Accumulation Of Defective Mitochondria Research Articles

Mitophagy in relation to chronic inflammation/ROS in aging.

Various assaults on mitochondria occur during the human aging process, contributing to mitochondrial dysfunction. This mitochondrial dysfunction is intricately connected with aging and diseases associated with it. In vivo, the accumulation of defective mitochondria can precipitate inflammatory and oxidative stress, thereby accelerating aging. Mitophagy, an essential selective autophagy process, plays a crucial role in managing mitochondrial quality control and homeostasis. It is a highly specialized mechanism that systematically removes damaged or impaired mitochondria from cells, ensuring their optimal functioning and survival. By engaging in mitophagy, cells are able to maintain a balanced and stable environment, free from the potentially harmful effects of dysfunctional mitochondria. An ever-growing body of research highlights the significance of mitophagy in both aging and age-related diseases. Nonetheless, the association between mitophagy and inflammation or oxidative stress induced by mitochondrial dysfunction remains ambiguous. We review the fundamental mechanisms of mitophagy in this paper, delve into its relationship with age-related stress, and propose suggestions for future research directions.

USP8 Down-Regulation Promotes Parkin-Independent Mitophagy in the Drosophila Brain and in Human Neurons.

Stress-induced mitophagy, a tightly regulated process that targets dysfunctional mitochondria for autophagy-dependent degradation, mainly relies on two proteins, PINK1 and Parkin, which genes are mutated in some forms of familiar Parkinson's Disease (PD). Upon mitochondrial damage, the protein kinase PINK1 accumulates on the organelle surface where it controls the recruitment of the E3-ubiquitin ligase Parkin. On mitochondria, Parkin ubiquitinates a subset of mitochondrial-resident proteins located on the outer mitochondrial membrane, leading to the recruitment of downstream cytosolic autophagic adaptors and subsequent autophagosome formation. Importantly, PINK1/Parkin-independent mitophagy pathways also exist that can be counteracted by specific deubiquitinating enzymes (DUBs). Down-regulation of these specific DUBs can presumably enhance basal mitophagy and be beneficial in models in which the accumulation of defective mitochondria is implicated. Among these DUBs, USP8 is an interesting target because of its role in the endosomal pathway and autophagy and its beneficial effects, when inhibited, in models of neurodegeneration. Based on this, we evaluated autophagy and mitophagy levels when USP8 activity is altered. We used genetic approaches in D. melanogaster to measure autophagy and mitophagy in vivo and complementary in vitro approaches to investigate the molecular pathway that regulates mitophagy via USP8. We found an inverse correlation between basal mitophagy and USP8 levels, in that down-regulation of USP8 correlates with increased Parkin-independent mitophagy. These results suggest the existence of a yet uncharacterized mitophagic pathway that is inhibited by USP8.

Defective PTEN-induced kinase 1/Parkin mediated mitophagy and neurodegenerative diseases.

The selective degradation of mitochondria through mitophagy is a crucial process for maintaining mitochondrial function and cellular health. Mitophagy is a specialized form of selective autophagy that uses unique machinery to recognize and target damaged mitochondria for mitophagosome- and lysosome-dependent degradation. This process is particularly important in cells with high metabolic activity like neurons, and the accumulation of defective mitochondria is a common feature among neurodegenerative disorders. Here, we describe essential steps involved in the induction and progression of mitophagy, and then highlight the various mechanisms that specifically contribute to defective mitophagy in highly prevalent neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and Amyotrophic Lateral Sclerosis.

Mitochondria-affecting small molecules ameliorate proteostasis defects associated with neurodegenerative diseases

Macroautophagic recycling of dysfunctional mitochondria, known as mitophagy, is essential for mitochondrial homeostasis and cell viability. Accumulation of defective mitochondria and impaired mitophagy have been widely implicated in many neurodegenerative diseases, and loss-of-function mutations of PINK1 and Parkin, two key regulators of mitophagy, are amongst the most common causes of heritable parkinsonism. This has led to the hypothesis that pharmacological stimulation of mitophagy may be a feasible approach to combat neurodegeneration. Toward this end, we screened ~ 45,000 small molecules using a high-throughput, whole-organism, phenotypic screen that monitored accumulation of PINK-1 protein, a key event in mitophagic activation, in a Caenorhabditis elegans strain carrying a Ppink-1::PINK-1::GFP reporter. We obtained eight hits that increased mitochondrial fragmentation and autophagosome formation. Several of the compounds also reduced ATP production, oxygen consumption, mitochondrial mass, and/or mitochondrial membrane potential. Importantly, we found that treatment with two compounds, which we named PS83 and PS106 (more commonly known as sertraline) reduced neurodegenerative disease phenotypes, including delaying paralysis in a C. elegans β-amyloid aggregation model in a PINK-1-dependent manner. This report presents a promising step toward the identification of compounds that will stimulate mitochondrial turnover.

Abstract P400: Treatment With The AMPK Agonist AICAR Alleviates Age-associated Cardiac Defects In The Mouse By Distinct Sex-specific Mechanisms

Heart failure is a major cause of mortality in the elderly. Features of cardiac aging include diastolic dysfunction and interstitial fibrosis with sex-specific differences. We treated old male and female mice (21 months-old) 3 times a week for 3 months with the AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR, 0.166 mg/g BW). We previously reported that AICAR normalizes the aged fibroblast phenotype. In a longitudinal study, we found that AICAR attenuates the age-associated increase in left atrial volume, an indirect indicator of diastolic dysfunction, in female AICAR-treated mice (-29% ±5%) but not in males (-7% ±6%). Cardiac fibroblasts from AICAR-treated mice expressed decreased pro-collagen levels by 42% (mean fluorescence intensity, P=0.0003 for females, and P=0.05 for males). Cardiac fibroblasts cultured on decellularized cardiac matrices also had a reduced expression of pro-collagen when treated with AICAR (0.5mM, 7 days). Myocardial hydroxyproline level (an indicator of total collagen content) was reduced in female hearts (from 0.83 ± 0.07 in controls to 0.48 ± 0.05 in AICAR-treated, P=0.006). Female cells also exhibited a reduced expression of periostin after treatment (by 65%, P=0.02). By contrast, age-matched control males had a lower cardiac level of hydroxyproline (-65%, P=0.0002) and periostin (-45%, P=0.0004) than females, and were not affected by the treatment. Accumulation of defective mitochondria is a hallmark of aging. Since AICAR can favor mitophagy, we isolated mitochondrial fractions from the hearts of old mice undergoing treatment. We found that AICAR decreased Parkin level in the small mitochondria fraction (0.75±0.09, P=0.028) in males, but no significant change was found between the groups of females. The reduction of Parkin may suggest improved clearance of defective mitochondria.For all experiments, we used 4-10 animals per group. One-way Anova or student’s T-test evaluated statistical significance. In conclusion, age-associated cardiac remodeling leads to distinct patterns between male and female mice. AICAR treatment can have sex-specific effects: it reduces fibrosis in females but may promote mitophagy in males, translating into an improvement of heart function via distinct mechanisms.

Coenzyme Q redox signalling and longevity

Mitochondria are the powerhouses of the cell. They produce a significant amount of the energy we need to grow, survive and reproduce. The same system that generates energy in the form of ATP also produces Reactive Oxygen Species (ROS). Mitochondrial Reactive Oxygen Species (mtROS) were considered for many years toxic by-products of metabolism, responsible for ageing and many degenerative diseases. Today, we know that mtROS are essential redox messengers required to determine cell fate and maintain cellular homeostasis. Most mtROS are produced by respiratory complex I (CI) and complex III (CIII). How and when CI and CIII produce ROS is determined by the redox state of the Coenzyme Q (CoQ) pool and the proton motive force (pmf) generated during respiration. During ageing, there is an accumulation of defective mitochondria that generate high levels of mtROS. This causes oxidative stress and disrupts redox signalling. Here, we review how mtROS are generated in young and old mitochondria and how CI and CIII derived ROS control physiological and pathological processes. Finally, we discuss why damaged mitochondria amass during ageing as well as methods to preserve mitochondrial redox signalling with age.

Aberrant mitochondrial morphology and function associated with impaired mitophagy and DNM1L-MAPK/ERK signaling are found in aged mutant Parkinsonian LRRK2R1441G mice

ABSTRACT Mitochondrial dysfunction causes energy deficiency and nigrostriatal neurodegeneration which is integral to the pathogenesis of Parkinson disease (PD). Clearance of defective mitochondria involves fission and ubiquitin-dependent degradation via mitophagy to maintain energy homeostasis. We hypothesize that LRRK2 (leucine-rich repeat kinase 2) mutation disrupts mitochondrial turnover causing accumulation of defective mitochondria in aging brain. We found more ubiquitinated mitochondria with aberrant morphology associated with impaired function in aged (but not young) LRRK2R1441G knockin mutant mouse striatum compared to wild-type (WT) controls. LRRK2R1441G mutant mouse embryonic fibroblasts (MEFs) exhibited reduced MAP1LC3/LC3 activation indicating impaired macroautophagy/autophagy. Mutant MEFs under FCCP-induced (mitochondrial uncoupler) stress showed increased LC3-aggregates demonstrating impaired mitophagy. Using a novel flow cytometry assay to quantify mitophagic rates in MEFs expressing photoactivatable mito-PAmCherry, we found significantly slower mitochondria clearance in mutant cells. Specific LRRK2 kinase inhibition using GNE-7915 did not alleviate impaired mitochondrial clearance suggesting a lack of direct relationship to increased kinase activity alone. DNM1L/Drp1 knockdown in MEFs slowed mitochondrial clearance indicating that DNM1L is a prerequisite for mitophagy. DNM1L knockdown in slowing mitochondrial clearance was less pronounced in mutant MEFs, indicating preexisting impaired DNM1L activation. DNM1L knockdown disrupted mitochondrial network which was more evident in mutant MEFs. DNM1L-Ser616 and MAPK/ERK phosphorylation which mediate mitochondrial fission and downstream mitophagic processes was apparent in WT using FCCP-induced stress but not mutant MEFs, despite similar total MAPK/ERK and DNM1L levels. In conclusion, aberrant mitochondria morphology and dysfunction associated with impaired mitophagy and DNM1L-MAPK/ERK signaling are found in mutant LRRK2 MEFs and mouse brain. Abbreviations: ATP: adenosine triphosphate; BAX: BCL2-associated X protein; CDK1: cyclin-dependent kinase 1; CDK5: cyclin-dependent kinase 5; CQ: chloroquine; CSF: cerebrospinal fluid; DNM1L/DRP1: dynamin 1-like; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; LAMP2A: lysosomal-associated membrane protein 2A; LRRK2: leucine-rich repeat kinase 2; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MEF: mouse embryonic fibroblast; MFN1: mitofusin 1; MMP: mitochondrial membrane potential; PAmCherry: photoactivatable-mCherry; PD: Parkinson disease; PINK1: PTEN induced putative kinase 1; PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; RAB10: RAB10, member RAS oncogene family; RAF: v-raf-leukemia oncogene; SNCA: synuclein, alpha; TEM: transmission electron microscopy; VDAC: voltage-dependent anion channel; WT: wild type; SQSTM1/p62: sequestosome 1.

Regulation of Mitophagy By O-Linked N-Acetylglucosamine Transferase Is Essential for Hematopoietic Stem Cell Maintenance

Hematopoietic stem cells (HSCs) reside in hypoxic niche in the bone marrow (BM), where they are maintained in quiescent state. To adapt to this hypoxic microenvironment, HSCs generate ATP mainly from glycolysis, while suppressing oxidative phosphorylation in mitochondria. Through this metabolic regulation, HSCs protect themselves from oxidative stress caused by reactive oxygen species (ROS). ROS levels are controlled by various mechanisms including autophagy, which is one of the critical mechanisms regulating HSC function mainly through maintaining mitochondrial homeostasis. Glycosylation of serine or threonine residues with O-linked N-acetylglucosamine (O-GlcNAcylation) by O-linked N-acetylglucosamine transferase (OGT) is crucial for regulating various protein functions, and dysregulation of O-GlcNAcylation is critically linked to metabolic or degenerative diseases (i.e. diabetes, Alzheimer disease), tumorigenesis and aging. In hematopoietic system, OGT is essential for differentiation and proliferation of T and B cells. However, a role for OGT in the function of hematopoietic stem and progenitor cells (HSPCs) remains elusive. To elucidate a role for OGT in HSPCs, we analyzed the effect of Ogt loss on HSPCs using Ogt-conditional knockout mice. Conditional disruption of Ogt in adult mice led to pancytopenia and significantly reduced numbers of HSPCs in the BM. In addition, Ogt-deficient HSCs exhibited loss of quiescence, increased apoptosis and impaired long-term repopulation as well as self-renewal capacities by serial competitive repopulation assays. Interestingly, ROS levels were significantly increased in Ogt-deficient HSPCs, and the treatment of mice with N-acetyl cysteine (NAC), a scavenger for ROS, at least partially rescued the phenotype of Ogt-deficient hematopoietic cells in in vivo settings. MitoSOX analysis showed that excessive ROS was generated mainly from mitochondria in Ogt-deficient HSCs. In addition, mitochondrial mass was significantly increased in Ogt-deficient HSCs. Assessment of mitochondrial quality by its membrane potential and extracellular flux analysis revealed that mitochondria of Ogt-deficient HSPCs had significantly lower membrane potential and lower spare respiratory capacity. Morphologically, mitochondria in Ogt-deficient HSPCs were enlarged and swollen with disorganized cristae. These findings suggest that inactivation of Ogt leads to loss of HSPCs and impaired HSC homeostasis, which is due, at least in part, to elevated ROS levels with accumulation of defective mitochondria. Since mitophagy (mitochondria-specific autophagy) plays an important role in quality control of mitochondria, we examined gene expressions of mitophagy regulators by RNA sequencing. This revealed that disruption of Ogt led to down-regulation of key mitophagy inducers, Pink1 and Bnip3. These findings together with the accumulation of defective mitochondria indicated that inactivation of Ogt impairs mitophagy probably due to reduced expression of Pink1 and Bnip3. OGT is known to regulate gene expressions by modifying trimethylation of lysine 4 on the histone H3 (H3K4me3) through proteolytic activation of HCF-1, which is a critical component of SET1/COMPASS complex. Chromatin immunoprecipitation (ChIP) assay showed significantly reduced levels of H3K4me3 at the transcriptional start sites of Pink1 and Bnip3 in Ogt-deficient HSPCs. Next we asked whether defective HSPC homeostasis caused by Ogt disruption could be rescued by restoring mitophagy in Ogt-deficient HSPCs through overexpressing Pink1, a critical initiator of mitophagy. As expected, Pink1 overexpression efficiently restored mitophagy in Ogt-deficient HSCs as shown by normalization of mitochondrial mass. Interestingly, a number and apoptosis of Ogt-deficient HSCs were restored to levels similar to those of wild-type HSCs. These data strongly indicate that Pink1 functions as a key downstream effector of OGT in HSC maintenance. In summary, our results revealed that OGT plays an essential role in HSC maintenance by assuring mitochondrial quality through mitophagy. DisclosuresNo relevant conflicts of interest to declare.

Abstract 3963: Cystine depleting human enzyme combined with auranofin synergistically inhibits pancreatic cancer cell growth in vitro and in vivo

Abstract Pancreatic ductal adenocarcinoma (PDAC) has one of the lowest 5-year survival rates of all cancers at 8%. Upregulated mechanisms to curb reactive oxygen species (ROS) accumulation are thought to help the growth of PDACs. Hence, perturbation of oxidative balance might be an effective therapeutic strategy for pancreatic cancer. To achieve this end, we used a human enzyme called Cyst(e)inase that degrades extracellular cysteine (Cys) and cystine (CSSC) - substrates that are necessary for synthesis of the antioxidant glutathione (GSH). Since tumor cells need to supplement intracellular Cys synthesis with extracellular import in order to fulfil their increased demand for GSH and protein synthesis, Cys/CSSC depletion can provide a therapeutic window. We validated this idea in our previous study where Cyst(e)inase treatment markedly inhibited tumor growth in several cancer models in vivo without any toxicity. In this study, we assessed the efficacy of Cyst(e)inase alone as well as in combination with auranofin in reducing pancreatic tumor growth. Cyst(e)inase treatment of cultured pancreatic cancer cells (Panc1, MIA-PaCa2, BxPC3) decreased intracellular Cys and GSH, and inhibited cell growth. Panc1 was very sensitive to Cyst(e)inase in vitro, exhibiting ROS accumulation and activation of stress-related signaling as well as G2-arrest and apoptotic cell death whereas BxPC3 and MIA-PaCa2 showed only a G1-arrest. Inhibition of the mTORC1-p70S6K-S6 ribosomal protein signaling pathway via the eIF2α-ATF4-Sestrin2 axis was observed in all 3 cell lines. In addition, MIA-PaCa2 but not Panc1 exhibited a rewiring of mitochondrial metabolism upon Cyst(e)inase treatment that bypassed succinate dehydrogenase. Combining auranofin and Cyst(e)inase caused synergistic increase in mitochondrial ROS and apoptotic cell death in the resistant cell lines MIA-PaCa2 and BxPC3 with associated activation of oxidative stress signaling. Surprisingly, the thioredoxin reductase system (the putative target of auranofin) was dispensable in producing synergy with Cyst(e)inase. Moreover, auranofin and Cyst(e)inase combination led to inhibition of mitophagy and accumulation of defective mitochondria. Supplementation with the antioxidant glutathione ethyl ester rescued the combination's cytotoxic effects. Biweekly intraperitoneal Cyst(e)inase treatment of nude mice harboring pancreatic cancer xenografts roughly recapitulated the in vitro sensitivity profile with Panc1 showing the most growth inhibition, albeit modest. BxPC3 xenografts were completely resistant to Cyst(e)inase alone but adding auranofin significantly inhibited their growth without causing any systemic toxicity. Collectively, the current data suggest that combining Cyst(e)inase and auranofin, an approved drug for rheumatoid arthritis, might constitute an effective therapy for pancreatic cancer. Citation Format: Sabin Kshattry, George Georgiou, Stefano Tiziani, Achinto Saha, Everett Stone, John DiGiovanni. Cystine depleting human enzyme combined with auranofin synergistically inhibits pancreatic cancer cell growth in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3963.

Regulation of mitophagy by the ubiquitin pathway in neurodegenerative diseases.

Mitophagy is a cellular process by which dysfunctional mitochondria are degraded via autophagy. Increasing empirical evidence proposes that this mitochondrial quality-control mechanism is defective in neurons of patients with various neurodegenerative diseases such as Ataxia Telangiectasia, Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis. Accumulation of defective mitochondria and the production of reactive oxygen species due to defective mitophagy have been identified as causes underlying neurodegenerative disease pathogenesis. However, the reason mitophagy is defective in most neurodegenerative diseases is unclear. Like mitophagy, defects in the ubiquitin/26S proteasome pathway have been linked to neurodegeneration, resulting in the characteristic protein aggregates often seen in neurons of affected patients. Although initiation of mitophagy requires a functional ubiquitin pathway, whether defects in the ubiquitin pathway are causally responsible for defective mitophagy is not known. In this mini-review, we introduce mitophagy and ubiquitin pathways and provide a summary of our current understanding of the regulation of mitophagy by the ubiquitin pathway. We will then briefly review empirical evidence supporting mitophagy defects in neurodegenerative diseases. The review will conclude with a discussion of the constitutively elevated expression of ubiquitin-like protein Interferon-Stimulated Gene 15 (ISG15), an antagonist of the ubiquitin pathway, as a potential cause of defective mitophagy in neurodegenerative diseases. Impact statement Neurodegenerative diseases place an enormous burden on patients and caregivers globally. Over six million people in the United States alone suffer from neurodegenerative diseases, all of which are chronic, incurable, and with causes unknown. Identifying a common molecular mechanism underpinning neurodegenerative disease pathology is urgently needed to aid in the design of effective therapies to ease suffering, reduce economic cost, and improve the quality of life for these patients. Although the development of neurodegeneration may vary between neurodegenerative diseases, they have common cellular hallmarks, including defects in the ubiquitin-proteasome system and mitophagy. In this review, we will provide a summary of our current understanding of the regulation of mitophagy by the ubiquitin pathway and discuss the potential of targeting mitophagy and ubiquitin pathways for the treatment of neurodegeneration.

Abstract IA05: Autophagy prevents fatal nucleotide pool depletion in Ras-driven cancer cells

Abstract Autophagy degrades and recycles proteins and organelles essential for quality control and survival in starvation. In genetically engineered mouse models (GEMMs) for Kras-driven lung cancer, autophagy prevents the accumulation of defective mitochondria and promotes tumor growth and malignancy. Autophagy-deficient tumor-derived cell lines (TDCLs) are respiration impaired, starvation sensitive, and glutamine-dependent. To survive starvation, autophagy may eliminate defective mitochondria or supply mitochondrial substrates. Here, we sequenced the mitochondrial genome from Kras-driven lung tumors and found higher mutant allele frequencies without autophagy-related-7 (Atg7). But the low pathogenic allele frequency was insufficient to account for metabolic defects. In starvation, Atg7 deficiency attenuated substrate supply for mitochondrial oxygen consumption and increased nucleotide degradation. The resulting nucleotide pool depletion and energy crisis in starvation was rescued by nucleoside supplementation. Thus the role for autophagy in Kras-driven tumor cells is to provide substrates to mitochondria to maintain nucleotide pools and energy homeostasis, which promotes survival to nutrient limitation. Citation Format: Eileen White. Autophagy prevents fatal nucleotide pool depletion in Ras-driven cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA05.

Mitochondrial DNA mutations distinguish bilateral multifocal renal oncocytomas from familial Birt–Hogg–Dubé tumors

Oncocytomas are mostly benign tumors characterized by accumulation of defective mitochondria, and in sporadic cases, are associated with disruptive mitochondrial DNA (mtDNA) mutations. However, the role mtDNA mutations have in renal tumors of Birt–Hogg–Dubé (BHD) patients and other renal oncocytomas with an apparent genetic component has not been investigated to date. Here we characterize the mitochondrial genome in different renal tumors and investigate the possibility of employing mtDNA sequencing analyses of biopsy specimens to aid in the differential diagnosis of oncocytomas. The entire mitochondrial genome was sequenced in 25 samples of bilateral and multifocal (BMF) renal oncocytomas, 30 renal tumors from BHD patients and 36 non-oncocytic renal tumors of different histologies as well as in biopsy samples of kidney tumors. mtDNA sequencing in BMF oncocytomas revealed that all tumors carry disruptive mutations, which impair the assembly of the NADH-ubiquinone oxidoreductase. Multiple tumors from a given BMF oncocytoma patient mainly harbor the same somatic mutation and the kidneys of these patients display diffuse oncocytosis. In contrast, renal oncocytomas of patients with BHD syndrome and renal tumors with different histologies do not show disruptive mtDNA mutations. Moreover, we demonstrate that it is feasible to amplify and sequence the entire mtDNA in biopsy specimens, and that these sequences are representative of the tumor DNA. These results show that pathogenic mtDNA mutations affecting complex I of the respiratory chain are strongly correlated with the oncocytoma phenotype in non-BHD-related renal tumors and that mtDNA sequences from biopsies are predictive of the tumor genotype. This work supports a role for mtDNA mutations in respiratory chain complexes as diagnostic markers for renal oncocytomas.

A REDD1/TXNIP pro-oxidant complex regulates ATG4B activity to control stress-induced autophagy and sustain exercise capacity.

Macroautophagy (autophagy) is a critical cellular stress response; however, the signal transduction pathways controlling autophagy induction in response to stress are poorly understood. Here we reveal a new mechanism of autophagy control whose deregulation disrupts mitochondrial integrity and energy homeostasis in vivo. Stress conditions including hypoxia and exercise induce reactive oxygen species (ROS) through upregulation of a protein complex involving REDD1, an mTORC1 inhibitor and the pro-oxidant protein TXNIP. Decreased ROS in cells and tissues lacking either REDD1 or TXNIP increases catalytic activity of the redox-sensitive ATG4B cysteine endopeptidase, leading to enhanced LC3B delipidation and failed autophagy. Conversely, REDD1/TXNIP complex expression is sufficient to induce ROS, suppress ATG4B activity and activate autophagy. In Redd1−/− mice, deregulated ATG4B activity and disabled autophagic flux cause accumulation of defective mitochondria, leading to impaired oxidative phosphorylation, muscle ATP depletion and poor exercise capacity. Thus, ROS regulation through REDD1/TXNIP is physiological rheostat controlling stress-induced autophagy.

Mask loss-of-function rescues mitochondrial impairment and muscle degeneration of Drosophila pink1 and parkin mutants.

PTEN-induced kinase 1 (Pink1) and ubiquitin E3 ligase Parkin function in a linear pathway to maintain healthy mitochondria via regulating mitochondrial clearance and trafficking. Mutations in the two enzymes cause the familial form of Parkinson's disease (PD) in humans, as well as accumulation of defective mitochondria and cellular degeneration in flies. Here, we show that loss of function of a scaffolding protein Mask, also known as ANKHD1 (Ankyrin repeats and KH domain containing protein 1) in humans, rescues the behavioral, anatomical and cellular defects caused by pink1 or parkin mutations in a cell-autonomous manner. Moreover, similar rescue can also be achieved if Mask knock-down is induced in parkin adult flies when the mitochondrial dystrophy is already manifested. We found that Mask genetically interacts with Parkin to modulate mitochondrial morphology and negatively regulates the recruitment of Parkin to mitochondria. We also provide evidence that loss of Mask activity promotes co-localization of the autophagosome marker with mitochondria in developing larval muscle, and that an intact autophagy pathway is required for the rescue of parkin mutant defects by mask loss of function. Together, our data strongly suggest that Mask/ANKHD1 activity can be inhibited in a tissue- and timely-controlled fashion to restore mitochondrial integrity under PD-linked pathological conditions.

Role of autophagy in K-RAS- and B-RAF-driven lung cancers

Autophagy degrades and recycles proteins and organelles to support metabolism and survival starvation. Oncogenic RAS upregulates autophagy required for mitochondrial function, stress survival, and engrafted tumor growth. We deleted an essential autophagy gene, autophagy-related-7 (Atg7), concurrently with Kras G12D activation with or without intact Trp53 in two mouse models for non-small-cell lung cancer (NSCLC). In both models, Atg7 deficiency caused tumor cells to accumulate dysfunctional mitochondria, and acquire metabolic, growth and survival defects associated with reduction in tumor burden. Importantly, Atg7 deficiency altered the fate of Kras G12D -induced carcinomas to that of oncocytomas, rare, predominantly benign tumors characterized by the accumulation of defective mitochondria. Surprisingly, lipid accumulation was observed in Atg7-deficient tumors only when Trp53 was deleted. Atg7-deficient tumor-derived cell lines (TDCLs) had compromised starvation survival and formed lipidic cysts instead of tumors, suggesting defective utilization of lipid stores. Atg7 deficiency reduced fatty acid oxidation (FAO) and increased sensitivity to FAO inhibition, indicating that with Trp53 loss, RAS-driven tumors require autophagy for mitochondrial function and lipid catabolism. Thus, autophagy is required for carcinoma fate, and cancers require autophagy for distinct roles in metabolism that are oncogene- and tumor suppressor gene-specific. To test the role of autophagy in oncogenic signaling pathways downstream of RAS, Atg7 was deleted in a mouse model of BRAF V600E -induced lung cancer in the presence or absence of the tumor suppressor Trp53. Atg7 deletion initially induced oxidative stress and accelerated tumor cell proliferation in a manner indistinguishable from Nrf2 ablation. Compound deletion of Atg7 and Nrf2 had no additive effect suggesting that both genes modulate tumorigenesis by regulating oxidative stress, revealing a potential mechanism of autophagy-mediated tumor suppression. At later stages of tumorigenesis, Atg7 deficiency resulted in an accumulation of defective mitochondria, proliferative defects, reduced tumor burden, conversion of adenomas and adenocarcinomas to oncocytomas, and increased mouse lifespan. Autophagy-defective tumor-derived cell lines were defective in their ability to respire, survive starvation and were glutamine-dependent, suggesting that autophagy-supplied substrates from protein degradation sustains Braf V600E -tumor growth and metabolism.

Autophagy Sustains Mitochondrial Glutamine Metabolism and Growth of BrafV600E–Driven Lung Tumors

Autophagic elimination of defective mitochondria suppresses oxidative stress and preserves mitochondrial function. Here, the essential autophagy gene Atg7 was deleted in a mouse model of BrafV600E-induced lung cancer in the presence or absence of the tumor suppressor Trp53. Atg7 deletion initially induced oxidative stress and accelerated tumor cell proliferation in a manner indistinguishable from Nrf2 ablation. Compound deletion of Atg7 and Nrf2 had no additive effect, suggesting that both genes modulate tumorigenesis by regulating oxidative stress and revealing a potential mechanism of autophagy-mediated tumor suppression. At later stages of tumorigenesis, Atg7 deficiency resulted in an accumulation of defective mitochondria, proliferative defects, reduced tumor burden, conversion of adenomas and adenocarcinomas to oncocytomas, and increased mouse life span. Autophagy-defective tumor-derived cell lines were impaired in their ability to respire and survive starvation and were glutamine-dependent, suggesting that autophagy-supplied substrates from protein degradation sustains BrafV600E tumor growth and metabolism. The essential autophagy gene Atg7 functions to promote BrafV600E-driven lung tumorigenesis by preserving mitochondrial glutamine metabolism. This suggests that inhibiting autophagy is a novel approach to treating BrafV600E-driven cancers.

Autophagy suppresses progression of K-ras-induced lung tumors to oncocytomas and maintains lipid homeostasis

Macroautophagy (autophagy hereafter) degrades and recycles proteins and organelles to support metabolism and survival in starvation. Oncogenic Ras up-regulates autophagy, and Ras-transformed cell lines require autophagy for mitochondrial function, stress survival, and engrafted tumor growth. Here, the essential autophagy gene autophagy-related-7 (atg7) was deleted concurrently with K-ras(G12D) activation in mouse models for non-small-cell lung cancer (NSCLC). atg7-deficient tumors accumulated dysfunctional mitochondria and prematurely induced p53 and proliferative arrest, which reduced tumor burden that was partly relieved by p53 deletion. atg7 loss altered tumor fate from adenomas and carcinomas to oncocytomas-rare, predominantly benign tumors characterized by the accumulation of defective mitochondria. Surprisingly, lipid accumulation occurred in atg7-deficient tumors only when p53 was deleted. atg7- and p53-deficient tumor-derived cell lines (TDCLs) had compromised starvation survival and formed lipidic cysts instead of tumors, suggesting defective utilization of lipid stores. atg7 deficiency reduced fatty acid oxidation (FAO) and increased sensitivity to FAO inhibition, indicating that with p53 loss, Ras-driven tumors require autophagy for mitochondrial function and lipid catabolism. Thus, autophagy is required for carcinoma fate, and autophagy defects may be a molecular basis for the occurrence of oncocytomas. Moreover, cancers require autophagy for distinct roles in metabolism that are oncogene- and tumor suppressor gene-specific.

Abstract LB-107: Mitochondrial DNA mutations are diagnostic markers of renal oncocytomas.

Abstract Oncocytomas are mostly benign tumors of epithelial origin, characterized by dense accumulation of defective mitochondria. To date, no nuclear gene has been found to be responsible for these tumors, but a correlation of the oncocytic phenotype with the occurrence of disruptive mitochondrial DNA (mtDNA) mutations, mostly affecting genes encoding for proteins of Complex I of the respiratory chain (NADH dehydrogenase), has been shown in sporadic oncocytomas of different organs, including renal oncocytomas (RO). Kidney cancer is made up of a number of tumors that present with a different histology and clinical course. They are caused by different genes, and therefore therapy needs to be adapted for each type of renal tumor. Imaging techniques are usually not sufficient to distinguish between benign and malignant forms of renal cell cancer (RCC). In addition, it is difficult to differentiate benign ROs from malignant forms of RCCs in samples from biopsies, because they share ultrastructural appearances, including abnormal mitochondria with altered cristae. Therefore, a reliable diagnostic tool would be invaluable to guide clinicians in the management of patients with RCC of unclear phenotype. In our current work we tested whether mtDNA mutations can be used as diagnostic tools to distinguish between benign ROs and more aggressive forms of RCC. We sequenced the entire mtDNA in 36 samples that included sporadic, bilateral and multifocal as well as familial ROs and 30 non-oncocytic RCCs of different histologies. Moreover, the mitochondrial copy number was determined in all samples by real-time PCR. Furthermore, we sequenced the mtDNA from biopsies and compared it to the mtDNA sequence of the resected tumor. We found a strong correlation between the presence of disruptive mtDNA mutations affecting complex I genes and the oncocytic phenotype in renal tumors. Additionally, oncocytomas showed significantly higher mtDNA copy number when compared to other RCCs. We also show that the mtDNA sequences from the biopsies correspond to the sequence of the tumor itself and are predictive of the phenotype. Hence, we propose that sequencing of the mitochondrial genome, supported by the analysis of the mtDNA copy number, may be used as a diagnostic tool to distinguish between benign and aggressive forms of RCCs to guide physicians in choosing the appropriate treatment for patients. Citation Format: Martin Lang, Cathy D. Vocke, Maria J. Merino, Laura S. Schmidt, Marston W. Linehan. Mitochondrial DNA mutations are diagnostic markers of renal oncocytomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-107. doi:10.1158/1538-7445.AM2013-LB-107

Abstract 17276: Cyclophilin D is Required for Mitophagy-Regulating Parkin Translocation to Mitochondria

INTRODUCTION Cyclophilin D (CyD), encoded by the Ppif gene, is a mitochondrial protein that regulates the Mitochondria Permeability Transition Pore (MPTP) and is necessary for the removal of damaged mitochondria via autophagy in cardiac cells. Ppif -/- mice show an increase in the mitochondrial calcium retention capacity and a desensitization of MPTP opening. These features are associated with protection against ischemia/reperfusion injury. However recent finding show that CyD is involved in different physiological pathways important for cell homeostasis. HYPOTHESIS We tested the hypothesis that the CyD mediates mitochondrial removal by controlling Parkin translocation to the mitochondrial outer membrane. METHODS Cardiac mitochondria were isolated from wild type and Ppif -/- young (2 months) and old (20 months) mice according to standard protocol using a sucrose based buffer. RESULTS Western Blot analysis of total heart tissue lysate showed a 1.8 fold increase in the microtubule-associated protein1 light chain 3 (LC3) and a 2.2 fold increase in the total Parkin protein levels in the Ppif -/- young mice compared to age matched wild type mice. Parkin in the mitochondrial fraction was 2.0 fold less in the samples from young and old Ppif -/- mice compared to age-matched wild type mice. The diminished activity in the Parkin-dependent mitophagy results in accumulation of defective mitochondria, leading to metabolic reprogramming followed by pathological hypertrophy. While young Ppif -/- mice show normal phenotype, old mice develop cardiac hypertrophy with elevated HW/BW ratio and ANF compared to age-matched wild type mice. CONCLUSIONS The decrease in the Parkin-dependent mitophagy machinery activity observed in the Ppif -/- mice describe for the first time the requirement for CyD in the Parkin-dependent mitophagy and give a further explanation for the cardiac hypertrophic phenotype in old Ppif -/- mice.

Microtubule-associated Protein 1S (MAP1S) Bridges Autophagic Components with Microtubules and Mitochondria to Affect Autophagosomal Biogenesis and Degradation

The ubiquitously distributed MAP1S is a homologue of the exclusively neuronal distributed microtubule-associated protein 1A and 1B (MAP1A/B). They give rise to multiple isoforms through similar post-translational modification. Isoforms of MAP1S have been implicated in microtubule dynamics and mitotic abnormalities and mitotic cell death. Here we show that ablation of the Map1s gene in mice caused reduction in the B-cell CLL/lymphoma 2 or xL (Bcl-2/xL) and cyclin-dependent kinase inhibitor 1B (P27) protein levels, accumulation of defective mitochondria, and severe defects in response to nutritive stress, suggesting defects in autophagosomal biogenesis and clearance. Furthermore, MAP1S isoforms interacted with the autophagosome-associated light chain 3 of MAP1A/B (LC3), a homologue of yeast autophagy-related gene 8 (ATG8), and recruited it to stable microtubules in a MAP1S and LC3 isoform-dependent mode. In addition, MAP1S interacted with mitochondrion-associated leucine-rich PPR-motif containing protein (LRPPRC) that interacts with the mitophagy initiator and Parkinson disease-related protein Parkin. The three-way interactions of MAP1S isoforms with LC3 and microtubules as well as the interaction of MAP1S with LRPPRC suggest that MAP1S isoforms may play positive roles in integration of autophagic components with microtubules and mitochondria in both autophagosomal biogenesis and degradation. For the first time, our results clarify roles of MAP1S in bridging microtubules and mitochondria with autophagic and mitophagic initiation, maturation, trafficking, and lysosomal clearance. Defects in the MAP1S-regulated autophagy may impact heart disease, cancers, neurodegenerative diseases, and a wide range of other diseases.