Does mitochondrial DNA play a role in Parkinson's disease? A review of cybrid and other supportive evidence

Abstract

Significance: Mitochondria are currently believed to play an important role in the neurodysfunction and neurodegeneration that underlie Parkinson's disease (PD).

Recent advances: While it increasingly appears that mitochondrial dysfunction in PD can have different causes, it has been proposed that mitochondrial DNA (mtDNA) may account for or drive mitochondrial dysfunction in the majority of the cases. If correct, the responsible mtDNA signatures could represent acquired mutations, inherited mutations, or population-distributed polymorphisms.

Critical issues and future directions: This review discusses the case for mtDNA as a key mediator of PD, and especially focuses on data from studies of PD cytoplasmic hybrid (cybrid) cell lines.

FIG. 1.

Stages of mitochondrial enrichment. When measuring mitochondrial ETC enzyme activities from a tissue section the first step is homogenization. There is no mitochondrial enrichment in the homogenate, and most of the protein in the homogenate is not mitochondrial. The first enrichment step involves the removal of nuclei to produce a postnuclear fraction. The supernatant from the postnuclear fraction can be centrifuged to pellet mitochondria and other similar-sized organelles. This stage is called a crude mitochondria fraction. The highest degree of enrichment results after the mitochondria have been separated from other organelles via density gradient centrifugation. With each enrichment step the assayed signal is greater and therefore more sensitive to subtle differences between samples.

FIG. 2.

Complex I is part of the ETC. Complex I, also known as NADH:ubiquinone oxidoreductase, accepts electrons from NADH and delivers those electrons to coenzyme Q. Electrons eventually reach complex IV, also called cytochrome oxidase, where they are used to reduce oxygen and form water. As electrons are passed from higher to lower energy states, the energy that is released is used to pump protons from the mitochondrial matrix and into the mitochondrial intermembrane space. These protons re-access the matrix through a non-ETC enzyme called complex V, or the ATP synthase, and energy from that proton flux is used to generate ATP. Complexes I through V are all multimeric enzymes, and complexes I, III, IV, and V are bi-genomically encoded and contain subunits encoded on both mtDNA and nuclear DNA. Complex I contains 7 mtDNA-encoded subunits and at least 39 nuclear DNA-encoded subunits. ETC, electron transport chain; mtDNA, mitochondrial DNA.

FIG. 3.

Cybrid cell lines are created by mixing the cytosolic components of a nucleated cell with the cytosolic contents of a non-nucleated cell or cytoplast. When control over a cybrid cell line's mtDNA content is desired, an mtDNA-containing cytoplast can be mixed with a ρ0 cell line that was previously depleted of its endogenous mtDNA. In this figure the cell line's original endogenous mtDNA is shown as open circles within mitochondria, and after conversion to a ρ0 cell the mtDNA-less mitochondria are shown to lack mtDNA. The cytoplast's mtDNA is shown as filled circles. During a transfer process some of the cytoplast's mtDNA-containing mitochondria mix with the cytosolic contents of some of the ρ0 cells. Because ρ0 cells lack 13 key components of the ETC and ATP synthase that are normally encoded by mtDNA, placing the cells in a selection medium that will not support ρ0 cell survival removes nontransformed cells. Cells that incorporated mtDNA from the cytoplast, though, are restored to aerobic competency and expand during the selection process. The result is a unique cell line that has the nucleus of the original ρ0 cell line and the mtDNA from the cytoplast donor.

FIG. 4.

Cybrid clonal selection facilitates testing of threshold effects. After ρ0 cells are mixed with cytoplasts that contain an mtDNA heteroplasmy, it is possible to isolate the individual colonies that arise through the expansion of cells that have incorporated cytoplast mtDNA. The fusion process can therefore lead to cybrid sub-cell lines that have different heteroplasmic ratios. Biochemical assays of each sub-cell line can reveal the mutational burden, or threshold, that is required to cause biochemical consequences. Results must be carefully interpreted since mutations may have greater effects in primary tissues than they do in cell culture.

FIG. 5.

Model via which mtDNA heteroplasmy can produce sporadic-appearing phenotypes through threshold effects and mitotic segregation. An ova with a heteroplasmic mtDNA mutation is fertilized to produce a zygote. As the zygote gives rise to a multicellular organism, its mitochondria are distributed randomly so that different tissues end up containing unequal amounts of the mutation. This process is called “mitotic segregation.” The individual's subsequent risk for developing a central nervous system mitochondriopathy disease depends on the burden and severity of the mutation in the central nervous system, a concept known as “threshold.” In this model individuals with low burdens of nondeleterious mutations would avoid disease, whereas individuals with higher burdens of the same mutation or lower burdens of more deleterious mutations would manifest a disease phenotype.

FIG. 6.

Inferences about a subject's mtDNA can be made by comparing a cybrid cell line made with that subject's mtDNA to cybrid cell lines containing mtDNA from different subjects. Because the nuclear genetic composition and the environment are equivalent between the different cybrid lines, differences in function or structure most likely arise from differences between mtDNA. This type of cybrid approach can be used to screen for functional mtDNA differences between individuals.

FIG. 7.

PD cybrid cell lines demonstrate numerous functional and structural perturbations. Many of these perturbations recapitulate phenomena observed in persons with PD. Documenting these functional phenomena in PD cybrid cell lines suggests that the complex I defect seen in sporadic, idiopathic PD subjects is able to cause the phenomena. PD, Parkinson's disease.

FIG. 8.

Heteroplasmy may cause disease-free mothers to transmit a high risk of disease to their offspring, and cause disease-affected mothers to transmit a low risk of disease to their offspring. During early development a stem cell gives rise to a female embryo's oogonia. A stem cell with heteroplasmic mtDNA could produce oogonia with varying amounts of a heteroplasmic mutation. The disease risk of that female's offspring would in turn depend on the amount of mtDNA mutation in the eggs that are fertilized. The disease risk of the mother herself, meanwhile, depends not on the mutational burden in her oogonia but rather on the mutational burden in her central nervous system (see Fig. 5). Mechanisms such as this would enable mtDNA-inherited diseases to appear as sporadic diseases as opposed to matrilineal diseases, and diseases that are influenced by mtDNA inheritance to show non-Mendelian epidemiology.