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, 36 (9), 2617-27

Genetic Reduction of Mitochondrial Complex I Function Does Not Lead to Loss of Dopamine Neurons in Vivo

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Genetic Reduction of Mitochondrial Complex I Function Does Not Lead to Loss of Dopamine Neurons in Vivo

Hyung-Wook Kim et al. Neurobiol Aging.

Abstract

Inhibition of mitochondrial complex I activity is hypothesized to be one of the major mechanisms responsible for dopaminergic neuron death in Parkinson's disease. However, loss of complex I activity by systemic deletion of the Ndufs4 gene, one of the subunits comprising complex I, does not cause dopaminergic neuron death in culture. Here, we generated mice with conditional Ndufs4 knockout in dopaminergic neurons (Ndufs4 conditional knockout mice [cKO]) to examine the effect of complex I inhibition on dopaminergic neuron function and survival during aging and on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in vivo. Ndufs4 cKO mice did not show enhanced dopaminergic neuron loss in the substantia nigra pars compacta or dopamine-dependent motor deficits over the 24-month life span. These mice were just as susceptible to MPTP as control mice. However, compared with control mice, Ndufs4 cKO mice exhibited an age-dependent reduction of dopamine in the striatum and increased α-synuclein phosphorylation in dopaminergic neurons of the substantia nigra pars compacta. We also used an inducible Ndufs4 knockout mouse strain (Ndufs4 inducible knockout) in which Ndufs4 is conditionally deleted in all cells in adult to examine the effect of adult onset, complex I inhibition on MPTP sensitivity of dopaminergic neurons. The Ndufs4 inducible knockout mice exhibited similar sensitivity to MPTP as control littermates. These data suggest that mitochondrial complex I inhibition in dopaminergic neurons does contribute to dopamine loss and the development of α-synuclein pathology. However, it is not sufficient to cause cell-autonomous dopaminergic neuron death during the normal life span of mice. Furthermore, mitochondrial complex I inhibition does not underlie MPTP toxicity in vivo in either cell autonomous or nonautonomous manner. These results provide strong evidence that inhibition of mitochondrial complex I activity is not sufficient to cause dopaminergic neuron death during aging nor does it contribute to dopamine neuron toxicity in the MPTP model of Parkinson's disease. These findings suggest the existence of alternative mechanisms of dopaminergic neuron death independent of mitochondrial complex I inhibition.

Keywords: Dopamine neuron; Mitochondrial complex I; Parkinson's disease.

Conflict of interest statement

The authors claim no conflict of interest.

Figures

Figure 1
Figure 1. Generation of transgenic mice with targeted ndufs4 gene deletion in dopaminergic neurons
(A) Breeding scheme for generating Ndufs4 cKO mice in which the Ndufs4 gene is conditionally deleted in dopaminergic neurons. (B) Representative fluorescent immunostaining images of the SNpc regions from Ndufs4 cKO mouse brain. Bottom panels are higher magnification images of the corresponding boxed areas. TH: tyrosine hydroxylase (red color); β-gal: β galactosidase (green color). (C) Western blotting analysis demonstrating that Ndufs4 protein is expressed in dopaminergic synaptosomes (DAT+) prepared from control but not Ndufs4 cKO mice. Non-dopaminergic synaptosomes (DAT) were used as controls. β-actin was used as a protein loading control. Similar results were obtained from three independent experiments. (D) Complex I activity in purified DAT+ synaptosomes from the striatums of from control or Ndufs4 cKO mouse brains was measured by the polarography method.
Figure 2
Figure 2. Effect of Ndufs4 deletion on the number of dopaminergic neurons in the SNpc and dopamine content in the striatum during aging
(A) Number of TH+ cells in the SNpc of 3-, 12-, and 24-month-old control or cKO mice. n = 6 mice/genotype. (B) Aging-related decrease of dopamine content in the striatum of cKO mice. n = 3 mice/genotype. *, p < 0.05; n.s. not statistically significant.
Figure 3
Figure 3. Age-related changes in motor activity in control and Ndufs4 cKO mice
(A) Latency to fall in accelerated rotarod test during a 2-year lifespan. The same cohort of mice was tested at different ages as indicated. (B) Latency to fall in 2-year-old control and cKO mice treated with or without L-Dopa. (C) Locomotor activity in open field test, quantified as total distance traveled. (D) Total number of rearing in the open field test with 2-year-old mice. n = 6 mice/genotype; *, p < 0.05, **, p <0.01; n.s. not statistically significant.
Figure 4
Figure 4. Catwalk assay to evaluate locomotion of 24-month-old control and Ndufs4 cKO mice
(A) Maximum contact area; (B) maximum contact intensity; (C) minimum contact intensity; (D) mean contact intensity; (E) print length; (F) print width; (G) print area; (H) stride length; (I) swing speed. RF, right forepaw; RH, right hind paw; LF, left forepaw; LH, left hind paw. n = 6 mice/genotype; *, p < 0.05; n.s. not statistically significant.
Figure 5
Figure 5. Age-related changes in levels of phospho-α-synuclein in control and Ndufs4 cKO mice
(A) Representative TH (green) and phospho (p) -α-synuclein (red) immunostaining in the SNpc of 24-month-old mice. Scale bar = 20 µm. (B) Quantification of the number of phospho-α-synuclein+ cells among TH+ cell population. (C) Representative western blot analysis for phospho-α-synuclein in the SNpc of 2-year-old mice, n=3 for each genotype. (D) Quantification of phospho-α-synuclein protein, normalized to β-actin. *, p < 0.05; **, p <0.01.
Figure 6
Figure 6. Conditional Ndufs4 deletion in dopaminergic neurons does not affect their susceptibility to MPTP
(A–D) Representative photomicrographs of TH immunostaining of SNpc from control mice treated with vehicle (A), MPTP (B), or from Ndufs4 cKO mice treated with vehicle (C) or MPTP (D). Scale bar = 200 µm. (E) Quantification of the total number of TH+ neurons in the SNpc of mice treated with vehicle or MPTP. n = 5–6 mice/genotype/treatment; *, p < 0.05; **, p < 0.01; n.s. not statistically significant.
Figure 7
Figure 7. Inducible Ndufs4 deletion in all cells of adult mice does not affect their susceptibility to MPTP
(A) Scheme for generating control and Ndufs4 iKO mice in which the Ndufs4 gene is deleted in the whole body upon tamoxifen treatment. (B) Representative Western blots of Ndufs4 proteins from the brains of two control and two Ndufs4 iKO mice. Beta-actin was used as a loading control. (C) Quantification of Ndufs4 protein, normalized to β-actin. (D) Complex I activity in mitochondria purified from control or Ndufs4 iKO mouse brains was measured by the polarography method. (E) State 2 and state 3 oxygen consumption rates in purified mitochondria. (F) Quantification of the total number of TH+ neurons in the SNpc of mice treated with vehicle or MPTP. n = 6 mice/genotype/treatment. *, p < 0.05; **, p <0.01.

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