukuhara et al. 1980; Silvestri et al. 1993).

Complex IV deficiency is the most prominent biochemical finding in patient’s

muscle with MERRF. In some cases, complex I is also affected. Most patients

with MERRF do harbor a heteroplasmic A8344G mtDNA mutation in tRNA-

lysine gene (Shoffner et al. 1990). Clinical, biochemical and molecular studies

on large pedigrees with A8344G mutation have shown a positive correlation

between the severity of the disease, age at onset, mtDNA heteroplasmy and

reduced activity of respiratory chain enzymes in skeletal muscle (Zeviani and

Di Donato 2004).

5.1.6. Neuropathy, ataxia retinitis pigmentosa (NARP) The neuropathy, ataxia, retinitis pigmentosa (NARP) syndrome, first

described in 1990 (Holt et al. 1990), is characterized by weakness due to

motor neuropathy, sensory disturbances, cerebellar ataxia and retinitis

pigmentosa. Additional features may be developmental delay, mental

retardation, dementia, ataxia, cardiomyopathy and epilepsy. There is no

Chapter 1


clinical or histological evidence of myopathy in NARP. The disease is caused

due to mutations in ATPase 6 gene of mtDNA (McFarland et al. 2002).

5.1.7. Leigh syndrome Leigh syndrome is the most common pediatric phenotype of isolated complex

I deficiency (Loeffen et al. 2000). The disease is a progressive

neurodegenerative disorder involving encephalopathy with lactic acidosis,

occasionally complicated by cardiomyopathy or a multisystemic presentation

(van Erven et al. 1987; Robinson 1998). The onset is usually in the first year

of life, and the children present with developmental delay and failure to

thrive. Motor and intellectual retardation, ataxia, dystonia, hypotonia, and

optic atrophy are frequently encountered. Neuroimaging shows symmetrical

lesions in the basal ganglia, midbrain and brainstem. On pathological

examination, a bilateral symmetrical focus of spongy necrosis is detected with

myelin degeneration, vascular proliferation and gliosis in the thalami, brain

stem and spinal cord. Patients with similar symptoms but with a typical or

unknown neuropathology are referred to as Leigh-like cases (LLS) (Rahman

et al. 1996; Morris et al. 1996). Besides complex I deficiency, LS can have

several other biochemical causes, and it has been described in association

with defects of all OXPHOS system enzymes (Dahl 1998). Mutations in both

mtDNA and nDNA may lead to LS, but a severe form of the disease, called

maternally inherited Leigh syndrome (MILS), is caused by mutations in

mtDNA. The T8993G mutation in the ATPase 6 gene is a common cause of

MILS (Holt et al. 1990). The degree of heteroplasmy of the T8993G mutation

correlates well with the severity of the disease. High levels of mutated

mtDNA (>95%) lead to Leigh’s phenotype, whereas low levels of mutation

load can cause NARP syndrome (Tatuch et al. 1992).

Chapter 1


5.1.8. Leber hereditary optic neuropathy Leber’s hereditary optic neuropathy is the commonest cause of maternally

inherited blindness particularly in young men. The unique anatomical and

physiological features of the optic nerve seem to make it especially vulnerable

to complex I deficiency (Bristow et al. 2002). The main phenotype of LHON is

a tissue-specific, subacute, painless visual loss characterized by central

scotomas, abnormal color vision and optic atrophy. Vision deteriorates over a

period of days or weeks in one eye followed by the other eye. LHON typically

affects young adults, the average age being 20 years. Men are affected three

to four times more often than women (Simon and Johns 1999; Man et al.

2002; Zeviani and Di Donato 2004). Approximately 90% of LHON cases carry

one of the three mtDNA mutations, G3460A (Howell et al. 1991), G11778A

(Wallace et al. 1988) or T14484C (Chinnery et al. 2001). All these mutations

are residing on genes encoding complex I subunits and are usually

homoplasmic. Interestingly, only 50% of men and 10% of women harboring a

pathogenic mtDNA mutation develop the optic neuropathy. This marked

incomplete penetrance and gender bias imply that additional genetic or

environmental factors modulate the phenotypic expression of LHON (Hudson

et al. 2005; Man et al. 2002).

5.1.9. Myoneurogastrointestinal encephalopathy (MNGIE) Myoneurogastrointestinal encephalopathy (MNGIE) is a multisystem

disorder (Ionasescu 1983) characterized by gastrointestinal dysmotility,

manifesting before age of 20 years as episodic nausea, vomiting,

gastroparesis, progressive intestinal pseudo-obstruction, abdominal pain,

dilation and dysmotility of oesophagus, stomach and small intestine,

diarrhoea, and malabsorption with progressive malnutrition, leading to death

around 40 years of age. Additional features include generalized myopathy

with CPEO, cognitive decline due to leucencephalopathy, retinitis pigmentosa,

deafness, hoarseness, dysarthria and polyneuropathy. Post-mortem changes

Chapter 1


include visceral neuropathy (loss of neurons and fibrosis in the coeliac,

mesenteric and Auerbach plexuses) and scleroderma-like changes (Schapira

and Cock 1999). MNGIE has been shown to result from mutations in the

nDNA encoded gene for the thymidine-phosphorylase (Nishino et al. 2001;

DiMauro and Schon 2003) (Table 1.2).

6. Treatment of mitochondrial disorders Treatment options for mitochondrial encephalomyopathies are very limited

and there is no cure available. Some effects such as cardiac arrhythmia,

seizure disorders, renal bicarbonate loss and hypoglycemia can be treated.

Seizures usually respond to conventional anticonvulsants. However, valproic

acid should be used with caution and in association with L-carnitine, because

it inhibits carnitine uptake (Tein et al. 1993). In patients with CPEO,

improvement in severe ptosis is possible by surgery; congenital cataracts are

also treated surgically. A specific lactic acid lowering agent is dichloroacetate

(DCA), which acts by inhibiting pyruvate dehydrogenase (PDH) kinase, thus

keeping PDH in the dephosphorylated, active form and favoring pyruvate

metabolism and lactate oxidation (Stacpoole 1989). DCA should not be used

long term in patients with mitochondrial disorders, who are already prone to

development of peripheral neuropathy by virtue of their mitochondrial

dysfunction. Since defects of the respiratory chain result in the increased

production of free radicals, the use of antioxidants do play a significant role.

N-acetylcysteine and coenzyme Q10, both antioxidants, improved OXPHOS

function and reduced free radical production in cybrid cells carrying the

T8993G mutation that causes NARP or MILS (Mattiazzi et al. 2004).

However, the use of antioxidants in mtDNA disease has yet to be tested in a

clinical trial. In vitro studies have revealed a potentially useful therapeutic

approach to a fatal infantile form of encephalocardiomyopathy associated

with COX deficiency due to mutations in the SCO2 gene. When copper was

Chapter 1


added to the medium of cultured COX-deficient myoblasts harboring SCO2

mutations, COX activity was restored (Jaksch et al. 2001; Salviati et al. 2002).

This suggests that copper supplementation should be tried in infants with

cardiopathy and SCO2 mutations.

Various strategies are being assessed to modify the mtDNA mutant load in

cells and tissues of patients. An obvious target would be the preferential

expansion of wildtype mtDNA or the suppression of mutant mtDNA

expansion. A few approaches are:

Inhibiting Replication of Mutant Genomes Selective hybridization of nucleic acid derivatives to mutant mtDNA would

inhibit their replication while allowing propagation of wild-type genomes,

thus causing the proportion of mutant genomes to fall below the pathogenic

threshold. Some success has ben obtained in decreasing the ratio of A8344G

MERRF mutants in vitro (Taylor et al. 1997), but there were problems with

the delivery of peptide nucleic acids to human mitochondria (Chinnery et al.

1999). Importing RNAs into Mitochondria Normal yeast tRNAs can be imported from the cytoplasm to compensate for

mutant mitochondrial tRNAs, and human mitochondria can internalize yeast

tRNA derivatives in the presence of a specific yeast transport factor

(Kolesnikova et al. 2000). Interestingly, the same yeast tRNA-lysine

derivatives expressed in cybrid human cell lines harboring the MERRF

mutation (A8344G in tRNA-lysine) were imported into mitochondria and

were able to partially restore mitochondrial function (Kolesnikova et al. 2004).

Recently, import of cytosolic tRNAs by Leishmania RNA import complexes

into mitochondria restored mitochondrial function in MERRF cybrids

harboring a mutant mitochondrial tRNA lysine (Mahata et al. 2006).

Chapter 1


Importing Polypeptides into Mitochondria This therapeutic strategy is based on the phenomenon of allotropic

expression and xenotopic expression, and import of restriction endonucleases.

Allotopic expression refers to a strategy aimed at reducing the load of mutant

proteins by importing a normal version of a mutant mtDNA-encoded protein

from a gene ‘‘snuck’’ into the nucleus. For example, the ATPase 6 gene of

mtDNA can be converted from the mitochondrial into the nuclear genetic

code. To be sure that the novel nuclear protein encoded by the converted gene

is recognized by, and transported into mitochondria, it has to be provided

with a leader peptide, whose genetic sequence can be ‘‘borrowed’’ from

another mtDNA encoded protein. This approach has been realized in vitro to

correct the biochemical defect in cybrid cells harboring the T8993G

NARP/MILS mutation (Manfredi et al. 2002) and in cybrids harboring the

G11778A LHON mutation (Guy et al. 2002).

Still another molecular ‘‘trick’’ is to correct a respiratory chain defect due to a

mtDNA mutation by transfecting affected mammalian cells with either

mitochondrial or nuclear genes from other organisms but encoding cognate

proteins (‘‘xenotopic expression’’) (Seo et al. 1998; Bai et al. 2001; Ojaimi et al.

2002). A more direct molecular approach is to import specific restriction

endonucleases as ‘‘magic bullets’’ to selectively destroy mutant mtDNAs. This

approach has proven successful in cybrid cell lines harboring the T8993G

NARP/MILS mutation in the ATPase 6 gene, which creates a unique SmaI

site in human mtDNA. The gene for SmaI was fused to mitochondrial

targeting sequences and transiently expressed in heteroplasmic cybrids,

which lost mutant mtDNAs (Tanaka et al. 2002). Genetic counselling Prenatal diagnosis for tRNA point mutations, including the more common

ones associated with MELAS and MERRF is practically impossible by two

concerns. First, the mutation load in amniocytes or chorionic villi does not

Chapter 1


necessarily correspond to that of other fetal tissues. Second, mutation load

measured in prenatal samples may shift in utero or after birth due to mitotic

segregation. At the other end of the spectrum, large-scale deletions of mtDNA

as a rule are neither inherited nor transmitted and either arise de novo in

oogenesis or in early embryogenesis. Mitochondrial diseases caused by

nuclear gene mutations will be transmitted by Mendelian inheritance.

Genetic counselling in mitochondrial disorders is a considerable challenge

given the diversity of the clinical manifestations and the poor link between

phenotype and genotype. Practical advice can include the possibility of in vitro fertilization with a donor egg, but another possibility for the future is of

nuclear transfer from a maternal egg and fertilization in a donor cytoplasm

using paternal sperm. The recurrence risks for the relative of an individual

with LHON are 30% for brothers, 8% for sisters, 46% for nephews, 10% for

nieces, and 31% and 6% for male and female cousins, respectively (Harding et

al. 1995).

Advances in molecular and diagnostic technology will help us better

understand these complex diseases and help medical practitioners in better

managing their patients.

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