Induced pluripotent stem cells (iPSCs) have fewer and immature mitochondria than somatic cells and mainly rely on glycolysis for energy source. During somatic cell reprogramming, somatic mitochondria and other organelles get remodeled. However, events of organelle remodeling and interaction during somatic cell reprogramming have not been extensively explored. We show that both SKP/SKO (Sox2, Klf4, Pou5f1/Oct4) and SKPM/SKOM (SKP/SKO plus Myc/c-Myc) reprogramming lead to decreased mitochondrial mass but with different kinetics and by divergent pathways. Rapid, MYC/c-MYC-induced cell proliferation may function as the main driver of mitochondrial decrease in SKPM/SKOM reprogramming. In SKP/SKO reprogramming, however, mitochondrial mass initially increases and subsequently decreases via mitophagy. This mitophagy is dependent on the mitochondrial outer membrane receptor BNIP3L/NIX but not on mitochondrial membrane potential (ΔΨ) dissipation, and this SKP/SKO-induced mitophagy functions in an important role during the reprogramming process. Furthermore, endosome-related RAB5 is involved in mitophagosome formation in SKP/SKO reprogramming. These results reveal a novel role of mitophagy in reprogramming that entails the interaction between mitochondria, macroautophagy/autophagy and endosomes.

Heteroplasmic cells, harboring both mutant and normal mitochondrial DNAs (mtDNAs), must accumulate mutations to a threshold level before respiratory activity is affected. This phenomenon has led to the hypothesis of mtDNA complementation by inter-mitochondrial content mixing. The precise mechanisms of heteroplasmic complementation are unknown, but it depends both on the mtDNA nucleoid dynamics among mitochondria as well as the mitochondrial dynamics as influenced by mtDNA. We tracked nucleoids among the mitochondria in real time to show that they are shared after complete fusion but not 'kiss-and-run'. Employing a cell hybrid model, we further show that mtDNA-less mitochondria, which have little ATP production and extensive Opa1 proteolytic cleavage, exhibit weak fusion activity among themselves, yet remain competent in fusing with healthy mitochondria in a mitofusin- and OPA1-dependent manner, resulting in restoration of metabolic function. Depletion of mtDNA by overexpression of the matrix-targeted nuclease UL12.5 resulted in heterogeneous mitochondrial membrane potential (ΔΨm) at the organelle level in mitofusin-null cells but not in wild type. In this system, overexpression of mitofusins or application of the fusion-promoting drug M1 could partially rescue the metabolic damage caused by UL12.5. Interestingly, mtDNA transcription/translation is not required for normal mitochondria to restore metabolic function to mtDNA-less mitochondria by fusion. Thus, interplay between mtDNA and fusion capacity governs a novel 'initial metabolic complementation'.

Mitochondrial DNA depletion syndrome (MDS) is a group of severe inherited disorders caused by mutations in genes, such as deoxyribonucleoside kinase (DGUOK). A great majority of DGUOK mutant MDS patients develop iron overload progressing to severe liver failure. However, the pathological mechanisms connecting iron overload and hepatic damage remains uncovered. Here, two patients' skin fibroblasts are reprogrammed to induced pluripotent stem cells (iPSCs) and then corrected by CRISPR/Cas9. Patient-specific iPSCs and corrected iPSCs-derived high purity hepatocyte organoids (iHep-Orgs) and hepatocyte-like cells (iHep) are generated as cellular models for studying hepatic pathology. DGUOK mutant iHep and iHep-Orgs, but not control and corrected one, are more sensitive to iron overload-induced ferroptosis, which can be rescued by N-Acetylcysteine (NAC). Mechanically, this ferroptosis is a process mediated by nuclear receptor co-activator 4 (NCOA4)-dependent degradation of ferritin in lysosome and cellular labile iron release. This study reveals the underlying pathological mechanisms and the viable therapeutic strategies of this syndrome, and is the first pure iHep-Orgs model in hereditary liver diseases.

The mechanisms of somatic cell reprogramming have been revealed at multiple levels. However, the lack of tools to monitor different reactive oxygen species (ROS) has left their distinct signals and roles in reprogramming unknown. We hypothesized that mitochondrial flashes (mitoflashes), recently identified spontaneous bursts of mitochondrial superoxide signaling, play a role in reprogramming. Here we show that the frequency of mitoflashes transiently increases, accompanied by flash amplitude reduction, during the early stages of reprogramming. This transient activation of mitoflashes at the early stage enhances reprogramming, whereas sustained activation impairs reprogramming. The reprogramming-promoting function of mitoflashes occurs via the upregulation of Nanog expression that is associated with decreases in the methylation status of the Nanog promoter through Tet2 occupancy. Together our findings provide a previously unknown role for superoxide signaling mediated epigenetic regulation in cell fate determination.

Reprogramming of somatic cells to induced pluripotent stem cells reconfigures chromatin modifications. Whether and how this process is regulated by signals originating in the mitochondria remain unknown. Here we show that the mitochondrial permeability transition pore (mPTP), a key regulator of mitochondrial homeostasis, undergoes short-term opening during the early phase of reprogramming and that this transient activation enhances reprogramming. In mouse embryonic fibroblasts, greater mPTP opening correlates with higher reprogramming efficiency. The reprogramming-promoting function of mPTP opening is mediated by plant homeodomain finger protein 8 (PHF8) demethylation of H3K9me2 and H3K27me3, leading to reduction in their occupancies at the promoter regions of pluripotency genes. mPTP opening increases PHF8 protein levels downstream of mitochondrial reactive oxygen species production and miR-101c and simultaneously elevates levels of PHF8's cofactor, α-ketoglutarate. Our findings represent a novel mitochondria-to-nucleus pathway in cell fate determination by mPTP-mediated epigenetic regulation.

Mitochondrial quality control plays an important role in maintaining mitochondrial homeostasis and function. Disruption of mitochondrial quality control degrades brain function. We found that flunarizine (FNZ), a drug whose chronic use causes parkinsonism, led to a parkinsonism-like motor dysfunction in mice. FNZ induced mitochondrial dysfunction and decreased mitochondrial mass specifically in the brain. FNZ decreased mitochondrial content in both neurons and astrocytes, without affecting the number of nigral dopaminergic neurons. In human neural progenitor cells, FNZ also induced mitochondrial depletion. Mechanistically, independent of ATG5- or RAB9-mediated mitophagy, mitochondria were engulfed by lysosomes, followed by a vesicle-associated membrane protein 2– and syntaxin-4–dependent extracellular secretion. A genome-wide CRISPR knockout screen identified genes required for FNZ-induced mitochondrial elimination. These results reveal not only a previously unidentified lysosome-associated exocytosis process of mitochondrial quality control that may participate in the FNZ-induced parkinsonism but also a drug-based method for generating mitochondria-depleted mammal cells.

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Our study reveals a dynamic remodeling of the ER-mitochondria interface underlying neuritic degeneration.