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Critical review

Licensee OA Publishing London 2013. Creative Commons Attribution License (CC-BY)

For citation purposes: Frye RE, Rossignol D. Mitochondrial physiology and autism spectrum disorder. OA Autism 2013 Mar 01;1(1):5.

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Mitochondrial physiology and autism spectrum disorder RE

Frye1*, D Rossignol2

AbstractIntroductionRecent studies have suggested that mitochondrial dysfunction is rela-tively common in many children with autism spectrum disorder (ASD) and appears to be the most prevalent metabolic disorder associated with ASD. However, the exact prevalence of mitochondrial disease in ASD is not clear as the classic criteria for diagnosing mitochondrial disease in ASD appear to underestimate the true prevalence. Indeed, the preva-lence of biomarkers of mitochondrial dysfunction and abnormal electron transport chain function appears to be high in ASD. In addition, recent studies have uncovered novel forms of mitochondrial dysfunction in ASD that may not be readily recog-nized by classic diagnostic criteria. This critical review provides a brief overview of mitochondrial function and its influence on other cellular systems in the context of ASD. The mitochondria’s role in producing en-ergy is complex and linked to other metabolic systems. Mitochondrial energy production is a result of the tricarboxylic acid cycle, fatty-acid oxidation and the electron transport chain working in concert. The unique architecture of the mitochondria facilitates the function of these sys-tems. The function and architecture of the mitochondria for producing energy with a special reference to the source of biomarkers of mitochon-drial dysfunction is reviewed. Non-energy functions of the mitochon-dria including calcium buffering,

heat production and apoptosis are outlined. Interactions with other systems, including the porphyrin pathway, urea cycle and glutathione metabolism, are also outlined, fol-lowed by a discussion of mitochon-drial control and regulation. Finally, the recommended algorithm for the diagnosis of mitochondrial disorders in children with ASD is discussed.Conclusion The mitochondrial is critically im-portant in understanding the physio-logical abnormalities associated with ASD. By providing details regarding mitochondrial function, this critical review aims to provide a better un-derstanding of the importance of mi-tochondria as it is related to ASD.

IntroductionMitochondria are complex subcellu-lar organelles that are essential for cellular function. The primary role of the mitochondria is energy produc-tion. Thus, the number of mitochon-dria per cell is proportional to the energy requirements of the cell. For example, skin cells have several hun-dred mitochondria, whereas muscle cells have several thousand mito-chondria. However, mitochondria have many roles aside from energy production, and mitochondrial me-tabolism interacts with several other critical cellular systems, potentially

influencing, and being influenced by, these systems. In addition, regul-ation and control of mitochondria is complex (Table 1). This critical review provides an overview of m-itochondrial function in the cont-ext of autism spectrum disorder.

DiscussionMitochondrial medicineMitochondrial medicine is a relative-ly new and evolving field. Although the clinical and histological features of mitochondrial disease (MD) were recognized in the 1960s, it was not until 1988 that specific MD could be linked to a causative genetic muta-tion1. From that point on, MDs have primarily been described in reference to their associated genetic altera-tions. However, as the wide variation in the phenotypic presentation of MD is being appreciated, it is becoming clear that many cases of MD cannot be simply linked to a single specific causative genetic abnormality.

MD and ASDRecent studies have suggested that mitochondria are not functioning properly in many children with ASD2 and in genetic diseases associated with ASD3. Of the many metabolic disorders associated with ASD, mito-chondrial dysfunction appears to be the most prevalent4. Table 2 lists the

* Corresponding author Email: [email protected] Arkansas Children’s Hospital Research

Institute, Little Rock, AR, USA2 Rossignol Medical Center, Irvine, CA, USA

Table 1 Important aspects of mitochondrial function

Mitochondrial functions

Metabolic system that interact with mitochondria

Mitochondrial control and regulation systems

• Carbohydrate oxidation• FAO• ATP production• Calcium buffering• Apoptosis• Heat production• Inflammation

• Porphyrin pathway• Urea cycle• Glutathione metabolism

• mtDNA• nDNA• Epigenetics• Membrane potential

regulation• Redox regulation• Regeneration

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diagnosing MD may underestimate the number of ASD children with mitochondrial dysfunction2. Studies that have examined biomarkers for MD suggest that the percentage of children with ASD who have abnor-mal mitochondrial function may be as high as 30% to 50%2,13. Further-more, one study found that 80% of ASD children demonstrated lower than normal electron transport chain (ETC) function in lymphocytes14. Interestingly, several studies have pointed to novel forms of mitochon-dria dysfunction in ASD, including ETC overactivity rather than defi-ciencies15,16, abnormalities in super-complex assembly17 and unique ele-vations in acyl-carnitines associated with ETC, tricarboxylic acid (TCA) cy-cle and glutathione abnormalities18. These novel forms of mitochondrial dysfunction would be missed by the classic criteria for diagnosing MD, but it is not clear that such forms of mito-chondrial dysfunction should be cat-egorized as classic MD. Thus, at this point, it is uncertain if these novel forms of mitochondrial dysfunction associated with ASD are equivalent to classic MD, or whether they repre-sent a more widespread novel form

cause neurodegenerative events11. The ability to assess this potential re-lationship is further obscured by the lack of the differentiation between true MD and mitochondrial dysfunc-tion (refer to the next paragraph) in ASD. Since the association between an inflammatory/infectious trigger and neurodevelopmental regression has only been demonstrated in children with MD, the same association may not hold true for mitochondrial dys-function.

Despite this interesting connection between ASD and classically defined MD, the proportion of children with ASD and classically defined MD ap-pears to be low. A recent meta-anal-ysis found that 5% of children with ASD met strict criteria for classic MD. One of the major studies that this prevalence estimate was based on was a population-based prospective study conducted in Portugal and the Azores, making the validity of this es-timate particularly strong12. The clas-sic criteria for diagnosing MD relies heavily on diagnosing genetic abnor-malities, but genetic abnormalities are only found in the minority of cases of children with MD and ASD, suggesting that the classic criteria for

forms of mitochondrial dysfunction that have been associated with ASD.

The suggestion that abnormalities in carbohydrate metabolism could be involved in ASD was first proposed over 25 years ago when an unusual high rate of lactic acidosis (a biomark-er of MD) was noticed in a case series of children with ASD5. Since then, several other notable reports have associated MD with ASD. In 2002, the HEADD syndrome, an association of hypotonia, epilepsy, autism and de-velopmental delay, was described in a series of ASD children with respira-tory chain disorders6. Although this association appears common in chil-dren with ASD and MD, the HEADD acronym has never really been com-monly applied clinically or in the re-search literature. More recently, the association of MD and ASD with vac-cines gained controversial attention. In one case that received consider-able notoriety, a girl with underlying MD underwent rapid regression into an ASD diagnosis following a febrile episode induced by multiple vacci-nations given on the same day7. This connection between infectious/in-flammatory triggers with ASD and MD was further substantiated by a case series of 28 children with ASD and MD. Shoffner et al.8 found that 71% of the children in this case series who manifested regression into ASD re-gressed within 2 weeks of an episode of fever greater than 101°F. Interest-ingly in 33% of these cases, the fever was associated with routine child-hood vaccinations. This should not be completely surprising as it is widely believed that infections can result in metabolic decompensation in some children with underlying metabolic disease such as MD9, and studies have verified this association in children with an underlying MD10. However, the true prevalence of this association is unknown. Many practitioners sug-gest vaccination as a way to prevent severe illnesses and point to the fact that, for the large majority of indi-viduals with MD, vaccination does not

Table 2 Mitochondrial disorders reported in ASD

Functional abnormalities

ETC deficiency in complex I, II, III, IV and V Co-enzyme Q deficiencyFAO

Mitochondrial DNA abnormalities

A3243A>G: mitochondrial encephalopathy with lactic acidosis and seizures (MELAS) syndrome3397A>G: complex I8363G>A, 10406G>A, 4295A>G: tRNALarge-scale mtDNA deletionsDNA depletion

Novel mitochondrial abnormalities

Complex I overactivityComplex IV overactivityAbnormal supercomplex assemblyUnique acyl-carnitine abnormalities (short- and long- but not medium-chain elevations)

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functions such as calcium buffer-ing and mediating programmed cell death (also known as apopto-sis). The important functions of the mitochondria will be reviewed here.

Energy productionThe pathways for energy produc-tion are outlined in Figure 1 and are discussed in detail below.

Electron carriers between mitochondrial metabolic reactionsElectrons are derived from the oxida-tion of carbohydrates and fatty acids

MatrixThe matrix contains the majority of the enzymes that are responsible for primary functions of the mitochon-dria including TCA cycle and fatty-acid oxidation (FAO) enzymes as well as mitochondrial DNA (mtDNA) and the enzymes and machinery nec-essary to transcribe and translate mtDNA into proteins.

Mitochondrial functionsAlthough mitochondria are the primarily powerhouse of the cell, they serve several other important

of mitochondrial dysfunction that could be secondary to other, possibly amenable, factors. Using a broader criterion for diagnosing MD, such as the Morava et al. criteria (refer to the Conclusion section)19,20, could allow for the diagnosis of these novel forms of mitochondrial dysfunction as MD but could also blur the line between an important distinction of different types of MD.

Mitochondrial structureThe double membrane of the mito-chondrion creates two separate com-partments, each of which has distinct structure and functions.

Outer membraneThe outer membrane encloses the entire mitochondria and is made of a phospholipid bilayer with integral proteins called porins. Porins are large channels that are permeable to molecules of about 5,000 Da, allow-ing ions, nutrients and other impor-tant small molecules to pass through the membrane.

Inner membraneThe inner membrane is a special-ized membrane that is relatively impermeable to most molecules. This facilitates the ability of the ETC to cre-ate an electrochemical proton gradi-ent across the inner membrane. Many proteins reside in the inner membrane such as the ETC and specialized trans-port proteins. The inner membrane has a very high protein-to-phospho-lipid ratio and unique phospholipids, such as cardiolipin, which help make the inner membrane impermeable.

CristaeThe inner membrane is folded into lamellae called the cristae. These folds greatly increase the inner mem-brane surface area, allowing a greater number of ETC complexes to exist in the mitochondria and decreasing the distance between the ETC complexes and important enzymes that reside in the matrix.

Figure 1: Energy production pathways of the mitochondria. Biomarkers for MD are coloured red. Pyruvate dehydrogenase complex is divided into three subunits: E1, pyruvate dehydrogenase; E2, dihydrolipoyl transacetylase; E3, dihydrolipoyl dehydrogenase. TCA cycle contains multiple enzymes: CS, citrate synthase; AC, aconitase; ID, isocitrate dehydrogenase; KD, α-ketoglutarate dehydrogenase; SS, succinyl-CoA synthetase; SD, succinate dehydrogenase; FM, fumarase; MD, malate dehydrogenase. FAO requires the carnitine shuttle to bring fatty acids into the mitochondrial matrix: CPT I, carnitine palmitoyltransferase; CACT, carnitine-acylcarnitine translocase; CPT II, carnitine palmitoyltransferase II. ETC is composed of five enzyme complexes bound to the inner membrane. Complex II is also known as SD and contains several subunits. Subunit A of SD catalyses succinate to fumarate, while subunits B/C/D of SD oxidize FADH2 in order to reduce coenzyme Q10. Two electron carriers, the reduced forms of NADH and FADH2, carry electrons from TCA and beta-oxidation to the ETC complexes.

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acids) or until a propionyl-CoA re-mains (for odd-chain length fatty ac-ids). Propionyl-CoA is further metab-olized to succinyl-CoA so it can enter the TCA cycle.

ATP productionAdenosine triphosphate (ATP) is produced by the ETC. The majority of reported cases of MD in ASD involve dysfunction of the ETC2.

ETCA series of five enzyme complexes oxi-dize the electron carriers NADH and FADH2 to create ATP. Complexes I, III and IV (collectively known as the su-percomplex) pump protons from the matrix into the intermembrane space. mtDNA mutations potentially linked to abnormal supercomplex assem-bly have been reported in relation to ASD17. The electrochemical gradient produced by pumping protons across the membrane results in a proton mo-tive force, which is utilized by complex V (ATP synthase) to produce ATP from adenosine diphosphate. Complex II transfers electrons to the ETC electron carrier coenzyme Q10 (CoQ), which is further utilized by complex III.

Electron carriers of the ETCTwo electron carriers are used to transfer electrons between ETC complexes.

Coenzyme Q and the Q cycleCoenzyme Q10 carries electrons from complex I and II to complex III. A complicated sequence of oxidation and reduction of CoQ, which is called the Q cycle, occurs at complex III. In brief, a reduced CoQ carrying two electrons and two protons is joined by another reduced CoQ derived from the Q cycle to donate four elec-trons and four protons to complex III. The protons are pumped into the intermembrane space to increase the proton gradient while two electrons reduce cytochrome b and the other two reduce the Rieske iron-sulphur protein. The reduced cytochrome b is

build up in the form of organic ac-ids that are biomarkers of MD. One case series has specifically reported dysfunction in the TCA cycle in ASD18.

FAOFatty acids are oxidized in the mito-chondrial matrix. While short-chain and medium-chain fatty acids can freely diffuse across the mitochon-drial membranes, long-chain fatty acids (greater than 10 carbons in length) require the carnitine shuttle. Very long-chain fatty acids (greater than 22 carbons in length) are ini-tially oxidized in peroxisomes rather than the mitochondria until they are shortened to long-chain fatty acids. One case series18 and one case re-port2 has specifically reported poten-tial abnormalities in FAO pathways in ASD.

Carnitine shuttleThree enzymes facilitate the trans-portation of fatty acids into the matrix. Carnitine palmitoyltransferase I (CPT I), which sits on the outer mitochon-drial membrane, transfers an acyl group (the fatty acid) from acyl-CoA to Carnitine, producing an acyl-car-nitine molecule. The acyl-carnitine crosses the inner mitochondrial membrane using the transporter carnitine-acylcarnitine translocase. Inside the mitochondrial matrix, the acyl is transferred back to a CoA by CPT II. Total carnitine and specific acyl-carnitines are helpful biomark-ers of mitochondrial dysfunction. Ab-normalities in total carnitine as well as acyl-carnitines have been reported in ASD2,18.

Beta-oxidationFatty acids are oxidized in the ma-trix by a series of reactions that in-crementally decrease the length of the fatty acid by two carbons. Each time a fatty acid is shortened, one acetyl-CoA and one NADH and one FADH2 are produced. This reaction continues until only one acetyl-CoA remains (for even-chain length fatty

through the TCA cycle and FAO, re-spectively. These electrons are trans-ported to the ETC, the final common pathway for producing energy, using two electron carriers, the reduced form of nicotinamide adenine dinu-cleotide (NADH) and flavin adenine dinucleotide (FADH2).

Carbohydrate oxidationOne of the main functions of the mi-tochondria is the oxidation of glu-cose, the main carbohydrate energy source of the cells. Glucose is initially metabolized by glycolysis to form pyruvate.

PyruvateIf the mitochondria are function-ing adequately, pyruvate crosses the mitochondrial membranes into the matrix where it is metabolized into acetyl-CoA through a complex of three enzymes called the pyruvate dehydrogenase complex. Abnor-malities in pyruvate dehydrogenase complex function are known to cause childhood MD21 but, to date, have not been reported in ASD. Pyruvate is an important biomarker for diagnosing MD. If the mitochondria are dysfunc-tional, the TCA cycle will slow or shut down, resulting in a build-up of pyru-vate. Two metabolites of pyruvate, namely lactate and alanine, also build up if pyruvate is elevated. These are important biomarkers of MD.

Tricarboxylic acid cycleThe TCA cycle combines the two carbon acetyl-CoA (derived from pyruvate) with the four carbon ox-aloacetate (a metabolite in the TCA cycle). Through a series of reactions, a total of two carbons are removed and converted to CO2. The electrons from these reactions are transferred to two electron carriers, NADH and FADH2. The TCA cycle comes full cir-cle to reproduce oxaloacetate, which is used in the next cycle. Dysfunction of the mitochondria can slow down or stop the TCA cycle. In such a case, the metabolites of the TCA cycle can

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Porphyrin pathwayPorphyrins are aromatic hetero-cyclic macrocycles, which are best known for their ability to hold met-als within their ring. The best known porphyrin complex is heme. Heme is widely used in many critical enzymes including cytochromes a, b and c, which are components of ETC com-plex III and IV. Porphyrin production occurs both inside and outside the mitochondria and requires a special transporter that is ATP dependent (see Figure 2A). Thus, mitochondrial dysfunction can decrease porphyrin production, which can further lead to inadequate ETC function and a further decrease in porphyrin pro-duction, resulting in a vicious cycle. This may be an important area of research as abnormalities in porphy-rins23 appear to be associated with ASD.

the outer mitochondrial membrane allows small mitochondria-derived activator of caspases to be released from the mitochondria where it modulates apoptosis.

Heat productionMitochondria, primarily in brown adipose tissue, can produce heat, instead of ATP, by allowing the in-ner membrane proton gradient to leak through uncoupling protein 1 (UCP1).

Critical systems that interact with the mitochondriaMany metabolic systems feed their biochemical products into mito-chondrial pathways and/or derive their biochemical substrates from mitochondrial pathways; thus, mi-tochondrial dysfunction can affect non-energy-producing systems.

then used to reduce CoQ along with two protons derived from the ma-trix. In this way, the Q cycle assists complex III to indirectly pump pro-tons across the inner mitochondrial membrane. Lower than normal CoQ (ubiquinone) concentrations have been reported in children with ASD as compared to control children2.

Cytochrome CCytochrome C carries electrons from complex III to complex IV. The re-duced Rieske iron-sulphur protein in complex III reduces cytochrome C which is oxidized by complex IV and recycled. Cytochrome C also has an important role in apoptosis.

Calcium bufferingCalcium is transported in and out of the intermembranous space by the voltage-dependent anion-selective channel VDAC1. The behaviour of this channel is dependent on the mito-chondrial membrane potential. Calci-um fluxes into the intermembranous space can stimulate ATP production. Regulation of calcium metabolism by the mitochondria is part of the mi-tochondria-associated endoplasmic reticulum membrane, a structure in which the mitochondria is tethered to the endoplasmic reticulum by a spe-cial protein tethering complex. Calci-um is critical for neurotransmitter re-lease at synapses, and many different types of calcium metabolism abnor-malities have been reported in ASD22.

ApoptosisApoptosis, or programmed cell death, occurs through non-mitochondrial (extrinsic) and/or mitochondrial (intrinsic) pathways. Two molecules are released from the mitochondria that influence cytosolic apoptosis pathways. First, the mitochondrial apoptosis-induced channel (MAC) in the outer mitochondrial membrane allows cytochrome C to leak into the cytosol where it interacts with cellu-lar regulators of apoptosis. Second, an increase in the permeability of

Figure 2: Interaction between the mitochondria and other important metabolic systems. (a) The initial and final steps in the porphyrin pathway are located in the mitochondria. One of the products of the porphyrin pathway, heme, is essential for subunits of ETC complexes. (b) Portions of the urea cycle are located in the mitochondria. Notably, function of the ornithine transporter is dependent on the inner mitochondrial membrane potential. (c) Generation of glutathione de novo requires ATP, which is derived from the mitochondria. Mitochondria are dependent on glutathione for redox regulation.

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Urea cycleThe urea cycle maintains the concen-tration of the toxic ammonium ion in a narrow, tolerable range by convert-ing ammonia into urea, which can be excreted by the kidney. The urea cy-cle resides partially in the mitochon-dria matrix (see Figure 2B). Trans-portation of ornithine, one of the key amino acids in the urea cycle, across the inner mitochondrial membrane is dependent on the proton gradient. A reduction in the inner mitochondrial membrane potential, as occurs with ETC dysfunction, limits the ability of ornithine to be transported across the inner mitochondrial membrane. Increased ammonia is toxic and has been reported in some individuals with MD and also in individuals with ASD2. Urea cycle disorders have been reported in only a few cases of ASD4.

Glutathione metabolism Glutathione is important for main-taining the normal redox state by eliminating reactive oxygen and ni-trogen species. As mitochondria are the source of these toxic species, proper mitochondrial function is de-pendent on glutathione. Glutathione cannot be made in the mitochondria but is rather transported into the mi-tochondria from the cytosol through a special carrier. The production of glutathione requires ATP. Thus, mi-tochondrial dysfunction can reduce the ability of the cell to produce new glutathione, thus leaving the mitochondria vulnerable to reactive species, some of which it produces itself (see Figure 2C). Abnormalities in glutathione metabolism have been frequently reported in children with ASD24.

Mitochondrial control and regulationMitochondrial DNAMitochondria have their own DNA, which provides genetic information to produce several ETC subunits; the remaining ETC subunits are coded by nuclear DNA (nDNA) (Table 3). Many

Figure 2: (Continued)

MDs, particularly those that are com-mon in adults, have been linked to abnormalities in genes in the mtD-NA. A minority of children with ASD and MD have been reported to have abnormalities in mtDNA2.

The existence of mtDNA has sev-eral consequences. First, a sepa-rate system for translation and transcription of mtDNA genes into proteins is needed for the mito-chondria. Some components of this separate system are also coded for by mtDNA. Because this system is not as robust as the nuclear system, mtDNA is much more vulnerable to

intrinsic and extrinsic factors that cause mutations. Second, since mi-tochondria are inherited through maternal cell lines (specifically the ovum), MD does not follow a pattern of Mendelian inheritance. In addi-tion, only some of the mitochondria inherited may manifest abnormal mtDNA, leading to heteroplasmy. This may result in only one portion of the body or one organ system be-ing affected by MD.

nDNA and epigeneticsIn addition to the genetic informa-tion in mtDNA, 1500 or more nu-

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et al.19 suggest an MDC score ≥3 as criterion for performing a fur-ther workup, including a mus-cle biopsy, in order to confirm a diagnosis of MD.

It is believed that there are no clear cures for MD but with supportive treatment, the usual downhill course associated with MD may be miti-gated, and it is possible that recov-ery from the neurodevelopmental effects of MD may be somewhat

can be used in association with the Morava et al. criterion19. The Morava et al. criterion is based on several objective clinical, histologi-cal, biochemical, molecular, neu-roimaging and enzymatic findings (Table 4). The likelihood of an MD can be determined by the MD crite-ria (MDC) score. An MDC score pre-dicts MD as: not likely (≤1), possible (2–4 points), probable (5–7 points) or definite (≥8 points). Morava

clear genes control mitochondrial function. These include genes en-coding ETC complexes, membrane transporters, FAO and TCA en-zymes as well as genes for mtDNA replication and maintenance, mito-chondrial quality control and ETC complex assembly. Mitochondria play a critical role in controlling nuclear genes through epigenet-ics. Products and metabolites de-rived from mitochondria, includ-ing ATP, acetyl-CoA and reducing equivalents, modulate cytosolic and nuclear pathways that control nuclear gene activity25. Because nDNA is involved in some portions of mitochondria function, some forms of MD may exhibit Mendelian inheritance.

Mitochondrial membrane potentialThe inner mitochondrial membrane potential results from the proton gradient created by the ETC. Large electrochemical gradients can be as-sociated with increased oxidative stress. At least two mechanisms are used to reduce this oxidative stress: calcium fluctuations and uncoupling proteins (UCP1, UCP2, UCP3), which allow protons to leak across the inner mitochondrial membrane without going through complex V.

Mitochondrial quality controlMitochondria can reproduce, re-generate and terminate through the processes of fission, fusion and autophagy. These processes are important for controlling the qual-ity of mitochondrial function. For example, mitochondria with ab-normal mtDNA can combine with mitochondria without such abnor-malities to reduce the influence of such abnormalities26.

Diagnosing and treating MDThe workup for MD can be quite complex27,28. One recent review has outlined an evaluation algorithm for children with ASD2 (Figure 3). The findings from this workup

Table 3 Origin of genes for ETC complex subunits

ETC complex nDNA subunits mtDNA subunits

I 38 7

II 0 4

III 10 1

IV 10 3

V 14 2

Figure 3: Recommended algorithm for the metabolic workup of patients with ASD. First, patients are screened with biomarkers of abnormal mitochondrial function in the fasting state. Abnormalities may need to be verified with repeat testing. For patients with biomarkers that point to an FAO defect, other disorders of fatty-acid metabolism may need to be ruled out before further workup for a mitochondrial disorder is conducted. Patients with consistent biomarkers for mitochondrial dysfunction may initially be investigated for genetic causes before a muscle and/or skin biopsy is considered.

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in some cases, the diet can be modi-fied to optimize mitochondrial func-tion. Some have found that diets such as a ketogenic diet or a modified At-kins diet can be helpful. Last, com-mon secondary effects of MD such as gastrointestinal disturbances, thyroid dysfunction, cerebral folate

alleviated. Treatment for MD is fo-cused on four strategies11,29. First, precautions must be implemented to prevent metabolic decompensation and further developmental regres-sion. This involves avoiding environ-mental triggers (such as environ-mental toxicants) and medications

that are known to be harmful to the mitochondria as well as avoiding ill-ness, dehydration and fever. Second, supplementation with specific vita-mins can help support mitochondrial function. These include carnitine, CoQ, B vitamins, vitamin C and vita-min E as well as antioxidants. Third,

Table 4 Diagnostic worksheet for MD

Section I: Clinical signs and symptoms

(a) Muscular presentation (points)

• Ophthalmoplegia (2) • Exercise intolerance (1)

• Facies myopathica (1)• Rhabdomyolysis (1)

• Abnormal EMG (1) • Muscle weakness (1)

Total Points I(a): (maximum score 2 points)

(b) CNS presentation (points)

• Developmental delay (1)• Loss of skills/regression (1)• Seizures (1)• Extrapyramidal signs (1)

• Pyramidal signs (1)• Stroke-like episodes (1)• Migraine (1)

• Brainstem dysfunction (1)• Cortical blindness (1)• Myoclonus (1)

Total Points I(b): (maximum score 2 points)

(c) Multisystem disease (points)

• Haematology (1)• GI tract (1)• Endocrine/growth (1)

• Recurrent/familial (1)• Heart (1)• Hearing (1)

• Vision (1)• Kidney (1)• Neuropathy (1)

Total Points I(c): (maximum score 3 points)

I(a) + I(b) + I(c) Total points Section I: (maximum score 4 points)

Section II: Metabolic/imaging studies (points)

• Elevated lactate (2)/alanine (2)• Elevated lactate/pyruvate ratio (1)• Urinary tricarbon acid excretion (2)• Ethylmalonic aciduria (1)

• Elevated CSF lactate (2), protein (1), alanine (2)

• Stroke-like MRI picture on MRI (1) • Leigh syndrome on MRI (2)• Elevated lactate on MRS (1)

Total points Section II: (maximum score 4 points)

Section III: Morphology from muscle biopsy (points)

• Reduced succinic dehydrogenase staining (1)• Abnormal mitochondria on electron microscopy (2)• Reduced cytochrome oxidase staining (4)• Ragged red/blue fibres (4) • Cox-negative fibres (4) • Succinic dehydrogenase-positive blood vessels (2)

Total points Section III: (maximum score 4 points)

Total score (I + II + III): (maximum score 12 points)

Interpretation: 1: Unlikely 2–4: Possible 5–7: Probable 8–12: Definite

This worksheet, which is based on the Morava et al. criterion, can be used to diagnose MD. The points from Section I: Clinical signs and symptoms, Section II: Metabolic/imaging studies and Section III: Morphology from muscle biopsy are added together; the total provides a probability of a diagnosis of MD.

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deficiency and seizures should be addressed to optimize health.

ConclusionMitochondrial dysfunction and ASDIt appears that mitochondrial dys-function, rather than classic MD, is more prevalent in children with ASD. Children with mitochondrial dysfunction may have a very differ-ent developmental trajectory than children with classic MD, especially if the mitochondrial dysfunction is secondary to an amendable factor. For example, the mitochondrion is an adaptable organelle which can modify its function depending on the intracellular and extracellular envi-ronment. Specific conditions could be inhibiting the function of the mito-chondria in some children with ASD, causing them to be dysfunctional. If this is the case, changing the intracel-lular and extracellular environment may restore mitochondrial function, at least theoretically. In addition, the mitochondrion is capable of repair and regeneration. In the absence of a genetic defect, it is possible that the function of the mitochondria could be restored by these repair mecha-nisms, and it might be possible in the future to enhance these repair mech-anisms. Although we are just begin-ning to understand the significance of mitochondrial dysfunction and MD in children with ASD, this new under-standing of the molecular mecha-nisms associated with ASD provides a pathway for discovering new treat-ments. In fact, several studies have now reported clinical improvements in children with ASD using certain treatments that may target the mi-tochondria such as certain antioxi-dants, vitamins and minerals2,11,30.

Abbreviations listASD, autism spectrum disorder; ATP, adenosine triphosphate; CoQ, co-enzyme Q10; CPT, carnitine palmi-toyltransferase; ETC, electron trans-port chain; FADH2, flavin adenine

dinucleotide; FAO, fatty-acid oxida-tion; HEADD, hypotonia, epilepsy, au-tism and developmental delay; MAC, mitochondrial apoptosis-induced channel; MD, mitochondrial disease; MDC, MD criteria; mtDNA, mitochon-drial DNA; NADH, nicotinamide ad-enine dinucleotide; nDNA, nuclear DNA; TCA, tricarboxylic acid; UCP, uncoupling protein

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