OXPHOS mutations and neurodegeneration

Abstract

Mitochondrial oxidative phosphorylation (OXPHOS) sustains organelle function and plays a central role in cellular energy metabolism. The OXPHOS system consists of 5 multisubunit complexes (CI-CV) that are built up of 92 different structural proteins encoded by the nuclear (nDNA) and mitochondrial DNA (mtDNA). Biogenesis of a functional OXPHOS system further requires the assistance of nDNA-encoded OXPHOS assembly factors, of which 35 are currently identified. In humans, mutations in both structural and assembly genes and in genes involved in mtDNA maintenance, replication, transcription, and translation induce 'primary' OXPHOS disorders that are associated with neurodegenerative diseases including Leigh syndrome (LS), which is probably the most classical OXPHOS disease during early childhood. Here, we present the current insights regarding function, biogenesis, regulation, and supramolecular architecture of the OXPHOS system, as well as its genetic origin. Next, we provide an inventory of OXPHOS structural and assembly genes which, when mutated, induce human neurodegenerative disorders. Finally, we discuss the consequences of mutations in OXPHOS structural and assembly genes at the single cell level and how this information has advanced our understanding of the role of OXPHOS dysfunction in neurodegeneration.

Figure 1

Energy metabolism in a typical mammalian cell. To meet cellular energy demands, ATP is generated by the glycolysis pathway (blue), the tricarboxylic acid (TCA) cycle and the oxidative phosphorylation (OXPHOS) system. The main energy substrate glucose (GLC) enters the cell via GLC transporters (GLUTs) and is converted into pyruvate (PYR). Alternatively, surplus GLC can be stored as glycogen for later use or enter the pentose phosphate pathway (green). PYR can have two different fates: either it is converted into lactate (LAC) that leaves the cell, or it enters the mitochondrion (yellow) to form Acetyl coenzyme A (AcCoA). The latter is processed by the TCA cycle to yield NADH and FADH₂, which are substrates of the OXPHOS system. In addition to GLC also fructose (FRC), galactose (GAL), fatty acids (FAs) and glutamine (GLN) can enter the ATP producing system (see text for details). 6PG, 6-phosphogluconate; 6PGL, 6-phosphogluconolactone; DHAP, dihydroxyacetone phosphate; FRU6P, fructose 6-phosphate; FRUBP, fructose 1,6-bisphosphate; GA3P, glyceraldehyde 3-phosphate; GLU, glutamate; G6PDH, glucose 6-phosphate dehydrogenase; G6PGDH, 6-phosphogluconate dehydrogenase; GT, glutamine transporter; GPI, phosphoglycose isomerase; HK, hexokinase; LDH, lactate dehydrogenase; MCT, monocarboxylate transporter; PDH, pyruvate dehydrogenase; PFK, phospofructokinase; RL5P, ribulose 5-phosphate; R5P, ribose 5-phosphate; TA, transaldolase; TK, transketolase; TPI, triosephosphate isomerase.

Figure 2

Genetic origin and functional interaction of the mitochondrial oxidative phosphorylation (OXPHOS) complexes. The mitochondrial OXPHOS system consists of five multisubunit complexes (CI–CV) that reside in the mitochondrial inner membrane (MIM). The MIM encloses the mitochondrial matrix and is surrounded by the mitochondrial outer membrane (MOM). An inter-membrane space (IMS) is located between the MIM and MOM. The subunits of CI, CIII, CIV and CV are encoded by the mitochondrial (mtDNA; red) and nuclear DNA (nDNA; blue), whereas CII exclusively consists of nDNA-encoded subunits (table at the top). OXPHOS biogenesis is mediated by nDNA-encoded assembly factors (green). The nDNA-encoded proteins are imported into the mitochondrial matrix via the TOM (translocator of the inner membrane) and TIM (translocator of the inner membrane) systems. At CI and CII, NADH and FADH₂ are oxidized, respectively, and the released electrons are transported to CIII via Coenzyme Q₁₀ (CoQ₁₀; ‘Q’). From thereon, electrons are transported to CIV via cytochrome-c (cyt-c; ‘c’) and donated to oxygen (O₂). Together, CI–CIV constitute the electron transport chain (ETC). The energy derived from the electron transport is used to expel protons (H⁺) from the mitochondrial matrix across the MIM. This establishes an electrochemical proton-motive force, associated with an inside-negative mitochondrial membrane potential (Δψ) and increased matrix pH. The controlled backflow of H⁺ is used by CV to drive the production of ATP (see text for details).

Figure 3

Integration of the OXPHOS system and mitochondrial metabolism. The five OXPHOS complexes, depicted on the lower left of the figure (see also Figure 2), maintain the inside-negative mitochondrial membrane potential (Δψ) and generate reactive oxygen species (ROS; red) in the form of superoxide (O₂^·−) and hydrogen peroxide (H₂O₂). ROS can also be generated by the TCA cycle enzyme α-ketoglutarate dehydrogenase (αKGDH), under conditions of elevated NADH/NAD⁺ ratio. ROS are removed by several antioxidant systems (green). In addition to fuelling ATP generation by CV, a sufficiently negative Δψ is also crucial for import of nDNA-encoded mitochondrial preproteins (PreP) via the TIM system. Moreover, metabolite and ion exchange across the mitochondrial inner membrane (MIM; right part of the figure) is driven by Δψ (orange) or its associated pH gradient (ΔpH; blue) (see text for details). ANT, adenine nucleotide translocase; GR, glutathione reductase; GPX, glutathione peroxidase; GSH, glutathione; HCa, proton/calcium transporter; I_F, flavin site in CI; I_Q, CoQ₁₀-binding site in CI; KH, potassium/proton transporter; NaCa, sodium/calcium transporter; NaH, sodium/proton transporter; Pi, inorganic phosphate/proton transporter; PYR, pyruvate/proton transporter; Q_o, CoQ₁₀-binding site in CIII; SOD2, superoxide dismutase 2; TRXR, thioredoxin reductase; TIM, translocator of the inner membrane; UCP, uncoupling protein; UNI, mitochondrial calcium uniporter.

Figure 4

Biogenesis and neurodegeneration-associated mutations of the OXPHOS system. The mitochondrial DNA (mtDNA; red) encodes ribosomal RNAs (rRNAs), transfer RNAs (tRNAs) and OXPHOS subunits. Mitochondrial ribosomal proteins, tRNA synthetases, mtDNA repair proteins, dNTP (deoxynucleoside triphosphate) pool-maintaining proteins and proteins mediating mtDNA replication, transcription and translation are all encoded by the nuclear DNA (nDNA; purple). Also, OXPHOS assembly factors (green) and the remainder of the OXPHOS subunits (blue) are nDNA encoded. Mutated genes associated with neurodegeneration are indicated (italic). Gene names are given according to the HGNC (HUGO Gene Nomenclature Committee) standard (see text for details). cyt-c, cytochrome-c biogenesis; Fe-S, iron-sulphur cluster biogenesis; CoQ₁₀, CoQ₁₀ biogenesis.

Figure 5

Quantitative analysis of mitochondrial (dys)function at the live-cell level. (A) Flow scheme illustrating how live-cell microscopy techniques can be applied to study OXPHOS dysfunction. Topics/decisions associated with the corresponding box are indicated at the right. (B) Image processing strategy allowing quantification of mitochondrial structure and function in a primary human skin fibroblast (#5120) from a healthy individual. Living cells were stained with the Δψ-sensitive fluorescent cation tetramethylrhodamine (TMRM) and visualized using epifluorescence microscopy. The obtained image (RAW) was corrected for background fluorescence (COR) and binarized to highlight mitochondrial structures (BIN; white objects). By masking the COR image with the BIN image information about mitochondrial structure, number and position (BIN image) were combined with TMRM intensity information from the COR image. This allows simultaneous quantification of these parameters from the MSK image. In this example, the number of mitochondrial objects equals 341, the average size of a mitochondrion equals 69±7 (s.e.m.) pixels, the average formfactor F (a combined measure of mitochondrial length and degree of filamentation) equals 2.7±0.2 (s.e.m.) arbitrary units, and the average mitochondrial TMRM fluorescence intensity equals 100±0.2 (s.e.m.) grey values. (C) Mitochondrial objects sorted (column-wise from top to bottom and from top left to lower right) based upon their size. The colour coding indicates the TMRM intensity, suggesting that Δψ is heterogeneous between individual mitochondrial objects. (D) Relationship between mitochondrial size (x axis), TMRM intensity (y axis) and formfactor (z axis) allowing multivariate analysis and multiparameter classification. Dark-grey spheres represent the original data points (each representing a mitochondrial object in the MSK figure), blue dots represent a projection of the data on the yz plane, red dots represent an projection of the data on the xy plane and light-grey dots represent a projection of the data on the xz plane). The latter reveals a linear correlation (R=0.97, P<0.001) between mitochondrial size and mitochondrial form factor F.