lecture 28

Question 1

What are mitochondria?

Answer 1

• the power plants of the cell
• outer membrane and tightly folded inner membrane
• role in energy generation
  fuel + oxygen → energy (ATP)

Question 2

What is our power requirement at rest?

Answer 2

• 100kcal/hour = 116W
• O2 consumption 380L/day
• 65kg ATP/day (=$3m worth)
• MIM surface area = 14,000 m^2
• brain: 2% of weight, 20% of energy use

Question 3

What happens if they don’t work?

Answer 3

• excerise intolerance at mildest end
• lack of energy in the muscles/poor work capacity
• any symptom, any organ or tissue, any age, any mode of inheritence

hearing
- sensorineural deafness

visual

• death of optic nerve
• cataracts
• retinitis pigmentosa

bone marrow

• all blood cell types
• myelodysplasia
• siderblastic anaemia
• thrombocytopenia
• cyclic neutropenia

heart

• cardiomyopathy
• hypertrophic
• conduction defects
• fibrosis

kidney

• tubules
• glomeruli
• widespread

GIT

• liver failure
• chronic diarrhoea
• villous atrophy
• pseudo-obstruction
• paragangliomas: benign tumours of the carotid body (oxygen sensing organ)
• dysfunction in specific glands e.g. thyroid
• facial dysmorphism
• hypertrichosis (excess hair)

pancreas

• insulin secretion defects
• exocrine dysfunction

Question 4

What is leigh disease?

Answer 4

• the most common mitochondrial disease of childhood
• typically healthy until ~6 months
• progressive, episodic neurodegenerative disorder
• motor and or intellectual regression with signs of brainstem dysfunction
• focal symmetric spongiform lesions in CNS
  → demyelination, gliosis, capillary proliferation
• see holes lighting up e.g. in basal ganglia
• loss of myelin
• overgrowth of glia

Question 5

What do mitochondria look like?

Answer 5

• football shape in hepatocyte
• typical 0.4 x 2.3 µm length
• tightly folded inner membrane
• enourmous SA for proteins to sit on
• football structure from textbooks
• more like a filamentous network that is dynamic
• constantly undergoing fission and fusion

Question 6

What is the distribution of mitochondria in the muscle?

Answer 6

• w/i and w/o muscle fibre
• in subsarcolemmal space
• 2% fast muscle fibres
• 8% slow muscle fibres
• 20% liver

Question 7

What is the endosymbiont hypothesis?

Answer 7

• primitive aerobic bacterium phagocytosed by a fermentative eukaryotic cell
  → symbiosis and gradual loss of mtDNA functions
• mtDNA circular, lacks histones
• inner membrane bacterial-like
• rRNA is chloramphenical S, emetine R: i.e. ribosomes more like bacterial ribosomes than eukaryotic

Question 8

What is the genetic content of the nucleus?

Answer 8

• 23 chromosome pairs
• 20,000 genes
• inherit half from each parent

Question 9

What is the genetic content of mitochondria?

Answer 9

• 1 chromosome
• 37 genes
• inherited from mother only

Question 10

How many genes encode mitochondrial proteins?

Answer 10

• 37 mtDNA genes
• 1000-1500 genes
• 5-10% of our genes encode mitochondrial proteins

Question 11

WHy are mitochondrial disorders complicated?

Answer 11

consider other well known genetic disorders e.g. CF

• 1 protein, 1 gene, 1 common mutation
• 1 type of inheritance (autosomal recessive)
• screened for at birth

mito disorders comprise > 150 different monogenic diseases

adult-onset mito disorders

• ~70% maternally inherited mtDNA mutations
• autosomal dominant, sporadic, autosomal recessive

childhood-onset mito disorders

• ~30% maternally inherited mtDNA mutations
• mostly autosomal recessive
• some sporadic, X-linked, autosomal dominant

Question 12

What is the mitochondrial DNA?

Answer 12

• double stranded circle
• 16 569 base pairs
• 13 protein-coding genes
• 22 tRNA genes
• 2 rRNA genes
• genes in the mtDNA can’t be read properly by the cytosolic machiner
• chock full of genes
• some overlapping
• heavy strand
• no introns
• interspersed with tRNA
• 7 of the 44 subunits of complex I
• complex III and IV, 1 and 3 respectively
• complex V has a couple of subunits

Question 13

What is the mtDNA morbidity map?

Answer 13

• pathogenic point mutations found in 35 of 37 mtDNA genes
  MELAS: mito encephalomyopathy, lactic acidosis and stroke-like episodes (typically kids at puberty collapsing/constant seizures)
• 80% of the time caused a mutation in the teran A leucine gene
• bunch of other genes in which mutations can cause the same condition
• allelic heterogeneity
  LHON: leber’s hereditary optic neuropathy
• typically characterised by sudden onset of blindness
• most often caused by mutation in ND4 subunit gene, subunit of complex I, 50% of the time
• mutations in several other genes can cause same presentation

not just one gene causing one clinicical presentation, and not just one clinical presentation being caused by one clinical presentation

Question 14

What are unique features of mtDNA?

Answer 14

• maternal inheritance
• • mtDNA genes are identical
• • nucluear gene gene alleles only 1/256th of great^6 grandmother
• • haplogroup K - 16,000 ybp, northern europe
• • haplogroup U - 40000 yp, south central asia
• multiple copies (2 to 10/mito and 50, 200,000/cell)
• high mutation rate (~10-fold higher than nucleus)
• used to track female populations around the world
• mt haplogroups used in tracking human migration and in forensics
• heteroplasmy
  → co-existence of mutant and wildtype mtDNA
• threshold effect
  → minimum critical proportion of mutant mtDNAs needed for each tissue to become dysfunctional
• mtDNA bottleneck
  → in early oogenesis, a small number of genomes are “selected” to repopulate the oocyte, allowing rapid shifts in heterplasmy (resets the biological clock?)
  → mitochondrial numbers decrease and then increase, most of the time you’d clean out the mutant
• tissue specific segregation/selection
  → variation b/w tissues because amount of a mutation has increased in one cell lineage and not others in embryogenesis, or more oftenly selection for or against mutation

Question 15

Why is there maternal inheritance ?

Answer 15

• in ovum there are 200,000 mitochondira
• sperm 50mt DNA
• in midpiece of sperm
• paternal mitochondria are tagged with ubiquitin
• one documented incidence of paternally inherited mitochondrial disease: exception that proves the rule

Question 16

What is the probability of severe outcome in mutant load?

Answer 16

• 45% → < 5%
• 63% → 25%
• 71% → 50%
• 78% → 75%
• 92% → >95%

Question 17

What is OXPHOS genetics?

Answer 17

• mtDNA heteroplasmy, the bottleneck and threshold effect
• mother with mild or no symptoms → small number of mother’s mitochondria, selected randomly, goes into each early egg cell → contribution from mother + contribution from father = possible outcome
• mother’s cells may have 20% mutant mitochondria → cells that will become egg cells → mature egg cells (80% mutant, 50% mutant, 20% mutant) + sperm cells (no mitochondria) = child with severe disease? or child with mild disease? or child with no disease?

Question 18

What is Kearn-sayre syndrome?

Answer 18

• external ophthalmoplegia, retinitis pigmentosa, heart block or ataxia
• vastly different amounts of mutation in different tissues
• e.g. muscle 30%, liver 60%, fibroblast 1%

Question 19

What is the evidence for OXPHOS defects?

Answer 19

Evidence

• clinical
• metabolic
• histology
• OXPHOS enzymes
• DNA

Diagnosis

• definite
• probable
• possible
• unlikely

Question 20

What is histology of mito diagnosis?

Answer 20

• cytochrome c oxidase (Complex IV or COX) histochemistry and single fibre PCR

Question 21

What is an example of investigating a patient?

Answer 21

Patient:

• clinical: leigh syndrome
• metabolic: lactate 3.8mM (NR A

RFLP

tests aren’t 100% sensitive