Original Article

Mitochondrial Genetics and Disease

Journal of Child Neurology 2014, Vol. 29(9) 1208-1215 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0883073814539561 jcn.sagepub.com

Estela Area-Gomez, PhD1, and Eric A. Schon, PhD1,2

Abstract Mitochondrial disease resulting in reduced bioenergetic output can be due to mutations in either nuclear DNA–encoded or mitochondrial DNA–encoded gene products. We summarize some of the underlying principles of mitochondrial genetics that impact the diagnosis and pathogenesis of mitochondrial disorders. In addition, we present a brief overview of a new frontier in the field, namely, mitochondrial ‘‘dynamics,’’ which controls organellar fusion, fission, trafficking, and positioning, and exerts mitochondrial ‘‘quality control’’ by maintaining organellar integrity and viability. Analysis of mutations in gene products associated with this latter area has opened up new vistas in the study of disorders associated with compromised energy production. Keywords mitodynamics, mtDNA, oxidative phosphorylation, respiratory chain Received May 08, 2014. Received revised May 08, 2014. Accepted for publication May 13, 2014.

Our cells are informational composites. The DNA that encodes all that we are (but not who we are) resides not just in the nucleus but also in the numerous mitochondria located in the cytoplasm. This dual localization of information is responsible for 2 different types of genetics—mendelian genetics for nuclear DNA and population genetics for mitochondrial DNA—that is highly relevant to a group of human pathologies known as mitochondrial diseases. Given the numerous reviews that have been published on the subject in recent years,1 we will briefly summarize mitochondrial genetics as illuminated by examples of mitochondrial disorders of interest to clinicians, with a further focus on recent conceptual advances in the field.

Mitochondria and Bioenergetics Mitochondria are small, about the size of a typical bacterium (ie, *1 mm), and they have a double-membrane structure (the mitochondrial outer and inner membranes, enclosing the intermembrane space and the matrix). This small size and unique topology are no accidents, as, from an evolutionary standpoint, the precursors of mitochondria were, in fact, prokaryotes.2 Although the exact nature of the endosymbiotic event that gave rise to the eukaryotic cell is still contentious, there is general agreement that the fundamental reason mitochondria, and mitochondrial DNAs, have been retained in eukaryotic cells is their role in oxidative energy metabolism. In fact, of the approximately 1700 gene products present in the typical mammalian mitochondrion, more than 200 are devoted to producing energy (Table 1), via the respiratory chain/oxidative phosphorylation (OxPhos) system. The OxPhos system consists of 5 biochemically linked (and in some ways physically linked as well) complexes, all

embedded in the mitochondrial inner membrane (Figure 1A). Three of the 5—complexes I, III, and IV—take protons (hydrogen ions derived from the nicotinamide adenine dinucleotide [NADH] and flavin adenine dinucleotide [FADH2] that are, in turn, derived from the food we eat and that are produced mainly in the tricarboxylic acid cycle) and pump them ‘‘vertically’’ from the matrix to the intermembrane space, thereby generating a proton gradient across the inner membrane. That gradient is then used to synthesize adenosine triphosphate from adenosine diphosphate and free phosphate, by flowing in the opposite direction (ie, across the mitochondrial inner membranes from the intermembrane space to the matrix) through complex V, which is an adenosine triphosphate synthetase. Because all biochemical reactions must be electrically neutral, the protons are accompanied by an equal number of electrons flowing ‘‘horizontally’’ from complex I to complex IV (with the help of 2 small electron carriers, ubiquinone [also called coenzyme Q] and cytochrome c), and eventually to molecular oxygen to produce water (hence the term oxidative phosphorylation). Complexes I, III, IV, and V are composed of both nuclear DNA– and mitochondrial DNA–encoded

1

Department of Neurology, Columbia University Medical Center, New York, NY, USA 2 Department of Genetics and Development, Columbia University Medical Center, New York, NY, USA Corresponding Author: Estela Area-Gomez, PhD, Department of Neurology, P&S 4-443, Columbia University Medical Center, 630 West 168th Street, New York, NY 10032, USA. Email: [email protected]

Area-Gomez and Schon

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Table 1. Human Gene Products Present in Mitochondria.a Maintenance functions (829)

Specialized functions (984)

Protein translation and stability (260) Ribosomal components—large subunit assembly (52) Ribosomal components—small subunit assembly (39) Aminoacyl-tRNA synthetases (20) Transfer RNAs (45) Translation factors (27) Protein modification and stability (77)

Respiratory chain/oxidative phosphorylation (215) Complex I (62) Complex II (7) Complex III (18) Complex IV (50) Complex V (29) Respiratory supercomplex assembly (4) Heme, cytochrome, and iron-sulfur metabolism (29) Ubiquinone metabolism (16)

Organellar morphology and inheritance (163) Actin-associated proteins (12) Microtubule-associated proteins (41) Autophagy-related proteins (26) Mitochondrial shape and structure (42) Mitochondrial ‘‘germline-specific’’ proteins (13) Other ‘‘morphology-related’’ proteins (29)

Lipid metabolism (160) Cholesterol, steroid, and xenobiotic metabolism (35) Fatty acid metabolism (70) Phospholipid metabolism (41) Sphingolipid and glycolipid metabolism (14)

Carriers and transporters (152)

Signal transduction (152)

Nucleic acid metabolism (131) Nucleotide and phosphate metabolism (34) DNA replication (17) DNA plasticity, recombination, and repair (27) RNA transcription, processing, and maturation (53)

Intermediate metabolism (130)

Amino acid and nitrogen metabolism (74)

Stress response (65)

Miscellaneous/unknown (131)

Apoptosis (122)

Protein import and sorting machinery (58) Outer membrane components (22) Intermembrane space components (8) Inner membrane components (17) Matrix components (11) Total number of unique gene loci ¼ 1737 Abbreviation: tRNA, transfer RNA. a Numbers denote gene products in each category. Note that some gene products have functions in more than one category.

subunits, which is why the OxPhos system is also a biochemical composite (Figure 1A). In fact, the only polypeptides encoded by the mitochondrial genome (which is tiny—only 16.6-kb, as compared with the 3 000 000-kb nuclear genome) are subunits of these 4 complexes (Figure 1B). Interestingly, the fifth complex, complex II (succinate dehydrogenase, which is also a component of the tricarboxylic acid cycle), participates only ‘‘half-heartedly’’ in this process, as it transfers electrons to coenzyme Q but does not have a proton-pumping activity. This quirk is also reflected in SDH’s makeup: It contains only nuclear DNA–encoded subunits. Of the 1700-plus gene products targeted to mitochondria (Table 1), about half are required for the maintenance and well-being of the organelle itself (eg, organellar morphology, viability, and integrity; importation of proteins; replication, transcription, and translation of mitochondrial DNA; transport of small molecules via carriers and transporters); these are present in the mitochondria of essentially every cell in the body. On the other hand, the remaining gene products are associated with what we consider to be more specialized functions historically associated with mitochondria, which are required for the well-being of the cell as a whole (eg, amino acid,

intermediate, and lipid metabolism; OxPhos; and more recently, apoptosis and cell signaling), and often are present in the mitochondria of particular tissues but absent in others. For example, cytochrome P450 enzymes predominate in liver mitochondria, where they are used to degrade toxic agents. Obviously, mutations in any of these 1700 gene products could conceivably cause a pathologic condition, but for the purposes of this review, we will define mitochondrial diseases as those associated only with OxPhos dysfunction, either directly (eg, mutation in a respiratory chain subunit) or indirectly (eg, mutation in an OxPhos assembly protein). Given the composite makeup of the OxPhos system, it stands to reason that mutations in both mitochondrial DNA– and nuclear DNA–encoded genes can cause mitochondrial disorders.

Genetics of Mitochondrial Diseases By and large, mutations in nuclear DNA–encoded genes give rise to classical mendelian-inherited diseases.1 Most notable among these is Leigh syndrome, a fatal disorder of infancy or childhood. Children with Leigh syndrome have psychomotor retardation or regression, hypotonia, respiratory abnormalities,

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A Succinate

MATRIX INNER MEMBRANE

ND1 ND2 ND3 ND6 ND4 ND5 ND4L

Fumarate

e-

e-

CoQ

O2 Cytb

e-

INTERMEMBRANE

e-

e-

H 2O COX I COX II COXIII

ADP

ATP A8 A6

Cyt c

SPACE

Complex I nDNA-encoded subunits: mtDNA-encoded subunits: “Assembly” proteins:

H+

H+

H+

H+

38

CoQ Complex II

-

Complex III

4

11

Cyt c 1

Complex IV

11

Complex V

~16

7

0

0

1

0

3

2

13

16

2

6

2

29

4

B

Figure 1. The OxPhos system in humans. (A) The mitochondrial respiratory chain. Blue subunits are nuclear DNA–encoded; colored subunits are mitochondrial DNA–encoded, as in B. (B) Human mitochondrial DNA. The genes for the 12S and 16S ribosomal RNAs, the subunits of complexes I (nicotinamide adenine dinucleotide [NADH]–coenzyme Q oxidoreductase [ND]), III (cytochrome b [Cyt b]), IV (cytochrome c oxidase [CO]), and V (ATP synthase [A]), and 22 transfer RNAs (1-letter nomenclature), are shown. ‘‘Assembly proteins’’ are ancillary polypeptides required for the assembly and/or stability of the holocomplex; they are all nuclear DNA–encoded. Note. Figure is available in full color in the online version at jcn.sagepub.com

seizures, and a general failure to thrive. Leigh syndrome is defined neuropathologically and/or neuroradiologically by bilateral symmetrical lesions in the central nervous system, especially in the thalamus, brainstem, basal ganglia, and cerebellum; pathologically, there is neuronal loss, reactive astrocytosis, and proliferation of cerebral microvessels. Importantly, Leigh syndrome due to nuclear DNA mutations follows standard mendelian inheritance patterns, most frequently autosomal-recessive inheritance. As such, assuming that the culprit gene is known, diagnosis is straightforward, as is genetic counseling. Even if the gene is unknown, recent advances in whole-exome (and even whole-genome) sequencing technology may still make it possible to identify the culprit gene.3 The example of Leigh syndrome as a mitochondrial disorder is a useful one, because the syndrome can also be caused by

mutations in mitochondrial DNA, especially in those genes encoding respiratory chain structural subunits of complex I, IV (also known as cytochrome c oxidase, or COX), and V.1 In this case, however, the genetics can be truly confusing for the clinician, because we are now dealing with population genetics, not mendelian genetics, for the simple reason that mitochondrial DNAs are present in multiple copies in each cell. Although somatic cells contain only 2 copies of the autosomes, there may be hundreds or even thousands of copies of mitochondrial DNA, depending on the energetic requirements of that cell. Normally, all the mitochondrial DNAs in a cell or tissue are identical (ie, homoplasmy), but in the case of a disease due to a mutation in mitochondrial DNA, the patient may harbor both normal and mutated mitochondrial DNAs in the same cell or tissue (ie, heteroplasmy).

Area-Gomez and Schon Let us take the example of a baby with a mitochondrial DNA–encoded mutation in the COX II subunit of complex IV, causing Leigh syndrome. If we analyze the infant’s blood (or muscle, which is preferable for diagnostic purposes), we will find that the amount of mutation is generally greater than 80% of total mitochondrial DNA. Where did those mutated mitochondrial DNAs come from? Typically, they came from the mother, because human mitochondria (and mitochondrial DNAs) are maternally inherited. But if the mother is also heteroplasmic, why doesn’t she have the disease? In fact, she is heteroplasmic, but analysis of the mother’s blood will show a mutation load typically in the range of 50% to 70%, not 80%. This result demonstrates 2 key features of mitochondrial (ie, population) genetics: the threshold effect and the bottleneck effect. The threshold defines the level of mutation above which overt pathology ensues, because at that point there are not enough ‘‘good’’ genomes in the cells of a baby with Leigh syndrome to compensate for either missing COX II (eg, in the case of a stop-codon mutation) or functionally deficient COX II (eg, in the case of a missense mutation) derived from the ‘‘bad’’ genomes. Experience has shown that most mitochondrial DNA– based diseases are ‘‘recessive,’’ in the sense that the mutation load must be quite high—as we noted above, typically >80%—to induce pathology. This threshold applies even if the mutation is in a ‘‘protein synthesis’’ gene, such as a transfer RNA. Note that even though a mutation in COX II and a mutation in a transfer RNA both inhibit OxPhos, they do so by different routes (ie, mutated COX II compromises only complex IV function, whereas a mutated transfer RNA compromises global translation of all 13 mitochondrial DNA–encoded polypeptides). Mutations in transfer RNAs can cause distinctly different disorders, as exemplified by mutations in transfer RNALeu(UUR) (see Figure 1B) that typically cause mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS).4 So the mother is ‘‘normal’’ (or at worst oligosymptomatic, but surely healthy enough to have a baby) in spite of the fact that she is heteroplasmic, because her 50% mitochondrial DNA mutation load is below the threshold for dysfunction. But why does the baby have 80%? This demonstrates the bottleneck effect: Only a subset of the mother’s mitochondrial DNAs are ‘‘picked’’ to repopulate the child’s complement of mitochondrial DNA. For example, of the *1,000 mitochondrial DNAs in the mother’s primordial germ cells prior to oogenesis, only about 20 to 200 mitochondrial DNAs eventually wind up in the baby (actually we use the term segregating units rather than mitochondrial DNAs, because mitochondria contain *5 mitochondrial DNAs per mitochondrion, and it is possible that multiple mitochondrial DNAs can be inherited together as a ‘‘unit,’’ either as part of the organelle itself or as part of a mitochondrial DNA–protein complex called a nucleoid). This means that if the mother has 50% mutated mitochondrial DNAs in her oogonia, through the vagaries of random sampling the zygote may harbor 80%, in which case the baby likely will be affected. Of course, the baby may inherit 40% or even less through the germline, in which case the child likely will be normal. We say ‘‘likely will be normal’’ but not ‘‘will be normal’’ because there is a second mitochondrial bottleneck, in postnatal

1211 development. Following oogenesis, there are *100 000 mitochondrial DNAs in the oocyte (all amplified from those original 20-200 segregating units). At that point, mitochondrial division and mitochondrial DNA replication cease, and do not recommence until long after fertilization, after implantation of the blastocyst (64- to 128-cell stage). Because only a few cells will actually become the fetus (*90%-95% of the cells will give rise to the extraembryonic tissues, such as the placenta), the original *100 000 mitochondrial DNAs in the zygote are reduced *100-fold. Thus, any mutated mitochondrial DNAs that survived passage from the germline to the zygote will run a second gantlet of random selection through this postimplantation bottleneck. From an evolutionary and developmental standpoint, these bottlenecks make sense, as they, together with maternal inheritance, help ensure that deleterious mitochondrial DNA mutations do not spread through the population, thereby protecting the species from extinction. However, there is a price to be paid for this evolutionary ‘‘protection’’: the occurrence of rare pedigrees in which potentially fatal mitochondrial DNA mutations are indeed transmitted. Fortunately (if that is the right word), these mutations cannot spread ‘‘laterally’’ through the population, as they are confined to maternal lineages. We should note that how and where in development these bottlenecks operate are still under investigation, with much debate in the field.5,6 Even if a pathogenic mitochondrial DNA mutation passes into the fetus, we still cannot be sure whether the child will be affected in postnatal development, owing to yet another unique aspect of mitochondrial population genetics: mitotic segregation. Following cell division, the mitochondria are partitioned randomly into the 2 daughter cells, approximately 50:50, at which point the organelles and genomes ‘‘doubleup’’ to their pre-division copy number.7 Again, we use the word approximately and not exactly, as the partitioning of mitochondria and mitochondrial DNAs during cell division is essentially stochastic. In normal homoplasmic individuals, this is not an issue, as all progeny cells contain the same mitochondrial DNA genotype. However, in the case of heteroplasmy, the distribution of the mutation in daughter cells follows a bell-shaped curve, with most cells having approximately the same mutation load as the parental cell but with rarer daughter cells having significantly more, or fewer, mutated mitochondrial DNAs. As can be imagined, this ‘‘genetic drift’’ is far less pronounced in cells that divide rarely (eg, muscle, brain) than in those that divide relatively frequently (eg, blood, liver), and that can have real clinical consequences. A beautiful example of this phenomenon is the connection between Pearson marrow pancreas syndrome and KearnsSayre syndrome. Pearson marrow pancreas syndrome is a devastating pancytopenia resulting from a spontaneous largescale (kilobase-sized) partial deletion of mitochondrial DNA arising during oogenesis or even during early embryonic development, with the deletion (called D-mitochondrial DNA) present predominantly (but usually not exclusively) in the hematopoietic tissues. Because the D–mitochondrial DNA is present in a rapidly dividing tissue, the phenomena of mitotic segregation and genetic drift, coupled with a potential for the

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Table 2. The Next Frontier in Mitochondrial Diseases. Disorder

Gene

Protein

Diseases associated with defects in mitochondrial dynamics Charcot-Marie-Tooth disease type 2E NEFL Neurofilament light polypeptide, mito traffic Charcot-Marie-Tooth disease type 4A GDAP1 Ganglioside-induced differentiation-associated protein Dominant optic atrophy 1 OPA1 Optic atrophy protein 1 (dynamin-related GTPase) Fatal infantile encephalomyopathy DNM1L Dynamin-related protein 1 Fatal infantile encephalomyopathy MFF Mitochondrial fission factor Spastic paraplegia type 4 SPAST Spastin (microtubule-severing protein) Diseases associated with defects in mitochondrial quality control Parkinson disease type 2 PARK2 Parkin (E3 ubiquitin-protein ligase) Parkinson disease type 6 PINK1 PTEN-induced putative kinase 1 Spastic paraplegia type 7 SPG7 Paraplegin (metalloprotease) Spinocerebellar ataxia type 28 AFG3L2 Paraplegin-like protein (metalloprotease) Amyotrophic lateral sclerosis type 14 VCP Valosin-containing protein (ATPase) Diseases associated with defects in communication between mitochondria and ER Alzheimer disease type 3 PSEN1 Presenilin-1 (aspartyl protease) Alzheimer disease type 4 PSEN2 Presenilin-2 (aspartyl protease) Amyotrophic lateral sclerosis type 8 VAPB VAMP-associated protein B Amyotrophic lateral sclerosis type 16 SIGMAR1 Sigma 1 non-opioid receptor (calcium regulation) Charcot-Marie-Tooth disease type 2A MFN2 Mitofusin-2 (transmembrane GTPase) MEGDEL syndrome SERAC1 Serine active site-containing protein 1 Parkinson disease type 1 SNCA a-Synuclein Spastic paraplegia type 18 ERLIN2 ER lipid raft-associated protein-2

Reference 9,10 11,12 13,14 15 16 17 18 19 20 21 22 23,24 23,24 25,26 27 28,29 30 31,32 33

Abbreviations: ER, endoplasmic reticulum; MEGDEL, 3-methylglutaconic aciduria with deafness, encephalopathy, and Leigh-like syndrome; VAMP, vesicleassociated membrane protein.

D–mitochondrial DNAs to replicate at a rate different from that of normal mitochondrial DNAs, result in a slow but steady reduction of the mutation load in bone marrow and blood. This shift is accentuated and accelerated by clinical interventions to save the child, including blood transfusions containing normal mitochondrial DNAs from the blood donor. The irony is that although the child is slowly recovering because of the declining load of D–mitochondrial DNA in blood, the very same mechanisms of mitotic segregation and genetic drift are operating in the opposite direction in other, more slowly dividing tissues such as muscle and brain (in which mitochondria and mitochondrial DNAs still replicate even though their host cells do not), giving rise to Kearns-Sayre syndrome, the most florid expression of a systemic sporadic D–mitochondrial DNA disorder. Kearns-Sayre syndrome is typically fatal, with the patient suffering from progressive external ophthalmoplegia, diabetes, and heart conduction block, among other symptoms. These population genetic principles—maternal inheritance, mitotic segregation, the threshold effect, the bottleneck—all converge to explain the dynamic nature of heteroplasmy at the cellular, tissue, and organismic level. It is for this reason that prenatal diagnosis of a mitochondrial DNA–based disorder, with but a few exceptions, is fraught with difficulties, both technical and ethical.8

A New Frontier in Mitochondrial Disease Although the number of OxPhos disorders due to mutations in mitochondrial DNA and nuclear DNA will continue to grow, a new and exciting frontier has opened up that is redefining what

we mean by genetic disorders affecting bioenergetics. We are referring here to the aspect, long relatively unappreciated, of mitochondria as dynamic organelles that play a truly fundamental role in the life of the cell that goes beyond ‘‘mere’’ bioenergetics. A cursory look at Table 1 shows that mitochondria play key roles in lipid metabolism, in apoptosis, in signal transduction, in autophagy, and in other aspects of cellular life. Moreover, they are not the static organelles pictured in textbooks. Mitochondria can fuse and form tubular structures, they can divide and form punctate structures, they can travel throughout the cell on microtubules (for long distances) and on actin cables (for short distances), they can anchor themselves in specific regions of the cell, they are involved in the immune response and in protection from viral invaders, and they can signal the rest of the cell to initiate autophagy or, in extremis, apoptosis. In short, a mitochondrial disorder, including one that affects bioenergetics, can be based not only on what has gone wrong, but also on where, when, and how it has gone wrong. In other words, disorders associated with the genetic control of mitochondrial dynamics, quality control, and intracellular communication with other organelles are now falling under the rubric of mitochondrial diseases (Table 2). Interestingly, most of these disorders are associated with late-onset neurodegeneration, although some have been connected to juvenile forms of neurodegenerative diseases.34 We have classified the disorders into 3 groups, but in truth it is difficult to categorize these diseases neatly and there is much overlap among them. We will not discuss the entire list exhaustively (the reader is referred to the citations in Table 2 for

Area-Gomez and Schon further information), but will rather focus on 1 or 2 disorders in each group that illustrate salient points. The first group comprises diseases associated with defects in mitochondrial dynamics (ie, fusion, fission, anchorage, trafficking). Among them, one disorder, dominant optic atrophy, caused by mutations in a gene called OPA1, is essentially the mendelian version of an authentic mitochondrial DNA–based mitochondrial disease called Leber hereditary optic neuropathy, which was the first maternally inherited disorder to be described at the molecular level.35 Like Leber hereditary optic neuropathy, dominant optic atrophy is characterized by optic disc pallor, loss of visual acuity, and centrocecal scotoma, but the underlying biology is fundamentally different. Leber hereditary optic neuropathy is associated with mutations in mitochondrial DNA–encoded subunits of complex I, often in the homoplasmic state, whereas dominant optic atrophy is associated mutations in OPA1, a dynamin-related GTPase located in the mitochondrial inner membrane that, together with mitofusin-1, another GTPase located on the outer membrane, participates in mitochondrial fusion, and perhaps in other functions as well.13 Thus, a defect in mitochondrial fusion (resulting in an increase in fission; the 2 processes are inversely correlated) in every cell in the body can cause an ophthalmologic disorder as a result of a change in organellar shape (and to be sure, in bioenergetics as well). Interestingly, mutations in a mitochondrial fission gene, DNM1L, causes a Leigh-like fatal infantile encephalomyopathy.15 Why dominant optic atrophy (and Leber hereditary optic neuropathy, for that matter) is tissue-specific is a complete mystery at present. Other fission-related genes associated with mitochondrial disease include ganglioside-induced differentiation-associated protein (GDAP1)11,12,36 and mitochondrial fission factor (MFF).16 Besides fusion and fission, mitochondrial dynamics also encompasses motility. Thus, failure to deliver mitochondria to the proper place and in the proper amount to various regions of the cell can cause localized deficiencies in the production of adenosine triphosphate (and other mitochondrial-derived metabolites and ions), with deleterious consequences. Diseases associated with ‘‘trafficking’’ defects include those due to mutations in spastin,17,37 a microtubule-severing protein, and in neurofilament light chain.9,10,38 It is almost intuitive that ‘‘trafficking’’ defects would be most pronounced in neurons in which the distance from the cell body to axonal terminals and to dendrites are vast compared with those in somatic cells, and that is indeed the case. The second group comprises diseases associated with defects in mitochondrial quality control. Like any machine, mitochondria are subject to wear and tear and must be maintained in proper working condition, or failing that, eliminated. Thus, defects in proteins involved in these processes, which include the proper folding of proteins, the proteasomal degradation of improperly folded proteins,39 and mitochondrial autophagy (mitophagy),40 can cause disease. Perhaps the best-known among these are mutations in PINK1 (PTEN-induced putative kinase 1), a kinase, and

1213 parkin, an E3 ubiquitin ligase associated with proteasomal protein degradation. Mutations in both proteins cause familial forms of Parkinson disease, and in fact both proteins appear to operate in the same pathway to remove dysfunctional mitochondria via mitophagy.40 The last group comprises diseases associated with defects in the communication between mitochondria and other subcellular organelles, most prominently the endoplasmic reticulum. Endoplasmic reticulum–mitochondrial communication occurs in a specialized subcompartment of the endoplasmic reticulum called mitochondria-associated endoplasmic reticulum membranes.41 Interestingly, mitofusin-2, a mitochondrial fusion protein that is highly similar to mitofusin-1 noted above, also controls the apposition between the endoplasmic reticulum and mitochondria,28 and, when mutated, can cause disease.29 Because mitofusin-2 aids in the connection of the endoplasmic reticulum to mitochondria, one can well imagine how loss or impairment of this connectivity could affect numerous processes that might impinge on mitochondrial bioenergetics. To cite but one example, given that calcium is a key regulator of the pyruvate dehydrogenase complex and the tricarboxylic acid cycle,42,43 impairment of calcium trafficking from endoplasmic reticulum to mitochondria through the mitochondria-associated endoplasmic reticulum membrane–localized inositol triphosphate receptor-3 (IP3R3)44 will have both direct and indirect effects on bioenergetics. Although mitofusin-2 promotes endoplasmic reticulummitochondrial connectivity, presenilin-1 (PSEN1) and presenilin-2 (PSEN2), which are associated with Alzheimer disease, suppress it,23 perhaps helping to explain why OxPhos is compromised in this disorder.45 Because mitochondria also communicate with other organelles, including autophagosomes,46 endosomes,47 and peroxisomes,48 we can expect to see reports of mitochondrial disorders arising from mutations in genes associated with these processes in the near future.

Concluding Remarks For more than 20 years, genetic diseases due to deficiencies in OxPhos were associated with ‘‘simple’’ mutations in nuclear DNA– and mitochondrial DNA–encoded genes closely associated with the machinery of adenosine triphosphate production. In the past few years, however, our perspective on what causes a mitochondrial bioenergetic disorder has changed radically, based on a new understanding of the spatial and temporal localization of mitochondria in cells, and the other subcellular organelles with whom they partner. Our enhanced appreciation of the ‘‘4-dimensional’’ role this organelle plays in the overall life of the cell has expanded radically our definition and understanding of mitochondrial disease. Authors’ Note This work is based on a paper delivered at the 2013 Neurobiology of Disease in Children Symposium: Mitochondrial Disease, held in

1214 conjunction with the 42nd Annual Meeting of the Child Neurology Society, Austin, Texas, October 30, 2013.

Acknowledgments We thank Dr Salvatore DiMauro for helpful comments and Melanie Fridl Ross, MSJ, ELS, for editing assistance.

Author Contributions EA-G and EAS wrote the paper.

Declaration of Conflicting Interests The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from by the U.S. National Institutes of Health (HD32062), the Department of Defense (W911NF-12-10159), the Muscular Dystrophy Association, the Ellison Medical Foundation, the J. Willard and Alice S. Marriott Foundation, and the National Institute of Neurological Disorders and Stroke (R13 NS40925).

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