Nuclear encoding of mitochondrial DNA transgenes followed by mitochondrial targeting of the expressed proteins (allotopic expression; AE) represents a potentially powerful strategy for creating animal models of mtDNA disease. Mice were created that allotopically express either a mutant (A6M) or wildtype (A6W)
Throughout mitochondrial evolution, gene transfer from the mitochondrial compartment to the nucleus has been an ongoing process [
Transversion of T to G at position 8993 of the human mitochondrial genome causes substitution of arginine for a conserved leucine in residue 156 (L156R) of the mitochondrial encoded MT-ATP6 gene [
Two forms of the nuclear-coded mitochondrial
Synthesized
Schematic representation of AE DNA constructs. High-level transcription is driven by the human EF1
Electron microscopy was performed to analyze mitochondrial localization of allotopically expressed ATP6. Anaesthetized mice were perfused with 3% paraformaldehyde and 0.2% glutaraldehyde. Striatum was diced into 1-2 mm pieces and fixed in the same perfusion solution for 30–40 min. Tissue was rinsed, dehydrated in a graded ethanol series, and embedded in LR White Medium Grade Resin (Electron Microscopy Sciences). Immune labeling of sections utilized 1 : 4000 rabbit polyclonal anti-myc antibody (Abcam) with 1 : 100 donkey anti-rabbit Ultrasmall gold (Aurion) (diameter of average gold cluster <0.8 nm) as secondary label. R-Gent SE-EM (Aurion) was utilized for silver enhancement of samples.
Forty mice were subjected to all motor tests. Groups of 5 males and 5 females were hemizygous A6M and nontransgenic (C57BL/6), hemizygous A6W and nontransgenic (B6(B6SJL)). All neuromuscular and motor tests followed a paradigm outlined earlier [
Neuromuscular strength was measured using a wire hang test [
Motor coordination was tested using a balance beam [
Two analyses were performed using a Rota-Rod apparatus [
Motor coordination was evaluated with a pole test apparatus [
Gait was assessed as described [
Following motor testing, all mice were subjected to a series of biochemical tests. ATP synthesis and respirometry assays were performed the same day as mitochondrial isolation; remaining mitochondria and sera were frozen for lactate and SOD measurements. Mitochondrial isolation and ATP synthesis and respirometry tests were performed on 10 groups of four mice each. Each group contained one A6M hemizygous, one A6M nontransgenic, one A6W hemizygous, and one A6W nontransgenic mouse. A6M transgenic and wild-type mice were euthanized and samples taken at a mean age of
Mitochondria were isolated from whole brain, heart, and gastrocnemius muscle as previously reported [
Aerobic respiration of isolated mitochondria was measured using MitoXpress A65N-1 (Luxcel) [
ATP production rate was measured using the chemiluminescent ATP-consuming reaction of Luciferase-Luciferin [
Protein measurements of manganese superoxide dismutase (MnSOD, SOD2) were generated with a SOD2 Protein Quantity Microplate Assay Kit (MitoSciences) ELISA.
Lactate was measured in serum of all experimental mice using the Lactate Colorimetric Assay Kit (Abcam). All samples were measured in duplicate.
All statistical analyses were performed using SAS software (SAS Institute). Hazard ratios (HRs) with 95% confidence intervals were generated for all measurements that produced censored values (wire hang, balance beam time, Rota-Rod and pole measurements) by proportional hazards regression analysis using PROC PHREG. Analysis of gait measurements was performed with repeated measures modeling using PROC MIXED.
Two synthesized, nuclear-coded ATP6 genes were each cloned into vectors containing a high-level constitutive EF-1
The A6M expression construct was injected into 263 C57BL/6 and 97 B6(B6SJLF1) embryos from which 3 C57BL/6 and 7 B6(B6SJLF1) transgenic founder mice were derived. The A6W expression construct was injected into 112 C57BL/6 and 78 B6(B6SJLF1) embryos from which 0 C57BL/6 and 5 B6(B6SJLF1) transgenic founder mice resulted. One line of transgenic mice for each construct was selected for further characterization based on transgene expression and fertility in founder transgenic mice.
Allotopically expressed proteins from both A6M and A6W lineages were found to colocalize with mitochondria in electron micrographs of striatum sections (Figure
Mitochondrial localization of allotopically expressed ATP6 in brain sections of nontransgenic, A6M and A6W mice. The small amount of cytosolic nonmitochondrial staining is not unexpected as proteins are cytoplasmically translated prior to mitochondrial translocalization.
A6M and A6W mice were compared to nontransgenic control mice in a series of neuromuscular and motor assays (Table
Motor and biochemical analyses of A6M and A6W transgenic mice. Hazard and odds ratios are expressed with 95% confidence intervals (95% CI) and
A6M | A6W | |
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Wirehang hazard ratio (95% CI) | HR: 0.54 (0.376–0.770) | HR: 0.424 (0.246–0.731) |
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BB slips/crossing odds ratio (95% CI) | OR: 0.12 (0.021–0.709) | OR: 0.68 (0.141–3.276) |
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BB time hazard ratio (95% CI) | HR: 1.72 (1.274–2.309) | HR: 0.67 (0.506–0.896) |
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Constant Rota-Rod hazard ratio (95% CI) | HR: 2.28 (1.722–3.030) | HR: 2.09 (1.414–3.097) |
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Accelerating Rota-Rod hazard ratio (95% CI) | HR: 2.37 (1.589–3.523) | HR: 1.09 (0.681–1.739) |
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Poletest hazard ratio (95% CI) | HR: 1.50 (1.156–1.941) | HR: 1.10 (0.860–1.399) |
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Gait | ||
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LF-LF |
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LF-LR |
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LF-RF |
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LR-LR |
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LR-RR |
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RF-RF |
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RF-RR |
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RR-RR |
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Brain state II (nmol O2/min/mg |
Transgenic: |
Transgenic: |
Nontransgenic: |
Nontransgenic: |
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Heart state II (nmol O2/min/mg |
Transgenic: |
Transgenic: |
Nontransgenic: |
Nontransgenic: |
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Skeletal muscle state II (nmol O2/min/mg |
Transgenic: |
Transgenic: |
Nontransgenic: |
Nontransgenic: |
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Brain state III (nmol O2/min/mg |
Transgenic: |
Transgenic: |
Nontransgenic: |
Nontransgenic: |
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Heart state III (nmol O2/min/mg |
Transgenic: |
Transgenic: |
Nontransgenic: |
Nontransgenic: |
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Skeletal muscle state III (nmol O2/min/mg |
Transgenic: |
Transgenic: |
Nontransgenic: |
Nontransgenic: |
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Brain respiratory control ratio | Transgenic: |
Transgenic: |
Nontransgenic: |
Nontransgenic: |
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Heart respiratory control ratio | Transgenic: |
Transgenic: |
Nontransgenic: |
Nontransgenic: |
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Skeletal muscle respiratory control ratio | Transgenic: |
Transgenic: |
Nontransgenic: |
Nontransgenic: |
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Brain ATP synthesis (nmol/min/mg protein) | Transgenic: |
Transgenic: |
Nontransgenic: |
Nontransgenic: |
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Heart ATP synthesis (nmol/min/mg protein) | Transgenic: |
Transgenic: |
Nontransgenic: |
Nontransgenic: |
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Skeletal muscle ATP synthesis (nmol/min/mg protein) | Transgenic: |
Transgenic: |
Nontransgenic: |
Nontransgenic: |
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Brain MnSOD protein levels (arbitrary colorimetric values) | Transgenic: |
Transgenic: |
Nontransgenic: |
Nontransgenic: |
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Heart MnSOD protein levels arbitrary colorimetric values) | Transgenic: |
Transgenic: |
Nontransgenic: |
Nontransgenic: |
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Skeletal muscle MnSOD protein levels (arbitrary colorimetric values) | Transgenic: |
Transgenic: |
Nontransgenic: |
Nontransgenic: |
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Serum lactate (mM) | Transgenic: |
Transgenic: |
Nontransgenic: |
Nontransgenic: |
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LF: left front; LR: left rear; RF: right front; RR: right rear.
Of the parameters measured in this group of tests (wire hang, beam time, beam slips, constant Rota-Rod, accelerating Rota-Rod, pole test, gait), A6M mice displayed performance inferior to control mice in four out of seven tests while A6W mice displayed inferior performance to their controls in a single analysis. Additionally, both A6M and A6W mice displayed enhanced performance in two measures in comparison to their controls. The superior performance of A6M mice in all Rota-Rod analyses, while surprising, might be explained by differences in what is measured in each test on a physiological or tissue level. This discrepancy of A6M Rota-Rod results versus the other tests suggests that further study and characterization of the functional consequences of allotopic expression of mitochondrial genes is warranted.
A6M transgenic and wild-type mice were euthanized and samples taken at a mean age of
The absence of detectable differences in mitochondrial function between transgenic mice and their nontransgenic counterparts was somewhat surprising in light of the clear differences seen in motor function in A6M mice. These discrepancies may reflect a reduction of stress due to the lag time between the end of motor tests and commencement of mitochondrial isolation. Alternatively, levels of allotopically expressed L156R ATP6 protein in mitochondria might vary during an individual’s lifespan such that differences in ATP synthesis that are undetectable in adult mice are of sufficient magnitude in fetal and/or postnatal development to cause a change in developmental trajectory that results in the functional differences observed. Future experiments on mice that undergo motor analysis and functional strain immediately prior to biochemical analysis might yield different results. The experiments reported here using genetically defined mice might yield different results with modified genetic and/or environmental variables.
While mtDNA mutations are primary etiologic agents in mitochondrial disease, pathogenic phenotypes are intensified or attenuated by numerous secondary factors including background mtDNA sequence [
The results of these experiments have implications for the potential future use of allotopic expression as a strategy for gene therapy. Nuclear expression of one or more mitochondrial genes in a clinical setting could improve mitochondrial function in the context of mitochondrial disease. This modeling might also provide an effective method for protecting the 13 genes encoded on the mitochondrial genome from the oxidative damage that results from normal aging, age-related neurodegenerative diseases and other pathological states shown to have mitochondrial involvement.
The authors thank M. H. Irwin, M. V. Cannon, and K. Parameshwaran for helpful discussion and critical project reviews, S. Agnew, L. Buchanan, J. DeFoor, C. Goracke, R. McFarlin, P. Rubinstein, M. Isaacson, A. Parnell, C. Shafferman, S. Widick, and E. Zimmer for assistance with motor assays, and K. Wolfe for assistance with electron microscopy. This work was supported in part by grants from the MitoCure Foundation, National Institutes of Health (NIH) (HD053037 and RR16286), National Science Foundation (NSF) (EPS-0447675), the Alabama Agricultural Experiment Station, and Auburn University.