Introduction to Mitochondrial Disorders

Question 1

Outline mitochondria function.

Answer 1

• Involved in many cellular functions and processes.
• Major role in cellular energy production.
• Produce 90% of a typical cells ATP in the process of oxidative phosphorylation via the electron transport chain.
• Involved in apoptosis. The mitochondria is a critical location for many apoptotic calcium signals. Involved in apoptosis via the intrinsic pathway. Normally ATP production is coupled to oxygen consumption, during cellular stress which can occur as a result of fever, cancer or stroke the mitochondria become uncoupled leading to increased reactive oxygen species. Excess free radicals damage mitochondrial function leading to apoptosis which results in organ dysfunction.
• Mitochondria are also involved in cell specific functions. For example, they are required for steroid synthesis, haem synthesis, calcium homeostasis, fatty acid oxidation, detoxification of ammonia in the urea cycle and synthesis of Fe-S clusters.

Question 2

Overview the respiratory chain enzyme pathway.

Answer 2

• The electron transport / respiratory chain enzyme pathway is a system comprised of 5 complexes arranged across the inner mitochondrial membrane.
• Complex I = NADH Dehydrogenase Coenzyme Q Reductase.
• Complex II = Succinate Dehydrogenase.
• Complex III = Ubiquinin Cytochrome C Oxidoreductase.
• Complex IV = Cytochrome Oxidase.
• Complex V = ATP Synthase.
• In addition to this there are 2 enzyme carriers called Coenzyme Q (Ubiquinone) and Cytochrome C.
• Complexes I-IV pump NADH and FADH2 derived protons produced by the citric acid cycle and the Beta-oxidation of fatty acids from the matrix across the mitochondrial inner membrane to the inter-membrane space to generate a proton gradient. At the same time electrons get transferred to molecular oxygen to produce water.
• The proton gradient which makes up most of the mitochondrial membrane potential is used to do work by being dissipated across the inner membrane in the opposite direction through the Complex V ATP synthase.
• 92 structural subunits of the respiratory chain have been identified to date, of which 13 are encoded by mtDNA.
• The entire mitochondrial proteome is estimated to be approximately 1500 proteins.

Question 3

Give an overview of mitochondrial dynamics.

Answer 3

• Fusion and fission work in concert to maintain morphology, size, number of mitochondrion.
• Both processes are essential to the cell, and implicated in physiological functions including respiratory capacity, apoptosis and response to cellular stress.
• Fusion and fission under control of nuclear encoded genes.

Question 4

What is the function of mitochondrial fusion?

Answer 4

• Mitochondrial fusion promoted inter-mitochondrial cooperation.
• Allows sharing of mtDNA and mt proteins.
• Involves 3 major GTPases - mitofusin 1 and 2 are involved in the fusion of the outer membrane and OPA1 dynamin related GTPase is involved in the fusion of the inner membrane / intermembrane space.
• Depletion of the function of these genes results in decreased fusion and variants occurring in these important genes have been implicated in disease phenotypes. For example, pathogenic variants in mitofusin 2 (Mfn2) have been shown to be associated with the peripheral sensory neuropathy Charcot Marie Tooth Type 2 A. Pathogenic variants in OPA1 are associated with Dominant Optic Atrophy (DOA).

Question 5

Outline mitochondrial fission.

Answer 5

• Mitochondrial fission involves 2 classes of molecules. The central player appears to be Drp1, a dynamin related protein. Drp1 assembles on mitochondrial microtubules and is though to regulate constriction and scission. Drp1 is cytosolic and therefor a second class of proteins, Drp1 binding proteins are required to recruit Drp1 for fission. Candidate receptors include molecules such as Fis1, Mff, miD49 and MiD51 on mitochondrial surface.
• A single case has been reported in which a Drp1 mutation has been linked with neonatal lethality with multisystem failure.

Question 6

Describe the mitochondrial genome.

Answer 6

• 16,569 bp circular dsDNA molecule.
• Packaged in nucleoids (protein-DNA complexes).
• Encodes 13 subunits of the respiratory chain, 22 tRNAs and 2 rRNAs.
• Approximately 100-10,000 copies per cell.
• Maternally inherited (sperm mtDNA are thought to be removed by ubiquitination).

Question 7

Give an overview of mitochondrial disorders.

Answer 7

• Mitochondrial disorders or diseases are typically classified as disorders due to biochemical abnormalities of the mitochondrial respiratory chain enzyme pathway, otherwise known as oxidative phosphorylation (OXPHOS).
• This leads to insufficient cellular energy and hence the clinical phenotype.
• Phenotypes are extremely diverse, but typically affect tissues with a high energy demand.

Question 8

Outline the genetic classification of mitochondrial disease.

Answer 8

1) . Primary mtDNA disorders.
- Maternal inheritance - mtDNA point mutations such as m.3243A>G (MELAS), m.8344A>G (MERRF), m.8993T>C/G (NARP, Leigh Syndrome), m.1178G?A (LHON).
- Sporadic - mtDNA deletions (CPEO, Kearns-Sayre syndrome, Pearson syndrome).
2) . mtDNA maintenance disorders (secondary mtDNA disorders).
- Autosomal recessive mtDNA depletion syndrome, due to genes including POLG, DGUOK, TK2.
- Autosomal dominant or recessive PEO, due to genes including POLG, C10orf2 (Twinkle), RRM2B.
3) . Nuclear gene disorders with no effect on mtDNA.
- Autosomal disorders affecting subunits of the respiratory chain or assembly function, such as recessive Leigh syndrome due to SURF1.
- Autosomal disorders of impaired mitochondrial protein translation, such as recessive pontocerebellar hypoplasia due to RARS2.
- Autosomal disorders of impaired mitochondrial dynamics, such as dominant optic atrophy due to OPA1 - although note that mitochondrial dynamics appears to be important in mtDNA stability and so abnormal function including due to OPA1 mutation can also disrupt mtDNA maintenance).

Question 9

Give an overview of mtDNA heteroplasmy.

Answer 9

• Each cell contains hundreds or thousands of copies of mtDNA.
• Homoplasmy: all mtDNA copies are identical.
• Heteroplasmy: a mixture of 2 or more mtDNA species with different DNA sequences - e.g. point mutation at a particular nucleotide present in a proportion of the mtDNA copies.
• The majority of pathogenic mtDNA mutations are heteroplasmic in patients.
• Threshold effect:
• For any given mutation, there is a critical threshold level of heteroplasmy above which mitochondrion/cell/tissue function will be impaired.
• This threshold differs for different mutations and also for different tissues or cell types, but is typically 80-90% mutant at the cellular level.
• Heteroplasmy to phenotype correlation:
• There is a correlation between level of heteroplasmy and biochemical and clinical severity, but this is complicated by changing levels of heteroplasmy between tissues and over time.