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Defined neuronal populations drive fatal phenotype in a mouse model of Leigh syndrome

  1. Irene Bolea  Is a corresponding author
  2. Alejandro Gella
  3. Elisenda Sanz
  4. Patricia Prada-Dacasa
  5. Fabien Menardy
  6. Angela M Bard
  7. Pablo Machuca-Márquez
  8. Abel Eraso-Pichot
  9. Guillem Mòdol-Caballero
  10. Xavier Navarro
  11. Franck Kalume
  12. Albert Quintana  Is a corresponding author
  1. Center for Developmental Therapeutics, Seattle Children’s Research Institute, United States
  2. Institut de Neurociències, Universitat Autònoma de Barcelona, Spain
  3. Universitat Autònoma de Barcelona, Spain
  4. Center for Integrative Brain Research, Seattle Children’s Research Institute, United States
  5. Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain
  6. University of Washington, United States
Research Article
Cite this article as: eLife 2019;8:e47163 doi: 10.7554/eLife.47163
7 figures, 2 videos, 3 tables, 1 data set and 2 additional files

Figures

Figure 1 with 3 supplements
Generation of two mouse lines lacking Ndufs4 selectively in Vglut2-expressing glutamatergic neurons or Gad2-expressing GABAergic neurons.

(A) Breeding strategy to obtain mice with conditional Ndufs4 deletion in glutamatergic neurons (Vglut2:Ndufs4cKO) or GABAergic neurons (Gad2:Ndufs4cKO) and their respective controls (Vglut2:Ndufs4cCT, Gad2:Ndufs4cCT). (B) Kaplan-Meier survival curve for Vglut2:Ndufs4cKO (n = 96; blue), Gad2:Ndufs4cKO (n = 40; red) and cCT mice (n = 50; black). (C–D) Body weight curves for Vglut2:Ndufs4cKO (n = 57 males, n = 48 females; blue) and Vglut2:Ndufs4cCT (n = 52 males, n = 50 females; black). (E–F) Body weight curves for Gad2:Ndufs4cKO (n = 30 males, n = 32 females; red) and Gad2:Ndufs4cCT (n = 41 males, n = 34 females; black). Data are presented as the mean ± SEM. Statistical analysis was performed using a two-way ANOVA (***p<0.001). (G) Representative images showing reduced body size in Vglut2:Ndufs4cKO mice when compared to Vglut2:Ndufs4cCT mice at P65. (H) Gad2:Ndufs4cKO mice also display reduced body size when compared to control littermates (Gad2:Ndufs4cCT) at P70. Food intake values (kcal/day) for (I) Vglut2:Ndufs4cKO (n = 6; closed blue squares) and Vglut2:Ndufs4cCT mice (n = 7; open black squares) and (J) Gad2:Ndufs4cKO (n = 6; closed red circles) and Gad2:Ndufs4cCT mice (n = 5; open black circles) at 8 weeks of age. Data are presented as the mean ± SEM. Statistical analysis was performed using an unpaired t-Test (**p<0.01, *p<0.05).

https://doi.org/10.7554/eLife.47163.003
Figure 1—figure supplement 1
Generation and characterization of a conditional mouse line lacking Ndufs4 in ChAT-expressing neurons.

(A) Breeding strategy used to generate mice with selective Ndufs4 deletion in cholinergic neurons (ChAT:Ndufs4cKO mice) and its genetic control (ChAT:Ndufs4cCT mice). (B) Body weight and (C) rotarod performance in ChAT:Ndufs4cKO (n = 5; red) mice and their respective control (ChAT:Ndufs4cCT mice; n = 6, blue). Data are presented as the mean ± SEM. Statistical analysis was performed using a two-way ANOVA.

https://doi.org/10.7554/eLife.47163.004
Figure 1—figure supplement 2
NDUFS4 levels and food intake after conditional deletion of Ndufs4 in Vglut2- and Gad2-expressing cells.

(A) Western blot analysis for NDUFS4 and GAPDH (loading control) levels in the olfactory bulb and thalamus of late-stage Vglut2:Ndufs4cKO mice, Gad2:Ndufs4cKO mice, and their respective controls (Vglut2:Ndufs4cCT and Gad2:Ndufs4cCT mice) (n = 3). (B) Food intake (Kcal/day) corrected for body weight (g) in Vglut2:Ndufs4cKO mice (n = 6, (P65), Gad2:Ndufs4cKO mice (n = 5, (P70), and their respective age-matched controls (Vglut2:Ndufs4cCT, n = 7; Gad2:Ndufs4cCT, n = 4). Data are presented as the mean ± SEM. Statistical analysis was performed using an unpaired t-Test.

https://doi.org/10.7554/eLife.47163.005
Figure 1—figure supplement 3
Reactive gliosis in the spinal cord of Vglut2:Ndufs4cKO mice.

(A) Vglut2:Ndufs4cKO mice present hindlimb clasping starting at P40. (B) Percentage of Vglut2:Ndufs4cKO developing ataxia and clasping (n = 11). *p<0.05 ataxia vs. clasping. Log-rank (Mantel-Cox) test. (C) Western blot analysis and (D) band intensity quantification showing GFAP, Iba-1, NSE and GAPDH levels in spinal cords of Vglut2:Ndufs4cCT (n = 5) and late-stage Vglut2:Ndufs4cKO mice (n = 5). GAPDH was used as a loading control. Statistical analysis was performed using an unpaired t-Test (**p<0.01, ***p<0.001). (E) Immunofluorescence analysis for GFAP and Iba-1 (top), hematoxylin and eosin staining (H and E; middle), and Luxol fast blue staining (LFB; bottom) in spinal cord sections from Vglut2:Ndufs4cKO and Vglut2:Ndufs4cCT mice. Data are shown as mean ± SEM.

https://doi.org/10.7554/eLife.47163.006
Figure 2 with 2 supplements
Distinct histopathological pattern in Vglut2:Ndufs4cKO and Gad2:Ndufs4cKO mice.

(A) Whole-mount composite sagittal sections showing GFAP (red) and Iba-1 (green) staining in late-stage Vglut2:Ndufs4cKO mice and their respective controls (Vglut2:Ndufs4cCT). Affected areas are indicated: Vestibular nucleus (VN), inferior olive (IO) and fastigial nucleus (FN). (B) Whole-mount composite sagittal sections showing GFAP (red) and Iba-1 (green) staining in Gad2:Ndufs4cKO mice and their respective controls (Gad2:Ndufs4cCT). Affected areas are indicated: External globus pallidus (GPe), the sustantia nigra pars reticulata (SNr) and the olfactory bulb (OB). (C) Close-up micrographs showing GFAP and Iba1 staining in the VN, IO, and FN of late-stage Vglut2:Ndufs4cKO and Vglut2:Ndufs4cCT mice. (D) Close-up micrographs showing GFAP and Iba-1 staining in the GPe, SNr and OB in Gad2:Ndufs4cKO and Gad2:Ndufs4cCT mice. Scale bar A-B:1000 µm. C-D: 50 µm. (E–F) Western blot analysis (E) and quantification (F) for active caspase eight and β-Actin (loading control) levels in the vestibular nucleus of late-stage Vglut2:Ndufs4cKO mice (n = 3) and Vglut2:Ndufs4cCT mice (n = 3). (G) Slc17a6 (Vglut2) transcript levels in the vestibular nucleus of late-stage Vglut2:Ndufs4cKO mice (n = 3) and Vglut2:Ndufs4cCT mice (n = 3). (H) Gad2 transcript levels in the olfactory bulb of Gad2:Ndufs4cKO mice (n = 3) and Gad2:Ndufs4cCT mice (n = 3). Data are presented as the mean ± SEM. Statistical analysis was performed using an unpaired t-Test (*p<0.05).

https://doi.org/10.7554/eLife.47163.010
Figure 2—figure supplement 1
Histopathological pattern in Vglut2:Ndufs4cKO and Gad2:Ndufs4cKO mice.

(A) Close-up micrographs showing GFAP and Iba1 staining in the VN, IO, and FN of Gad2:Ndufs4cKO (n = 3) and Gad2:Ndufs4cCT mice (n = 3). (B) Close-up micrographs showing GFAP and Iba-1 staining in the GPe, SNr and OB in late-stage Vglut2:Ndufs4cKO (n = 3) and Vglut2:Ndufs4cCT mice (n = 3).

https://doi.org/10.7554/eLife.47163.011
Figure 2—figure supplement 2
Ndufs4 deficiency in Gad2-expressing GABAergic and Vglut2-expressing glutamatergic cells results in prominent neuroinflammation in specific areas.

(A) Immunofluorescence analysis for tyrosine hydroxylase (TH; dopaminergic neurons), GFAP (astrocytes) and Iba-1(microglia) in brain sections of P55 Gad2:Ndufs4cCT and Gad2:Ndufs4cKO mice containing the substantia nigra (SN). (B) Immunofluorescence analysis for Calbindin (Calb; Purkinje cells) and Iba-1 in brain sections of Gad2:Ndufs4cKO mice containing the cerebellar vermis (top) and the cerebellar flocculus (bottom). DAPI was used as a nuclear counterstain. (C) Volcano plot showing differentially expressed transcripts (p<0.05, Fold Change (FC) > 2) in the brainstem of Vglut2:Ndufs4cKO mice when compared to Vglut2:Ndufs4cCT mice. (D) Volcano plot showing the top 10 most enriched Biological Process (non-redundant) categories by Gene Ontology (GO) Overrepresentation Analysis (ORA) from differentially expressed genes (1.4-fold or higher) in the brainstem of late-stage (over P68) Vglut2:Ndufs4cKO mice (n = 4) when compared to Vglut2:Ndufs4cCT mice (n = 4). (E) ImmuCC analysis showing the relative immune cell abundance inferred from gene expression analysis in the brainstem of late-stage Vglut2:Ndufs4cKO and Vglut2:Ndufs4cCT mice.

https://doi.org/10.7554/eLife.47163.009
Figure 3 with 1 supplement
Motor impairment in Vglut2:Ndufs4cKO mice.

Latency to fall (seconds) in the Rotarod test for (A) Vglut2:Ndufs4cKO (n = 8) and Vglut2:Ndufs4cCT mice (n = 10), and (B) Gad2:Ndufs4cKO (n = 4) and Gad2:Ndufs4cCT mice (n = 10). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-test (*p<0.05, ***p<0.001). Representative mouse track plots, total distance traveled and average speed during 10 min in the open-field for (C–E) Vglut2:Ndufs4cKO (n = 36) and Vglut2:Ndufs4cCT mice (n = 70), and (F–H) Gad2:Ndufs4cKO (n = 11) and Gad2:Ndufs4cCT mice (n = 16). Statistical analysis was performed using an unpaired t-Test (***p<0.001). (I) Representative images of gastrocnemius neuromuscular junctions of Vglut2:Ndufs4cKO mice and Gad2:Ndufs4cKO mice and their respective controls (scale bar: 100 µm). (J) Proportion of fully innervated neuromuscular junctions in the gastrocnemius muscle of late-stage Vglut2:Ndufs4cKO and Vglut2:Ndufs4cCT mice (n = 3 mice per group). (K) Quantification of fully innervated neuromuscular junctions in the gastrocnemius muscle of the Gad2:Ndufs4cKO and Gad2:Ndufs4cCT mice (n = 3 mice per group). Statistical analysis was performed using a Mann-Whitney test. Data are presented as the mean ± SEM.

https://doi.org/10.7554/eLife.47163.012
Figure 3—figure supplement 1
Age-dependent motor decline in Vglut2:Ndufs4cKO mice.

(A) Total distance traveled and (B) speed was analyzed in the rotarod test for Vglut2:Ndufs4cKO (n = 16) and Vglut2:Ndufs4cCT (n = 36) at different weeks of age (6–9 weeks). Data are presented as the mean ± SEM. Statistical analysis was performed using two-way ANOVA with Bonferroni post-test (*p<0.05; ***p<0.001).

https://doi.org/10.7554/eLife.47163.013
Breathing alterations in Vglut2:Ndufs4cKO mice.

Representative 5 s plethysmographic recordings from (A) Vglut2:Ndufs4cCT mice (top), late-stage Vglut2:Ndufs4cKO mice (bottom), and (B) Gad2:Ndufs4cCT mice (top) and Gad2:Ndufs4cKO mice (bottom). Respiratory frequency (fR, breaths·min−1) and tidal volume (VT, μL·g−1) for (C–D) Vglut2:Ndufs4cCT (n = 12) and Vglut2:Ndufs4cKO mice at different stages of the disease (early, n = 7; mid, n = 8; late, n = 12) and (E–F) Gad2:Ndufs4cCT (n = 7) and Gad2:Ndufs4cKO mice (n = 7). Data are presented as the mean ± SEM. Statistical analysis was performed using (C–D) one-way ANOVA followed by Bonferroni post-test (*p<0.05, **p<0.01, ***p<0.001) and (E–F) an unpaired t-Test.

https://doi.org/10.7554/eLife.47163.014
Vglut2-expressing glutamatergic cells in the vestibular nucleus show reduced in vivo electrophysiological activity in Vglut2:Ndufs4cKO mice.

(A) Representative identification of two different glutamatergic cells with raster plots (upper part) and peri-stimulus histograms (PSTH, lower part) using optogenetic stimulation. 0 represents the onset of optogenetic stimulation. Red line represents mean firing rate from the 500 ms before the stimulus plus three time the SD. Blue shading indicates period of laser stimulation. (B) Firing rate of Vglut2-expressing glutamatergic cells (n = 17 from Vglut2:Ndufs4cCT mice and n = 12 from Vglut2:Ndufs4cKO mice) during a 10 min session in an open-field. Reduced electrophysiological activity was observed in freely-moving late-stage Vglut2:Ndufs4cKO mice when compared to Vglut2:Ndufs4cCT mice regardless behavioral state (resting or active). Data are presented as the mean ± SEM. Statistical analysis was performed using two-way repeated measures ANOVA with Bonferroni post-test (***p<0.001).

https://doi.org/10.7554/eLife.47163.015
Decreased body temperature in Vglut2:Ndufs4cKO and Gad2:Ndufs4cKO mice.

(A) Telemetric body temperature measurements in Vglut2:Ndufs4cKO (n = 4–8) and Vglut2:Ndufs4cCT mice (n = 7–10) at different stages of the disease. (B) Telemetric body temperature measurements in Gad2:Ndufs4cKO (n = 7–11) and Gad2:Ndufs4cCT mice (n = 5–7) at different ages. Data are presented as the mean ± SEM. Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-test (*p<0.05, ***p<0.001).

https://doi.org/10.7554/eLife.47163.016
Figure 7 with 2 supplements
Gad2:Ndufs4cKO mice present epileptic seizures.

(A) EEG-EMG recordings and spectrogram analysis showing the frequency and power density of a typical spontaneous seizure in a Gad2:Ndufs4cKO mouse. A preceding myoclonic seizure (MC) is marked with an asterisk. (B) Representative EEG-EMG traces and (C) number of epileptiform events (interictal spikes and myoclonic seizures) in Gad2:Ndufs4cKO (n = 9) and control (Gad2:Ndufs4cCT; n = 5) mice. Data are presented as the mean ± SEM. Statistical analysis was performed using two-way ANOVA (***p<0.001). (D) Local field potential (LFP) recordings in the globus pallidus of Gad2:Ndufs4cCT and Gad2:Ndufs4cKO mice at P35. Notice the presence of epileptic events (red lines) in the traces from Gad2:Ndufs4cKO mice. (E) Percentage of Gad2:Ndufs4cKO and Gad2:Ndufs4cCT mice remaining seizure-free after increasing body temperature. (F) Representative EEG-EMG recordings and spectrogram analysis of a thermally-induced seizure in a Gad2:Ndufs4cKO mouse. A preceding myoclonic seizure (MC) is marked with an asterisk. Lft Cx, left cortex; Rt Cx: right cortex; EMG: electromyography.

https://doi.org/10.7554/eLife.47163.017
Figure 7—figure supplement 1
Vglut2:Ndufs4cKO mice do not present spontaneous seizures.

(A–B) Representative EEG-EMG recordings (B is a magnification of the inset in A) and (C) spectrogram analysis in a late-stage Vglut2:Ndufs4cKO mouse.

https://doi.org/10.7554/eLife.47163.018
Figure 7—figure supplement 2
Lifespan of Gad2:Ndufs4cKO mice is not extended by antiepileptic treatment.

Survival rate curve for Gad2:Ndufs4cKO mice treated intraperitoneally (i.p) with vehicle, levetiracetam (60 mg/kg daily; n = 3), perampanel (0.75 mg/kg daily, n = 4) or carbamazepine (40 mg/kg daily; n = 5). Statistical analysis was performed using a Log-rank (Mantel Cox) test.

https://doi.org/10.7554/eLife.47163.019

Videos

Video 1
Breathing irregularities in a late-stage Vglut2:Ndufs4cKO mice.
https://doi.org/10.7554/eLife.47163.008
Video 2
Generalized tonic-clonic seizure without hypermotor (Racine scale stage 4) in Gad2:Ndufs4cKO mice.
https://doi.org/10.7554/eLife.47163.020

Tables

Table 1
Neurological signs observed in Vglut2:Ndufs4cKO mice according to the stage of disease.
https://doi.org/10.7554/eLife.47163.007
EarlyMidLate
Body tremor++++++
Motor alterations
 Decline in balance++++++
 Ataxia-+/+++++
 Loss of motor coordination-+/+++++
 Inability of righting-+++/+++
 Hindlimb clasping-+++
Respiratory abnormalities
 Breathing irregularities++++++
  1. -, absent; +, mild; ++, moderate; +++, severe.

Table 2
Clinical signs inNdufs4-LS patients, Ndufs4KO mice and conditional Vglut2:Ndufs4cKO and Gad2:Ndufs4cKO mice.
https://doi.org/10.7554/eLife.47163.021
Ndufs4-LS patientsNdufs4KO mouseVglut2:Ndufs4cKO mouse*Gad2:Ndufs4cKO mouse*
Decreased lifespan22/22YesYesYes
Feeding impairment8/22n.dYesYes
Reduced body weightn.a.YesYesYes
Brainstem lesions14/22YesYesNo
Basal ganglia lesions9/22YesNoYes
Ataxia, motor alterations22/22YesYesNo
Growth retardation11/22YesYesYes
Hypotonia22/22YesYesNo
Respiratory abnormalities22/22YesYesNo
Increased sensitivity to VAsn.a.YesYes†No†
Seizures4/22YesNoYes
Hypothermian.a.YesYes‡Yes
  1. n.d, not determined; n.a, not available. *Reported in this study. †Reported in Zimin et al. (2016). ‡Only in late-stage mice. VAs: volatile anesthetics. Adapted from Quintana et al. (2012).

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent (M. Musculus)Slc17a6Cre (BAC-Vglut2::Cre)Borgius L, et al. Mol Cell Neurosci. 2010; 45(3):245–57MGI:4881727Tg(Slc17a6-icre)1Oki
Genetic reagent (M. Musculus)Gad2Cre/+(Gad2-IRES-Cre)Taniguchi H, et al. Neuron. 2011;71(6):995–1013.MGI:4418713B6J.Cg-Gad2tm2(cre)Zjh/MwarJ
JAXs Stock No: 028867
Genetic reagent (M. Musculus)ChatCre/+(Chat-IRES-Cre)Rossi J, et al. Cell Metab. 2011;13(2):195–204MGI:6121363B6.129S-Chattm1(cre)Lowl/MwarJ
JAXs Stock No: 031661
Genetic reagent (M. Musculus)Ndufs4Δ/+Kruse SE, et al. Cell Metab. 2008; 7 (4):312–20MGI:5614215B6.129S4-Ndufs4tm1.1Rpa/J
JAXs Stock No: 027058
Genetic reagent (M. Musculus)Ndufs4lox/loxKruse SE, et al. Cell Metab. 2008; 7 (4):312–20MGI:5613135B6.129S4-Ndufs4tm1Rpa/J
JAXs Stock No: 026963

Data availability

Normalized and raw data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (accession number GSE125470).

The following data sets were generated
  1. 1
    NCBI Gene Expression Omnibus
    1. E Sanz
    2. A Quintana
    (2019)
    ID GSE125470. Gene expression analysis in the Brainstem of Vglut2:Ndufs4cKO mice.

Additional files

Supplementary file 1

Statistical parameters.

(A) Statistical parameters for two-way ANOVA. (B) Statistical parameters for two-tailed unpaired t-Tests. (C) Statistical parameters for one-way ANOVA. (D) Statistical parameters for Log-rank (Mantel-Cox) tests.

https://doi.org/10.7554/eLife.47163.022
Transparent reporting form
https://doi.org/10.7554/eLife.47163.023

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