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

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    This manuscript starts from the hypothesis that a model of mitochondrial disease, the NDUFS4 knockout mouse, causes iron dysregulation, and that iron status may modify the neurological phenotypes that result in the mouse. This study has the potential to inform how body iron homeostasis can modify neurological phenotypes caused by mitochondrial disease. This study will be of interest to a broad audience of neuroscientists, particularly those with an interest in mitochondrial diseases.

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Mitochondrial dysfunction caused by aberrant Complex I assembly and reduced activity of the electron transport chain is pathogenic in many genetic and age-related diseases. Mice missing the Complex I subunit NADH dehydrogenase [ubiquinone] iron-sulfur protein 4 (NDUFS4) are a leading mammalian model of severe mitochondrial disease that exhibit many characteristic symptoms of Leigh Syndrome including oxidative stress, neuroinflammation, brain lesions, and premature death. NDUFS4 knockout mice have decreased expression of nearly every Complex I subunit. As Complex I normally contains at least 8 iron-sulfur clusters and more than 25 iron atoms, we asked whether a deficiency of Complex I may lead to iron perturbations, thereby accelerating disease progression. Consistent with this, iron supplementation accelerates symptoms of brain degeneration in these mice, while iron restriction delays the onset of these symptoms, reduces neuroinflammation, and increases survival. NDUFS4 knockout mice display signs of iron overload in the liver including increased expression of hepcidin and show changes in iron-responsive element-regulated proteins consistent with increased cellular iron that were prevented by iron restriction. These results suggest that perturbed iron homeostasis may contribute to pathology in Leigh Syndrome and possibly other mitochondrial disorders.

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  1. eLife assessment

    This manuscript starts from the hypothesis that a model of mitochondrial disease, the NDUFS4 knockout mouse, causes iron dysregulation, and that iron status may modify the neurological phenotypes that result in the mouse. This study has the potential to inform how body iron homeostasis can modify neurological phenotypes caused by mitochondrial disease. This study will be of interest to a broad audience of neuroscientists, particularly those with an interest in mitochondrial diseases.

  2. Reviewer #1 (Public Review):

    Complex I deficiency is associated with multiple diseases ranging from devastating inborn errors of metabolism to more common ailments associated with aging. It has long been known that Complex I transports electrons through a series of iron-sulfur clusters; however, the consequences of the likely dysfunction of these cofactors in models of Complex I deficiency have been surprisingly unexplored. The authors of this manuscript explore the contributions of iron to pathophysiology seen in a mouse model of Complex I deficiency, the Ndufs4 knockout model. Specifically, the authors hypothesize "that Complex I deficiencies may alter normal cellular or regional iron distribution which contributes to mitochondrial disease progression."

    The authors begin by convincingly demonstrating that modulating iron availability affects clasping - a marker of neurodegeneration - as well as lifespan. Using either an iron-chelating agent or a low iron diet, the authors delay the onset of clasping (i.e., neurodegeneration) and extend lifespan in Ndufs4 knockout mice. As expected, a limited iron diet causes anemia, but does not affect overall weight in either wild type or Ndufs4 knockout mice, suggesting the diet is tolerated despite the hematological defects. To begin to understand the molecular mechanisms underlying these phenotypes, the authors quantify total iron levels across tissues, and surprisingly find tissue-specific effects of chelation and/or diet-induced modulation of total iron levels. These data suggest that a low iron diet rescues iron levels in the liver, kidney, and duodenum, but not in the brain, which is surprising given the rescue of the clasping phenotype. To follow up on this, the authors look at multiple metals whose uptake can be affected by altered iron homeostasis, and find select defects in other tissues, such as elevated zinc in the brains of knockout mice relative to wild-type controls and elevated manganese in Ndufs4 KO tissues. However, the effects of such metal imbalances were not further explored. The authors then show that the imbalance in liver free iron could cause oxidative stress and reprogram the cellular program of iron transport. Interestingly, these phenotypes are found in the liver but not the brain, consistent with the tissue-specific changes in metals found in previous experiments. Collectively, the data presented by the authors convincingly demonstrate that: 1. Complex I deficiency causes defects in metal homeostasis, albeit in a tissue-specific manner; 2. In tissues harboring elevated iron levels in Ndufs4 knockout mice, this leads to altered iron regulation pathways as well as elevated oxidative stress; and 3. That, despite the tissue specificity of the molecular changes in iron homeostasis, limiting iron uptake in Ndufs4 knockout mice rescues neurodegeneration and extends lifespan in mice. While these data support the authors' hypothesis that "Complex I deficiencies may alter... regional iron distribution which contributes to mitochondrial disease progression," the authors do not test the role of altered cellular distribution of iron within this model.

    Overall, the data presented strongly suggest that modulating iron intake may ameliorate the pathophysiology associated with Complex I dysfunction. However, the specific tissues in which these benefits may occur, and the molecular mechanisms underlying this therapeutic benefit are not fully established in this study. Furthermore, the benefits of modulating iron levels as a potential therapeutic strategy for Complex I dysfunction would need to be balanced with potential complications, such as anemia.

  3. Reviewer #2 (Public Review):

    The major strengths of the manuscript are 1) the widely used mouse model, 2) the extensive analysis of transition metals other than iron, 3) the molecular data providing evidence for the cellular effects of excess iron accumulation on gene expression and protein levels, and 4) the phenotypic and lifespan data in animals treated with diet manipulations.

    The major weakness of the manuscript is the lack of a link between the iron status of individual animals and their behaviors. The authors attempt to correlate molecular and behavioral parameters in Figure 5C, but the strength of this analysis (sample size and resolution) is modest.

    The conclusions are entirely justified by the data. Moreover, this study opens several questions that, if answered, would potentially have a major impact on mitochondrial disease research. As the authors note, whether patients with different genetic defects affecting the OxPhos complexes exhibit iron excess, and whether this is detectable in the blood or other biofluid is an important question.

  4. Reviewer #3 (Public Review):

    This manuscript uses the NDUFS4-/- mouse, which models severe mitochondrial disease Leigh Syndrome, to examine if changes in iron homeostasis modify disease progression. They report that iron limitation delays the phenotype of "clasping", a neurologic change associated with loss of NDUFS4. The study is mostly observational and has little mechanism regarding how possible alterations in iron homeostasis contribute to disease progression. Therefore, it does not advance our understanding of how changes in iron homeostasis add to the progression of Leigh Syndrome.


    The authors propose that iron homeostasis may be altered in the absence of NDUFS4 in mice, which is utilized as a model for the human disease Leigh Syndrome. To test this hypothesis, the authors show that limiting iron by either iron chelation or restriction in the diet delays disease progression (clasping is delayed and longer survival). They show by ICP-MS elevated iron in NDUFS4-/- mouse livers, kidney and duodenum and that "overall" tissue iron levels are elevated in the absence of NDUFS4. They show the predicted changes in iron levels in those tissues when the iron content in the diet is limited. They also show that other metals are changed in the absence of NDUFS4 and that when iron is limited in the diet there are increased levels of other metals with the most significant changes in Mn. They show a significant correlation between increased peroxidation of PUFAs in the liver of NDUFS4-/- mice and increased clasping, a neurologic measure of disease progression.

    Unfortunately, the authors do not detect changes in iron levels in neurologic tissues (brain) in the absence of NDUFS4 nor do they show changes in iron levels in the brain upon limiting iron in the diet. In addition, the authors do not provide any imaging of the brain or brain stem to support slowed progression of lesions in this model. That a change in iron in the diet affects RBC levels simply confirms that the diet is limiting for erythropoiesis and does not provide supporting evidence that iron levels may be changing in the brain.

    The authors spend a lot of experimental effort measuring metal levels in all tissues without evidence of changes in neurologic tissues and then focus on changes in metals in the liver and increased lipid peroxidation in the liver. It is unclear to this reviewer if the authors are suggesting that the iron loading in the liver is contributing to the neurologic phenotypes associated with loss of NDUFS4 or if they are suggesting that there must also be inappropriate iron loading in neurologic tissues (with no supportive data) that gives rise to disease progression. The authors did not measure if iron loading in the liver, kidney or duodenum in NDUFS4-/- mice resulted in decreased organ function thus leaving open the possibility that other organ dysfunction contributes to the observed neurologic phenotypes associated with this disease.

    It is unclear why the authors did not measure lipid peroxidation in the brain tissue or other neurologic tissues, nor did they measure lactate levels in blood and CSF upon dietary iron limitation.

    There is no mechanistic experimental data that inform on how iron changes accelerate the progression of disease.

    Fig 4 -They measure metal levels in different tissues, however, they do not show any changes in iron levels in neurologic tissues nor do they assess iron protein levels in neurologic tissues.

    Together, this study does not determine the how of increased iron in tissues of NDUFS4-/- mice, and if there are changes in mitochondrial function upon dietary iron restriction, whether the location of iron in tissues is different (e.g., it is unclear whether there is increased mitochondrial iron, often a phenotype associated with mitochondrial dysfunction).