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Disruption of mitochondrial complex I induces progressive parkinsonism

Abstract

Loss of functional mitochondrial complex I (MCI) in the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson’s disease1. Yet, whether this change contributes to Parkinson’s disease pathogenesis is unclear2. Here we used intersectional genetics to disrupt the function of MCI in mouse dopaminergic neurons. Disruption of MCI induced a Warburg-like shift in metabolism that enabled neuronal survival, but triggered a progressive loss of the dopaminergic phenotype that was first evident in nigrostriatal axons. This axonal deficit was accompanied by motor learning and fine motor deficits, but not by clear levodopa-responsive parkinsonism—which emerged only after the later loss of dopamine release in the substantia nigra. Thus, MCI dysfunction alone is sufficient to cause progressive, human-like parkinsonism in which the loss of nigral dopamine release makes a critical contribution to motor dysfunction, contrary to the current Parkinson’s disease paradigm3,4.

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Fig. 1: Loss of NDUFS2 function triggers metabolic adaptations in SN DA neurons.
Fig. 2: Loss of Ndufs2 induces early axonal dysfunction.
Fig. 3: Progressive loss of the somatodendritic phenotype, but not neuronal death.
Fig. 4: cNdufs2−/− mice present progressive, levodopa-responsive parkinsonism.
Fig. 5: Boosting mesencephalic dopamine levels reverses motor deficits in cNdufs2−/− mice.

Data availability

RNA-seq reads have been deposited at the NCBI Sequence Read Archive under BioProject accession number PRJNA752682. DA release data measured using fast-scan cyclic voltammetry were analysed using custom software that is available at GitHub (https://github.com/surmeierlab/imagej_macros)42. The mouse model will be made available on request. All data generated or analysed in this study are included in this published Article and the Supplementary Information). Source data are provided with this paper.

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Acknowledgements

We thank the staff at the Metabolomic Core Facility at Robert H. Lurie Comprehensive Cancer Center of Northwestern University for HPLC–MS experiments and staff at the NUSeq Core Facility for RNA-seq analysis; D.G. Galtieri for the original scripts in the Surmeier lab GitHub; and G. Yellen for insights on PercevalHR experiments. Electron microscopy tissue processing and imaging was performed at the Northwestern University Center for Advanced Microscopy, supported by NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center. This study was supported by grants from the Michael J. Fox Foundation (to D.J.S.), the JPB Foundation (to D.J.S.), the IDP Foundation (to D.J.S.), the Flanagan Fellowship (to P.G.-R.) and the European Research Council ERC Advanced Grant PRJ201502629 (to J.L.-B.).

Author information

Affiliations

Authors

Contributions

Conceptualization: P.G.-R., M.G.K., P.T.S. and D.J.S. Methodology: P.G.-R., E.Z., K.A.S., E.I., J.N.G., B.Y., T.T., L.G., M.A.S., D.L.W., J.L.-B., M.G.K., P.T.S. and D.J.S. Validation: P.G.-R., E.Z., K.A.S., J.N.G., E.I., T.T. and B.Y. Formal analysis: P.G.-R., E.Z., K.A.S., J.N.G., E.I., T.T. and B.Y. Investigation: P.G.-R., E.Z., K.A.S., J.N.G., E.I., T.T., B.Y., L.G., M.A.S., M.G.K., J.L.-B., P.T.S. and D.J.S. Resources: J.L.-B., M.G.K. and D.J.S. Writing: P.G.-R. and D.J.S. Visualization: P.G.-R., P.T.S. and D.J.S. Supervision: D.J.S. Funding acquisition: P.G.-R., J.L.-B. and D.J.S.

Corresponding author

Correspondence to D. James Surmeier.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature thanks Ken Nakamura and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Metabolic remodeling in cNdufs2−/− mice.

a, b, Ndufs2 was ablated specifically in dopaminergic neurons by selective breeding of mice expressing Cre under the control of the dopamine transporter (DAT) promoter with mice containing a floxed allele of the Ndufs2 gene. c, Cartoon representing the activity of the ETC (electron transport chain) and ANT following Ndufs2 deletion. d, Electron micrographs of SN DA neurons. The nucleus and the mitochondria are highlighted in green and red, respectively. Scale bar: 1μm. e, Box plots showing no differences in mitochondrial density (wildtype, n=21; cNdufs2−/−, n=21) where n is the number of cells. f, Box plots showing mitochondrial morphology in wildtype and MCI-Park SN neurons (wildtype, n=21; cNdufs2−/−, n=21). Insets: representative electron micrographs of mitochondria showing intact (top) or abnormal (bottom) morphology (wildtype, n=21; cNdufs2−/−, n=21). The percentage of abnormal mitochondria was calculated as the ratio between the area occupied by abnormal mitochondria over the total area occupied by mitochondria for each cell. Scale bar: 0.2μm. g, The schematic on top is a sagittal view of the brain, and the red line indicates the position at 3.52mm from bregma, which is shown as a coronal section in the bottom panel (modified from Allen Mouse Brain Atlas, online version 1, 2008). h, Box plots indicate that OXPHOS index (OXPHOS/ (OXPHOS + glycolysis)) is lower in Ndufs2 deficient neurons (wildtype, n=5; cNdufs2−/−, n=7). i, Confocal image of dopaminergic terminals in wildtype mice expressing PercevalHR in ex vivo brain slice at P40. Scale bar: 20μm. j, Box plots show decrease in the OXPHOS index in dopaminergic terminals of cNdufs2−/− mice (wildtype, n=4; cNdufs2−/−, n=5). Wildtype (grey); cNdufs2−/− (black). Two-tailed Mann-Whitney test; (e), (f), (h), (j). For the boxplots, the centre line indicates the median, the box limits indicate the first and third quartiles, and the whiskers indicate the data range. *P < 0.05; ****P < 0.0001.

Source data

Extended Data Fig. 2 Bar graph of enrichment analysis in MCI-Park mice.

Bar graph for viewing top 20 enrichment clusters, one per cluster, using a discrete color scale to represent statistical significance (Metascape). Wildtype, N=5; cNdufs2−/−, N=6 where N is the number of mice. a, Enriched Ontology cluster analysis of down-regulated genes. b, Gene Ontology biological processes analysis of down-regulated genes. c, Enriched Ontology cluster analysis of up-regulated genes. d, Gene Ontology biological processes analysis of up-regulated genes.

Extended Data Fig. 3 Enrichment network visualization in MCI-Park mice.

Network of enriched terms colored by cluster identity, where nodes that share the same cluster identity are typically close to each other (Metascape). Wildtype, N=5; cNdufs2−/−, N=6 where N is the number of mice. a, Enrichment network visualization in down-regulated genes in MCI-Park mice. b, Enrichment network visualization in up-regulated genes in MCI-Park mice.

Extended Data Fig. 4 TH expression in SNc and VTA dopaminergic neurons in wildtype and cNdufs2−/− mice.

a, Quantification of TH expression in SNc dopaminergic neurons at P30 and P60 (wildtype, N=5; cNdufs2−/−, N=5) where N is the number of mice. b, Quantification of TH expression in VTA dopaminergic neurons at P30 and P60 (wildtype, N =5; cNdufs2−/−, N =4). c, Representative images showing TH-IR in VTA and SN dopaminergic neurons in wildtype mouse at P60. Scale bar: 200μm. d, Magnified VTA region showing dopaminergic neurons in wildtype at P60. Scale bar: 15μm. e, Representative images showing TH-IR in VTA and SN dopaminergic neurons in cNdufs2−/−mouse at P60. Scale bar: 200μm. f, Magnified VTA region showing dopaminergic neurons in cNdufs2−/−mouse at P60. Scale bar: 15μm. Wildtype (grey); cNdufs2−/− (black). Two-tailed Mann-Whitney test (a) and (b). For the boxplots, the centre line indicates the median, the box limits indicate the first and third quartiles, and the whiskers indicate the data range. *P < 0.05; **P < 0.01.

Source data

Extended Data Fig. 5 Dopamine release is reduced in cNdufs2−/− mice.

Representative images from SN in P30 wildtype (a) and cNdufs2−/− (b) mice are shown. Scale =5µm. Representative images from dorsal striatum in wildtype (c) and cNdufs2−/− (d) mice are shown. Scale= 5µm (wildtype, N=5; cNdufs2−/−, N=5) where N is number of mice. Note that at P30, the TH promoter (not TH expression) was effectively driving the expression of a fluorescent reporter (ERtdTomato) in dopaminergic neurons, despite the down-regulation in TH expression in the dorsolateral striatum (shown in Fig. 2c). eg, Dopamine release was measured by fast-scan cyclic voltammetry in wildtype and cNdufs2−/− mice at P20. Representative colorplots (e) and traces (f) show dramatic reduction in evoked (1p, 350 nA, 2 ms) release in dorsal striatum of cNdufs2−/− mice. Scale: vertical =0.5 µM dopamine, horizontal = 1s. g, Summary data demonstrate dopamine release is significantly decreased by P20 (wildtype, N= 12, cNdufs2−/−, N= 4). Striatal dopamine release measured with dLight1.3b at P30 (h, j, l) and P60 (i, k, m). Traces are ΔF/F0 over time. Solid lines represent median trace, shaded area is 95% CI. Scale bars: (hi), vertical =200 % ΔF/F0, horizontal= 500ms. Quantification of dopamine release at P30 (j, l) and P60 (k, m) in dorsal striatum; striatal dLight1.3b responses were analyzed either by defining 16-pixel-wide line profiles, which provided high temporal resolution measurements of selected regions (j,k), or by averaging the entire field of view, with lower temporal resolution but broader sampling area (l, m). (j, wildtype N=5; cNdufs2−/− N=5; k, wildtype N=6; cNdufs2−/− N=6; l, wildtype N=4; cNdufs2−/− N=5; m, wildtype N=9; cNdufs2−/− N= 4). Wildtype (grey); cNdufs2−/− (black). Two-tailed Mann-Whitney test: (g), (j), (k), (l) and (m). For the boxplots, the centre line indicates the median, the box limits indicate the first and third quartiles, and the whiskers indicate the data range. *P < 0.05, ****P ≤ 0.0001.

Source data

Extended Data Fig. 6 Physiology remodeling in cNdufs2−/− mice.

Dopamine (a), DOPAC (b), serotonin (c), and acetylcholine (d) separated from wildtype and cNdufs2−/− striatum tissue lysate (P30 and P60, wildtype, N=4, cNdufs2−/−, N=4) where N is the number of mice. Note that elevation of striatal serotonin was detected at P120. This is a common feature of rodent PD models55. e, Heat maps illustrating the remodeling of ion channels in cNdufs2−/− mice; repeated samples are grouped horizontally (wildtype, N=5; cNdufs2−/−, N=6). f, qPCR analysis of RiboTag harvested mRNA showing a drop in hcn2 mRNA in cNdufs2−/− neurons (wildtype, N=4; cNdufs2−/−, N=4). g, Whole-cell somatic recording showing hyperpolarization-activated, cyclic nucleotide-gated currents from a wildtype and cNdufs2−/− neuron at P30. Scale bars: 100pA, 200ms. h, Cumulative probability plot of peak current from wildtype and cNdufs2−/− SN dopaminergic neurons (wildtype, n=12; cNdufs2−/−, n=10) where n is the number of cells. i, qPCR analysis of RiboTag harvested mRNA showing a drop in Cav1.3 mRNA in cNdufs2−/− SN dopaminergic neurons (wildtype, N=4; cNdufs2−/−, N=4). j, Cumulative probability plot of peak [Ca2+] at proximal dendrite (wildtype, n=8; cNdufs2−/−, n=6). k, Whole-cell somatic recordings showing the response to glutamate uncaging in wildtype (left) and cNdufs2−/− (right) mice. Scale bars: 20mV, 1s. Representative SN DA neuron filled with Alexa Flour 594 showing the location for uncaging in blue. Scale bar: 20μm. l, Spikes/burst - peak spiking rate plot showing the difference in response to uncaged glutamate between wildtype (n=5) and cNdufs2−/− (n=5). m, Representative traces showing spike width in SN neurons from wildtype and cNdufs2−/− at P30. n, Box plots indicate AP half width in wildtype and cNduf2−/− at P30 (wildtype n=6; cNdufs2−/−n=7). Wildtype (grey); cNdufs2−/− (black). Two-tailed Mann-Whitney test: (a-d, f and i). One tailed Mann-Whitney test: (h), (j), (l) and (n). For the boxplots, the centre line indicates the median, the box limits indicate the first and third quartiles, and the whiskers indicate the data range. *P < 0.05; **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 7 Physiological characterization of SN dopaminergic neurons in cNdufs2−/− mice at P60.

a, b, Electron micrographs of SN dopaminergic neurons at P60. The nucleus and the mitochondria are highlighted in green and red, respectively. Scale bar: 2µm. c, Box plots showing no differences in mitochondrial density (wildtype, n=14; cNdufs2−/−, n=23) where n is the number of cells. d, Box plots showing abnormal morphology in MCI-Park mitochondria (wildtype, n=14; cNdufs2−/−, n=23). e, Representative images showing normal (top) and abnormal (bottom) mitochondria. The percentage of abnormal mitochondria was calculated as the ratio between the area occupied by abnormal mitochondria over the total area occupied by mitochondria for each cell. Scale bar: 500nm. f, Schematic diagram of injection site (modified from Allen Mouse Brain Atlas, online version 1, 2008). TH-Fusion Red reporter was bilaterally injected into the SN of cNdufs2−/−mouse at P50. Experiments were done at P60 (+ 4 days). Representative image showing TH-Fusion Red expression in wildtype (g) and cNdufs2−/− (h) mice at P60. Scale bar: 20μm (wildtype, N=5; cNdufs2−/−, N=5) where N is the number of mice. i, Cell attached recordings from identified wildtype and cNdufs2−/− SN DA neurons at P60. Scale bars: 10pA, 1s. j, Cumulative probability plot of autonomous discharge rates (wildtype n=20; cNdufs2−/− n=25 cells). k, Whole-cell somatic recordings from a cNdufs2−/− SN DA neurons at P60 showing the response to glutamate uncaging. Representative SN DA neuron filled with Alexa Flour 594 is showing the location for uncaging in blue (n=4). Scale bars: 20mV, 2s, 20μm. l, Spikes/burst - peak spiking rate plot showing the difference in response to uncaged glutamate between wildtype (n=4) and cNdufs2−/− (n=5). Wildtype (grey); cNdufs2−/− (black). Two-tailed Mann-Whitney test (c, d). One-tailed Mann-Whitney test (j) and (l). For the boxplots, the centre line indicates the median, the box limits indicate the first and third quartiles, and the whiskers indicate the data range. *P < 0.05.

Source data

Extended Data Fig. 8 Behavioural phenotypes in cNdufs2−/− mice.

a, Schematic diagram of the experimental protocol for Y maze test. The scoring of 2 and 3 was counted as incorrect. b, Open-field traces in wildtype and cNdufs2−/− mice at P60 and P120 with and without levodopa (3mg/Kg) treatment. c, Representatives traces showing the effect of levodopa treatment on the speed in wildtype (N=5) and cNdufs2−/− (N=5) mice at P120 where N is the number of mice. d, Rearing test performance. e, Number of rearings in a 3-minute period in wildtype, cNdufs2−/− and cNdufs2−/− + 3mg/kg levodopa mice at different ages (P20-P120). Number of rearings begin to be impaired at P40. Levodopa did not rescue this deficit (N=11 per group) f, Rearing time in wildtype, cNdufs2−/− and cNdufs2−/− + 3mg/kg levodopa mice at different ages (P20-P120). At P60, cNdufs2−/− mice show difficulty transitioning between rearing and landing, spending much more time ‘stuck’ in an elevated posture. Levodopa did not rescue this deficit (N=11 per group). (g, h) Body weight was analyzed from P20 to P120. g, Body weight development of wildtype (N=10) and cNdufs2−/− (N=11) mice in males. h, Body weight development of wildtype (N=10) and cNdufs2−/− (N=11) mice in females. Wildtype (gray), cNdufs2−/− (black), cNdufs2−/− plus levodopa (red). One tailed Kruskal-Wallis with Dunn’s correction for multiple comparisons: (e, f). One-way ANOVA followed by Tukey’s post hoc test: (g, h). Data are presented as median and range (shaded area). *P < 0.05; **P < 0.01.

Source data

Extended Data Fig. 9 Gait analysis in cNdufs2−/− mice.

a, Pictures of wildtype and cNdufs2−/− mice footprints: RF (Right Fore), RH (Right Hind), LF (Left Fore) and LH (Left Hind) in yellow. Body length measurements (b) and hind limb stance width (c) for wildtype (N=7), cNdufs2−/− (N=7) and cNdufs2−/−+levodopa (N=7) where N is the number of mice. d, Representative graph of right hind paw area. Stride length (e) and swing duration (f) for wildtype N=7, cNdufs2−/− N=7 and cNdufs2−/−+levodopa N=7. g, Step sequence for wildtype (N=7), cNdufs2−/− (N=7) and cNdufs2−/−+levodopa (N=7). h, Stance duration for wildtype (N=7), cNdufs2−/− (N=7) and cNdufs2−/−+levodopa (N=7). i, Brake duration for wildtype (N=7), cNdufs2−/− (N=7) and cNdufs2−/−+levodopa (N=7). j, Paw area for wildtype (N=7), cNdufs2−/− (N=7) and cNdufs2−/−+levodopa (N=7). k, Stride frequency for wildtype (N=7), cNdufs2−/− (N=7) and cNdufs2−/−+levodopa (N=8). l, Propulsion time for wildtype (N=7), cNdufs2−/− (N=8) and cNdufs2−/−+levodopa (N=7). m, Gait symmetry for wildtype (N=7) and cNdufs2−/− (N=8) mice. n, Fore limb stance width for wildtype (N=7) and cNdufs2−/− (N=7) mice. o, Box-plots summarizing stereological estimate of the numbers of NeuN-immunopositive neurons in the SNC (o) and VTA (p), wildtype (N=5) and cNdufs2−/− (N=5). q, Representative images showing NeuN immunostaining in the midbrain in wildtype (top panel) and cNdufs2−/− mouse (bottom panel) in P120-150 mice. Scale bar:100µm. Wildtype(grey); cNdufs2−/−(black); cNdufs2−/−+levodopa (red). One-way ANOVA followed by Tukey’s post hoc test: (b), (c), (en), two-tailed Mann–Whitney test (o, p). Data from right hind paw: (b), (c), (en). Levodopa dosage: 6mg kg−1. For the boxplots, (b), (c), (e), (f), (o) and (p), the centre line indicates the median, the box limits indicate the first and third quartiles, and the whiskers indicate the data range. (gn): Solid lines represent median trace shaded area is the range. *P < 0.05; **P < 0.01.

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Extended Data Fig. 10 SN expression of AADC did not induce DA release in P100 cNdufs2−/− mice even after perfusion with dopamine.

a, Schematic diagram of injection site. AAV2-GFP-AADC was bilaterally injected into the striatum of cNdufs2−/−mouse at P60. Confocal image of coronal slice containing striatum (b) or SN (c) from cNdufs2-/ mouse. Scale bar: 200μm. d, Magnified view of SN showing no expression of AADC-GFP. Scale bar: 20μm. e, Schematic diagram of injection site. AAV2-GFP-AADC was bilaterally injected into the SN of cNdufs2−/−mouse at P60. Confocal image of coronal slice containing striatum (f) or SN (g) from cNdufs2−/−mouse. Scale bar: 200μm. h, Magnified view of SN showing expression of AADC- GFP. Scale bar: 20μm. Wildtype (N=5), cNdufs2−/−(N=5) where N is the number of mice. i, Quantification of dopamine in striatal tissue from wildtype (N=4), cNdufs2−/−+low levodopa (1.5mg/kg) with (N=4) or without AADC (N=4) and cNdufs2−/−+high levodopa (12mg kg−1) (N=4). j, Schematic diagram of the AAV-AADC injection into the SN and AAV-dLight injection into the striatum (P60). k, dLight fluorescence (raw in thin lines and average in thick lines) in response to a single electrical stimulus (350μA, 2ms). l, Summary of dLight responses (wildtype n=16, cNdufs2−/−-AADC n=9, cNdufs2−/−+AADC n=12 where n is the number of slices). m, An example recording of dLight fluorescence response upon bath application of dopamine (100µM) followed by washout in a cNdufs2−/− mouse injected with AADC in the SN. n-o, Example recordings of dLight fluorescence responses (raw in thin lines and average in thick lines) upon single electrical stimulus after dopamine washout. cNdufs2−/− mouse injected with AADC (n, n=4) or GFP (o, n=4) into the SN. Wildtype (grey); cNdufs2−/− (black); cNdufs2−/−+AADC (red); cNdufs2−/− +high levodopa (blue). (i), two-way ANOVA followed by Tukey’s post hoc test, *P< 0.05; **P< 0.01; (l), one-tailed Kruskal-Wallis with Dunn’s correction for multiple comparisons, ****P<0.0001. For the boxplots, the centre line indicates the median, the box limits indicate the first and third quartiles, and the whiskers indicate the data range. p, Schematic showing the cascade of events following to Ndufs2 deletion in MCI-Park mice. DA (dopamine), DLS (dorsolateral striatum). The schematics in (a), (e) and (j) are modified from the Allen Mouse Brain Atlas, online version 1, 2008 (http://mouse.brain-map.org).

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González-Rodríguez, P., Zampese, E., Stout, K.A. et al. Disruption of mitochondrial complex I induces progressive parkinsonism. Nature 599, 650–656 (2021). https://doi.org/10.1038/s41586-021-04059-0

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