ING2 Controls Mitochondrial Respiration via Modulating MRPL12 Ubiquitination in Renal Tubular Epithelial Cells

Mitochondrial injury of tubular epithelial cells (TECs) is the key pathogenic event underlying various kidney diseases and a potential intervening target as well. Our previous study demonstrated that ING2 is ubiquitously expressed at tubulointerstitial area within kidneys, while its role in regulating TEC mitochondrial respiration is not fully elucidated. To clarify the roles of ING2 in mitochondrial homeostasis of TECs and pathogenesis of acute ischemic kidney injury, Western blot, PCR, immunofluorescence, immunoprecipitation, and oxygen consumption rate assay were applied to address the roles of ING2 in modulating mitochondrial respiration. We further complemented these studies with acute ischemic kidney injury both in vitro and in vivo. In vitro study demonstrated ING2 could positively control TEC mitochondrial respiration. Concurrently, both mRNA and protein levels of mtDNA encoded respiratory chain components were altered by ING2, suggesting ING2 could regulate mtDNA transcription. In mechanism, ING2 could regulate the ubiquitination of a newly identified mitochondrial transcription factor MRPL12, thereby modulating its cellular stability and abundance. We also demonstrated ING2-mediated modulation on mtDNA transcription and mitochondrial respiration are involved in serum deprivation induced TEC injuries. Finally, immunohistochemistry study revealed that ING2 expression was significantly altered in kidney biopsies with acute ischemic kidney injury. In vivo study suggested that kidney specific ING2 overexpression could effectively ameliorate acute ischemic kidney injury. Our study demonstrated that ING2 is a crucial modulator of TEC mitochondrial respiration. These findings suggested a unrecognized role of ING2 in TEC mitochondrial energetic homeostasis and a potential intervening target for TEC mitochondrial injury associated pathologies.

Modulation of mitochondrial nucleoid structure during aging and by mtDNA content in Drosophila

ABSTRACT Mitochondrial DNA (mtDNA) encodes gene products that are essential for oxidative phosphorylation. They organize as higher order nucleoid structures (mtNucleoids) that were shown to be critical for the maintenance of mtDNA stability and integrity. While mtNucleoid structures are associated with cellular health, how they change in situ under physiological maturation and aging requires further investigation. In this study, we investigated the mtNucleoid assembly at an ultrastructural level in situ using the TFAM-Apex2 Drosophila model. We found that smaller and more compact TFAM-nucleoids are populated in the mitochondria of indirect flight muscle of aged flies. Furthermore, mtDNA transcription and replication were cross-regulated in the mtTFB2-knockdown flies as in the mtRNAPol-knockdown flies that resulted in reductions in mtDNA copy numbers and nucleoid-associated TFAM. Overall, our study reveals that the modulation of TFAM-nucleoid structure under physiological aging, which is critically regulated by mtDNA content.

Mitochondrial functional resilience after TFAM ablation in the adult heart

The nuclear genome-encoded mitochondrial DNA (mtDNA) transcription factor A (TFAM) is indispensable for mitochondrial energy production in the developing and postnatal heart; a similar role for TFAM is inferred in adult heart. Here, we provide evidence that challenges this long-standing paradigm. Unexpectedly, conditionalTfam ablation in vivo in adult mouse cardiomyocytes resulted in a prolonged period of functional resilience characterized by preserved mtDNA content, mitochondrial function, and cardiac function, despite mitochondrial structural alterations and decreased transcript abundance. Remarkably, TFAM protein levels did not directly dictate mtDNA content in the adult heart, and mitochondrial translation was preserved with acute TFAM inactivation, suggesting maintenance of respiratory chain assembly/function. Long-term Tfam inactivation, however, downregulated the core mtDNA transcription and replication machinery, leading to mitochondrial dysfunction and cardiomyopathy. Collectively, in contrast to the developing heart, these data reveal a striking resilience of the differentiated adult heart to acute insults to mtDNA regulation.

Mitochondrial STAT3 regulates proliferation of tissue stem cells

ABSTRACTThe STAT3 transcription factor, acting both in the nucleus and mitochondria, maintains embryonic stem cell pluripotency and promotes their proliferation. In this work, using zebrafish, we determined in vivo that mitochondrial STAT3 regulates mtDNA transcription in embryonic and larval stem cell niches and that this activity determines their proliferation rates. To dissect the molecular requirements for mitochondrial STAT3 functions, we used drugs and missense mutations to kinase-targeted STAT3 residues. As a result, we demonstrated that STAT3 import inside mitochondria requires Y705 phosphorylation by Jak2, while its mitochondrial transcriptional activity, as well as its effect on proliferation, depends on the MAPK target S727. Moreover, while STAT3-dependent mtDNA transcription is needed and sufficient to induce cell proliferation, it is not required to maintain a stem-like phenotype in the tectal niche. Surprisingly, STAT3-dependent increase of mitochondrial transcription seems independent from STAT3 binding to DNA and does not originate from STAT3 regulation of mtDNA replication.

Mitochondrial functional resilience after TFAM ablation in adult cardiomyocytes

The adult heart is a terminally differentiated tissue that depends on mitochondria for its energy supply. Respiratory chain energy supply deficits due to alterations in the mitochondrial genome (mtDNA) or in nuclear genome (nDNA)-encoded mtDNA regulators are associated with cardiac pathologies ranging from primary mitochondrial cardiomyopathies to heart failure. Mitochondrial transcription factor A (TFAM) is an nDNA-encoded regulator of mtDNA transcription, replication, and maintenance. Insufficiency of this protein in embryonic and postnatal cardiomyocytes causes cardiomyopathy and/or lethality, establishing TFAM as indispensable to the developing heart; its role in adult tissue has been inferred from these findings. Here, we provide evidence that challenges this long-standing paradigm using Tfam ablation in the adult heart. Unexpectedly, loss of Tfam in adult cardiomyocytes resulted in a prolonged period of functional resilience characterized by preserved mtDNA content, mitochondrial function, and cardiac function despite mitochondrial structural alterations and decreased transcript abundance. Remarkably, TFAM protein levels did not directly dictate mtDNA content in the adult heart, and mitochondrial translation was preserved with acute TFAM inactivation, suggesting a mechanism whereby respiratory chain assembly and function can be sustained, which we term ‘functional resilience’. Finally, long-term Tfam inactivation induced a coordinated downregulation of the core mtDNA transcription and replication machinery that ultimately resulted in mitochondrial dysfunction and cardiomyopathy. Taken together, adult-onset cardiomyocyte-specific Tfam inactivation reveals a striking resilience of the adult heart to acute insults to mtDNA regulatory mechanisms and provides insight into critical differences between the developing versus differentiated heart.