Mitochondrial Dysfunction

Mitochondria and Necrotic Cell Death

Disruption of the blood supply to the coronary

arteries of the heart (heart attack) or to
arteries supplying the brain (stroke) for more
than 3-4 mins results in irreversible damage to
the tissue.
 Mitochondria and Necrotic Cell Death
• In the heart damage occurs following the
resumption of the blood supply termed
reperfusion injury

• In the brain there is rapid death of neurons at

the central ischaemic core, while neurons
surrounding the core may undergo a delayed
death occurring after termination of the stroke
• The inflammatory response is partially
responsible for the damage of reperfusion injury.

• White blood cells, carried to the area by the

newly returning blood, release a host of
inflammatory factors such as interleukins as well
as free radicals in response to tissue damage.

• White blood cells may also bind to the

endothelium of small capillaries, obstructing
them and leading to more ischemia.
 However in both cases there is clear
involvement of mitochondrial dysfunction
 Permeability Transition Pore
• Mitochondria play a critical role in initiating both
apoptotic and necrotic cell death.

• A major player in this process is the mitochondrial

permeability transition pore (MPTP), a non-specific
pore, permeant to any molecule of < 1.5 kDa, that
opens in the inner mitochondrial membrane under
conditions of elevated matrix [Ca2+], especially when
this is accompanied by oxidative stress and
depleted adenine nucleotides.
 Evolutionary Role of the MPTP
• Some have speculated that the regulated
opening of the MPTP may minimize cell injury by
causing ROS-producing mitochondria to undergo
selective lysosome-dependent mitophagy during
nutrient starvation conditions.

• Under severe stress/pathologic conditions,

MPTP opening would trigger injured cell death
mainly through necrosis.
 May also Play a Role in Apoptosis
• MPTP opening has been suggested as one
mechanism that would lead to the release of
cytochrome c and other proteins from the
intermembrane space leading to PCD.

• However opening of the MTP would lead to a

reduction in ATP synthesis which would inhibit
apoptosis.
 A Possible “Low Conductance” State?
• There is controversy about the question of whether the
MPTP is able to exist in a harmless, "low-conductance" state.

• This low-conductance state would not induce MPT and

would allow certain molecules and ions to cross the
mitochondrial membranes. The low-conductance state may
allow small molecules like Ca2+ to leave mitochondria quickly,
in order to aid in the cycling of Ca2+ in healthy cells.

• If this is the case, MPT may be a harmful side effect of

abnormal activity of a usually beneficial MPTP.
 Reperfusion Injury and the Heart
• Reperfusion of the heart creates conditions
that will facilitate the permeability transition

• Namely
1. Matrix Ca2+ accumulation
2. Low ATP and high Pi
3. Oxidative stress
These conditions lead to the opening of the permeability transition
pore

• Which results in:

• A dramatic collapse of the proton motive force

(Dp)
• The release of accumulated Ca2+
• The swelling of the matrix
• The rupture of the outer membrane
• The resulting bioenergetic crisis caused by the
return of O2 leads to the rapid death of the
cardiomyocyte.
Evidence for the role of the PTP comes from the “Hot-Dog” experiment

• Hearts were perfused with radiolabeled 2-

deoxyglucose (DOG)

• DOG accumulates in the cytosol as DOG-6P but

cannot enter the matrix

• Following ischaemia-reperfusion subsequently

isolated mitochondria were found to contain DOG-
6P indicating permeabilization of the inner
membrane during the reperfusion
 Treatment with Cyclosporin A
• The one-time administration of cyclosporin A
at the time of percutaneous coronary
intervention (PCI) has been found to deliver a
40 percent reduction in infarct size in a small
group proof of concept study of human
patients with reperfusion injury published in
The New England Journal of Medicine in 2008.
 Treatment with Cyclosporin A
• Cyclosporin A inhibits the actions of cyclophilin
D, a protein which is induced by excessive
intracellular calcium flow to interact with other
pore components and help open the MPT pore.

• Inhibiting cyclophilin D has been shown to

prevent the opening of the MPT pore and
protect the mitochondria and cellular energy
production from excessive calcium inflows
 Similar mitochondrial dysfunction
caused by the permeability transition
can lead to neuronal damage during
and following stroke
 Mitochondrial Genetic Diseases
• Eukaryotic cells contain at least two genomes

• Just as mutation in the nuclear genome can result

in genetic disorders similarly problems in the
mitochondrial genome can result in disease states
 Mitochondrial Genetic Diseases
• A single cell can contain hundreds of mitochondria and
each mitochondria contains multiple copies of mtDNA.

• Thus the severity of a bioenergetic defect resulting from

mutation can differ between organs and individuals and
will only produce a phenotype when a critical threshold is
reached

• The proportion of mutant mtDNA can also vary with time

• Also mitochondria are maternally inherited

 Disease Location Result

Pearson’s syndrome, Deletion of 4977 bp All mt protein synthesis

Kearns-Sayre syndrome. between A9 and ND5 abolished due to lack of
CPEO tRNA’s
MELAS tRNAleu(UUR) All mt protein synthesis
abolished due to lack of
tRNA’s

MERRF tRNAlys All mt protein synthesis

abolished due to lack of
tRNA’s

Leber’s hereditary optic 6 point mutations in Loss of complex I activity

neuropathy (LHON) complex I ND genes
Neuropathy, ataxia and A6 Inhibition of ATP
retinitis pigmentiosa synthesis
(NARP)
 Mitochondria Involvement in
Neurodegenerative Diseases
• There is increasing evidence that some of the most
common neurodegenerative diseases are associated in
some way with mitochondrial dysfunction

• However cause and effect remains to be established in

most cases

• The general principle is that even a slight restriction in

respiratory chain activity may substantially increase
the chances of neuronal cell death
Friedreich’s Ataxia

• FRDA is an autosomal
recessive mitochondrial
disorder caused by a
decreased expression of
frataxin

• Frataxin is a mitochondrial
protein whose role is not
entirely clear but may be
involved in the assembly of
Fe-S clusters
 Friedreich’s Ataxia
• FRDA is characterized by degeneration of large
sensory neurons, cardiomyopathy and an
increased incidence of diabetes

• The mitochondrial disease caused by a

mutation in the nuclear genome (specifically,
expansion of an intronic GAA triplet repeat)
 Friedreich’s Ataxia
• Yeast knock out mutants accumulate Fe within
their mitochondria and show decreased
synthesis of Fe/S proteins resulting in
deficiencies in complexes I-III and aconitase

• An overexpression of frataxin in Drosophila

has shown an increase in antioxidant
capability, resistance to oxidative stress and
longevity
 Huntington’s Disease
• HD is a neurodegenerative genetic disorder
that affects muscle coordination and leads to
cognitive decline and psychiatric problems
Caused by an abnormal CAG expansion in the
gene encoding the protein huntingtin. A
chain of less than 38 glutamines gives no
pathology while above 40 the protein begins
to aggregate.
 Huntington’s Disease
• The protein is not mitochondrially targeted and
forms the aggregates within the nucleus and
cytosol, it is associated with a major deficiency
of complex II and III activity in the caudate
nucleus and a severe reduction in aconitase
activity.

• Inhibition of complex II with malonate or 3-

nitropropionic acid produced behavioral
changes and pathology in primates similar to
HD
Alzheimer’s Disease
 Alzheimer’s Disease
• In one study up to 39% reduction of complex IV
activity in a specific region of the cerebral cortex
• Both mtDNA and nDNA encoded complex IV
subunits are reduced in AD
• However this may reflect a non-specific decrease in
the degenerating brain
• Addition of aggregated b amyloid peptides to
isolated nerve terminals resulted in an increase in
Ca2+ entry, oxidative stress and mitochondrial
dysfunction
 Parkinson’s Disease
• The main clinical
characteristics are slowing of
movement, rigidity and
tremor.

• Later, thinking and

behavioural problems may
arise, with dementia
commonly occurring in the
advanced stages of the
Illustration of Parkinson's disease by William
Richard Gowers, first published in A Manual of disease, whereas depression
Diseases of the Nervous System (1886) is the most common
psychiatric symptom.
 Parkinson’s Disease
• These characteristics can be ascribed to the loss
of dopaminergic neurons within their cell bodies
in the substantia nigra (a region of the midbrain)

• The causes of the cell death is unknown; in

some instances there is evidence for a genetic
component, while chronic exposure to
environmental toxins has also been proposed as
a contributing factor
• PD traditionally has been considered a non-genetic
disorder; however, around 15% of individuals with PD have
a first-degree relative who has the disease. At least 5% of
people are now known to have forms of the disease that
occur because of a mutation of one of several specific
genes.

• Mutations in specific genes have been conclusively shown

to cause PD. These genes code for alpha-synuclein (SNCA),
parkin (PRKN), leucine-rich repeat kinase 2 (LRRK2 or
dardarin), PTEN-induced putative kinase 1 (PINK1), DJ-1 and
ATP13A2.

• In most cases, people with these mutations will develop PD.

 Environmental Effects
• A number of environmental factors have been associated with an
increased risk of Parkinson's including: pesticide exposure, head
injuries, and living in the country or farming. Rural environments
and the drinking of well water may be risks as they are an
indirect measures of exposure to pesticides.

• Implicated agents include insecticides, primarily chlorpyrifos and

organochlorines, pesticides, and herbicides such as Agent Orange
and paraquat.

• Heavy metals exposure has been proposed to be a risk factor,

through possible accumulation in the substantia nigra; however,
studies on the issue have been inconclusive.
 Good for the Scientists but Bad
for the Users
• A group of drug addicts in the early 1980s who
were using the synthetic opiate MPPP (a
Demerol analogue) developed Parkinson’s
disease. It was determined that the drug was
contaminated with MPTP
• MPTP was oxidized by monoamine oxidase-B in
glial cells to form MPP+

• MPP+ is transported by the dopamine

transporter in the plasma membrane of
surrounding dopaminergic neurons and in cells
behaves as a lipophilic cation and is accumulated
in the mitochondrial matrix in response to DΨ
• MPP+ is an inhibitor of complex I and is toxic in
vivo. The specificity of MPP+ for dopominergic
neurons is due to its selective uptake

The chloride of MPP+ has been used

as a herbicide under the trade name
cyperquat and is structurally similar
to the herbicide paraquat. NADH NAD+
• Interestingly rotenone also caused PD
symptoms when chronically administered to
rats

• There is good evidence for a selective partial

deficiency of complex I in the substantia nigra,
but not in other brain areas in some PD
patients
 Cytoplasmic Male Sterility
• Plant male sterility that is transmitted
through the female termed cytoplasmic male
sterility (CMS) has been found in more than
150 different plant species

• It has been intensively studied in maize,

sunflower, rice, petunia and bean.
• In all cases studied the sterility is due to the
expression of an abnormal protein in the
mitochondria

• Different CMS types express different

abnormal proteins

• Although the mutation is in all of the

mitochondria of the plant, only anthers display
a phenotype
• It is not fully understood
how all CMS proteins
disrupt mitochondrial
function.
• However it is suggested that
efficient mitochondrial
function is particularly
important during pollen
development
 Hybrid seed production can be facilitated
using male sterility
• CMS is commercially useful in that it does not
require the laborious emasculation of the
intended female parent

• To make hybrid seeds CMS plants are used as

the female parents and plants with the
nuclear restorer genes as the male resulting in
a male fertile F1 hybrid
 E.G. URF 13

T-type male sterility was used in maize

breeding programs in the 1960s.
 T-urf13 a chimeric protein

Origins of the T-urf13 gene. General structure of the maize mitochondrial 26S
rRNA gene is shown. Below (orange)shows a generic representation of the
T-urf13 protein.

Above blue shows parts of the 23S rRNA gene pieced together to yield T-
urf13. “?” Denotes apart of the Tuf13 protein whose origins are unknown.
Tassels of maize plants. T cytoplasm causes male
sterility in plants lacking the nuclear restorer
genes (right); plants carrying restorer genes are
male-fertile (left).
 The 1970’s Southern Corn Leaf Blight
Epidemic

A fungal disease of maize caused by the plant pathogen Bipolaris

maydis. The disease, which first appeared in the United States in
1968, reached epidemic status in 1970 and destroyed about 15%
of the corn belt's crop production that year.

The monetary value of the lost corn crop is estimated at one

billion US dollars
 Use of URF 13 CMS in Maize Breeding
• T URF 13 was used extensively in breeding until there
was an estimated 90% prevalence of Texas male sterile
cytoplasm (Tcms) maize, vulnerable to the blight.

• It appears that a toxin produced by the fungus

resulted in the URF13 chimeric protein acting as a
gated non selective ion pore in the inner
mitochondrial membrane and thus uncoupling the
mitochondria resulting in a bioenergetic crisis in the
maize plants,
Take Care Your Mitochondria and
They Will take Care of You!