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 paradigm^3,4.

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.

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 Ontologycluster 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.

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). e–g, 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: (h, i), 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.

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 models. 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.

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.

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.

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), (e–n), two-tailed Mann–Whitney test (o, p). Data from right hind paw: (b), (c), (e–n). 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. (g–n): Solid lines represent median trace shaded area is the range. *P < 0.05; **P < 0.01.

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 srecording 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. Fossr 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).

Fig. 1 |. Loss of NDUFS2 function triggers metabolic adaptations in SN DA neurons.

a, Schematic of a coronal section of the midbrain highlighting the sampled region of the SN pars compacta (SNc; orange). Schematic modified from the Allen Mouse Brain Atlas, online version 1, 2008 (http://mouse.brain-map.org ). SNr, SN pars reticulata; ml, medial lemniscus; VTA, ventral tegmental area. b, Meta-analysis of RNA-sequencing (RNA-seq) data obtained by Cre-dependent RiboTag mRNA precipitation from P40 wild-type mice. The heat map shows enrichment of dopaminergic-specific markers (N = 5), where N indicates the number of mice. GLUT, glutamatergic; GABA, gabaergic. c, Effective Ndufs2 knockout was verified by qPCR analysis of RiboTag-collected mRNA. N = 5 (wild type (WT)) and N = 5 (cNdufs2^−/−). d, TMRM-labelled mitochondria of an SN DA neuron in an ex vivo brain slice. Scale bar, 10 μm. e, Representative TMRM fluorescence time-series analysis after the application of carboxyatractyloside, an ANT inhibitor (iANT). f, Box plots of TMRM after blocking ANT and depolarizing mitochondria with FCCP. Wild type: n = 8 (P40); cNdufs2^−/−: n = 4 (P20) and n = 5 (P40), where n indicates the number of cells. g, h, Heat maps of expression determined using RNA-seq show the downregulation of OXPHOS (g) and upregulation of glycolysis (h) in cNdufs2^−/− mice. N = 5 (wild type) and N = 6 (cNdufs2^−/−). i, Representative image of SN DA neurons expressing PercevalHR and stained for TH in an ex vivo brain slice. Scale bar, 15 μm. N = 4. ROI, region of interest. j, Representative time-lapse measurements of the PercevalHR fluorescence ratio. Oligomycin and 2-deoxyglucose (2-DG) were applied to determine OXPHOS and glycolytic contributions to the ATP/ADP ratio. Scale bar, 10 min. k, The OXPHOS index (OXPHOS/(OXPHOS + glycolysis)). n = 6 (wild type) and n = 10 (cNdufs2^−/−). Statistical analysis was performed using two-tailed Mann–Whitney U-tests (c and k) and one-tailed Kruskal–Wallis tests with Dunn’s correction for multiple comparisons (f); *P < 0.05, **P < 0.01, ***P < 0.001. For the boxplots in c, f and k, the centre line indicates the median, the box limits indicate the first and third quartiles, and the whiskers indicate the data range.

Fig. 2 |. Loss of Ndufs2 induces early axonal dysfunction.

a, Heat map of RNA-seq analysis shows the downregulation of dopamine release and synaptic function in cNdufs2^−/− mice. N = 5 (wild type) and N = 6 (cNdufs2^−/−), where N indicates the number of mice. b, c, Representative images showing a significant reduction in TH staining in the dorsal striatum but not in the SNc in P30 wild-type (b) and cNdufs2^−/− (c) mice. Scale bars, 1 mm (top) and 200 μm (bottom). TH-IR, immunoreactivity. d, Quantification of TH expression in the dorsal striatum, SNc and VTA. N = 5 (wild type) and N = 5 (cNdufs2^−/−). e, Striatal (top) and dendritic (bottom) dopamine release measured with fast-scan cyclic voltammetry and dLight1.3b measurements. The solid lines represent the median trace and the shaded area indicates the 95% confidence intervals. Scale bars, 1 μM dopamine (vertical), 1 s (horizontal) (top); and 5% Δf/f₀ (vertical) 10 s (horizontal) (bottom). f, g, Quantification of dopamine release at P30 in the dorsal striatum (f; N = 4 (wild type) and N = 5 (cNdufs2^−/−) and in the SN (g; N = 6 (wild type) and N = 9 (cNdufs2^−/−). AUC, area under the curve. h, Cell-attached recordings from identified wild-type and cNdufs2^−/− SN DA neurons. Scale bars, 10 pA (vertical) and 1 s (horizontal). i, Cumulative probability plot of autonomous discharge rates. n = 21 (wild type) and n = 21 (cNdufs2^−/−), where n indicates the number of cells. j, Representative reconstruction of a Fura-2-filled SN neuron (left). Scale bar, 20 μm. Right, whole-cell recording and Fura-2 Ca²⁺ imaging of SN DA neurons (n = 5) at P30. Scale bars, 20 mV (vertical) and 1 s (horizontal). Statistical analysis was performed using two-tailed Mann–Whitney U-tests (d, f and g) and a one-tailed Mann–Whitney U-test (i); **P < 0.01. For the box plots in d, f and g, the centre line indicates the median, the box limits indicate the first and third quartiles, and the whiskers indicate the data range.

Fig. 3 |. Progressive loss of the somatodendritic phenotype, but not neuronal death.

a, b, Representative images showing TH immunostaining in the striatum in wild-type (a) and cNdufs2^−/− (b) mice at P60. White dashed line encloses the dorsal striatum; yellow dotted lines indicate areas used for quantification of TH expression Scale bar, 1 mm. c, Quantification of TH expression in the dorsal striatum. N = 5 (wild type) and N = 5 (cNdufs2^−/−), where N indicates the number of mice. d, e, Representative images showing a significant reduction in TH staining in the SNc of cNdufs2^−/− mice (e) compared with wild-type mice (d) at P60. Scale bars, 200 μm (top) and 15 μm (higher-magnification images of the SNc showing DA neurons in wild-type and cNdufs2^−/− mice). f, Quantification of TH expression in the SNc. N = 5 (wild type) and N = 5 (cNdufs2^−/−). g, Striatal (top) and dendritic (bottom) dopamine release measured using fast-scan cyclic voltammetry and dLight1.3b measurements. The solid lines represent the median trace and the shaded area shows the 95% confidence intervals. Scale bars, 1 μM of dopamine (vertical), 1 s (horizontal) (top); and 5% Δf/f₀ (vertical), 10 s (horizontal) (bottom). h, i, Quantification of dopamine release at P60 in the dorsal striatum (h; n = 4 (wild type) and n = 5 (cNdufs2^−/−)) and in the SN (i; n = 5 (wild type) and n = 9 (cNdufs2^−/−)), where n indicates the number of cells. j, Diagram of Fluoro-Gold injection for the analysis of retrograde transport. Representative images showing phenotypic downregulation but not cell loss in brain sections obtained from wild-type (N = 4) and cNdufs2^−/− (N = 4) mice at P60. Scale bars, 20 μm. Statistical analysis was performed using two-tailed Mann–Whitney U-tests (c, f, h and i); *P < 0.05, **P < 0.01. For the box plots in c, f, h and i, the centre line indicates the median, the box limits indicate the first and third quartiles, and the whiskers indicate the data range.

Fig. 4 |. cNdufs2−/− mice present progressive, levodopa-responsive parkinsonism.

a, The percentage of correct choices (Y-maze test) for wild-type mice (N = 8 (P30) and N = 6 (P60)), cNdufs2^−/− mice (N = 8 (P30) and N = 6 (P60)) and cNdufs2^−/− mice treated with 6 mg kg⁻¹ levodopa (N = 8 (P30) and N = 6 (P60)), where N indicates the number of mice. b, Striatal dopamine release measured using fast-scan cyclic voltammetry at P30 and P60. P30: N = 5 (vehicle) and N = 9 (levodopa); P60: N = 4 (vehicle) and N = 6 (levodopa). c, Adhesive removal test from P15 to P120. N = 10 (wild type), N = 11 (cNdufs2^−/−) and N = 10 (cNdufs2^−/− + 6 mg kg^–1 levodopa). d, Open-field traces in wild-type and cNdufs2^−/− mice at P30 and P100. e, The number of pauses during the open-field test. N = 10 (wild type), N = 11 (cNdufs2^−/−) and N = 10 (cNdufs2^−/− + 6 mg kg⁻¹ levodopa). m/5 min, metres per 5 min. f, The total distance travelled in 5 min. N = 11 (wild type), N = 13 (cNdufs2^−/−) and N = 11 (cNdufs2^−/− + 6 mg kg⁻¹ levodopa). g, Schematic of the behavioural differences between young and older cNdufs2^−/− mice. For a, c, e and f, statistical analysis was performed using one-tailed Kruskal–Wallis tests with Dunn’s correction for multiple comparisons; *P < 0.05, **P < 0.01 (wild type versus cNdufs2^−/−); ^#P < 0.01 (cNdufs2^−/− versus cNdufs2^−/− + 6 mg kg⁻¹ levodopa). For b, statistical analysis was performed using two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test; ***P < 0.001. For the box plots in a and b, the centre line indicates the median, the box limits indicate the first and third quartiles, and the whiskers indicate the data range. For c, e and f, the solid lines represent median trace and the shaded area shows the range.

Fig. 5 |. Boosting mesencephalic dopamine levels reverses motor deficits in cNdufs2−/− mice.

a, Schematic of the conversion of levodopa to dopamine by AADC (top). Bottom, the experimental timeline for the AADC experiments. b, Schematic of the striatal injection strategy. c, Confocal image of a coronal slice showing GFP expression in the striatum of cNdufs2^−/−mice (N = 4) injected with AAV2-GFP-AADC, where N indicates the number of mice. Scale bar, 200 μm. d, Magnified view of the dorsal striatum showing the expression of AADC–GFP in medium spiny neurons in cNdufs2^−/− mice. Scale bar, 20 μm. e, Schematic of the SN injection strategy. AAV2-GFP-AADC or AAV2-GFP was bilaterally injected into the SN of cNdufs2^−/− mice at P60. f, Confocal image of a coronal slice showing the expression of AADC–GFP in the SN of cNdufs2^−/− mice (N = 4) at P100. Scale bar, 500 μm. g, h, Magnified views of the SN showing GFP⁺ neurons in cNdufs2^−/− mice. Scale bars, 100 μm (g) and 20 μm (h). i, j, Representative traces of locomotor activity in the open-field test for striatal AADC (i) and SN AADC (j) before and after levodopa treatment (1.5 mg kg⁻¹). k, The total distance travelled during 5 min for cNdufs2^−/− mice infected with AAV2-GFP (black) or AAV2-GFP-AADC (red). A single AAV injection was performed for the SN. For the striatum, two injection strategies were tested: a single AAV injection (striatal) or three AAV injections to cover the whole striatum (×3 striatal). −AADC: N = 5 (striatal), N = 6 (SN) and N = 6 (×3 striatal); +AADC: N = 6 (striatal), N = 6 (SN) and N = 5 (×3 striatal). l, Schematic of our hypothesis of the cascade of events in the prediagnostic and clinical phase of PD. For k, statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test; *P < 0.05, ***P < 0.001. For the boxplots in k, the centre line indicates the median, the box limits indicate the first and third quartiles, and the whiskers indicate the data range. The schematics in b and e are modified from the Allen Mouse Brain Atlas, online version 1, 2008 (http://mouse.brain-map.org ).