Iron status influences mitochondrial disease progression in Complex I-deficient mice

  1. CJ Kelly
  2. Reid K Couch
  3. Vivian T Ha
  4. Camille M Bodart
  5. Judy Wu
  6. Sydney Huff
  7. Nicole T Herrel
  8. Hyunsung D Kim
  9. Azaad O Zimmermann
  10. Jessica Shattuck
  11. Yu-Chen Pan
  12. Matt Kaeberlein  Is a corresponding author
  13. Anthony S Grillo  Is a corresponding author
  1. Department of Laboratory Medicine & Pathology, University of Washington, United States
5 figures, 2 tables and 1 additional file

Figures

Figure 1 with 2 supplements
Iron restriction delays mitochondrial disease in mice.

(A) Correlation between the onset of clasping and survival. Each point represents data from a single mouse. p=0.0047, Pearson’s test. (B) Age at which Ndufs4−/ mice exhibited the clasping phenotype …

Figure 1—figure supplement 1
Brain-permeable iron chelators are effective in delaying mitochondrial disease.

(A) Age at which Ndufs4−/ mice exhibited the clasping phenotype on chow diet. Mice were treated with either vehicle, deferiprone (DFP) in the water (0.5 or 1 mg/mL), or deferoxamine (DFO) via daily …

Figure 1—figure supplement 2
Iron restriction induces iron-deficiency anemia.

Complete blood count in PND30 WT or Ndufs4−/ mice on normal (40 ppm) or low (8 ppm) AIN-93G synthetic diet to quantify (A) hematocrit, (B) hemoglobin, (C) red blood cell count which were used to …

Figure 2 with 2 supplements
Total iron quantification in tissues.

Quantification of total iron by ICP-MS from WT and Ndufs4−/ mice at PND35 fed control (40 ppm) or low (8 ppm) AIN-93G in (A) liver, (B) whole brain, (C) kidney, (D) heart, (E) quadricep, (F) …

Figure 2—figure supplement 1
Analysis of biologically-relevant transition metals.

(A) Left, Box, and whisker plot of combined z-score-normalized ICP-MS values of tissue iron, (B) manganese, (C) zinc, and (D) copper in WT and Ndufs4−/ mice on control (40 ppm) or low (8 ppm) …

Figure 2—figure supplement 2
Iron restriction has no effect on organ size.

(A) Weight at time of tissue collection (PND35) of WT or Ndufs4−/ mice on a normal (40 ppm) or low (8 ppm) AIN-93G synthetic diet. (B) Percent of tissue weight from mice in (A) relative to total …

Iron restriction reduces iron-dependent oxidative damage and neuroinflammation.

(A) Quantification of non-heme iron by ferrozine assay and (B) MDA-TBA adduct in livers from WT and Ndufs4−/ mice at PND35 that were fed control (40 ppm) or low (8 ppm) AIN-93G synthetic diet. (C) …

Figure 4 with 2 supplements
Changes in iron-dependent proteins suggest increased labile iron.

(A) Representative western blot images and (B) densitometry (relative to actin) of proteins involved in regulation of iron transport, storage, or metabolism in livers from PND35 WT and Ndufs4−/

Figure 4—figure supplement 1
Simplified schematic of IRE-dependent translational regulation of proteins involved in iron transport, storage, or metabolism.

Translation is blocked for 5′-IREs (e.g., Fth1, Ftl1, and Fpn1) when iron regulatory proteins (IRPs) are bound to the IRE in low iron conditions (top left). Protein expression increases due to mRNA …

Figure 4—figure supplement 2
Immunoblot of iron-dependent proteins in whole brain extracts.

(A) Representative western blot images and (B) densitometry (relative to actin) of proteins involved in regulation of iron transport, storage, or metabolism in whole brains from PND35 WT and Ndufs4−/

Expression profiling of IRE-containing genes by qPCR.

(A) Quantification of relative mRNA expression of Tfr1, (B) Dmt1, (C) Fpn1, (D) Fth1 (ferritin heavy chain 1), (E) Ftl1 (ferritin light chain 1), and (F) Hamp (hepcidin) in livers from PND35 WT and N…

Tables

Table 1
ICP-MS quantification of biologically relevant metals in WT and Ndufs4−/ tissues at PND35 fed a normal (40 ppm) and low (8 ppm) iron AIN-93G synthetic diet.

Metals were measured as µg metal relative to total dry weight of tissue. N=3–5 mice, - p<0.10, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ANOVA with post hoc Tukey.

Ndufs4+/+ MiceNdufs4−/ Micep Value
Metal (µg/g)AIN-93G normal ironAIN-93G
low iron
AIN-93G normal ironAIN-93G
low iron
WT-Con versus
KO-Con
KO-Con versus
KO-Low
LiverFe122.9±18.948.0±3.5352.5±55.562.5±3.6*****
Mn2.20±0.335.68±0.633.70±0.448.62±0.51****
Zn47.5±5.0762.0±5.860.9±5.071.2±4.0
Cu9.6±0.6412.7±0.913.9±1.515.6±1.2-
BrainFe39.3±0.7840.6±2.539.6±1.837.8±1.9
Mn1.46±0.041.94±0.051.71±0.091.91±0.09
Zn37.7±0.5348.3±1.644.1±1.849.1±3.1
Cu10.3±0.5211.4±0.511.8±0.412.1±0.7
KidneyFe101.9±6.477.1±12.4161.5±15.485.8±3.5***
Mn4.54±0.445.53±0.314.48±0.206.01±0.51*
Zn55.8±1.856.6±2.658.9±4.858.4±5.7
Cu15.1±0.315.3±0.917.5±0.718.0±0.9-
HeartFe226.8±6.9182.7±9.5259.2±26.9230.7±24.0
Mn2.51±0.093.00±0.052.59±0.193.72±0.35**
Zn46.6±3.047.7±1.531.5±3.041.3±5.3*
Cu30.5±1.230.5±0.131.7±2.940.4±2.8*
Skeletal MuscleFe31.2±1.423.3±2.135.4±3.933.0±5.3
Mn0.53±0.050.83±0.080.65±0.080.88±0.10
Zn20.7±0.721.8±1.422.5±2.818.8±1.8
Cu3.80±0.243.85±0.224.60±0.564.40±0.44
SpleenFe710.4±59.1316.9±22.3860.5±265.6507.1±43.7
Mn0.94±0.091.28±0.080.92±0.401.45±0.23
Zn110.5±7.5123.1±10.4109.0±38.7126.0±6.8
Cu5.13±0.556.43±0.433.84±1.265.29±0.57
DuodenumFe150.1±34.240.0±8.6243.0±66.351.7±6.2*
Mn6.78±1.212.88±2.636.76±1.2817.1±1.7**
Zn89.2±2.284.9±12.7101.4±9.6114.1±9.3
Cu9.4±0.38.4±1.310.8±0.911.2±0.8
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)Ndufs4tm1.1Rpa C57Bl/6NCrlPalmiter Laboratory Kruse et al., 2008
OtherPicoLab Mouse Diet 20LabDietCat. 5058Facility Chow, Mouse Diet Studies
OtherAIN-93G Growth Purified DietLabDietCat. 57W5Normal Iron (40 ppm), Mouse Diet Studies
OtherAIN-93G Growth Purified DietLabDietCat. 5SSULow Iron (8 ppm), Mouse Diet Studies
Chemical compound, drugDeferiprone (3-hydroxy-1,2-dimethyl-4(1H)-pyridone)Sigma-AldrichCat. 379409CAS 30652-11-0
Chemical compound, drugFerric hydroxide dextran complexSigma-AldrichCat. D8517CAS 9004-66-4
Chemical compound, drugTrace metal grade concentrated HNO3Thermo Fisher ScientificCat. A509P500CAS 7697-37-2
Chemical compound, drugLow trace metals 30% H2O2 solutionThermo Fisher ScientificCat. NC1199178
Chemical compound, drugUltra Trace Elemental Analysis Grade H2OThermo Fisher ScientificCat. W9-500
Chemical compound, drugFerrozine iron reagent, hydrate, 98% pureThermo Fisher ScientificCat. AC410570010CAS 1266615-85-3
Chemical compound, drugTrichloroacetic acid, 99%Thermo Fisher ScientificCat. AAA1115636CAS 76-03-9
Chemical compound, drugThioglycolic acidThermo Fisher ScientificCat. AAB2039122CAS 68-11-1
Chemical compound, drugHALT Protease and Phosphatase Inhibitor Cocktail (100X)Thermo Fisher ScientificCat. 78444
OtherBovine Serum Albumin, Heat Shock TreatedThermo Fisher ScientificBP1600-100Western Blot Assays
Chemical compound, drugRIPA Lysis BufferThermo Fisher ScientificCat. 89901
Chemical compound, drugRestorePlus Stripping BufferThermo Fisher ScientificCat. 46430
Commercial assay or kitTBARS Assay KitCayman ChemicalCat. 10009055
Commercial assay or kitSuperSignal West Pico PLUS Chemiluminescent SubstrateThermo Fisher ScientificCat. 34578
Commercial assay or kitSuperSignal West Femto Maximum Sensitivity SubstrateThermo Fisher ScientificCat. 34095
Commercial assay or kitPierce BCA Protein Assay KitThermo Fisher ScientificCat. 23225
Commercial assay or kitPureLink RNA Mini KitThermo Fisher ScientificCat. 12183025
Commercial assay or kitiTaq Universal SYBR Green One-Step KitBio-RadCat. 1725151
Commercial assay or kitPhire Tissue Direct PCR Master MixThermo Fisher ScientificCat. F170LGenotyping
Commercial assay or kitNo-Stain Protein Labeling ReagentThermo Fisher ScientificCat. A44717
AntibodyAnti-FTH1 (rabbit polyclonal)Cell Signaling TechnologyCat. cs-39981:5000
AntibodyAnti-TFR1 (rabbit monoclonal)AbcamCat. ab2140391:3000
AntibodyAnti-FPN1 (rabbit polyclonal)Thermo Fisher ScientificCat. PA5-774701:3000
AntibodyAnti-DMT1 (mouse monoclonal)Santa Cruz BiotechnologyCat. sc-1668841:3000
AntibodyAnti-IRP1 (rabbit monoclonal)Cell Signaling TechnologyCat. cs-202721:1000
AntibodyAnti-IRP2 (rabbit monoclonal)Cell Signaling TechnologyCat. cs-371351:1000 in 1% BSA
AntibodyAnti-GFAP (rabbit monoclonal)Cell Signaling TechnologyCat. cs-123891:5000
AntibodyAnti-Actin HRP conjugate (rabbit monoclonal)Cell Signaling TechnologyCat. cs-51251:5000
AntibodyAnti-rabbit IgG (H+L) secondary antibody, HRP (donkey polyclonal)Thermo Fisher ScientificCat. 314581:20,000
Otherm-IgGκ binding protein HRP conjugateSanta Cruz BiotechnologyCat. sc-5161021:2000, Western Blot Assays
Sequence-based reagent, primerForward: TCAAGCCAGATCAGCATTCTC
Reverse: AGCCAGTTTCATCTCCACATG
Integrated DNA TechnologiesTfr1
Sequence-based reagent, primerForward: TCCTCATCACCATCGCAGACACTT
Reverse: TCCAAACGTGAGGGCCATGATAGT
Integrated DNA TechnologiesDmt1A/B+IRE
Dmt1A/B-IRE
Sequence-based reagent, primerForward: TGGATGGGTCCTTACTGTCTGCTAC
Reverse: TGCTAATCTGCTCCTGTTTTCTCC
Integrated DNA TechnologiesFpn1
Sequence-based reagent, primerForward: CTCATGAGGAGAGGGAGCAT
Reverse: GTGCACACTCCATTGCATTC
Integrated DNA TechnologiesFth1
Sequence-based reagent, primerForward: GTCCCGTGGATCTGTGTCT
Reverse: AGGAGCTAACCGCGAAGAGA
Integrated DNA TechnologiesFtl1
Sequence-based reagent, primerForward: AAGCAGGGCAGACATTGCGAT
Reverse: CAGGATGTGGCTCTAGGCTATGT
Integrated DNA TechnologiesHamp
Sequence-based reagent, primerForward: GTGTGAACGGATTTGGCCGTATTGGGCG
Reverse: TCGCTCCTGGAAGATGGTGATGGGC
Integrated DNA TechnologiesGapdh
Sequence-based reagent, primerForward: GCTGAGAGGGAAATCGTGCGTG
Reverse: CCAGGGAGGAAGAGGATGCGG
Integrated DNA TechnologiesActb
OtherBlood collection tubeThermo Fisher ScientificCat. 02-669-33Lavendar Cap, CBC Assay
OtherMetal-Free Centrifuge Tube, 15 mLVWRCat. 89049-172ICP-MS Assay

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