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Mitochondria in Health and Disease

Course Authors

Claus Desler, Ph.D., and Lene Juel Rasmussen, Ph.D.

Dr. Desler is Assistant Professor and Dr. Rasmussen is Professor and Managing Director, Center for Healthy Aging, Department of Molecular and Cellular Medicine, University of Copenhagen, Denmark.

Within the past 12 months, Drs. Desler and Rasmussen have no conflicts of interest relevant to this activity.

Albert Einstein College of Medicine, CCME staff, and interMDnet staff have nothing to disclose relevant to this activity.

Estimated course time: 1 hour(s).

Albert Einstein College of Medicine – Montefiore Medical Center designates this enduring material activity for a maximum of 1.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only the credit commensurate with the extent of their participation in the activity.

In support of improving patient care, this activity has been planned and implemented by Albert Einstein College of Medicine-Montefiore Medical Center and InterMDnet. Albert Einstein College of Medicine – Montefiore Medical Center is jointly accredited by the Accreditation Council for Continuing Medical Education (ACCME), the Accreditation Council for Pharmacy Education (ACPE), and the American Nurses Credentialing Center (ANCC), to provide continuing education for the healthcare team.

 
Learning Objectives

Upon completion of this Cyberounds®, you should be able to:

  • Describe the role of mitochondria in humans;

  • Discuss the consequences of mitochondrial dysfunction in human diseases;

  • Discuss potential strategies for diagnosis and treatment of mitochondrial diseases.

 

Mitochondria are semiautonomous organelles present in almost all eukaryotic cells in quantities ranging from a single copy to several thousand per cell. Important mitochondrial functions include ATP production by oxidative phosphorylation, ?2-oxidation of fatty acids and metabolism of amino acids and lipids. Furthermore, mitochondria have a prominent role in the initiation of apoptosis (i.e., an organism's normal and controlled death of cells).

Due to their involvement in ATP production, dysfunction of mitochondria is involved in a range of pathologies known as mitochondrial cytopathies, which preferentially affect the muscle and nervous system, but also impact other high- energy-requiring tissues. Originally, it was believed that the condition was very rare and only affected high-energy-requiring tissues resulting in a few select pathologies. However, the understanding of mitochondrial cytopathies has evolved immensely and mitochondrial cytopathies are now known to be the largest group of metabolic diseases and to cause a wide variety of pathologies.

Mitochondrial Physiology and Genetics

In aerobic cells the majority of ATP is produced by oxidative phosphorylation. This process takes place in the mitochondria where electrons donated from the Krebs cycle are passed through the four complexes (complex I-IV) comprising the electron transport chain (ETC), eventually reducing oxygen and producing water. The flux of electrons creates an electrochemical potential between the inter-membrane space and the matrix of the mitochondria. This potential is utilized by the ATP synthase to phosphorylate ADP, thus producing ATP.

Dysfunction of mitochondria is involved in a range of pathologies known as mitochondrial cytopathies.

According to the mitochondrial theory of aging, functional alterations in mitochondria contribute to the aging process.(1). The human mitochondrial genome encodes 37 genes needed for mitochondrial protein synthesis and 13 essential subunits of the ETC and ATP synthase. The encoded polypeptides comprise few but essential subunits of complex I (7 peptides), complex III (1 peptide), complex IV (3 peptides) and ATP synthase (2 peptides).(2)(3) It has been demonstrated that brain, heart and skeletal muscle of aging humans harbor an increased mutational load of the mitochondrial genome compared to corresponding tissues of young.(4)(5)(6)

The circular mitochondrial DNA (mtDNA) is more susceptible to DNA damage in comparison to nuclear DNA (nDNA). Importantly, mtDNA molecules are not protected by histones; they are supported with only rudimentary DNA repair and are localized in close proximity to the electron transport chain (ETC), which continuously generates oxidizing products known as reactive oxygen species (ROS). Thus, the mutation rate of mtDNA has been reported to be up to 15-fold higher than the rate observed for nDNA in response to DNA damaging agents.

Mutations in mtDNA affect the function of the ETC, the electrochemical potential and the generation of ATP by oxidative phosphorylation. Furthermore, mutations of mtDNA can result in an elevated production of reactive oxygen species (ROS). ROS serve as signaling molecules but an overproduction can lead to unwanted oxidation of lipids, proteins and DNA which can further lead to cellular damage, mutations and cell death.(7) ROS generated by mitochondria have, therefore, numerous times been appointed as the mediator of the mitochondrial contribution to the aging process.(8)(9) Even though this relationship in some contexts has been validated, studies have demonstrated that mitochondrial generated ROS cannot be the sole explanation for the correlation between aging and mitochondria.

Mitochondrial Produced ROS Can Damage Cellular Component

More than 90% of cellular oxygen uptake is utilized in the process of oxidative phosphorylation, which continuously generates ROS such as superoxide anions, O2.-, hydrogen peroxide, H2O2, and hydroxyl radicals, OH.(10)(11) Complex I and especially complex III are the prime sites for electron leakage to molecular oxygen yielding O2.-.(12)(13) The production of ROS is inversely correlated with the rate of electron transport, increasing exponentially when complex I or III are impaired.(14) The main mediator of electron leakage is the reduced form of ubiquinone, ubiquinol, which is able to reduce molecular oxygen.(15)

In order to neutralize the produced ROS, a number of antioxidant defenses are active within the mitochondria. O2.- is neutralized by intramitochondrial Mn superoxide dismutase (SOD2) catalyzing the formation of H2O2. The latter either diffuses out of the mitochondria or is inactivated by reaction with glutathione catalyzed by glutathione peroxidase.(16)(17)(18) If the amount of produced ROS exceeds the capacity of SOD2 and glutathione peroxidase, O2.- and H2O2 levels will rise. In the presence of transition metals, such as iron or copper, highly reactive OH· can be produced by Haber – Weiss or Fenton reactions. OH· can in turn give rise to a plethora of ROS, which can further damage proteins, lipids, and DNA.(11)(19)

It has been demonstrated that inactivation of the Sod2 gene in mice leads to a two- to three-fold increase of oxidative damages of the nuclear DNA in heart and brain tissue when compared to mice expressing SOD2.(10) Furthermore, it has been demonstrated that an overexpression of a human catalase targeted for the mitochondria, prolonged median and maximal lifespan of mice by approximately 20% and enhanced exercise performance compared to wild type littermates.(20)(21) Mice overexpressing the mitochondrial targeted catalase did not display any adverse side effects, but rather a decrease in a select group of age-related pathologies.(22)

Induced mutations in genes encoding subunits for complex II have been demonstrated to result in increased production of O2.- and H2O2 in hamster fibroblasts. The increased production of ROS was co-occurring with an increase of aneuploidy (i.e., abnormal number of chromosomes in a cell) that could be reversed by expression of complex II subunits without mutations.(23) This led the authors to hypothesize that the increase of mitochondrial produced ROS resulted in genomic instability.

Mitochondrial ROS Functions As Second Messenger Molecules

Despite having being described mainly as a detrimental by-product of oxidative phosphorylation, it has become evident that certain types of ROS function as second messengers under subtoxic concentrations. As such, ROS has been demonstrated to regulate gene expression by controlling transcription factors and to affect protein activity by regulating kinases and phosphatases. The regulative effects of ROS are exerted through their redox potential. As an example, several ROS-sensitive molecules contain cysteine rich proteins, in which ROS-induced oxidation can result in the formation of disulfide bonds in the same molecule or between two cysteine rich molecules. The formation of disulfide bonds can, therefore, lead to conformational changes of a molecule, or result in the dimerizations (i.e., two monomers react to form a dimer) of two or more molecules, thereby modulating activation and activity of the molecules.(24)

Molecules that are regulated by ROS have been demonstrated to be involved in cell survival, cell cycle control, apoptosis, differentiation and several stress responses. Abnormal signaling elicited by aberrant ROS production can therefore affect essential pathways of the cell, potentially initiating an incorrect cellular response to a given situation, increasing the risk of senescence or tumorigenesis.(25)(26)

Mitochondrial-produced ROS has been demonstrated to affect the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 is a heterodimer consisting of the constitutively expressed (i.e., a gene continually transcribed) subunit HIF?2 and the oxygen sensitive subunit HIF-1?. Upon expression HIF-1? is marked by ubiquination (i.e., modification of a protein by covalent attachment of one or more ubiquitin molecules, small molecules present in all cells) and subsequent degradation by the proteasome.(27) However, an increased production of mitochondrial ROS has been demonstrated to stabilize HIF-1?, allowing the subunit to dimerize with HIF?2 and form the active nuclear transcription factor. Active HIF-1 is known to regulate the expression of glycolytic enzymes, the angiogenic factor VEGF and affect pathways promoting apoptosis.(28)(29) Furthermore, loss of HIF-1? has been demonstrated to accelerate premature cellular senescence in mice.(25)

The purpose of this regulation is most likely for the cell to react upon oxygen availability for oxidative phosphorylation, energetic yield, and to respond to increased oxidative load and mitochondrial stresses. As such, the targets of this regulation are therefore involved in critical cellular pathways, and it is no surprise that the majority of pathways that can be regulated by mitochondrial ROS have been correlated with cancer and senescence upon deregulation.

The circular mitochondrial DNA is more susceptible to DNA damage in comparison to nuclear DNA.

Mitochondria and Reserve Respiratory Capacity

Oxidative phosphorylation is an indispensable resource of ATP in tissues which require high energy. If the ATP demand is not met, this will lead to senescence and cell death in the affected tissue. The term reserve respiratory capacity, or spare respiratory capacity, is used to describe the amount of extra ATP that can be produced by oxidative phosphorylation in case there occurs a sudden increase in energy demand. Depletion of the reserve respiratory capacity has been related to a range of pathologies affecting high-energy-requiring tissues.

The energy requirement of different tissues and cells fluctuates constantly and the metabolism of ATP is correspondingly regulated to avoid futile energy expenditure and to cater to the specific needs of different tissues.

Short-term regulators of the oxidative phosphorylation exert their control in response to sudden changes of ATP demand. Cytochrome c oxidase (Complex IV of the ETC) has been demonstrated to be a potent facilitator of this short-term regulation.(30) Complex IV is the final electron acceptor of the ETC and catalyzes the reduction of O2 to H2O. The complex has been demonstrated to be allosterically (i.e., binding of regulators at a site other than the enzyme's active site) inhibited by ATP, whereby a futile overproduction of ATP is avoided.(31)(32)

Furthermore, the catalytic activity of complex IV is regulated by the mitochondrial electric membrane potential,(33)(34) balancing the activity of the ETC according to the needs, while avoiding a hyperpolarization of the mitochondrial membrane and, thereby, avoiding an excessive ROS production.

Several other targets exist for post-translational regulation of the ETC and the ATP synthase. Multiple phosphorylation sites have been demonstrated in complex IV(35)(36)(37) and phosphorylation of these sites has been shown to almost completely inhibit the catalytic activity of the complex.(36) Similarly, complex I and the ATP synthase have been demonstrated to contain phosphorylation sites.(38)(39) The mtNOS (mitochondrial nitric oxide synthase) is similarly post-transcriptionally regulated by both acylation and phosphorylation.(40)

Long-term regulators of the oxidative phosphorylation are effectors that alter the mitochondrial respiration in response to a changing role for the mitochondria in the tissue, for example, in skeletal muscles after endurance training(41) or as demonstrated as a consequence of caloric restriction.(42) Long-term regulators can also permanently change the properties of the mitochondrial respiration. This allows a differentiation of mitochondria, making them specialized for different cells and tissues. Accordingly, the ratio between produced ATP and consumed oxygen can vary greatly between different tissues. Heart and brain mitochondria of rats have been demonstrated to produce ATP faster than mitochondria of the liver, while liver mitochondria produce ATP more efficiently using less oxygen per produced ATP.(43)

Aging and Oxidative Phosphorylation

Aging impairs mitochondrial function by affecting both the capacity and the control of oxidative phosphorylation. Reduced activities of complexes I and IV, but not complexes II and III, have been found in aging mice and rats. In brain tissue of aging rats, a 22-35% reduction of complex IV activity was measured.(44) A similar trend has been demonstrated in humans where a reduced activity of complex IV has been found in skeletal muscle, heart and brain of aging subjects.(45)(46)(47) Correspondingly, an age-related decline of mitochondrial capacity for oxidative phosphorylation has been demonstrated in both human skeletal muscle and rat hearts.(5)(47)(48)

One factor affecting oxidative phosphorylation is an age-related accumulation of mtDNA mutations, resulting in an aberrant expression of mitochondrial encoded ETC subunits(5)(47) Increasing evidence suggests an important role of accumulating mtDNA mutations in the pathogenesis of many age-related neurodegenerative diseases, as well as a number of age-related pathological alterations of heart, skeletal muscle and the vascular system.

Cumulative damage to the mtDNA is, however, not the only contributor to the age-related decline of oxidative phosphorylation. Transcriptional profiling has revealed different regulation of nuclear genes encoding important peptides for oxidative phosphorylation when comparing young to old. In frontal cortex, the ?-subunit of the ATP synthase was found to be significantly down-regulated in old compared to young humans.(49) Using siRNA (i.e., synthetic RNA) to approximate same expression levels of ?-subunit of the ATP synthase in a neuroblastoma cell line resulted in a 24% reduction in cellular ATP levels.(49) In another study, skeletal muscle from aging rhesus monkeys displayed a selective down-regulation of nuclear encoded proteins involved in mitochondrial electron transport and oxidative phosphorylation.(50) These included subunits of complex I, complex IV and ATP synthase.

One mechanism that has been proposed to link aging with decreased transcription of nuclear genes encoding mitochondrial peptides is the shortening of telomeres.(51) Telomerase-deficient mice were intercrossed to produce successive generations with decreasing telomere reserves. The shortened telomeres of subsequent generations were demonstrated to be associated with reduced mtDNA content and impaired oxidative phosphorylation in the hematopoietic stem cells, liver and heart of the telomerase-deficient mice.(51) The telomere dysfunction was associated with a repression of the transcriptional coactivators, PGC-1? and PGC-1?2, and this repression was hypothesized to be responsible for the observed decline in mitochondrial function.(51)

These diseases can be caused by gene mutations in either nuclear DNA or mitochondrial DNA.

An age-related decline of oxidative phosphorylation can therefore be related to the decreased expression of both mitochondrial and nuclear expressed peptides functioning in the ETC. This decline affects most components of oxidative phosphorylation. Of the affected components, complex IV of the ETC stands out, as it has an important role in regulation of oxidative phosphorylation. Complex IV is the primary target of regulation by both short-term and long-term regulators, and the complex is therefore the main determinant of maximal, basal and, thereby, also the reserve respiratory capacity of mitochondria. Nuclear encoded subunits of complex IV have been shown to be down-regulated in an age-related manner.(50) Of the 13 peptides constituting complex IV, three are mitochondrial encoded. Consequently, an age-related decline of mtDNA fidelity also impairs the activity and the regulatory properties of complex IV and subsequently impairs or inactivates the ETC.

Mitochondrial Diseases

Mitochondrial diseases are a clinically heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrion. These diseases can be caused by gene mutations in either nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). As mtDNA is inherited exclusively from the mother, only mutations in the maternal mtDNA will potentially cause disease in offspring, whereas nDNA-encoded gene mutations can be both paternally and maternally inherited.

The clinical features of mitochondrial diseases are often quite complex. Some mitochondrial disorders only affect a single organ (e.g., the eye in Leber hereditary optic neuropathy [LHON]) while others show multiple organ systems defects often characterized by neurologic and myopathic features [e.g., Kearns-Sayre syndrome (KSS), chronic progressive external ophthalmoplegia (CPEO), mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), myoclonic epilepsy with ragged-red fibers (MERRF), neurogenic weakness with ataxia and retinitis pigmentosa (NARP) or Leigh syndrome (LS)].(52)

Mitochondrial dysfunction has also been implicated in premature aging, age-related diseases, and tumor initiation and progression.(7)

Diagnosis and Treatment of Mitochondrial Diseases

Symptoms of mitochondrial disease include poor growth, loss of muscle coordination, muscle weakness, visual problems, hearing problems, learning disabilities, heart disease, liver disease, kidney disease, gastrointestinal disorders, respiratory disorders, neurological problems, autonomic dysfunction and dementia. Diagnosis of mitochondrial diseases can be very difficult. Clinical tools include investigation of creatine kinase, lactate and glucose levels in the blood, histochemistry of muscle biopsies, PCR and RFLP analysis of mtDNA, sequencing mtDNA and electron microscopy of mitochondria.

Despite rapid advances of the understanding of the molecular basis of mitochondrial disorders, treatment remains limited and is restricted to reducing the effects of the mitochondrial disorder through exercise and through supplements. Current offered supplements include vitamins and antioxidants and adherence to a ketogenic diet. The success of treatments is very varied from patient to patient.

Summary

Ever since the first diagnosis of a mitochondrial disease in 1959,(53) the interest in mitochondrial cytopathies has continued to increase. Over the years, the understanding of mitochondrial cytopathies has evolved immensely and mitochondrial cytopathies are now known to be the largest group of metabolic diseases, causing a wide variety of pathologies. This range of pathologies includes general signs of premature aging, neuromuscular dysfunctions, cancer, diabetes, various heart diseases, inflammation and other conditions not previously known to be related to mitochondrial function.

A better understanding of mitochondria, therefore, allows a deeper understanding of related pathologies. We can now track mitochondrial function as biomarkers for the diseases and, most importantly, we will, hopefully, soon be able to apply this deeper understanding to finding potential treatments or cures for mitochondrial diseases.


Footnotes

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