Mitochondria: Function, Genetics, Diseases

Question 1

What effect will the insufficiency of oxygen have on the mitochondria? (Or in mito disease)

Answer 1

The reactions of oxidative metabolism will change

i.e. Something will go wrong with:
Pyruvate conversion to acetyl coA

Running of TCA cycle to make CO2 and capture electrons from acetyl coa

NADH and FADH providing electrons to ETC to pump protons across inner membrane

Question 2

What happens when the muscle is out of oxygen?

Answer 2

Pyruvate is converted to Lactate through lactate dehydrogenase

Question 3

Glycolysis

Answer 3

6-C sugar is oxidized and split into 2x 3-C pyruvates

Makes 2 ATP

Question 4

Fate of Pyruvate (aerobic and anerobic)

Answer 4

Aerobic: Pyruvate oxidized and converted to Acetyl CoA. NADH produced, CO2 produced

Anaerobic: (also in RBCs which lack mito)
Pyruvate reduced to lactate, NAD+ produced
NAD+ keeps glycolysis going

Question 5

Fate of lactate

Answer 5

Sent via blood to liver where it’s converted to pyruvate and then glucose (Cori cycle)

Question 6

Heteroplasmy of mtDNA

Answer 6

Causes:
Fission and fusion
Accumulated mutations
Random segregation during mitosis
Proximity to ETC (ROS)
Limited protection and repair mechanisms

Question 7

Threshold Effect

Answer 7

70-90% mutation load is associated with clinical manifestations

Question 8

PTEN-induced Putative Kinase Protein 1 (PINK1)

Answer 8

Remove damaged mitochondria

Accumulates following mitochondrial damage

Recruits E3 ubiquitin kinase PARKIN

Question 9

Parkin

Answer 9

E3 ubiquitin kinase
ubiquitylates mito proteins causing mito to be engulfed by isolation membrane that fuses with lysosomes

Autophagosome around mito

Question 10

PGC1a (and why it’s increased)

Answer 10

Regulation of mito biogenesis

Increased by:
endurance exercise
caloric restriction
energy deprivation
cold exposure
neuro-hormonal signaling
oxidative damage

Question 11

Mutations in mtDNA

Answer 11

Sporadic or maternal inheritance

Small mutations or large deletions

Cause defects in:
ETC components
rRNA or tRNA for synthesis in mito

Question 12

Mutations in nuclear DNA

Answer 12

Sporadic or Mendelian inheritance

Small mutations or large deletions

Cause defects in:
ETC complexes/assembly 
mtDNA maintenance
protein import
lipid dynamics
biosynthesis of CoQ10

Question 13

MELAS (mito encephalomyopathy, lactic acidosis, stroke-like episodes)

Answer 13

Point mutations in tRNA genes in mtDNA

Clinical findings:
CNS (seizures, ataxia, cortical blindess, headahces, dystonia)
Muscle weakness

Question 14

Kearns-Sayre Syndrome

Answer 14

Large deletions in mtDNA

Clinical findings:
Ataxia
Muscle weakenss (ophthalmoplegia and ptosis)
Pigmentary retinopathy
Endocrine, blood, cardiac

Question 15

PEO (progressive external ophthalmoplegia)

Answer 15

Autosomal dominant or recessive

Point mutations in nuclear gene encoding mtDNA polymerase (POLG)

Ptosis
Myopathy
Sensorineural hearing loss
Ataxia
Hypogonadism
Cataracts
Onset adolescence/adulthood

Question 16

Cytochrome C Oxidase (COX)

Answer 16

Complex IV

Absence of COX activity reveals SDH staining

Brown: Cox working
Blue: Cox absence, shows SDH

Question 17

Succinate Dehydrogenase (SDH)

Answer 17

Transfers electrons to complex II via FADH

Question 18

Mito/mtDNA in the cell

Answer 18

Each mitochondrion has 2-10 copies of mtDNA

Heart
Skeletal muscle
Brain
Lung

Question 19

Genetic bottleneck during oogenesis

Answer 19

Explains why we see difference in mutation load among children

Only a subset of heterogeneous mitochondria is passed on to a given oocyte

Mitotic segregation

Question 20

Tricarboxylic Acid (TCA) Cycle or Krebs Cycle

Answer 20

Stepwise oxidation of acetyl coA to CO2

Electrons are harvested by reducing NAD and FAD

Products:
3NADH
1FADH
1ATP
2CO2

Question 21

Where can the TCA cycle get acetyl-coa?

Answer 21

From pyruvate (glycolysis)
Beta-oxidation of fats
Breakdown of AA

Question 22

ATP Production (glycolysis and oxidative phosphorylation)

Answer 22

2: glycolysis
24: oxidative phosphorylation

Question 23

Location of ETC, TCA, b-oxidation, ATP synthase, membrane transporters

Answer 23

ETC: mito inner membrane on cristae

TCA: matrix

b-oxidation: matrix

membrane transporters: cristae

ATP synthase: cristae

Question 24

Pyruvate dehydrogenase

Answer 24

Pyruvate > Acetyl CoA

Happens in matrix

Question 25

TIM

Answer 25

Inner membrane transporters
Signal peptide determines where it goes in mito

TIM22: insert proteins into membrane; internal signal peptide

TIM23: insert proteins into matrix; pre-sequence signal peptide

Question 26

TOM

Answer 26

Outer membrane receptor (R)
+
General import pore (GiP)

Question 27

Red-ragged fibers

Answer 27

Seen in most patients with mtDNA defects and some with nDNA mutations

Due to accumulation of abnormal mitochondria below plasma membrane

Can lead outline of muscle fiber to become irregular “ragged”

NOT universal in mito disorders
NOT specific for primary mito diseases

Question 28

mtDNA vs nDNA

Answer 28

mtDNA:
replicates autonomously
DNA polymerase gamma (POLG)

nDNA:
encodes >85% of mito proteins

Question 29

DNA polymerase gamma (POLG)

Answer 29

nuclear encoded
structurally similar to bacteria DNA polymerases
Less accurate than nDNA polymerases

Question 30

Diagnostic criterion for mtDNA mutation

Answer 30

1. Disease expressed in children of both sexes or affected mother
2. No evidence of paternal inheritance

Question 31

NADH in metabolism

Answer 31

Oxidized by complex I in ETC under normal mitochondrial function

Oxidized when pyruvate is converted to lactate in defective respiratory metabolism

NAD+ needs to be regenerated to recycle back to glycolysis, where it is reduced again when electrons are harvested from C-C bonds in glucose

Question 32

Serum levels

Answer 32

Pyruvate: unchanged in MD

Lactate: elevated in MD

Lactate:Pyruvate ratio is normal/lower (<20) in patients with pyruvate dehydrogenase deficiency (pyruvate accumulates) in comparison to those with MD, where Lactate:Pyruvate is elevated (>20) because pyruvate does not accumulate

Question 33

SDH vs COX staining

Answer 33

SDH: shows blue
COX: shows brown

Brown covers up blue. When COX isn’t working, blue will show.