Mitochondrial dysfunction abrogates dietary lipid processing in enterocytes.

Digested dietary fats are taken up by enterocytes where they are assembled into pre-chylomicrons in the endoplasmic reticulum followed by transport to the Golgi for maturation and subsequent secretion to the circulation1. The role of mitochondria in dietary lipid processing is unclear. Here we show that mitochondrial dysfunction in enterocytes inhibits chylomicron production and the transport of dietary lipids to peripheral organs. Mice with specific ablation of the mitochondrial aspartyl-tRNA synthetase DARS2 (ref. 2), the respiratory chain subunit SDHA3 or the assembly factor COX10 (ref. 4) in intestinal epithelial cells showed accumulation of large lipid droplets (LDs) in enterocytes of the proximal small intestine and failed to thrive. Feeding a fat-free diet suppressed the build-up of LDs in DARS2-deficient enterocytes, which shows that the accumulating lipids derive mostly from digested fat. Furthermore, metabolic tracing studies revealed an impaired transport of dietary lipids to peripheral organs in mice lacking DARS2 in intestinal epithelial cells. DARS2 deficiency caused a distinct lack of mature chylomicrons concomitant with a progressive dispersal of the Golgi apparatus in proximal enterocytes. This finding suggests that mitochondrial dysfunction results in impaired trafficking of chylomicrons from the endoplasmic reticulum to the Golgi, which in turn leads to storage of dietary lipids in large cytoplasmic LDs. Taken together, these results reveal a role for mitochondria in dietary lipid transport in enterocytes, which might be relevant for understanding the intestinal defects observed in patients with mitochondrial disorders5.

Apoptotic stress causes mtDNA release during senescence and drives the SASP.

Senescent cells drive age-related tissue dysfunction partially through the induction of a chronic senescence-associated secretory phenotype (SASP)1. Mitochondria are major regulators of the SASP; however, the underlying mechanisms have not been elucidated2. Mitochondria are often essential for apoptosis, a cell fate distinct from cellular senescence. During apoptosis, widespread mitochondrial outer membrane permeabilization (MOMP) commits a cell to die3. Here we find that MOMP occurring in a subset of mitochondria is a feature of cellular senescence. This process, called minority MOMP (miMOMP), requires BAX and BAK macropores enabling the release of mitochondrial DNA (mtDNA) into the cytosol. Cytosolic mtDNA in turn activates the cGAS-STING pathway, a major regulator of the SASP. We find that inhibition of MOMP in vivo decreases inflammatory markers and improves healthspan in aged mice. Our results reveal that apoptosis and senescence are regulated by similar mitochondria-dependent mechanisms and that sublethal mitochondrial apoptotic stress is a major driver of the SASP. We provide proof-of-concept that inhibition of miMOMP-induced inflammation may be a therapeutic route to improve healthspan.

AIFM1 is a component of the mitochondrial disulfide relay that drives complex I assembly through efficient import of NDUFS5.

The mitochondrial intermembrane space protein AIFM1 has been reported to mediate the import of MIA40/CHCHD4, which forms the import receptor in the mitochondrial disulfide relay. Here, we demonstrate that AIFM1 and MIA40/CHCHD4 cooperate beyond this MIA40/CHCHD4 import. We show that AIFM1 and MIA40/CHCHD4 form a stable long-lived complex in vitro, in different cell lines, and in tissues. In HEK293 cells lacking AIFM1, levels of MIA40 are unchanged, but the protein is present in the monomeric form. Monomeric MIA40 neither efficiently interacts with nor mediates the import of specific substrates. The import defect is especially severe for NDUFS5, a subunit of complex I of the respiratory chain. As a consequence, NDUFS5 accumulates in the cytosol and undergoes rapid proteasomal degradation. Lack of mitochondrial NDUFS5 in turn results in stalling of complex I assembly. Collectively, we demonstrate that AIFM1 serves two overlapping functions: importing MIA40/CHCHD4 and constituting an integral part of the disulfide relay that ensures efficient interaction of MIA40/CHCHD4 with specific substrates.

The Mouse Heart Mitochondria N Terminome Provides Insights into ClpXP-Mediated Proteolysis.

The mammalian mitochondrial proteome consists of more than 1100 annotated proteins and their proteostasis is regulated by only a few ATP-dependent protease complexes. Technical advances in protein mass spectrometry allowed for detailed description of the mitoproteome from different species and tissues and their changes under specific conditions. However, protease-substrate relations within mitochondria are still poorly understood. Here, we combined Terminal Amine Isotope Labeling of Substrates (TAILS) N termini profiling of heart mitochondria proteomes isolated from wild type and Clpp-/- mice with a classical substrate-trapping screen using FLAG-tagged proteolytically active and inactive CLPP variants to identify new ClpXP substrates in mammalian mitochondria. Using TAILS, we identified N termini of more than 200 mitochondrial proteins. Expected N termini confirmed sequence determinants for mitochondrial targeting signal (MTS) cleavage and subsequent N-terminal processing after import, but the majority were protease-generated neo-N termini mapping to positions within the proteins. Quantitative comparison revealed widespread changes in protein processing patterns, including both strong increases or decreases in the abundance of specific neo-N termini, as well as an overall increase in the abundance of protease-generated neo-N termini in CLPP-deficient mitochondria that indicated altered mitochondrial proteostasis. Based on the combination of altered processing patterns, protein accumulation and stabilization in CLPP-deficient mice and interaction with CLPP, we identified OAT, HSPA9 and POLDIP2 and as novel bona fide ClpXP substrates. Finally, we propose that ClpXP participates in the cooperative degradation of UQCRC1. Together, our data provide the first landscape of the heart mitochondria N terminome and give further insights into regulatory and assisted proteolysis mediated by ClpXP.

CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels.

Despite being one of the most studied proteases in bacteria, very little is known about the role of ClpXP in mitochondria. We now present evidence that mammalian CLPP has an essential role in determining the rate of mitochondrial protein synthesis by regulating the level of mitoribosome assembly. Through a proteomic approach and the use of a catalytically inactive CLPP, we produced the first comprehensive list of possible mammalian ClpXP substrates involved in the regulation of mitochondrial translation, oxidative phosphorylation, and a number of metabolic pathways. We further show that the defect in mitoribosomal assembly is a consequence of the accumulation of ERAL1, a putative 12S rRNA chaperone, and novel ClpXP substrate. The presented data suggest that the timely removal of ERAL1 from the small ribosomal subunit is essential for the efficient maturation of the mitoribosome and a normal rate of mitochondrial translation.

CLPP deficiency protects against metabolic syndrome but hinders adaptive thermogenesis.

Mitochondria are fundamental for cellular metabolism as they are both a source and a target of nutrient intermediates originating from converging metabolic pathways, and their role in the regulation of systemic metabolism is increasingly recognized. Thus, maintenance of mitochondrial homeostasis is indispensable for a functional energy metabolism of the whole organism. Here, we report that loss of the mitochondrial matrix protease CLPP results in a lean phenotype with improved glucose homeostasis. Whole-body CLPP-deficient mice are protected from diet-induced obesity and insulin resistance, which was not present in mouse models with either liver- or muscle-specific depletion of CLPP However, CLPP ablation also leads to a decline in brown adipocytes function leaving mice unable to cope with a cold-induced stress due to non-functional adaptive thermogenesis. These results demonstrate a critical role for CLPP in different metabolic stress conditions such as high-fat diet feeding and cold exposure providing tools to understand pathologies with deregulated Clpp expression and novel insights into therapeutic approaches against metabolic dysfunctions linked to mitochondrial diseases.

Human R1441C LRRK2 regulates the synaptic vesicle proteome and phosphoproteome in a Drosophila model of Parkinson's disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset, autosomal dominant familial Parkinson`s disease (PD) and variation at the LRRK2 locus contributes to the risk for idiopathic PD. LRRK2 can function as a protein kinase and mutations lead to increased kinase activity. To elucidate the pathophysiological mechanism of the R1441C mutation in the GTPase domain of LRRK2, we expressed human wild-type or R1441C LRRK2 in dopaminergic neurons of Drosophila and observe reduced locomotor activity, impaired survival and an age-dependent degeneration of dopaminergic neurons thereby creating a new PD-like model. To explore the function of LRRK2 variants in vivo, we performed mass spectrometry and quantified 3,616 proteins in the fly brain. We identify several differentially-expressed cytoskeletal, mitochondrial and synaptic vesicle proteins (SV), including synaptotagmin-1, syntaxin-1A and Rab3, in the brain of this LRRK2 fly model. In addition, a global phosphoproteome analysis reveals the enhanced phosphorylation of several SV proteins, including synaptojanin-1 (pThr1131) and the microtubule-associated protein futsch (pSer4106) in the brain of R1441C hLRRK2 flies. The direct phosphorylation of human synaptojanin-1 by R1441C hLRRK2 could further be confirmed by in vitro kinase assays. A protein-protein interaction screen in the fly brain confirms that LRRK2 robustly interacts with numerous SV proteins, including synaptojanin-1 and EndophilinA. Our proteomic, phosphoproteomic and interactome study in the Drosophila brain provides a systematic analyses of R1441C hLRRK2-induced pathobiological mechanisms in this model. We demonstrate for the first time that the R1441C mutation located within the LRRK2 GTPase domain induces the enhanced phosphorylation of SV proteins in the brain.

Loss of CLPP alleviates mitochondrial cardiomyopathy without affecting the mammalian UPRmt.

The mitochondrial matrix protease CLPP plays a central role in the activation of the mitochondrial unfolded protein response (UPR(mt)) in Caenorhabditis elegans Far less is known about mammalian UPR(mt) signaling, although similar roles were assumed for central players, including CLPP To better understand the mammalian UPR(mt) signaling, we deleted CLPP in hearts of DARS2-deficient animals that show robust induction of UPR(mt) due to strong dysregulation of mitochondrial translation. Remarkably, our results clearly show that mammalian CLPP is neither required for, nor it regulates the UPR(mt) in mammals. Surprisingly, we demonstrate that a strong mitochondrial cardiomyopathy and diminished respiration due to DARS2 deficiency can be alleviated by the loss of CLPP, leading to an increased de novo synthesis of individual OXPHOS subunits. These results question our current understanding of the UPR(mt) signaling in mammals, while introducing CLPP as a possible novel target for therapeutic intervention in mitochondrial diseases.

Different faces of mitochondrial DNA mutators.

A number of studies have shown that ageing is associated with increased amounts of mtDNA deletions and/or point mutations in a variety of species as diverse as Caenorhabditis elegans, Drosophila melanogaster, mice, rats, dogs, primates and humans. This detected vulnerability of mtDNA has led to the suggestion that the accumulation of somatic mtDNA mutations might arise from increased oxidative damage and could play an important role in the ageing process by producing cells with a decreased oxidative capacity. However, the vast majority of DNA polymorphisms and disease-causing base-substitution mutations and age-associated mutations that have been detected in human mtDNA are transition mutations. They are likely arising from the slight infidelity of the mitochondrial DNA polymerase. Indeed, transition mutations are also the predominant type of mutation found in mtDNA mutator mice, a model for premature ageing caused by increased mutation load due to the error prone mitochondrial DNA synthesis. These particular misincorporation events could also be exacerbated by dNTP pool imbalances. The role of different repair, replication and maintenance mechanisms that contribute to mtDNA integrity and mutagenesis will be discussed in details in this article. This article is part of a Special Issue entitled: Mitochondrial Dysfunction in Aging.

Suppressed basal mitophagy drives cellular aging phenotypes that can be reversed by a p62-targeting small molecule.

Selective degradation of damaged mitochondria by autophagy (mitophagy) is proposed to play an important role in cellular homeostasis. However, the molecular mechanisms and the requirement of mitochondrial quality control by mitophagy for cellular physiology are poorly understood. Here, we demonstrated that primary human cells maintain highly active basal mitophagy initiated by mitochondrial superoxide signaling. Mitophagy was found to be mediated by PINK1/Parkin-dependent pathway involving p62 as a selective autophagy receptor (SAR). Importantly, this pathway was suppressed upon the induction of cellular senescence and in naturally aged cells, leading to a robust shutdown of mitophagy. Inhibition of mitophagy in proliferating cells was sufficient to trigger the senescence program, while reactivation of mitophagy was necessary for the anti-senescence effects of NAD precursors or rapamycin. Furthermore, reactivation of mitophagy by a p62-targeting small molecule rescued markers of cellular aging, which establishes mitochondrial quality control as a promising target for anti-aging interventions.

A cluster of pathogenic mutations in the 3'-5' exonuclease domain of DNA polymerase gamma defines a novel module coupling DNA synthesis and degradation.

Mutations in DNA polymerase gamma (pol g), the unique replicase inside mitochondria, cause a broad and complex spectrum of diseases in human. We have used Mip1, the yeast pol g, as a model enzyme to characterize six pathogenic pol g mutations. Four mutations clustered in a highly conserved 3'-5' exonuclease module are localized in the DNA-binding channel in close vicinity to the polymerase domain. They result in an increased frequency of point mutations and high instability of the mitochondrial DNA (mtDNA) in yeast cells, and unexpectedly for mutator mutations in the exonuclease domain, they favour exonucleolysis versus polymerization. This trait is associated with highly decreased DNA-binding affinity and poorly processive DNA synthesis. Our data show for the first time that a 3'-5' exonuclease module of pol g plays a crucial role in the coordination of the polymerase and exonuclease functions and they strongly suggest that in patients the disease is not caused by defective proofreading but results from poor mtDNA replication generated by a severe imbalance between DNA synthesis and degradation.

Mitochondrial matrix proteases: quality control and beyond.

To ensure correct function, mitochondria have developed several mechanisms of protein quality control (QC). Protein homeostasis highly relies on chaperones and proteases to maintain proper folding and remove damaged proteins that might otherwise form cell-toxic aggregates. Besides quality control, mitochondrial proteases modulate and regulate many essential functions, such as trafficking, processing and activation of mitochondrial proteins, mitochondrial dynamics, mitophagy and apoptosis. Therefore, the impaired function of mitochondrial proteases is associated with various pathological conditions, including cancer, metabolic syndromes and neurodegenerative disorders. This review recapitulates and discusses the emerging roles of two major proteases of the mitochondrial matrix, LON and ClpXP. Although commonly acknowledge for their protein quality control role, recent advances have uncovered several highly regulated processes controlled by the LON and ClpXP connected to mitochondrial gene expression and respiratory chain function maintenance. Furthermore, both proteases have been lately recognized as potent targets for anticancer therapies, and we summarize those findings.

Ovarian carcinoma immunoreactive antigen-like protein 2 (OCIAD2) is a novel complex III-specific assembly factor in mitochondria.

Assembly of the dimeric complex III (CIII2) in the mitochondrial inner membrane is an intricate process in which several accessory proteins are involved as assembly factors. Despite numerous studies, this process has yet to be fully understood. Here we report the identification of human OCIAD2 (ovarian carcinoma immunoreactive antigen-like protein 2) as an assembly factor for CIII2. OCIAD2 was found to be deregulated in several carcinomas and also in some neurogenerative disorders; however, its nonpathological role had not been elucidated.  We have shown that OCIAD2 localizes to mitochondria and interacts with electron transport chain (ETC) proteins. Complete loss of OCIAD2 using gene editing in HEK293 cells resulted in abnormal mitochondrial morphology, a substantial decrease of both CIII2 and supercomplex III2+IV, and a reduction in CIII enzymatic activity. Identification of OCIAD2 as a protein required for assembly of functional CIII2 provides a new insight into the biogenesis and architecture of the ETC. Elucidating the mechanism of OCIAD2 action is important both for the understanding of cellular metabolism and for an understanding of its role in malignant transformation.

CLUH controls astrin-1 expression to couple mitochondrial metabolism to cell cycle progression.

Proliferating cells undergo metabolic changes in synchrony with cell cycle progression and cell division. Mitochondria provide fuel, metabolites, and ATP during different phases of the cell cycle, however it is not completely understood how mitochondrial function and the cell cycle are coordinated. CLUH (clustered mitochondria homolog) is a post-transcriptional regulator of mRNAs encoding mitochondrial proteins involved in oxidative phosphorylation and several metabolic pathways. Here, we show a role of CLUH in regulating the expression of astrin, which is involved in metaphase to anaphase progression, centrosome integrity, and mTORC1 inhibition. We find that CLUH binds both the SPAG5 mRNA and its product astrin, and controls the synthesis and the stability of the full-length astrin-1 isoform. We show that CLUH interacts with astrin-1 specifically during interphase. Astrin-depleted cells show mTORC1 hyperactivation and enhanced anabolism. On the other hand, cells lacking CLUH show decreased astrin levels and increased mTORC1 signaling, but cannot sustain anaplerotic and anabolic pathways. In absence of CLUH, cells fail to grow during G1, and progress faster through the cell cycle, indicating dysregulated matching of growth, metabolism, and cell cycling. Our data reveal a role of CLUH in coupling growth signaling pathways and mitochondrial metabolism with cell cycle progression.

FGF21 modulates mitochondrial stress response in cardiomyocytes only under mild mitochondrial dysfunction.

The mitochondrial integrated stress response (mitoISR) has emerged as a major adaptive pathway to respiratory chain deficiency, but both the tissue specificity of its regulation, and how mitoISR adapts to different levels of mitochondrial dysfunction are largely unknown. Here, we report that diverse levels of mitochondrial cardiomyopathy activate mitoISR, including high production of FGF21, a cytokine with both paracrine and endocrine function, shown to be induced by respiratory chain dysfunction. Although being fully dispensable for the cell-autonomous and systemic responses to severe mitochondrial cardiomyopathy, in the conditions of mild-to-moderate cardiac OXPHOS dysfunction, FGF21 regulates a portion of mitoISR. In the absence of FGF21, a large part of the metabolic adaptation to mitochondrial dysfunction (one-carbon metabolism, transsulfuration, and serine and proline biosynthesis) is strongly blunted, independent of the primary mitoISR activator ATF4. Collectively, our work highlights the complexity of mitochondrial stress responses by revealing the importance of the tissue specificity and dose dependency of mitoISR.

Remission of obesity and insulin resistance is not sufficient to restore mitochondrial homeostasis in visceral adipose tissue.

Metabolic plasticity is the ability of a biological system to adapt its metabolic phenotype to different environmental stressors. We used a whole-body and tissue-specific phenotypic, functional, proteomic, metabolomic and transcriptomic approach to systematically assess metabolic plasticity in diet-induced obese mice after a combined nutritional and exercise intervention. Although most obesity and overnutrition-related pathological features were successfully reverted, we observed a high degree of metabolic dysfunction in visceral white adipose tissue, characterized by abnormal mitochondrial morphology and functionality. Despite two sequential therapeutic interventions and an apparent global healthy phenotype, obesity triggered a cascade of events in visceral adipose tissue progressing from mitochondrial metabolic and proteostatic alterations to widespread cellular stress, which compromises its biosynthetic and recycling capacity. In humans, weight loss after bariatric surgery showed a transcriptional signature in visceral adipose tissue similar to our mouse model of obesity reversion. Overall, our data indicate that obesity prompts a lasting metabolic fingerprint that leads to a progressive breakdown of metabolic plasticity in visceral adipose tissue.

[The assessment of cynacalcet (Mimpara) accompanied by alfacalcidol treatment efficacy in haemodialysis patients with different secondary hyperparathyroidism severity recognized by iPTH]. / Ocena skutecznosci cynakalcetu (Mimpara) i alfakalcydolu w leczeniu róznych stadiów zaawansowania wtórnej nadczynnosci przytarczyc monitorowanych stezeniami iPTH u chorych przewlekle hemodializowanych.

INTRODUCTION: Calcimimetics are highly efficient drugs in treatment of secondary hyperparathyroidism (sHPT) in patients on haemodialysis. The effect and the dose of cinacalcet may depend on severity of sHPT, and alfacalcidol supplementation helps in the treatment optimization. The study evaluated cinacalcet and alfacalcidol treatment efficacy in haemodialysis patients with different secondary hyperparathyroidism severity recognized by iPTH. MATERIAL AND METHODS: The study group comprised of 82 participants (male 67 and 34 female) in aged from 36 to 75 years, on haemodialysis. All patients were divided into two groups: the study group--40 participants treated with cinacalcet accompanied by alfacalcidol started after 8 months of the study (0.25 to 0.5 microg/day) and the control group--42 patients. The study group comprises of two subgroups: I--moderate sHPT with iPTH 500 to 800 pg/ml and II--severe sHPT with iPTH > 800 pg/ml. The basic phosphate binder treatment throughout the study period in all groups was calcium carbonate. RESULTS: In the subgroup I initial mean iPTH 700 +/- 129 pg/ml was reduced to 550 +/- 61 pg/ml (p < 0.05) in the third month with no need of the Mimpara dose change. No further iPTH decrease up to eighth month of the treatment was observed despite the cinacalcet dose increase to 53 mg (p < 0.05). The alfacalcidol supplementation decreased iPTH to 331 +/-55 pg/ml (p < 0.05) and the cynacalcet dose to 42 mg (p < 0.05). In the II subgroup iPTH was reduced from 1035 +/- 149 pg/ml to 885 +/- 101 pg/ml (p < 0.05) in the third month of the treatment and Mimpara dose changed to 90 mg. Up to eighth month iPTH did not change (790 +/- 92 pg/ml; p > 0.05) despite the cinacalcet dose increase to 122 mg (p < 0.05). The alfacalcidol supplementation induced iPTH reduction to 622 +/- 71 pg/ml (p < 0.05) and the cinacalcet dose to 100 mg (p < 0.05). CONCLUSIONS: Cinacalcet convinced its effectiveness in the iPTH serum concentration control in haemodialysis patients independently of secondary hyperparathyroidism severity and alfacalcidol supplementation enhanced its efficacy. Still in case of the late introduction of Mimpara this drug was recognized as potent however the efficient dose was mandatory multiply.

Tune instead of destroy: How proteolysis keeps OXPHOS in shape.

Mitochondria are highly dynamic and stress-responsive organelles that are renewed, maintained and removed by a number of different mechanisms. Recent findings bring more evidence for the focused, defined, and regulatory function of the intramitochondrial proteases extending far beyond the traditional concepts of damage control and stress responses. Until recently, the macrodegradation processes, such as mitophagy, were promoted as the major regulator of OXPHOS remodelling and turnover. However, the spatiotemporal dynamics of the OXPHOS system can be greatly modulated by the intrinsic mitochondrial mechanisms acting apart from changes in the global mitochondrial dynamics. This, in turn, may substantially contribute to the shaping of the metabolic status of the cell.

Adaptation to mitochondrial stress requires CHOP-directed tuning of ISR.

In response to disturbed mitochondrial gene expression and protein synthesis, an adaptive transcriptional response sharing a signature of the integrated stress response (ISR) is activated. We report an intricate interplay between three transcription factors regulating the mitochondrial stress response: CHOP, C/EBPß, and ATF4. We show that CHOP acts as a rheostat that attenuates prolonged ISR, prevents unfavorable metabolic alterations, and postpones the onset of mitochondrial cardiomyopathy. Upon mitochondrial dysfunction, CHOP interaction with C/EBPß is needed to adjust ATF4 levels, thus preventing overactivation of the ATF4-regulated transcriptional program. Failure of this interaction switches ISR from an acute to a chronic state, leading to early respiratory chain deficiency, energy crisis, and premature death. Therefore, contrary to its previously proposed role as a transcriptional activator of mitochondrial unfolded protein response, our results highlight a role of CHOP in the fine-tuning of mitochondrial ISR in mammals.