FNDC-1-mediated mitophagy and ATFS-1 coordinate to protect against hypoxia-reoxygenation.

Mitochondrial quality control (MQC) balances organelle adaptation and elimination, and mechanistic crosstalk between the underlying molecular processes affects subsequent stress outcomes. FUNDC1 (FUN14 domain containing 1) is a mammalian mitophagy receptor that responds to hypoxia-reoxygenation (HR) stress. Here, we provide evidence that FNDC-1 is the C. elegans ortholog of FUNDC1, and that its loss protects against injury in a worm model of HR. This protection depends upon ATFS-1, a transcription factor that is central to the mitochondrial unfolded protein response (UPRmt). Global mRNA and metabolite profiling suggest that atfs-1-dependent stress responses and metabolic remodeling occur in response to the loss of fndc-1. These data support a role for FNDC-1 in non-hypoxic MQC, and further suggest that these changes are prophylactic in relation to subsequent HR. Our results highlight functional coordination between mitochondrial adaptation and elimination that organizes stress responses and metabolic rewiring to protect against HR injury.Abbreviations: AL: autolysosome; AP: autophagosome; FUNDC1: FUN14 domain containing 1; HR: hypoxia-reperfusion; IR: ischemia-reperfusion; lof: loss of function; MQC: mitochondrial quality control; PCA: principle component analysis; PPP: pentonse phosphate pathway; proK (proteinase K);UPRmt: mitochondrial unfolded protein response; RNAi: RNA interference.

Optogenetic control of mitochondrial protonmotive force to impact cellular stress resistance.

Mitochondrial respiration generates an electrochemical proton gradient across the mitochondrial inner membrane called protonmotive force (PMF) to drive diverse functions and synthesize ATP. Current techniques to manipulate the PMF are limited to its dissipation; yet, there is no precise and reversible method to increase the PMF. To address this issue, we aimed to use an optogenetic approach and engineered a mitochondria-targeted light-activated proton pump that we name mitochondria-ON (mtON) to selectively increase the PMF in Caenorhabditis elegans. Here we show that mtON photoactivation increases the PMF in a dose-dependent manner, supports ATP synthesis, increases resistance to mitochondrial toxins, and modulates energy-sensing behavior. Moreover, transient mtON activation during hypoxic preconditioning prevents the well-characterized adaptive response of hypoxia resistance. Our results show that optogenetic manipulation of the PMF is a powerful tool to modulate metabolism and cell signaling.

Cardioprotection by the mitochondrial unfolded protein response requires ATF5.

The mitochondrial unfolded protein response (UPRmt) is a cytoprotective signaling pathway triggered by mitochondrial dysfunction. UPRmt activation upregulates chaperones, proteases, antioxidants, and glycolysis at the gene level to restore proteostasis and cell energetics. Activating transcription factor 5 (ATF5) is a proposed mediator of the mammalian UPRmt. Herein, we hypothesized pharmacological UPRmt activation may protect against cardiac ischemia-reperfusion (I/R) injury in an ATF5-dependent manner. Accordingly, in vivo administration of the UPRmt inducers oligomycin or doxycycline 6 h before ex vivo I/R injury (perfused heart) was cardioprotective in wild-type but not global Atf5-/- mice. Acute ex vivo UPRmt activation was not cardioprotective, and loss of ATF5 did not impact baseline I/R injury without UPRmt induction. In vivo UPRmt induction significantly upregulated many known UPRmt-linked genes (cardiac quantitative PCR and Western blot analysis), and RNA-Seq revealed an UPRmt-induced ATF5-dependent gene set, which may contribute to cardioprotection. This is the first in vivo proof of a role for ATF5 in the mammalian UPRmt and the first demonstration that UPRmt is a cardioprotective drug target.NEW & NOTEWORTHY Cardioprotection can be induced by drugs that activate the mitochondrial unfolded protein response (UPRmt). UPRmt protection is dependent on activating transcription factor 5 (ATF5). This is the first in vivo evidence for a role of ATF5 in the mammalian UPRmt.

Fndc-1 contributes to paternal mitochondria elimination in C. elegans.

Paternal mitochondria are eliminated following fertilization by selective autophagy, but the mechanisms that restrict this process to sperm-derived organelles are not well understood. FUNDC1 (FUN14 domain containing 1) is a mammalian mitophagy receptor expressed on the mitochondrial outer membrane that contributes to mitochondrial quality control following hypoxic stress. Like FUNDC1, the C. elegans ortholog FNDC-1 is widely expressed in somatic tissues and mediates hypoxic mitophagy. Here, we report that FNDC-1 is strongly expressed in sperm but not oocytes and contributes to paternal mitochondria elimination. Paternal mitochondrial DNA is normally undetectable in wildtype larva, but can be detected in the cross-progeny of fndc-1 mutant males. Moreover, loss of fndc-1 retards the rate of paternal mitochondria degradation, but not that of membranous organelles, a nematode specific membrane compartment whose fusion is required for sperm motility. This is the first example of a ubiquitin-independent mitophagy receptor playing a role in the selective degradation of sperm mitochondria.

Ubiquitination is required for the initial removal of paternal organelles in C. elegans.

Elimination of paternal mitochondria after fertilization occurs in many species using the process of selective autophagy. The mechanism for targeting paternal mitochondria, but not maternal mitochondria, for elimination in the early embryo is not well understood. The results in this paper suggest that there are at least two different mechanisms for targeting paternal mitochondria for elimination: the first involving ubiquitination and a second involving a mitochondrial associated autophagy receptor, fndc-1. Elimination of paternal mitochondria can be visualized in embryos of the nematode, C. elegans. Paternal mitochondria enter the zygote at fertilization. Initially, they are closely associated with another sperm organelle, the membraneous organelle (MO). The MOs become ubiquitinated within minutes after fertilization. Simultaneous RNAi knockdown of two ubiquitin conjugating enzymes, ubc-18 and ubc-16, reduces MO ubiquitination. Loss of function of ubc-18 alone leads to loss of K48-linked polyubiquitin chains and halts the recruitment of proteasome to MOs. Interestingly, knockdown of ubc-18 or ubc-16 or the combination does not reduce the localization of K63-linked ubiquitin chains to MOs suggesting that some ubiquitin structure other than K63 chains is responsible for recruiting the autophagy machinery to MOs. Double knockdown (ubc-18/ubc-16) inhibits the recruitment of the autophagy protein, LGG-1 (homolog of LC3/GABARAP), to paternal organelles and causes the persistence of paternal mitochondria into the two cell stage. If paternal mitochondria are not eliminated via this early process, they are eventually removed from the embryo in a process that depends on the mitophagy adaptor protein, fndc-1. Thus, there are two redundant, but temporally distinct mechanisms that target paternal mitochondria for elimination in C. elegans. In addition to the involvement of ubiquitination in the elimination of paternal mitochondria, two subunits of the proteasome, rpn-10 and rad-23, are required for elimination of paternal mitochondria. These subunits are known to function as ubiquitin receptors and knockdown of either inhibits the recruitment of proteasome to ubiquitinated MOs. Their knockdown does not affect the localization of LGG-1 to paternal structures indicating that the proteasome is not required for autophagy membrane recruitment but might be involved in autophagosome maturation or its fusion with the lysosome.

Structure and function of the N-terminal domain of the human mitochondrial calcium uniporter.

The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti-/pro-apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N-terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake-1 and mitochondrial calcium uptake-2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca(2+) uptake in a stable MCU knockdown HeLa cell line and exerted dominant-negative effects in the wild-type MCU-expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.

Interplay between the oxidoreductase PDIA6 and microRNA-322 controls the response to disrupted endoplasmic reticulum calcium homeostasis.

The disruption of the energy or nutrient balance triggers endoplasmic reticulum (ER) stress, a process that mobilizes various strategies, collectively called the unfolded protein response (UPR), which reestablish homeostasis of the ER and cell. Activation of the UPR stress sensor IRE1α (inositol-requiring enzyme 1α) stimulates its endoribonuclease activity, leading to the generation of the mRNA encoding the transcription factor XBP1 (X-box binding protein 1), which regulates the transcription of genes encoding factors involved in controlling the quality and folding of proteins. We found that the activity of IRE1α was regulated by the ER oxidoreductase PDIA6 (protein disulfide isomerase A6) and the microRNA miR-322 in response to disruption of ER Ca2+ homeostasis. PDIA6 interacted with IRE1α and enhanced IRE1α activity as monitored by phosphorylation of IRE1α and XBP1 mRNA splicing, but PDIA6 did not substantially affect the activity of other pathways that mediate responses to ER stress. ER Ca2+ depletion and activation of store-operated Ca2+ entry reduced the abundance of the microRNA miR-322, which increased PDIA6 mRNA stability and, consequently, IRE1α activity during the ER stress response. In vivo experiments with mice and worms showed that the induction of ER stress correlated with decreased miR-322 abundance, increased PDIA6 mRNA abundance, or both. Together, these findings demonstrated that ER Ca2+, PDIA6, IRE1α, and miR-322 function in a dynamic feedback loop modulating the UPR under conditions of disrupted ER Ca2+ homeostasis.

Sumoylation regulates ER stress response by modulating calreticulin gene expression in XBP-1-dependent mode in Caenorhabditis elegans.

Excessive accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen causes ER stress, which induces a set of genes, including those encoding ER-resident chaperones, to relieve the detrimental effects and recover homeostasis. Calreticulin is a chaperone that facilitates protein folding in the ER lumen, and its gene expression is induced by ER stress in Caenorhabditis elegans. Sumoylation conjugates small ubiquitin-like modifier (SUMO) proteins with target proteins to regulate a variety of biological processes, such as protein stability, nuclear transport, DNA binding, and gene expression. In this study, we showed that C. elegans X-box-binding protein 1 (Ce-XBP-1), an ER stress response transcription factor, interacts with the SUMO-conjugating enzyme UBC-9 and a SUMOylation target. Our results indicated that abolishing sumoylation enhanced calreticulin expression in an XBP-1-dependent manner, and the resulting increase in calreticulin counteracted ER stress. Furthermore, sumoylation was repressed in C. elegans undergoing ER stress. Finally, RNAi against ubc-9 mainly affected the expression of genes associated with ER functions, such as lipid and organic acid metabolism. Our results suggest that sumoylation plays a regulatory role in ER function by controlling the expression of genes required for ER homeostasis in C. elegans.

Pharmacological activation of Sirt1 ameliorates polyglutamine-induced toxicity through the regulation of autophagy.

Intracellular accumulation of polyglutamine (polyQ)-expanded Huntingtin (Htt) protein is a hallmark of Huntington's disease (HD). This study evaluated whether activation of Sirt1 by the anti-cancer agent, ß-lapachone (ß-lap), induces autophagy in human neuroblastoma SH-SY5Y cells, thereby reducing intracellular levels of polyQ aggregates and their concomitant cytotoxicity. Treatment of cells with ß-lap markedly diminished the cytotoxicity induced by forced expression of Htt exon 1 containing a pathogenic polyQ stretch fused to green fluorescent protein (HttEx1(97Q)-GFP). ß-lap increased autophagy in SH-SY5Y cells, as evidenced by the increased formation of LC3-II and autolysosomes. Furthermore, ß-lap reduced HttEx1(97Q)-GFP aggregation, which was significantly prevented by co-incubation with 3-methyladenine, an inhibitor of autophagy. ß-lap increased Sirt1 activity, as shown by the increased deacetylation of the Sirt1 substrates, PARP-1 and Atg5, and the nuclear translocation of FOXO1. Both the induction of autophagy and attenuation of HttEx1(97Q)-GFP aggregation by ß-lap were significantly prevented by co-incubation with sirtinol, a general sirtuin inhibitor or by co-transfection with shRNA against Sirt1. The pro-autophagic actions of ß-lap were further investigated in a transgenic Caenorhabditis elegans (C. elegans) line that expressed Q67 fused to cyanine fluorescent protein (Q67). Notably, ß-lap reduced the number of Q67 puncta and restored Q67-induced defects in motility, which were largely prevented by pre-treatment with RNAi against sir-2.1, the C. elegans orthologue of Sirt1. Collectively, these data suggest that ß-lap induces autophagy through activation of Sirt1, which in turn leads to a reduction in polyQ aggregation and cellular toxicity. Thus, ß-lap provides a novel therapeutic opportunity for the treatment of HD.