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Mitochondrial Disorders: An Update

I Smuts

Department of Paediatrics and Child Health

University of Pretoria

Steve Biko Academic Hospital

Mitochondrial structure

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1: Outer membrane i. Porins 5000 daltons molecules diffuse ii. Translocase of the OM larger molecules iii. Mitochondria-associated ER membrane

2: Intermembrane space i. High concentration ions and sugar ii. Cytochrome c

3: Inner membrane i. OXPHOS ii. ATP production iii. Transport proteins regulating metabolite passage (TIM) iv. Protein import v. Fusion and fission proteins

4: Cristae i. Surface area ii. Chemiosmotic

5: Matrix i. ATP production ii. Oxidation of pyruvate and fatty acids iii. Krebs cycle iv. RNA and protein production v. mtDNA

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1 i. Porins 5000 daltons molecules diffuse ii. Translocase of the OM larger molecules iii. Mitochondria-associated ER membrane (MAM)

Importance of MAM • Signalling and trafficking hub • Ca2+ signalling • Critical in intrinsic apoptosis • Lipid biosynthesis

Fission and fusion Interorganelle traficking for

Phosholipid biosynthesis Ceramide Cholestrol Glycosphingolipids

Functions in summary

• Energy conversion

• Heat production

• Storage of calcium

• Signalling through ROS

• Apoptosis

• Regulation of cell metabolism

• Steroid synthesis

• Hormonal signalling

Dual genetic control

1 500 genes involved in energy metabolism

16.5kb circular double stranded molecule 37 genes encoding:

13 structural proteins 2 rRNA 22 tRNA

Definition of mitochondrial disease

Two definitions

Lombѐs et al. Biochimie 100:1171-176, 2014.

1. Alteration to the OXPHOS pathway

2. All the diseases due to an alteration of a mitochondrial component

Primary mitochondrial disorder

Diseases associated with defects in OXPHOS function attributable to genetic differences in mtDNA or nDNA

UMDF/NIH workgroup. Mitochondrion 13:945-952, 2013.

Secondary mitochondrial disorder

Degenerative conditions with significant mitochondrial dysfunction

– Autism

– Alzheimer’s disease

– Parkinsonism

– ALS

– Muscle disorders

– Etc…..

How do mitochondrial disorders present?

• Any age

• Any organ or tissue

• Enormous variation Muscle Exercise intolerance Ocular myopathy Axial/limb weakness Myolysis

Brain Cognitive impairment Epilepsy Movement disorders Cerebellar syndrome Pseudo-strokes Leukodsytrophy

Peripheral nerves Sensory neuropathy Demyelinating neuropathy Axonal motor neuropathy

Heart Cardiomyopathy Conduction defects

Sensory organs Pigment retinopathy Optic atrophy Deafness

Organs Liver (Failure, cholestasis) Kidney (Proximal tubulopathy, glomerulopathy) Endocrine (Diabetes, hypogonadism, growth hormone defect…)

Bone marrow Sideroblastic anaemia Pancytopaenia

Lombѐs et al. Biochimie 100:1171-176, 2014.

Phenotypic diversity

• Heteroplasmy

• Restricted tissue expression of an OXPHOS defect

• Modifying factors

– Nuclear genes encoding mtDNA replication factors

– mtDNA variation/Haplogroups • Haplogroup L0: G263A C1048T C3516a T5442C T6185C

C9042T A9347G G10589A G12007A A12720G

• L0a1a1: T2759C

Lombѐs et al. Biochimie 100:1171-176, 2014.

mtDNA variation

• mtDNA accumulated variants over time

• Ancient variants extant over time in populations

• Haplogroups are defined

– Geographically

– Ethnically

http:www.mitomap.org

Human mtDNA migrations

http:www.mitomap.org

Simplified mt Lineages

Example

A10398G

• MTND3 (Complex I)

• Homoplastic on haplogroup J and K

Effects

• Reduces CI activity

• Reduces cytosolic calcium levels

• Reduces mitochondrial membrane potential

• Reduces level of ROS

Result • Reduces the risk of Parkinson’s disease

Genetic basis of mitochondrial diseases

mt-DNA defects (37 genes) – Point mutations (maternally inherited)

• tRNA

• Subunits

• rRNA

– Rearrangements • Deletions

• Duplications

DiMauro et al., 2013

Reported mutations on Mitomap 2015 864

• Apoptosis 50 • Metabolism 400 • Proteases 25 • aa-tRNA synthesis 23 • Chaperone 25 • Dynamics 12 • Nucleases 5 • Transporters 75 • Cytochromes 15 • Replication 1 • RNA modification 5 • Protein modification 15 • Cytoskeletal 10 • Transcription 30 • Translation 15

• Protein import: TIMMs and TOMMs 30 • DNA repair 12 • Regulation 10 • MRPLs 55 • RC complex and assembly 150 • Fe–S cluster 5 • Porins 4 • MRPSsc 36 • RNA modification/processing 15 • Immune 3 • ROS 15 • Others 150 • Unknown 150

Wong. Mitochondrion 13:379-387, 2013.

Genetic basis of mitochondrial disorders nDNA~1500 nuclear genes involved in mitochondrial function

Genetic basis of mitochondrial disease

117 nuclear genes

Nuclear encoded subunits

27/80 genes

Import, processing assembly

38 genes

mtDNA replication 5

genes

Nucleotide transport synthesis

9 genes

Nucleotide transport, synthesis

9 genes

mtDNA expression 24 genes

95: autosomal recessive 10:autosomal dominant

5: recessive or dominant 7: X-linked

Ohtake et al. Biochimica et Biophysica Acta 1840:1355-13596, 2014.

? Parents insist on answers

Money

Availability of facilities

Clinicians

Just write a script!!!

Clinicians

Explain

Epilepsy

Autism

Neuromuscular disease

Patients

Mitochondrial dysfunction in autism

• Biochemical marker suggestive of abnormal mt function • ETC deficiencies

– 14 different studies (2000-2012) • 131 patients • 100 ETC deficiencies

• mtDNA mutations – 8 different studies (1999-2013)

• 139 patients • 49 mtDNA mutations (355)

• nDNA mutations – 6 different studies – Gene expression in post mortem brains

Legido et al. Semin Pediatr Neurol 20:163-175, 2013.

Mitochondrial dysfunction in autism

• Prevalence of autism: 2:100

• Prevalence of MD: 1:2000

• Then expected:

– 1:110 pt with MD will have autism

– 1:2000 pt with ASD would have MD

Legido et al. Semin Pediatr Neurol 20:163-175, 2013.

Mitochondrial dysfunction in autism

Legido et al. Semin Pediatr Neurol 20:163-175, 2013.

Conclusion

• Genetic and biochemical evidence for mt role in ASD of a subset of children

• Mt dysfunction may still be secondary

• Further studies required

Mitochondrial dysfunction in neuromuscular disorders

Muscular dystrophies

Congenital muscular

dystrophies (CMD)

Duchene muscular

dystrophy (DMD)

Limb girdle muscular dystrophy

(LGMD)

Myotonic dystrophy (MD)

Congenital myopathies

Centronuclear myopathies

Core myopathies

Inflammatory myopathies

Inclusion body myositis (IBM)

Polymyositis

Spinal muscular atrophy –Motor neuron disease

Spinal muscular atrophy (SMA)

Amyotrophic lateral sclerosis

(ALS)

Peripheral neuropathies

Hereditary peripheral

neuropathies

Friedreich’s ataxia

Diabetic neuropathy

Drug induced neuropathies

Muscular dystrophies

Congenital muscular

dystrophies (CMD)

Duchene muscular

dystrophy (DMD)

Limb girdle muscular dystrophy

(LGMD)

Myotonic dystrophy (MD) • Disrupted Ca2+ homeostasis + impaired response to

oxidative stress underlie muscle cell death • Permeability transition pore (PTP) dysregulation • Defective autophagy

• CsA • Cyclophilin D

inhibitors

PTP

Congenital myopathies

Centronuclear myopathies

Core myopathies

Ryanodine receptor 1 (RYR1) is a skeletal muscle calcium release channel

Inflammatory myopathies

Inclusion body myositis

(IBM)

Polymyositis

• Ragged red fibres in IBM

• COX-deficient fibres

• Multiple mtDNA deletions

• Mt abnormalities

• Mt changes triggered by immune-mediated mechanisms of cellular injury

Spinal muscular atrophy –Motor neuron disease

Spinal muscular

atrophy (SMA)

Amyotrophic lateral

sclerosis (ALS)

SMA

– CI-IV deficiencies

– mtDNA in muscle decreased

– Decreased SDH activity

– Diminution of COX subunits

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Peripheral neuropathies

Hereditary peripheral

neuropathies

Friedreich’s ataxia

Diabetic neuropathy

Drug induced neuropathies

• Abnormal fission and fusion and mt movement

– e.g CMT2A

• Mt dysfunction in Schwann cells contribute to the pathogenesis of demyelination and axonal degeneration

• Hyperglycaemia induce abn neuronal Ca2+ handling and mt dysfunction. ATP exhausted and then axonal degeneration

• NRTI anti-retrovirals different mechanisms

Mitochondrial dysfunction in epilepsy

MD vs Epilepsy

Seizures vs

Mt function

AED vs Mt function

Relationship of mitochondria and epilepsy

Energy depletion

Homeostasis affected

Tissue excitability

Seizures

ROS

Mt dysfunction

Khurana et al. Semin Pediatr Neurol 20:163-175176-187, 2013.

Anti-epileptic drugs and mitochondrial function

AED (Examples) Mt impairment

Valproic acid CI+IV activity Inhibits O2 consumption ATP synthesis Inhibits ß-oxidation Impairs structural organization of the inner membrane

Carbamazepine Inhibits ATPase Impairs respiration Impairs calcium uptake or release

Lamotrigine CI activity CII, CII+III and CIV activities

Vigabatrin Induces apoptosis in the developing brain

Topiramate Zonisamide

Inhibits isoenzyme human carbonic anhydrase V

Khurana et al. Semin Pediatr Neurol 20:163-175176-187, 2013.

Anti-epileptic drugs and mitochondrial function

AED (Examples) Beneficial effects

Valproic acid Rotenone-induced toxicity Inhibits histone deacetylases

Carbamazepine Protects against CI inhibitor rotenone

Lamotrigine Neuro-protective Increases ATP production

Topiramate Inhibits PTP Increase CI activity Neuro-protective

Levetiracetam Anti-oxidant effect Neuro-protective

Khurana et al. Semin Pediatr Neurol 20:163-175176-187, 2013.

Other potential treatment modalities

• Antioxidant supplementations

– Vitamin E

– Melatonin

– Resveratrol

– Lipoic acid

– N-acetyl-cystein

– CoQ10

– Vitamin C

? Evidence

Khurana et al. Semin Pediatr Neurol 20:163-175176-187, 2013.

Conclusion