Mitochondrial Diseases Flashcards
What are mitochondrial diseases?
Diseases caused by qualitative or quantitative abnormalities of mtDNA
Describe mitochondrial structure
They have an outer membrane and an inner membrane that folds into cristae. Between these = intermembrane space
The matrix is on the inside of the mitochondrion
Mitochondria also contain ribosomes and mitochondrial DNA
Are mitochondria static?
No, they are highly dynamic, undergoing continual fission and fusion
What are the 2 functions all mitochondria can carry out
- The generation of ATP coupled to electron transport (oxidative phosphorylation)
- The expression of an integral genome.
Describe the structure of mtDNA
Closed-circular, double-stranded DNA molecule of ~16.6 kb (heavy strand/light strand)
arranged in nucleoids (nucleoprotein complexes)
Nucleoids are dispersed throughout the mitochondrial network
How does nuclear DNA compare to mtDNA?
Nuclear DNA vs mt: >3 billion base pairs vs 16.6kb 2 copies vs somatic cells have thousands of copies of mtDNA encodes many proteins vs encodes 13 nucleosomes vs nucleoids
How many propteins does mtDNA encode?
13
What do the proteins encoded by mtDNA do?
They are essential subunits of the oxidative phosphorylation enzymes
How are other mitochondrial proteins (not made from mtDNA) made?
They are made by the nucleus and sent into the mt
How many tRNA genes does mtDNA have?
22
What are the proteins involved in oxidative phosphorylation?
Complexes I‒V
Coenzyme Q10
Cytochrome-c
What does each protein complex do in oxidative phosphrylateion
I-II Take electrons from NADH and succinate
III-IV Receive electrons (IV donates the electrons to oxygen, reducing it to wate)
Complex V synthesises ATP using the H+ gradient made by the other complexes
What is another name for complex IV?
Cytochrome C oxidase
No nuclear DNA is needed for the 5 main oxidative phosphorylation complexes. TRUE or FALSE
FALSE
Compare mtDNA replication to nDNA
mtDNA replication occurs at a constant rate throughout the cell cycle and mtDNA continues to be replicated in post-mitotic cells.
nDNA replicates only during S-phase of mitotic cells
How was it found out that mtDNA always replicates independent of cell cycle?
Bromo-deoxyuridine (binds to replicating DNA) was given to cells
Cells which had divided during the experiment had MrdU in their nuclei, however all cells (divided and not) had BrdU staining in their mitochondria
Why are the 2 mtDNA strands called heavy and light?
Two strands of the mtDNA duplex have distinct buoyant densities (“heavy” and “light”) as result of their different base content and can be resolved by equilibrium CsCl gradient centrifugation.
What is the D-loop/displacement loop?
A short, three-stranded structure in which a short nucleic acid strand, complementary to the L-strand, displaces the H-strand
A major control site for mtDNA replication and transcription.
Describe mtDNA replication initiation and the proteins involved
RNA polymerase (POLRMT), TFAM, TFB2M and TEFM make the RNA primer and add it to the heavy origin of replication
What are the functions of TFAM?
transcription factor
packaging role
Describe mtDNA replication
- Twinkle (TWNK) to unwind the DNA duplex
- mitochondrial topoisomerase 1 (TOP1MT) to relax DNA supercoils
- mitochondrial single-stranded binding protein (SSBP1) to stabilize single-stranded regions of mtDNA replicative intermediates
- Synthesis of the mtDNA strands is catalyzed by DNA polymerase gamma
- Once DNA polymerase gamma comes full circle, it encounters the 5’-end of the nascent mtDNA strand. RNA primers are removed by RNase H1 (RNASEH1).
- Further processing of the 5’-end by mitochondrial genome maintenance exonuclease 1 (MGME1).
- gaps are filled in and ligated by DNA ligase III (LIG3)
- the daughter duplexes are topologically linked at the OH region as hemicatenanes, i.e. two circular DNA duplexes bound together via a single-stranded linkage.
- decatenation of the daughter mtDNA molecules is catalyzed by the mitochondrial isoform of topoisomerase 3alpha (TOP3A).
What are the subunits of DNA polymerase gamma?
one POLG subunit and two POLG2 subunits
What does POLG do?
POLG is the catalytic subunit, containing the 5’→3’ DNA polymerase activity as well as a proofreading 3’→5’ exonuclease activity
How is mtDNA inherited?
maternally
Why is mtDNA inheritied maternally?
- The mtDNA copy number in an egg cell (150,000‒700,000) is much higher than in a sperm cell (<10).
- Shortly before or just after fertilisation, sperm mtDNA is largely degraded by endonuclease G.
- After fertilisation, sperm mitochondria are coated with ubiquitin inside the zygote cytoplasm. The ubiquitination selectively earmarks sperm mitochondria for degradation by the proteasomal and autophagosomal systems.
How many proteins are needed for a perfectly functioning mitochondrion?
15,000
What kind of disorders are caused when nuclear genes get involved in depletion of mtDNA in infants?
Alpers disease, myocerebrohepatopathy specturm (MCHS), multi-organ or isolated hepatic disease, myopathy, encephalomyopathy
What causes Alpers disease?
A mutation in the POLG gene leads to mtDNA depletion in the liver, brain and muscle
What causes myocerebrohepatopathy?
A mutation in the POLG, TWNK, or TFAM genes which code for POLG, twinkle and TFAM proteins leads to mtDNA depletion in muscle, brain and liver cells
What kind of inheritance do Alpers disease and Myocerebrohepatopathy have?
autosomal recessive
What are the symptoms of Alpers disease?
Severe and lethal
get a severe encephalopathy, developmental delay, epilepsy, later get liver failure (cause of death)
When is the onset of Alpers disease?
childhood
When is the onset of MCHS?
childhood - die earlier than with alpers
What are the symptoms of MCHS?
Myotonia, myopathy, developmental delay, encephalopathy, liver failure
How much mtDNA do normal do patients with Alpers diseas and MCHS have?
<5% of normal mtDNA
What is another name for complex 4?
Cytochrome oxidase
What do you see in the liver of someone with Alper’s?
No cytochrome oxidase, baloon shaped mitochondria with no cristae
Name ways defects can arise in mtDNA?
Nuclear nuclear mutations
- Gene mutations (mtDNA replication)
- Genes responsible for deoxyribonucleoside synthesis
Not caused by nuclear mutations
- Single deletions in mtDNA
- Point mutations in mtDNA
What causes Multi-organ or isolated hepatic disease?
A mutation in DGUOK which codes for deoxyguanosine kinase (dGK) - mitochondrial enzyme which is responsible for the phosphorylation of deoxyguanosine (dGuo–>dGMP) and deoxyadenosine (dAdo–>dAMP)
What causes myopathy?
A mutation in TK2 (thimidine kinase 2), a protein involved in the phosphorylation of deoxycitidine (dCyt–>dCMP) and deoxythymidine (dThd–>dTMP)
What causes encephalomyopathy?
A mutation in RRM2B gene which codes for p53R2 (a subunit for ribonucleotide reducase
What does ribonucleotide reductase do?
Enzyme in cytosol which can convert NDPs to dNDPs (can be imported to mitochondria to make mtDNA)
How are deoxyribonucleosides made to get into the mitochondria in S phase?
1.dGuo and dAdo are converted to dGMP and dAMp by dGK
dCyt and dThd are converted to dCMP and dTMP by TK2
- dNMP kinase conversty these into dNDPs
- Together with the dNDPs made by ribonucleotide reductase these dNDPs are converted to dNTP by dNDP kinase and become part of mtDNA
Where are dGK, TK2 and p53R2 found?
dGK and TK2 in the mitochondrion
p53R2 in the cytosol
How do dividing cells with dGK deficiency compare to normal cells when stained with BrdU?
There is only mitochondrial replication in the cells that are in S phase (and to a significantly lesser extent)
Why do the cells in S phase with dGK deficiency have mitochondrial replication
Many nucleotides are present in S-phase so some of these enter the mitochondria and take part in mtDNA replication
What happens if you take cells from a p53R2, TK2, or dGK deficient patient and culture them without growth factors?
the amount of mtDNA decreases (if it were normal it would stay the same)
What happens if you take cells from a TK2 deficient patient and culture them without growth factors but with dCMP and dTMP?
the mtDNA deficiency decreases
this is the basis of nucleoside replacement therapy
Name diseases which are caused by mutations in nuclear genes involved in multiple deletions in mtDNA in adults
adPEO (autosomal dominant progressive external ophthalmoplegia) with or without Parkinsonism and Premature ovarian failure
arPEO(autosomal dominant progressive external ophthalmoplegia)with or without Parkinsonism and Premature ovarian failure
what causes adPEO?
A mutation in TWNK or POLG
what causes arPEO?
A mutation in POLG
what is PEO?
Progressive paralysis of eye muscles (move head not eyes) and ptosis
What kind of mutation in POLG lead to multiple deletion mutations?
missense
How common are mutations in mtDNA?
relatively common cause of human disorders (over 1/5000)
Which organs are affected most in mitochondrial diseases and why?
Mitochondria make ATP. Thus organs most reliant of mitochondrial ATP production are most affected (brain and muscle)
People with mitochondrial diseases have only mitochondria with the mutated mtDNA. TRUE or FALSE
FALSE
Most mtDNA mutations co-exist with wild-type mtDNA at varying
proportions in the cell. Only when the mutation load exceeds a critical threshold
. will the mutation result in a clinical phenotype.
What kind of range (related to affected organs) do mitochondrial diseases have?
Large range: eyes, brain, muscles, diabetes (pancreas), cardiomyopathy, eyes, kidney defects
What percentage of diabetes is caused by mitochondrial problems?
1.5% is caused by mt problem
What does homoplasmic mean?
It means that the cell has all its mtDNA the same
What does heteroplasmic mean?
It means that the cell a mixture of variant and wild type mtDNA
What is the result of the relaxed nature of mtDNA replication?
mtDNA deletions and point mutation accumulate with age through clonal expansion and random intracellular drift in post-mitotic cells
how many mtDNA deletions have been found to be associated with disease?
> 130
Name diseases that are caused by mtDNA deletions
PEO
Kearns-Sayre syndrome
Pearson bone marrow pancrease syndrome
What is Kearns-Sayre syndrome?
It is like PEO but worse. In addition to ptosis, they get heart disease and blindness
What is Pearson bone marrow pancrease syndrome
children have disfunctional pancreas and don’t form healthy RBC (form sideroblasts) - causes anemia
How many different mtDNA point mutations have been found to be associated with disease?
> 650
What are the most frequently occuring point mutations in mtDNA?
m. 3243A>G mutation in the gene coding for tRNA -leucine (MTTL1)
m. 8344A>G om gene coding for tRNA - Lys (MTTK)
m. 8993T>C/G in the gene coding for a complex V subuni (MTATP6)
m.3460A>G, m.11778A>G, m.14484T>C
in genes coding for subunits of complex I (MTND1, MTND4, MTND6)
m.1555A>G in the gene for 12S rRNA (MTRNR1)
Where was m.3243A>G (tRNA leu) first found?
Patients presenting with mitochondrial encephalomyopathy, lactic acidosis and stroke like episodes (melas)
What kind of range does m.3243A>G (tRNA leu) have?
Very broad: Diabetes, with or w/o deafness to hypertrophic cardiomyopathy and retinitis pigmentosa (blindness)
What can m.8344A (tRNA lys) cause?
MERRF: myoclonic epilepsy, associated with ragged-red fibres
What do the red fibres in MERRF mean?
An accumulation of defective mitochondria
What can m.8993T>C/G ( complex V subunit) cause?
NARP: Nauropathy, ataxia and retinis pigmentosa
Maternally inherited Leigh syndrome (MILS) - if >90% mutation load (other mutations can cause leighs)
What is Leigh syndrome ?
serious condition in children which causes a degenration of the brain
where are M.3460A>G, m.11778A>G, m.1444T>C (complex 1 subunit) and what do they do?
they are in genes coding for subunits of complex I (MTND1, MTND4, MTND6)
They cause Leber hereditary optic neuropathy (LHON)
What are the characteristics of Leber Hereditary Optic Neuropathy?
Acute loss of vision in teens
What does m.1555A>G in the gene for 12S rRNA (MTRNR1) do?
When you carry this mutation and you have sepsis and are given aminoglycosides, which usually binds to bacterial 16S ribosomal RNA, the aminoglycosides instead bind to the patients mitochondrias 12S rRNA
This leads to hearing loss as aminoglycosides accumulate in the ear
All mitochondrial DNA in ppl with point mutations heteroplasmic. TRUE or FALSE?
FALSE
MOST mtDNA in people with point mutation heteroplasmic. But not for example m.3460A>G or m.1555A>G
Why is the mutation load in children highly variable?
The pattern of mtDNA segregation is largely determined by random genetic drift during oogenesis.
During female germ line development, a small number of mtDNA molecules is sampled from a larger population for transmission and amplification.
Is there a cure for mtDNA diseases?
No
How do you treat mtDNA diseases?
Aerobic endurance training: stimulates mitochondrial biogenesis
Coenzyme Q10 and its water soluble analogue idebenone: antioxidants (not shown to be useful)
Arginine and citrulline in MELAS patients: raise production of NO (vasodilator to treat stroke-like episodes)
Nucleoside therapy in thymidine kinase 2 deficient patients
Name some emerging therapeutic approaches to mtDNA diseases?
Allotopic expression of genes of mitochondrial origin in the nuclear genome (make the nucleus do the job of mtDNA)
Modulation of heteroplasmy levels with engineered nucleases (cause a double stranded break in the mutated mtDNA and mitochondria cannot repair this):
- Mitochondrially targeted zinc finger nucleases (mtZFNs)
- Mitochondrially targeted transcription activator-like effector nucleases (mtTALENs)
What are difficulties with the allotopic expression of genes of the mitochondrial origin in the nuclear genome
You need to correct the gene with the virus
Mitochondria have different DNA so you have to turn it into universal genetic code
The protein has to get into the mitochondrion
What are the options with assisted reproductive techniques to prevent mtDNA transmission?
Egg donation
Prenatal diagnosis (PND): Amniocentesis or chorion villus sampling Mutation load in extra-foetal tissue may not accurately represent that of foetal tissue
Preimplantation genetic diagnosis (PGD) to select embryos created by in vitro fertilisation (IVF)
Mitochondrial replacement therapies (Pronuclear transfer or Maternal spindle transfer)
What is a pronuclear transfer?
transplantation of the nuclear genome after fertilisation
Transfer of the pronuclei from a zygote with mutated mtDNA to a donor zygote
with healthy mtDNA from which the pronuclei have been removed.
What is a maternal spindle transfer
transplantation of the nuclear genome before fertilisation
Transfer of the spindle with chromosomes from an unfertilised egg with mutated mtDNA to an unfertilised donor egg with healthy mtDNA from which the chromosomal spindle has been removed.
Which, pronuclear transfer or maternal spindle transfer is better?
Maternal spindle transfer - It is doesn’t involve killing half the embryos but more difficult to perform
