MITOCHONDRIAL DISEASES - StFX



MITOCHONDRIAL DISEASES

Textbook: p 86; pp 537- 538; pp. 703-705

There are four categories of mitochondrial diseases, or mitochondrial pathologies as they are often called:

1) Genetic diseases due to mutations in nuclear DNA) coding for mitochondrial proteins.. These diseases follow normal Mendelian genetics. Most are autosomal recessive.

2) Genetic disease due to mutations in mitochondrial DNA (mtDNA). These diseases do not follow Mendelian genetics and their degree of inheritance is hard to predict.

3) Disease due various pharmaceuticals, such as the drug AZT used against AIDS. These are not genetic diseases and can sometimes be reversed upon withdrawal of the drug.

4) Diseases, especially if one considers “ageing” a disease, due to production of reactive O2 species (ROS) by the mitochondrial electron transport chain. Some forms, at least, of ALS (amylotropic lateral sclerosis) are caused by ROS. About 5% of such cases are due to a mutated enzyme required for protection against ROS. The enzyme is called superoxide dismutase.

GENETICS OF MITOCHONDRIA

To understand these diseases, and how their genetics differs, we need to consider how mitochondria are made (i.e. their biogenesis) and how they divide. The biogenesis of mitochondria requires both nuclear genes and mitochondrial genes. About 1500 nuclear genes are directly required whereas there only 35 mitochondrial genes in total, in humans.. The diagram below shows how proteins that are coded by nuclear genes must be imported into mitochondria after they are synthesized.

[pic]

Notice how this import of proteins into an organelle, that is into the mitochondria, differs from protein import into the RER. Import into the RER occurs as the polypeptide is being made (apart from the polypeptide synthesis required for exit of the signal sequence from the ribosome). Such protein import is said to be co-translational. But for mitochondrial (and for chloroplasts and peroxisomes) the polypeptide is completely synthesized in the cytosol before being imported into the mitochondria. Such protein import is said to be post-translational. (Look at the previous diagram to remind yourself about the term cytosol!).

Mitochondria have some DNA (mtDNA), in humans enough to code for 13 polypeptides, all the tRNAs (22) used by the mitochondrial ribosomes, and the ribosomal RNAs.. This mtDNA is devoid of histones and is circular. There are multiple copies of these DNA circles in each mitochondrion and they are not assorted equally when the mitochondria divide. Neither are they all necessarily equally replicated. It is for these reasons, the multiple copies, lack of equal replication and lack of equal rates of replication that make mitochondrial genetics so complex.

[pic]

The above diagram shows the human mitochondrial genome. You should know the following details:

1) There are genes coding for 13 different polypeptides.

2) All these proteins are integral membrane proteins and are all members of the mitochondrial electron transport chain.

3) There are no genes for the succinate dehydrogenase (SDH) .

4) Complex I, III, and IV as well as the ATP synthase are assembled from both polypeptides synthesized in the mitochondria, using mitochondrial genes, and by proteins synthesized in the cytosol and then imported into the mitochondria. The latter are coded by nuclear genes. This is summarized in the diagram on the next page.

(5) Each DNA circle has an origin of replication sequence so that it can be replicated by

the mitochondrial DNA polymerase.

[pic]

Interestingly, this exact pattern does not hold for all organism and different protein subunits can be coded for in the mitochondrial versus the nuclear genome.

Mitochondria of some organisms do not use the universal gentic code! For example UGA, which is read as a “stop codon” for mRNA synthesized in the nucleus, is read as “trytophan” in the mitochondria of mammals, fungi, and invertebrates. This relaxed codon usage is possible because of the relatively small amount of coding attributable to the mtDNA. Thus a change in this codon, for example, did not cause a lethal mutation in any of the mitochondrial genes. Such a change in codon usage in the nuclear genome would certainly be lethal. In agreement with this, the normal genetic code is used by the protozoan Reclinomonas which has the largest number of known mitochondrial genes. Clearly, in this species, modification of codon usage would cause a lethal mutation in some genes.

Unlike bacteria, the mRNAs of mitochondria have a poly-A tail, as do the mRNAs produced in the nucleus! But they lack the methylG cap at the 5-prime end.

The ribosomes of mitochondrial are more Eubacteria-like than those found in the cytosol of eukaryotes. Mitochondrial ribosomes are inhibited by some of the same antibiotics that inhibit the Eubacterial ribosomes. Such antibiotics include streptomycin, eryhtomycin, and tetracycline. Use of such antibiotics in experimental systems allows one to selectively inhibit protein synthesis in the mitochondria (or chloroplasts).

[pic]

The diagram on the previous page shows why prediction of the inheritance of mitochondrial diseases caused by mutations in mitochondrial diseases is so difficult. In organism with obvious female gametes (eggs) and male gametes (sperm) it is common that the sperm makes no mitochondrial contribution to the zygote. Thus all the mitochondrial genes are from the egg. This one-sided contribution of mitochondrial genes is called maternal inheritance. Eggs from the same mother can have widely different “mutational load” in their mitochondria because:

A higher rate of repliaction of non-mutated mtDNA (blue

circles) than mutated DNA (red circles). Presumably just due to

chance. Mitochondrial DNA is replicated throughout the cell not

in S-phase like the nuclear DNA. This continuous DNA

replication in mitochondria because the mitochondria themselves

must be able to divide at any time during the cell cycle, as the

demand for mitochondria can increase depending on conditions.

Unequal assortment during mitochondrial division

This diagram represents the division of an egg cell. Notice that each daughter cell does not necessarily get an equal number of mitochondria (although with the much greater number of mitochondria that exist in normal cells, the discrepancy will not be as large as I have drawn. Note that the top egg is considerably more likely to result in an offspring with a mitochondrial pathology than the bottom egg.

Not surprisingly, those tissues that require the most rapid rates of ATP synthesis are those most seriously affected by mitochondrial pathology. Cardiac and skeletal muscle, and the brain, are thus most commonly the most seriously affected. If the mutation is in the mitochondrial DNA then whether the brain or the muscles are most affected is a matter of chance, depending upon the unpredictable genetics of mitochondria.

An example of mitochondrial pathology due to a nuclear mutation.

The most common Mendelian disease of mitochondria (i.e. caused by a mutation of a nuclear gene) is Leigh’s Disease, which is subacute (life is possible) necrotizing (involves cell death i.e. necrosis) encephalomyelitis ( inflammation of the brain and spinal cord) Actually, Leigh’s disease is more a collection of symptoms (a syndrome) than one specific disease, because it can be caused by a variety of different nuclear mutations. It is the most common mitochondrial disorder of childhood. It is an autosomal. recessive disease. Amongst the mutant genes that can cause Leigh’s disease are the genes involved for :

Pyruvate dehydrogenase

Complex I (the NADH dehydrogenase)

Complex IV (the cytochrome c oxidase)

ATP synthase

It is clear that a defect in any of these four complexes will result in reduced rates of ATP synthesis. Clearly, the mutations must be such that they only reduce the efficiency of mitochondrial ATP synthesis, not prevent it completely. The main symptoms, seen already in early childhood are developmental problems, blindness, poor motor control, muscle weakness, increasing intellectual deficiency, and respiratory abnormalities. Notice that since this is a Mendelian disease. Affecting all nuclei in the body equally, the disease affects both muscle and nervous tissues. Death usually follows in 2-5 years due to the breathing problems.

An example of mitochondrial pathology due to a mitochondrial mutation

An example of a disease caused by a mutation in a mitochondrial DNA is the disease called MELAS. This is a disease of skeletal muscle and is due to a lack of functioning Complex IV (the cytochrome c oxidase). There are a number of different muscle fibers that make up skeletal muscle. Two basic categories are the fast twitch fibers and the slow twitch fibers. The fast twitch fibers respond rapidly to a nerve stimulus but their contraction period is short. They are powered mainly by ATP made in glycolysis (anaerobic metabolism). Hence these fibers (or muscle cells) have fewer mitochondria. The slow twitch fibers are used for more sustained muscle contraction and these muscle cells use mainly mitochondrial ATP. So these muscle cells are richly supplied with mitochondria which in normal cells are positioned to be as close as possible to the contractile fibers where the myosin ATPase is located. Clearly, problems with cytochrome c oxidase will have most effect on the slow twitch fibers.

One imporatnt diagnostic tool for any myopathology is that of the muscle biopsy. Biochemical and cell biological observations of the tissue so obtained is necessary for any final diagnosis.

Because one wants to know if functioning cytochrome c oxidase is present in the sampled muscle, it is common to use activity stains.

[pic]

A section from muscle of a patient with no myopathology would show no fibers completely lacking in COX activity. A biopsy from a child with a more advanced case of the disease is shown below on the left. Notice that COX activity is low in all the fibers, especially when compared to the sample from a patient without the disease.

[pic]

The diagram shown on the next page shows how one stains for cytochrome c oxidase (COX) activity in muscle biopsy sections (or any other tissue section). I want you to know this because is a nice example between what is known in cell biology about mitochondrial function and a practical application. It also shows how ingenious humans are!

In the diagram on the next page the following key applies:

[pic]

The production of reactive active O2 species (ROS) in mitochondria

It is estimated that about 1-2% of the electron flow down the mitochondrial electron transport chain is diverted (accidentally) to O2 before the electrons reach the cytochrome c oxidase. It is also clear that this amount is variable and can also be higher under some circumstances. The main culprit in diverting electrons, inappropriately, to O2 seems to be Complex I (NADH dehydrogenase or NDH)). The most abundant ROS in the cell are the superoxide radical (O2.-) and hydrogen peroxide (H2O2).

O2 + electron from NDH O2.- superoxide radical formation

The enzyme superoxide dismutase (SOD) catalyzes the formation of hydrogen peroxide from superoxide radicals.

SOD

O2.- H2O2

The enzyme catalase (CAT) then catalyzes the decomposition of H2O2 to harmless H2O and O2.

CAT

2 H2O2 H2O + O2

In spite of these two protective enzymes, tissue damage due to ROS builds up with time and undoubtedly is a major cause of ageing.

In the disease called ALS, a muscle wasting disease, the patients have a mutated superoxide gene that cannot convert superoxide radicals to hydrogen peroxide. In fact, the SOD works in reverse, converting H2O2 to superoxide radicals.

-----------------------

NADH DEHYDROGENASE

UBQ

CYTOCHROME bc1

COMPLEX

CYTOCHROME c

OXIDASE

ATP SYNTHASE

Xs. PROBABLE FAST TWITCH FIBER (showing less COX staining than the slow twitch fiber).

Xs. SLOW TWITCH FIBER (quite heavily stained, indicating high COX activity)

FIBER SHOWING NO COX ACTIVITY (Due to a absence of mitochondria with functioning COX)

From a child with MELAS

From a child without MELAS

DAB (red)

CYT C (red)

COX

CYT C (ox)

DAB (ox)

Brown ppt.

O2

H2O

OXIDIZED CYTOCHROME C

REDUCED CYTOCHROME C

REDUCED DIAMINOBENZIDINE (DAB) - the “stain” –an artificial electron donor to cytochrome c i.e. reduced DAB can reduce oxidized cytochrome c.. Reduced DAB is colourless.

OXIDIZED DIAMINOBENZIDINE- is brown in colour and forms a brown

precipitate as more and more oxidized DAB is formed

................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download