Mechanism of neurodegeneration of neurons with mitochondrial DNA mutations

Abstract

Mutations of mitochondrial DNA are associated with a wide spectrum of disorders, primarily affecting the central nervous system and muscle function. The specific consequences of mitochondrial DNA mutations for neuronal pathophysiology are not understood. In order to explore the impact of mitochondrial mutations on neuronal biochemistry and physiology, we have used fluorescence imaging techniques to examine changes in mitochondrial function in neurons differentiated from mouse embryonic stem-cell cybrids containing mitochondrial DNA polymorphic variants or mutations. Surprisingly, in neurons carrying a severe mutation in respiratory complex I (<10% residual complex I activity) the mitochondrial membrane potential was significantly increased, but collapsed in response to oligomycin, suggesting that the mitochondrial membrane potential was maintained by the F(1)F(o) ATPase operating in 'reverse' mode. In cells with a mutation in complex IV causing approximately 40% residual complex IV activity, the mitochondrial membrane potential was not significantly different from controls. The rate of generation of mitochondrial reactive oxygen species, measured using hydroethidium and signals from the mitochondrially targeted hydroethidine, was increased in neurons with both the complex I and complex IV mutations. Glutathione was depleted, suggesting significant oxidative stress in neurons with a complex I deficiency, but not in those with a complex IV defect. In the neurons with complex I deficiency but not the complex IV defect, neuronal death was increased and was attenuated by reactive oxygen species scavengers. Thus, in neurons with a severe mutation of complex I, the maintenance of a high potential by F(1)F(o) ATPase activity combined with an impaired respiratory chain causes oxidative stress which promotes cell death.

Figure 1

Characteristics of mitochondrial membrane potential (Δψ_m) in cells with mitochondrial mutations. (A–B) Neurons and astrocytes with severe mutation in complex I (CY3-I) showed a significant increase (P < 0.001) in Δψ_m compared with control cells. The mitochondrial potential in cells with a mutation in complex IV (CY2-I) showed no significant difference from control. Non-differentiated CY3-I cells also exhibited a 26% increase (P < 0.05) in tetramethylrhodamine methylester (TMRM) fluorescence (i.e. an increased Δψ_m) compared with controls. In control and CY2-I neurons (C–D), oligomycin did not affect Δψ_m; rotenone induced a partial depolarization; FCCP induced complete depolarization. In CY3-I neurons (E), oligomycin caused a mitochondrial depolarization. In the CY3-I stem cells (F) the Δψ_m was not maintained by reverse electron flow since oligomycin did not cause mitochondrial depolarization. *P < 0.05; **P < 0.001.

Figure 2

Effect of mitochondrial substrates on mechanism of maintenance of Δψ_m in cells with mitochondrial mutations. Application of pyruvate (5 mM) or methyl succinate (5 mM) to neurons increased Δψ_m, but increased substrate provision did not prevent the oligomycin induced mitochondrial depolarization in CY3-I neurons (C). In the presence of pyruvate, methyl-succinate induced further hyperpolarization of mitochondria in ES-I and CY1-I neurons (A, B, D), but a small mitochondrial depolarisation in CY3-I cells. TMRM = tetramethylrhodamine methylester.

Figure 3

Calcium homeostasis. Simultaneous measurements of [Ca²⁺]_c and ΔΨ_m were made from neurons (A–B) and astrocytes (C–D) in mixed culture co-loaded with fura-2 and rhodamine 123. Traces are shown from single cells in each case. Mitochondrial mutations did not induce pathological changes in their mitochondrial responses to [Ca²⁺]_c signals under these conditions.

Figure 4

Mitochondrial and cytosolic reactive oxygen species production in cells with mitochondrial mutations. CY3-I neurons displayed a significantly higher basal rate of increase in mitochondrially targeted hydroethidine (Mitosox) and hydroethidine ratio, demonstrating a higher basal production of intra-mitochondrial and extra-mitochondrial reactive oxygen species compared with control (A). Histogram demonstrating percentage values of the rate of mitochondrially targeted hydroethidine or hydroethidine ratio compared with 100% for control neurons. (B–E) show increase of ΔΨ_m by mitochondrial substrates (pyruvate and TMPD/ascorbate) or inhibition of complex 1 with rotenone demonstrated the dependence of reactive oxygen species production on ΔΨ_m. *P < 0.05; **P < 0.001.

Figure 5

Glutathione in both neurons and astrocytes. MCB was used to assess astrocyte and neuronal glutathione concentration. Mean intensities of MCB-glutathione adduct fluorescence (arb.U) at a steady state are presented. In CY3-I glutathione concentration was decreased compared with other cell lines in both astrocytes and neurons.

Figure 6

Cell death in neurons and astrocytes. The number of functional neurons and astrocytes in a co-culture was estimated by counting cells that show [Ca²⁺]_c signals in response to physiological stimuli, which give characteristic and distinct responses in either neurons (glutamate, 50 μM) (A) or astrocytes (ATP, 100 μM) (B). With increasing time in culture, there was a progressive increase in cell death, seen predominantly in neurons, resulting in a change in the proportion between cell types (C–E). The antioxidant MnTBAP significantly protected CY3-I cells against the progressive increase in cell death; the number of dead neurons in this experiment was estimated as percentage of propidium iodide-positive neurons in the culture (F). *P < 0.05.