1. Neuroscience

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
https://doi.org/10.7554/eLife.47163
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Cite this article
  1. Irene Bolea
  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
(2019)
Defined neuronal populations drive fatal phenotype in a mouse model of Leigh syndrome
eLife 8:e47163.
https://doi.org/10.7554/eLife.47163
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7 figures, 2 videos, 3 tables and 2 additional files

Figures

Figure 1 with 3 supplements
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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 …

https://doi.org/10.7554/eLife.47163.003
Figure 1—figure supplement 1
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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 …

https://doi.org/10.7554/eLife.47163.004
Figure 1—figure supplement 2
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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 …

https://doi.org/10.7554/eLife.47163.005
Figure 1—figure supplement 3
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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) …

https://doi.org/10.7554/eLife.47163.006
Figure 2 with 2 supplements
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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 …

https://doi.org/10.7554/eLife.47163.010
Figure 2—figure supplement 1
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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 …

https://doi.org/10.7554/eLife.47163.011
Figure 2—figure supplement 2
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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 …

https://doi.org/10.7554/eLife.47163.009
Figure 3 with 1 supplement
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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 …

https://doi.org/10.7554/eLife.47163.012
Figure 3—figure supplement 1
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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 …

https://doi.org/10.7554/eLife.47163.013
Figure 4
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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). …

https://doi.org/10.7554/eLife.47163.014
Figure 5
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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 …

https://doi.org/10.7554/eLife.47163.015
Figure 6
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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 …

https://doi.org/10.7554/eLife.47163.016
Figure 7 with 2 supplements
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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 …

https://doi.org/10.7554/eLife.47163.017
Figure 7—figure supplement 1
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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
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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;…

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

Videos

Video 1
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Breathing irregularities in a late-stage Vglut2:Ndufs4cKO mice.
https://doi.org/10.7554/eLife.47163.008
Video 2
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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

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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