Mitochondrial disorders anddb.phm.utoronto.ca/Dojo Soeandy lecture 6.pdf · mitochondrial dysfunction are often confusingly cell type-specific, as is the case for many known mitochondrial

Mitochondrial disorders and

treatmentsLecture 6- Dojo Soeandy’s section

1

Mitochondrial homeostasis

• Mitochondria possess many of the same defense systems present elsewhere in the cell, including proteases, lipases, antioxidant enzymes and molecules, chaperones, etc.

• However, although mitochondria have multiple copies of their own genome, they lack nucleotide excision repair, a pathway responsible for the repair of DNA damage

• So instead, mitochondria and mtDNA are removed and replaced via mitochondrial fusion, fission, autophagy, and biogenesis, to allow them to tolerate and slowly remove otherwise irreparably damaged DNA

2

Mitochondrial homeostasis• Mitochondrial fusion-

• induced by increased energy demand and by stress (such as ROS)

• Mitochondrial fission-• induced by the need for a new organelle (mitochondria) and for

facilitation of quality control.

• Mitophagy-• Mitophagy is the mechanism by which impaired or superfluous

mitochondria are engulfed in autophagosomes to be then degraded into lysosomes (specialized autophagy).

3

Youle & van der Bliek, 2012

Mitochondrial dysfunction

• On the one hand, many mitochondrial effects are systemic, as in the case of exercise, which induces mitochondrial biogenesis in the brain and liver as well as muscle

• However, the effects of mitochondrial dysfunction are often confusingly cell type-specific, as is the case for many known mitochondrial diseases, in which a mutation results in a pathology in only one or a few tissues.

• Every 30 minutes, a child is born with mitochondrial disease and about 1 in 4,000 people has the disease.

• They can be caused by mutation of genes encoded by either nuclear DNA or mitochondrial DNA (mtDNA).

4

Mitochondrial dysfunction• Diagnostic tests:

• Biochemical markers: lactate, pyruvate, lactate-to-pyruvate ratio, ubiquinone, alanine, alanine-to-lysine ratio, ATP, acyl-carnitine, all of which are produced at some stage of respiration

• A physical exam to test strength and endurance (e.g. exercise test)

• A neurological exam- tests of reflexes, vision, speech, and basic cognitive (thinking) skills

• Muscle biopsy• When treated with dye that stains mitochondria and

other cell components (Gomori trichrome stain), muscles affected by mitochondrial disease often show ragged red fibers — muscle cells (fibers) that have excessive mitochondria.

• Stains can detect the absence of essential mitochondrial enzymes in the muscle.

• Can also extract mitochondrial proteins from the muscle and measure their activity (enzymology).

• A genetic test to determine if someone has a genetic mutation that causes mitochondrial disease 5

lhsc.on.ca


• Primary mitochondrial disease (PMD): genetically inherited and diagnosed by identifying mutations on mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) that directly affects mitochondrial metabolism (i.e. affecting oxidative phosphorylation).

• Secondary mitochondrial dysfunction: Mitochondrial dysfunction is also seen in a number of different genetic disorders. These are not strictly mitochondrial diseases.

6


• Examples:• Leber’s Hereditary Optic Neuropathy (LHON)

• Leigh syndrome

• Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS)

• Mitochondria and autism spectrum disorders (ASD)

• Mitochondria and Parkinson’s disease

7

Leber’s Hereditary Optic Neuropathy (LHON)• A maternally-inherited form of vision loss

• This condition usually begins in a person's teens or twenties; rare cases may appear in early childhood or later in adulthood

• For unknown reasons, males are affected much more often than females.

• Vision loss results from the death of cells that relays visual information from the eyes to the brain (the optic nerve)

8santhera.com

Maternal inheritance

9

mitochondrialdisease.nhs.uk

Ortiz, Mireles-Ramirez, et al., 2018

Leber’s Hereditary Optic Neuropathy (LHON)

• Signs and symptoms include:• Blurring and clouding of vision (usually the first

symptoms) affecting the central visual field

• Severe loss of visual acuity (sharpness of vision) and color vision over time

• Loss of ability to complete visual tasks such as reading, driving, and recognizing faces

• A growing, dense central scotoma (blind spot) seen during visual field testing

• Development of optic atrophy

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• Ophthalmological evaluation (fundus analysis)• Pale optic disc

• Border of disc margin not as well defined

• Hyperemia

• But the fundus can look normal in 20%–40% of those in the active stage of vision loss

11

lhsc.on.ca

Meyerson, Stavern and McClelland, 2015


• The most common mitochondrially inherited disease

• Arise primarily from mutations in mtDNA-encoded ETC components

• Over 95% of LHON patients harboured one of three mtDNA point mutations, G3460A (MT-ND1), G11778A (MT-ND4), and T14484C (MT-ND6) (all of which affect complex I of ETC)

• However, more than 50 percent of males with a mutation and more than 85 percent of females with a mutation never experience vision loss or related health problems 12

ghr.nlm.nih.gov


13


• One could argue that only retinal ganglion cells are affected because they require a sustained level of ATP for normal function

• It is also alternatively possible that retinal ganglion cells are preferentially involved because they are somehow exquisitely sensitive to subtle imbalances in cellular redox state or increased level of free radicals

14

ghr.nlm.nih.gov


• Treatment:• Currently no treatment available that improves the final visual

outcome in LHON

• Some studies have reported a benefit from using idebenone (a synthetic analogue of ubiquinone- i.e. Coenzyme Q10) and vitamin supplementation (B12 and C) to speed up visual recovery

15

santhera.com



• Leigh syndrome




16

Leigh syndrome

• A severe neurological disorder that usually becomes apparent in the first year of life.

• Characterized by progressive but rapid loss of mental and movement abilities (psychomotor regression) and typically results in death within two to three years, usually due to respiratory failure.

• A small number of individuals do not develop symptoms until adulthood or have symptoms that worsen more slowly.

• It affects about 1 in 40,000 newborns.

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Leigh syndrome• Signs/symptoms:

• Early symptoms may include poor sucking ability; loss of head control and motor skills; loss of appetite due to difficulty swallowing (dysphagia); vomiting; and seizures.

• As the condition progresses, symptoms may include weakness and lack of muscle tone; spasticity; movement disorders; cerebellar ataxia; and peripheral neuropathy.

• Complications can lead to impairment of respiratory, heart and kidney function.

• Signs and symptoms are caused in part by patches of damaged tissue (lesions) that develop in the brains of people with this condition (usually accompanied by loss of the myelin coating around nerves- i.e. demyelination).

• These regions include: • Basal ganglia- help control movement• Cerebellum- controls the ability to balance and coordinate

movement • Brainstem- connects the brain to the spinal cord and

controls functions such as swallowing and breathing. 18

Koenig, 2018

Leigh syndrome• Caused by mutations in one of more than 75 different genes

(either in nuclear DNA or mtDNA)

• Most genes are associated with ETC: disrupt the function of proteins in these complexes, how the complexes form, or additional steps related to energy production

• Common genes include:• NDUFAF2- encodes a complex I assembly factor (long arm of

chromosome 5 at position 12.1• SURF1*- encodes an assembly factor of mitochondrial complex IV

(on long arm of chromosome 9 at position 34.2)• LRPPRC- plays a role in translation or stability of mitochondrially

encoded cytochrome c oxidase subunits. May be involved in transcription regulation of both nuclear and mitochondrial genes. Mutations in this gene are associated with the French-Canadian type of Leigh syndrome (short arm of chromosome 2 at position 21)

• BCS1L- adds iron-Sulphur (Fe-S) protein to complex III (long arm of chromosome 2 at position 35)

• MT-ATP6*- provides instructions for making a piece of complex V, also known as the ATP synthase (on mtDNA)

19

Leigh syndrome• It is most commonly inherited in an autosomal

recessive manner.• In about 20% of cases, when Leigh syndrome is due to

mutations in mitochondrial DNA, it is inherited in a mitochondrial pattern (maternal inheritance).

• In a few cases of Leigh syndrome due to mutations in nuclear DNA, inheritance is X-linked recessive.

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Leigh syndrome• Treatment:

• Currently no good treatment options• Other than supportive care like treatment of acidosis,

seizures, cardiomyopathy, etc., common treatment include administration of various OXPHOS cofactors and antioxidants (like coenzyme Q10, thiamine).

• In FDA Expanded Access protocol, children may be treated with EPI-743 (a new drug that is based on vitamin E)

• Research also shows that treatment using rapamycin may slow progression of the disease. • Protein synthesis for proliferation requires large amounts of

energy. mTOR inhibition reduces protein synthesis and proliferation, redirecting that energy to other functions like the maintenance of neurons

21



• Leigh syndrome




22

MELAS

• MELAS: Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes

• Affects many of the body's systems, particularly the brain (nervous system) and muscles

• MELAS syndrome is a rare disorder that affects males and females in equal numbers

23

MELAS

• Signs and symptoms most often appear in childhood following a period of normal development (>2yrs), although they can begin at any age:• Early symptoms may include muscle weakness and pain,

recurrent headaches, loss of appetite, vomiting, and seizures.

• Most affected individuals experience stroke-like episodes beginning before age 40:• Often involve temporary muscle weakness on one side of the

body (hemiparesis), altered consciousness, vision abnormalities, seizures, and severe headaches resembling migraines.

• Repeated stroke-like episodes can progressively damage the brain, leading to vision loss, problems with movement, and a loss of intellectual function (dementia).

24

MELAS

• Other diagnostic features:• Lactic acidosis both in blood

and CSF• Ragged red fibers in stained

muscle biopsy• Brain injury as observable

by MRI (increased T2 signal), with distribution of lesions that do not follow vascular territories, with often no associated vascular pathology

• Genetic testing showing mutation in mtDNA

25

medcell.med.yale.edu

Yoshida, Ouchi, et al. 2013

ghr.nlm.nih.gov

MELAS

• MELAS is caused by mutations in mitochondrial DNA: in one of several genes, including MT-ND1, MT-ND5, MT-TH, MT-TL1, and MT-TV.

• Most common: A3243G pathogenic variant in the mitochondrial gene MT-TL1 (a transfer RNA gene) is present in approximately 80% of individuals with MELAS

26

Watanabe, 2010

MELAS

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MELAS

• Treatment:• No specific treatment is available for MELAS syndrome.

• Usually treat symptoms. E.g.• Anti-convulsant drugs (e.g. lamotrigine) to help prevent and

control seizures associated with MELAS

• Cochlear implants to treat sensorineural deafness

• Coenzyme q10 and L-carnitine have been beneficial in some patients

• Once an individual with MELAS has the first stroke-like episode, arginine has been reported to decrease the recurrence of stroke-like episodes

28



• Leigh syndrome




29

Mitochondria and ASD• Autism spectrum disorders (ASD): neurodevelopmental

disorders which include autistic disorder, Asperger’s syndrome, pervasive developmental disorder and childhood disintegrative disorder.

• According to the Centers for Disease Control, autism affects an estimated 1 in 59 children in the United States today

• Diagnosed on the basis of behavioural observations such as impaired social interaction, diminished verbal and non-verbal communication and repetitive behaviours.

• Over recent years, there is increasing evidence of comorbidities such as mitochondrial dysfunction, oxidative stress, gastrointestinal abnormalities and abnormalities in regulation of the immune system.

• So, autism may involve, or be a result of, systemic physiological abnormalities rather than entirely being a neurodevelopmental disorder.

30

Mitochondria and ASD

31Frye and Rossignol, 2011

Mitochondria and ASD• Estimates of the prevalence of

mitochondrial dysfunction in ASD vary widely:• a large population-based study estimated the

prevalence of mitochondrial disease in ASD as 4.2% (Oliviera, et al., 2005); based on elevated lactate and muscle biopsy analyses

• in a more controlled study, it is suggested that mitochondrial dysfunction may be present in up to 80% of children with ASD (Giulivi, et al., 2010); examined mitochondrial dysfunction and mtDNAabnormalities in lymphocytes• Found that children with autism were more

likely to have mitochondrial dysfunction, mtDNAoverreplication, and mtDNA deletions than typically developing children.

• low NADH oxidase (complex I) activity was the most common deficiency (8 of 10), followed by succinate oxidase (complex II) (6 of 10)

32

Slide adapted from Hampson 2017

Mitochondria and ASD• Mitochondrial ETC abnormalities in children with

autistic spectrum disorders

33Legido, Jethva, Goldenthal, 2013


• Genetic evidence of a link between mitochondrial dysfunction and autism

34Legido, Jethva, Goldenthal, 2013

Mitochondria and ASD• Mechanisms underlying the

relationship of mitochondria and autism (hypotheses)• An abnormal robust neuroimmune

response elevate intracellular Ca2+ levels, deranging neurodevelopment, driving oxidative stress, and ultimately affecting synaptic function and neural connectivity

• Mitochondrial dysfunction can lead to reduced synaptic neurotransmitter release in neurons that have high firing rates, such as inhibitory GABA interneurons; an imbalance in the excitatory (glutamate) and inhibitory (GABA) neurotransmitter systems has been implicated in the pathogenesis of ASD.

35

Legido, Jethva, Goldenthal, 2013


• Treatments:• Currently no one standard treatment for autism

spectrum disorder (ASD)

• Behavior and communication approaches- i.e. various types of therapy as necessary, e.g. speech therapy, occupational therapy, etc.

• Dietary approaches- removing certain types of foods from a child’s diet and using vitamin or mineral supplements.

• Medication- treat related symptoms; for example, medication might help manage high energy levels, inability to focus, depression, or seizures.

36



• Leigh syndrome




37

Mitochondria and Parkinson’s disease

• Parkinson’s disease (PD) is a slowly progressive, neurodegenerative disorder characterized by resting tremor, stiffness (rigidity), slow and decreased movement (bradykinesia), and gait and/or postural instability.

• Parkinson disease affects about• 0.4% of people > 40 yr• 1% of people ≥ 65 yr• 10% of people ≥ 80 yr

• The mean age at onset is about 60 years old.

• Usually idiopathic38

‘Diseases of the Nervous System' by William Gowers

Mitochondria and Parkinson’s disease• Hoehn and Yahr Staging of Parkinson's Disease:

• Stage one – symptoms on one side of the body only.

• Stage two – symptoms on both sides of the body. No impairment of balance.

• Stage three – balance impairment. Mild to moderate disease. Physically independent.

• Stage four – severe disability, but still able to walk or stand unassisted.

• Stage five – wheelchair-bound or bedridden unless assisted.

• The average life expectancy of a person with PD is generally the same as for people who do not have the disease.

39

Mitochondria and Parkinson’s disease• Movement is normally mediated by dopaminergic

neurons.

• When cells that normally produce dopamine (especially in the Substantia Nigra) die, the symptoms of Parkinson’s appear.

40Bazazeh, Shubair, Malik, et al., 2016

neurologicalsurgery.insaintlukeskc.org


41basicmedicalkey.com


• Pathological changes in Parkinson’s disease:• Low dopamine as per FDOPA/PET

• FDOPA= 18Fluoro-DOPA

• PET= Positron emission tomography

• Increased levels of a protein called α-synuclein, which accumulates in Lewy bodies

42

bic.mni.mcgill.ca


43

Irwin, Lee and Trojanowski, 2013


• Supporting evidence linking mitochondrial dysfunction and sporadic Parkinson’s disease:• MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)

causes the clinical development of progressive and irreversible parkinsonism in young drug addicts in the late 1970s/early 1980s in the United States • Post-mortem displayed degeneration of the substantia nigra

without the presence of Lewy bodies

• Both the beneficial and long-term adverse effects of L-dopa therapy in MPTP-intoxicated patients are comparable to those in patients suffering from sporadic PD

44


• Supporting evidence linking mitochondrial dysfunction and sporadic Parkinson’s disease:• MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)

• Mechanism: • MPTP crosses the blood–brain barrier and is oxidized by monoamine

oxidase (MAO-B) in glial cells

• It is then further oxidized to the toxic molecule MPP+, which is then taken up into dopaminergic neurons via the dopamine transporter.

• Within neurons MPP+ is concentrated in mitochondria and inhibits Complex I of the electron transport chain

• So mitochondrial ATP production is decreased while the generation of ROS is increased

• The downstream events leading ultimately to neuronal cell death seem to be quite complex and involve everything from apoptotic pathway members to inflammation

45

Mitochondria and Parkinson’s disease• Supporting evidence linking mitochondrial

dysfunction and sporadic Parkinson’s disease:• Rotenone

• Specific inhibitor of Complex I• Results in degeneration of subset of nigrostriatal dopaminergic

neurons; formation of cytoplasmic inclusion; and the development of parkinsonian motor behaviour in animal models

• 6-Hydroxydopamine (6-OHDA)• Hydroxylated analogue of dopamine• Potent inhibitor of the mitochondrial respiratory chain

complexes I and IV• Generates PD like phenotypes (e.g. impaired motor function),

degeneration of neurons and decreased dopamine content in substantia nigra

46

Mitochondria and Parkinson’s disease• Indeed, Complex I activities were reported to be

significantly reduced (in the range of about 30%) in post-mortem substantia nigra of PD patients

47Alagapan, 2011

Mitochondria and Parkinson’s disease• Supporting evidence linking mitochondrial dysfunction

and familial Parkinson’s disease:• Many pathogenic PD mutations are directly linked to

mitochondrial dysfunction

• SNCA gene-• Encodes α-Synuclein (α-Syn)

• The main component of Lewy bodies

• Mutation in this gene is autosomal dominant

• It has been reported to mediate neurotransmitter release at presynaptic terminals and interact with membranes of various organelles, including mitochondria, where it is said to regulate mitochondrial morphology and biogenesis among others

48


• Supporting evidence linking mitochondrial dysfunction and familial Parkinson’s disease:• LRRK2 gene-

• Most common cause of familial PD, but have variable penetrance

• Encodes for Leucine Rich Repeat Kinase 2, which is a multifunctional protein kinase

• Mutation in this gene is autosomal dominant

• Physiological levels of the common LRRK2 G2019S mutant were found in association with mitochondrial abnormalities in patient-derived dopaminergic neurons

• LRRK2 has been shown to cause mitochondrial fragmentation, increased proton leak and loss of mitochondrial membrane potential (ΔΨm), and defective mitophagy

49


• Supporting evidence linking mitochondrial dysfunction and familial Parkinson’s disease:• Parkin gene-

• Most common autosomal recessive PD

• Encodes cytosolic E3 ubiquitin ligase that ubiquitinates target proteins for signalling or proteasomal degradation

• Parkin primarily functions in association with mitochondria; Parkin has diverse functions in maintaining healthy mitochondria by regulating their biogenesis and degradation via mitophagy

50


• Supporting evidence linking mitochondrial dysfunction and familial Parkinson’s disease:• PINK1 gene-

• Second most common autosomal recessive PD; functions in the same pathway as Parkin

• Encodes PTEN-induced kinase 1 (PINK1), a mitochondrial serine/threonine kinase that plays a crucial role in maintaining mitochondrial homeostasis.

• Loss of PINK1 impairs various aspects of mitochondrial biology, including degradation, morphology and trafficking.

• The most widely studied of these is the function of PINK1 in mitophagy; facilitating removal of damaged mitochondria by recruiting and activating Parkin.

• PINK1 activates Parkin by a twofold mechanism: 1. Direct phosphorylation of Parkin at S65 2. Trans-activation by phosphorylation of ubiquitin at S65 and

subsequent binding to Parkin51


52

Youle & van der Bliek, 2012

• PINK1 normally imported from the outermitochondrial membrane to the inner mitochondrial membrane- dependent on mitochondrial potential.

• It is constitutively degraded by the protease PARL at the inner mitochondrial membrane and maintained at low levels on healthy mitochondria.

• When a mitochondrion becomes damaged to the point of depolarization across the inner membrane, PINK1 import to the inner membrane is prevented.

• PINK1 accumulates on the outer membrane and recruits the E3 ligase Parkin from the cytosol via PINK1 kinase activity.

• Parkin conjugates ubiquitin (Ub) to a variety of proteins on the outer mitochondrial membrane and mediates the proteosomal elimination of mitofusins 1 and 2.

• Lastly, Parkin induces autophagic elimination of the dysfunctional mitochondria.


53Park, Davis and Sue, 2018


• Treatment: • Levodopa/Carbidopa- main treatment

• Cannot simply take dopamine pills because dopamine does not easily pass through the blood-brain barrier.

• Instead, Levodopa, the metabolic precursor of dopamine, crosses the blood-brain barrier into the basal ganglia, where it is decarboxylated to form dopamine.

• Coadministration of the peripheral decarboxylase inhibitor carbidopa prevents levodopa from being decarboxylated into dopamine outside the brain (peripherally), thus lowering the levodopa dosage required to produce therapeutic levels in the brain and minimizing adverse effects due to dopamine in the peripheral circulation.

• Levodopa is most effective at relieving bradykinesia and rigidity, although it often substantially reduces tremor.

54

Mitochondria and Parkinson’s disease• Treatment:

• Dopamine agonists-• Include apomorphine, pramipexole, ropinirole, and rotigotine• Activate dopamine receptors in basal ganglia

• MAO-B inhibitors-• Include selegiline and rasagiline• Inhibit the enzyme monoamine oxidase B, or MAO-B, which breaks

down dopamine in the brain.• MAO-B inhibitors cause dopamine to accumulate in surviving

nerve cells and reduce the symptoms of PD, and when used withlevodopa, prolonge the action of each dose

• COMT inhibitors-• E.g. entacapone, tolcapone• COMT stands for catechol-O-methyltransferase, another enzyme

that breaks down dopamine.• The drug thus prolong the effects of levodopa by preventing the

breakdown of dopamine.

55

Mitochondria and Parkinson’s disease• Treatment:

• Amantadine-• An antiviral drug that can help reduce symptoms of PD.• It is often used alone in the early stages of the disease, or used

with an anticholinergic drug or levodopa.• Researchers are not certain how amantadine works in PD, but it

may increase the effects of dopamine.

• Anticholinergic-• Include trihexyphenidyl, benztropine, and ethopropazine• Decrease the activity of the neurotransmitter acetylcholine and

can be particularly effective for tremor.• Usually used only in young patients with tremor-predominant

Parkinson disease or with some dystonic components, because adverse effects may include cognitive impairment and dry mouth, which are particularly troublesome for the elderly

56

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Mitochondrial disorders anddb.phm.utoronto.ca/Dojo Soeandy lecture 6.pdf · mitochondrial dysfunction are often confusingly cell type-specific, as is the case for many known mitochondrial