Final Report Summary - CELLSPEX (Multiwavelength cell spectroscopy to define the pathophysiology of mitochondrial disorders in living cells.)
Leber’s hereditary optic neuropathy (LHON) is the most common cause of blindness in young men. It presents clinically as bilateral visual loss, typically in males between 15 and 30 years old, with a mean interval of 2 months between eyes. Histopathology reveals a dramatic loss of retinal ganglion cells, which are neurons whose axons form the optic nerve and transmit information from the photoreceptors to the brain. The optic nerve displays atrophy and severe diffuse demyelination but the retina is otherwise normal. It has been found that 90% of LHON cases arise from one of three point mutations in the mitochondrial DNA (mtDNA). Mitochondria are cellular organelles whose primary role is to generate energy for the cell in the form of ATP by a process called oxidative phosphorylation. Each mitochondrion is bounded by two membranes: an outer membrane that is freely permeable to small molecules and an impermeable inner membrane that is folded into cristae and encloses the matrix. The matrix contains the mtDNA which encodes the mitochondrial 12S and 16S ribosomal RNA and 22 transfer RNAs used for intra-mitochondrial protein synthesis as well as 13 polypeptide chains that are used for oxidative phosphorylation. While nuclear DNA (nDNA) is inherited from both parents by meiosis, mtDNA is exclusively maternally inherited as the mitochondria from the sperm do not enter the egg. Typically there are hundreds of mitochondria present in each cell and thousands of copies of mtDNA.
Oxidative phosphorylation (Figure 1) is carried out on the proton-impermeable inner mitochondrial membrane. Electrons from NADH and succinate, generated in the matrix by the tricarboxylic acid (TCA) cycle, enter the electron transfer chain at complexes I and complex II, respectively, and are used to reduce membrane-bound ubiquinone to ubiquinol. Complex III (the bc1 complex) reoxidizes the ubiquinol and passes the electrons to cytochrome c (Cytc) which diffuses in the intermembrane space and passes them to complex IV (cytochrome oxidase, CytOx) where they are used to reduce oxygen to water. Complexes I, III and IV couple electron transfer to proton translocation across the inner membrane and conserve much of the redox potential energy as a proton motive force (ΔP). ΔP is composed of the membrane potential (ΔΨ) and proton concentration difference (ΔpH, 60mV ≈ 1pH unit) across the membrane. Complex V (F1Fo ATP synthase) uses the energy released as protons are transferred back into the matrix to regenerate ATP from ADP and inorganic phosphate. ATP hydrolysis is the principal energy source that drives active cellular processes, and ATP deficiency leads to cellular dysfunction and ultimately cell death. Besides ATP production, ΔP is used for a number of additional purposes, including the generation of heat, Ca2+ trafficking, mitochondrial protein translocation, and metabolite uptake.
See Figure 1 in the attached document
The complexes of the electron transport chain are multi-subunit enzymes and the majority of the subunit polypeptides are coded on nDNA, synthesized in the cytosol and imported into the mitochondria. Of the 13 mtDNA encoded polypeptides, 7 form the core subunits of the membrane component of complex I, which is believed to be responsible for the proton pumping, 1 forms the core component of the bc1 complex that is responsible for proton pumping, 3 form the core subunits of cytochrome oxidase which include all the redox co-factors and the proton pump, and 2 form major components of the stator of the ATP synthase. Thus the mtDNA codes proteins which are essential for the function and proton pumping of the electron transport chain. The three mtDNA mutations which lead to LHON affect the genes which code for subunits of complex I. The G11778A mutation, which causes the R340V substitution in ND4, is responsible for the most severe form of disease, the T14484C mutation (M64V substitution in ND6 subunit) produces the least severe disease with the greatest chance of visual recovery, and the G3460A mutation (A52T in ND1) is intermediate. Although theses mutations are typically homoplasmic (they are present in all the copies of mtDNA in each cell) and are present in all the cells of the body, it is not known why these point mutations only affect the retinal ganglion cells and why the onset is so acute. Furthermore, the penetrance is quite low: many people carrying the mutations do not develop LHON. Finally, over the course of human existence, non-pathological single point mutations have accumulated in the mtDNA and have been inherited to the present day such that many different haplogroups exist. These haplogroups have been used to track human migration over the globe. Of the European haplogroups, it has been found that the T14484C mutation is only found to cause symptoms of LHON in the J haplogroup, which is an offshoot of the more common H haplogroup.
Prior to this grant, the Incoming Fellow developed unique instrumentation that can measure mitochondrial function with greater finesse in living cells than could previously be achieved in isolated mitochondria. Specifically, we can simultaneously quantitate (1) the oxygen consumption, (2) the content of the bc1 complex, cytochrome c and cytochrome oxidase, (3) the redox potentials of the NADH, ubiquinone and cytochrome c pools and (4) the membrane potential (ΔΨ) and pH gradient (ΔpH) which sum together to give the proton motive force (ΔP). The goal of this fellowship was to bring this technology to Europe and use it to discover how the LHON mutations lead to the selective loss of retinal ganglion cells. To this end, we used 143B osteosarcoma cybrid cells that have had their original mitochondria ablated by treatment with ethidium bromide and then mitochondria with either wild type or mutated mtDNA reintroduced. This allows the effects of the mtDNA mutation to be observed on the same nDNA background. Cells with wild type mtDNA were obtained from haplogroups H13b, J1c5, J1c7, J2a1 and J2b1, with the T14484C mutation in haplogroups J1c3 and J2a2, with the G3460A mutation in J1c2 and the G11778A mutation in U5a1 and J1c1 so that we have the T14484C mutation in haplogroups J1c and J2a with matching controls, the G3460A mutation in J1c with a matching control and the G11778A mutation in H and J1c with matching controls (U5 is sufficiently similar to H to be a control).
Under normal conditions we found that the oxygen consumption of all the cell lines carrying wild type mtDNA were not significantly different but the H13b cells had a ≈20% lower content of the bc1 complex and CytOx, than the J haplogroup cells and ≈20% higher electron flux suggesting they had slightly less mitochondria but that the mitochondria were working slightly faster. The T14484C mutation did not affect electron flux whereas the G3460A and G11778A mutations decreased electron flux by 33% and 50%, regardless of the background haplogroup.
The proton motive force (ΔP) provides the energy to generate ATP and varied very little between the H13 and J1c haplogroups and was only ≈5mV higher in the J2 haplogroup. The LHON mutations had little effect on ΔP with the G11778A mutation causing a 5mV decrease. The most striking changes were in the components of ΔP, which have not be quantitated before The T14484C had little effect whereas the G3460A and G11778A increase ΔΨ and decreases ΔpH by 15 and 25mV, respectively, independent of the background haplogroup. Another surprising observation is that the J1c and J2 haplogroups, which are believed to increase the penetrance of LHON, had a higher ΔΨ and lower ΔpH than the parent H haplogroup cells. From this data, we hypothesis that the inability to maintain a high ΔΨ and/or ΔpH sensitizes the retinal ganglion cells to apoptosis such that they are more likely to undergo apoptosis under certain developmental and/or environmental conditions. While this remains a tentative hypothesis, and based on limited numbers of cells lines, we believe these are the first steps in understanding the link between mutation and phenotype in LHON.
See Figure 2 in the attached document