Defective Mitophagy Research Articles

Mechanisms of Selectivity during Stationary Phase Mitophagy

The autophagic degradation of mitochondria is an important housekeeping function in eukaryotic cells. Defects in mitophagy occur in late-onset degenerative disorders, and decreases in the levels of mitophagy correlate with aging phenomena. Currently, it is not known whether a small cohort of defective molecules will cause the ablation of an entire mitochondrial compartment, or whether an active process segregates defective molecules from functional ones to allow a more efficient culling mechanism. We used SILAC-based proteomics combined with a molecular genetic and cell biology approach to determine whether such a segregation process occurs in yeast mitochondria. We find that different mitochondrial matrix proteins undergo mitophagic degradation at distinctly different rates, supporting the active segregation hypothesis. These differential degradation rates depend on mitochondrial dynamics, suggesting a mechanism that couples a weak physical segregation with mitochondrial fission and fusion to achieve a distillation-like effect. In agreement with this interpretation, the differential rates of mitophagy that we observed for different mitochondrial matrix proteins strongly correlate with the degree of physical segregation observed for these proteins, within the matrix. In addition, mutant cells lacking the mitochondrial protein phosphatase Aup1 are affected in the mitophagy of specific mitochondrial matrix proteins but not others, implying a role for protein phosphorylation in the selectivity of mitophagy. Our results suggest mechanisms which combine mitochondrial dynamics and matrix protein phosphorylation, in determining the selectivity of mitochondrial protein turnover via mitophagy.

The Mitochondrial Quality Control Protein Yme1 Is Necessary to Prevent Defective Mitophagy in a Yeast Model of Barth Syndrome

The Saccharomyces cerevisiae TAZ1 gene is an orthologue of human TAZ; both encode the protein tafazzin. Tafazzin is a transacylase that transfers acyl chains with unsaturated fatty acids from phospholipids to monolysocardiolipin to generate cardiolipin with unsaturated fatty acids. Mutations in human TAZ cause Barth syndrome, a fatal childhood cardiomyopathy biochemically characterized by reduced cardiolipin mass and increased monolysocardiolipin levels. To uncover cellular processes that require tafazzin to maintain cell health, we performed a synthetic genetic array screen using taz1Δ yeast cells to identify genes whose deletion aggravated its fitness. The synthetic genetic array screen uncovered several mitochondrial cellular processes that require tafazzin. Focusing on the i-AAA protease Yme1, a mitochondrial quality control protein that degrades misfolded proteins, we determined that in cells lacking both Yme1 and Taz1 function, there were substantive mitochondrial ultrastructural defects, ineffective superoxide scavenging, and a severe defect in mitophagy. We identify an important role for the mitochondrial protease Yme1 in the ability of cells that lack tafazzin function to maintain mitochondrial structural integrity and mitochondrial quality control and to undergo mitophagy.

Mitochondria Homeostasis Plays a Key Role in the Age‐related Susceptibility to Lung Epithelial Injury and Fibrosis

RationaleAging is associated with increased susceptibility to lung pathologies. The prevalence of idiopathic pulmonary fibrosis (IPF) has been found to increase with age. While familial disease has been related to disordered telomerase activity and mutations of SPC, the effects of advancing age and lung epithelium dysfunction and fibrosis remains unclear.MethodsLung fibrosis and mitochondria function, morphology and dynamics were analyzed in alveolar epithelial cell type II (AECII) of patients with IPF, aging mice and mice deficient in PINK1 (PTEN‐induced putative kinase 1).ResultsWe found that AECII in IPF lung have marked accumulation of dysmorphic and dysfunctional mitochondria. These mitochondrial abnormalities were recapitulated in normal mice with advancing age and ER stress stimulation. We found that impaired mitochondria in IPF and aging lungs were associated with low expression of the PINK1. Knockdown of PINK1 expression in lung epithelial cells resulted in mitochondria depolarization, and expression of pro‐fibrotic factors. Moreover, young mice deficient in PINK1 develop similar dysmorphic dysfunctional mitochondria in the AECII and are highly vulnerable to apoptosis and development of lung fibrosis.ConclusionOur data indicates that PINK1 deficiency and the subsequent swollen mitochondria dysfunction in the presence of ER stress causes defective mitophagy and promotion of fibrosis in the aging lung.

Nitric Oxide Induction of Parkin Translocation in PTEN-induced Putative Kinase 1 (PINK1) Deficiency: FUNCTIONAL ROLE OF NEURONAL NITRIC OXIDE SYNTHASE DURING MITOPHAGY

The failure to trigger mitophagy is implicated in the pathogenesis of familial Parkinson disease that is caused by PINK1 or Parkin mutations. According to the prevailing PINK1-Parkin signaling model, mitophagy is promoted by the mitochondrial translocation of Parkin, an essential PINK1-dependent step that occurs via a previously unknown mechanism. Here we determined that critical concentrations of NO was sufficient to induce the mitochondrial translocation of Parkin even in PINK1 deficiency, with apparent increased interaction of full-length PINK1 accumulated during mitophagy, with neuronal nitric oxide synthase (nNOS). Specifically, optimum levels of NO enabled PINK1-null dopaminergic neuronal cells to regain the mitochondrial translocation of Parkin, which appeared to be significantly suppressed by nNOS-null mutation. Moreover, nNOS-null mutation resulted in the same mitochondrial electron transport chain (ETC) enzyme deficits as PINK1-null mutation. The involvement of mitochondrial nNOS activation in mitophagy was further confirmed by the greatly increased interactions of full-length PINK1 with nNOS, accompanied by mitochondrial accumulation of phospho-nNOS (Ser(1412)) during mitophagy. Of great interest is that the L347P PINK1 mutant failed to bind to nNOS. The loss of nNOS phosphorylation and Parkin accumulation on PINK1-deficient mitochondria could be reversed in a PINK1-dependent manner. Finally, non-toxic levels of NO treatment aided in the recovery of PINK1-null dopaminergic neuronal cells from mitochondrial ETC enzyme deficits. In summary, we demonstrated the full-length PINK1-dependent recruitment of nNOS, its activation in the induction of Parkin translocation, and the feasibility of NO-based pharmacotherapy for defective mitophagy and ETC enzyme deficits in Parkinson disease.

Defects in mitophagy promote redox-driven metabolic syndrome in the absence of TP53INP1.

The metabolic syndrome covers metabolic abnormalities including obesity and type 2 diabetes (T2D). T2D is characterized by insulin resistance resulting from both environmental and genetic factors. A genome-wide association study (GWAS) published in 2010 identified TP53INP1 as a new T2D susceptibility locus, but a pathological mechanism was not identified. In this work, we show that mice lacking TP53INP1 are prone to redox-driven obesity and insulin resistance. Furthermore, we demonstrate that the reactive oxygen species increase in TP53INP1-deficient cells results from accumulation of defective mitochondria associated with impaired PINK/PARKIN mitophagy. This chronic oxidative stress also favors accumulation of lipid droplets. Taken together, our data provide evidence that the GWAS-identified TP53INP1 gene prevents metabolic syndrome, through a mechanism involving prevention of oxidative stress by mitochondrial homeostasis regulation. In conclusion, this study highlights TP53INP1 as a molecular regulator of redox-driven metabolic syndrome and provides a new preclinical mouse model for metabolic syndrome clinical research.

The kinase Jnk2 promotes stress-induced mitophagy by targeting the small mitochondrial form of the tumor suppressor ARF for degradation.

Mitophagy is essential for cellular homeostasis but the regulatory mechanism is largely unknown. Here we report that the kinase Jnk2 is required for stress-induced mitophagy. Jnk2 promoted ubiquitination and proteasomal degradation of small mitochondrial form of ARF (smARF). Loss of Jnk2 led to accumulation of smARF, which in turn induced excessive autophagic activity, resulting in lysosomal degradation of the mitophagy adaptor p62 in the steady state. The depletion of p62 prevented Jnk2-deficient cells from mounting mitophagy upon stress. Jnk2-deficient mice displayed defective mitophagy, resulting in tissue damage under hypoxic stress, as well as hyperactivation of inflammasome and increased mortality in sepsis. Our finding defines a unique mechanism of maintaining immune homeostasis that protects the host from tissue damage and mortality.

Impaired mitophagy leads to cigarette smoke stress-induced cellular senescence: implications for chronic obstructive pulmonary disease.

Cigarette smoke (CS)-induced cellular senescence is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). The molecular mechanism by which CS induces cellular senescence is unknown. Here, we show that CS stress (exposure of primary lung cells to CS extract 0.2-0.75% with a half-maximal inhibitory concentration of ∼0.5%) led to impaired mitophagy and perinuclear accumulation of damaged mitochondria associated with cellular senescence in both human lung fibroblasts and small airway epithelial cells (SAECs). Impaired mitophagy was attributed to reduced Parkin translocation to damaged mitochondria, which was due to CS-induced cytoplasmic p53 accumulation and its interaction with Parkin. Impaired Parkin translocation to damaged mitochondria was also observed in mouse lungs with emphysema (6 months CS exposure, 100 mg TPM/m(3)) as well as in lungs of chronic smokers and patients with COPD. Primary SAECs from patients with COPD also exhibited impaired mitophagy and increased cellular senescence via suborganellar signaling. Mitochondria-targeted antioxidant (Mito-Tempo) restored impaired mitophagy, decreased mitochondrial mass accumulation, and delayed cellular senescence in Parkin-overexpressing cells. In conclusion, defective mitophagy leads to CS stress-induced lung cellular senescence, and restoring mitophagy delays cellular senescence, which provides a promising therapeutic intervention in chronic airway diseases.

Temporal dynamics of PARK2/parkin and OPTN/optineurin recruitment during the mitophagy of damaged mitochondria

Damaged mitochondria are selectively degraded via autophagy in a regulated pathway known as mitophagy. Parkinson disease-linked proteins PINK1 (PTEN induced putative kinase 1) and PARK2 (parkin RBR E3 ubiquitin protein ligase) are recruited to the outer mitochondrial membrane upon mitochondrial damage, leading to the PARK2-mediated ubiquitination of mitochondrial proteins. Here, we discuss our recent work demonstrating that OPTN (optineurin) is recruited to damaged mitochondria, serving as an autophagy receptor for autophagosome formation around mitochondria. Using high-resolution live-cell imaging, we find that OPTN is recruited to ubiquitinated mitochondria downstream of PARK2, and induces autophagosome assembly around mitochondria via its LC3-interacting region. Mutations in OPTN are linked to both glaucoma and ALS (amyotrophic lateral sclerosis), and an ALS-associated E478G mutation in OPTN's ubiquitin binding domain leads to defective mitophagy and accumulation of damaged mitochondria. Importantly, our results highlight a role for mitophagy defects in ALS pathogenesis, and demonstrate that defects in the same pathway for mitochondrial homeostasis are causal for both familial Parkinson disease and ALS.

ALCAT1 controls mitochondrial etiology of fatty liver diseases, linking defective mitophagy to steatosis.

Defective autophagy is implicated in the pathogenesis of nonalcoholic fatty liver diseases (NAFLD) through poorly defined mechanisms. Cardiolipin is a mitochondrial phospholipid required for bioenergetics and mitophagy from yeast to mammals. Here we investigated a role for ALCAT1 in the development of NAFLD. ALCAT1 is a lysocardiolipin acyltransferase that catalyzes pathological cardiolipin remodeling in several aging-related diseases. We show that the onset of diet-induced NAFLD caused autophagic arrest in hepatocytes, leading to oxidative stress, mitochondrial dysfunction, and insulin resistance. In contrast, targeted deletion of ALCAT1 in mice prevented the onset of NAFLD. ALCAT1 deficiency also restored mitophagy, mitochondrial architecture, mitochondrial DNA (mtDNA) fidelity, and oxidative phosphorylation. In support of a causative role of the enzyme in a mitochondrial etiology of the disease, hepatic ALCAT1 expression was significantly up-regulated in mouse models of NAFLD. Forced expression of ALCAT1 in primary hepatocytes led to multiple defects that are highly reminiscent of NAFLD, including steatosis, defective autophagy, and mitochondrial dysfunction, linking pathological cardiolipin remodeling by ALCAT1 to the pathogenesis of NAFLD.

Mitochondrial Dysfunction and Autophagy in Hepatic Ischemia/Reperfusion Injury.

Ischemia/reperfusion (I/R) injury remains a major complication of liver resection, transplantation, and hemorrhagic shock. Although the mechanisms that contribute to hepatic I/R are complex and diverse involving the interaction of cell injury in hepatocytes, immune cells, and endothelium, mitochondrial dysfunction is a cardinal event culminating in hepatic reperfusion injury. Mitochondrial autophagy, so-called mitophagy, is a key cellular process that regulates mitochondrial homeostasis and eliminates damaged mitochondria in a timely manner. Growing evidence accumulates that I/R injury is attributed to defective mitophagy. This review aims to summarize the current understanding of autophagy and its role in hepatic I/R injury and highlight the various therapeutic approaches that have been studied to ameliorate injury.

Mitophagy: therapeutic potentials for liver disease and beyond.

Mitochondrial integrity is critical for maintaining proper cellular functions. A key aspect of regulating mitochondrial homeostasis is removing damaged mitochondria through autophagy, a process called mitophagy. Autophagy dysfunction in various disease states can inactivate mitophagy and cause cell death, and defects in mitophagy are becoming increasingly recognized in a wide range of diseases from liver injuries to neurodegenerative diseases. Here we highlight our current knowledge on the mechanisms of mitophagy, and discuss how alterations in mitophagy contribute to disease pathogenesis. We also discuss mitochondrial dynamics and potential interactions between mitochondrial fusion, fission and mitophagy.

PINK1 deficiency impairs mitochondrial homeostasis and promotes lung fibrosis.

Although aging is a known risk factor for idiopathic pulmonary fibrosis (IPF), the pathogenic mechanisms that underlie the effects of advancing age remain largely unexplained. Some age-related neurodegenerative diseases have an etiology that is related to mitochondrial dysfunction. Here, we found that alveolar type II cells (AECIIs) in the lungs of IPF patients exhibit marked accumulation of dysmorphic and dysfunctional mitochondria. These mitochondrial abnormalities in AECIIs of IPF lungs were associated with upregulation of ER stress markers and were recapitulated in normal mice with advancing age in response to stimulation of ER stress. We found that impaired mitochondria in IPF and aging lungs were associated with low expression of PTEN-induced putative kinase 1 (PINK1). Knockdown of PINK1 expression in lung epithelial cells resulted in mitochondria depolarization and expression of profibrotic factors. Moreover, young PINK1-deficient mice developed similarly dysmorphic, dysfunctional mitochondria in the AECIIs and were vulnerable to apoptosis and development of lung fibrosis. Our data indicate that PINK1 deficiency results in swollen, dysfunctional mitochondria and defective mitophagy, and promotes fibrosis in the aging lung.

Abstract 313: Essential role of ataxia telangiectasia in mitochondrial autophagy in mantle cell lymphoma

Abstract Ataxia telangiectasia mutated (ATM) is obligatory to initiate cellular responses to DNA double-strand breaks and DNA repair to preserve genomic integrity while loss of ATM leads to increased intracellular reactive oxygen species (ROS), accumulation of aberrant mitochondria, leading to abnormal mitochondrial homeostasis thereby exasperate cancer progression. ATM is frequently altered in several human cancers including 20-50% in mantle cell lymphoma (MCL). Despite ATM is known to induce global autophagy in certain cancer cells, the role of ATM in mitochondrial autophagy (mitophagy) in human cancer is imprecise. Here, we characterize a panel of MCL cell lines for their ability to trigger mitophagy induced by the mitochondrial uncoupler CCCP/FCCP. While Granta-519 lacks a functional ATM, both Jeko-1 and Mino cells exemplify as ATM proficient. Treatment with CCCP/FCCP (75µM for 4hr) elicits rapid up-regulation in ATM ser1981 phosphorylation in both Jeko-1 and Mino cells, while Granta-519 cells are mostly non-responsive. Cells were stained with TMRE (to detect mitochondrial membrane potential) or Mitotracker deep Red (to measure total mitochondrial mass) and analyzed by flow cytometry. Cells were also stained to detect both global and mitochondrial ROS (mROS) levels. CCCP/FCCP treatment resulted in rapid loss of (depolarized) membrane potential, total mitochondria mass, mROS and global ROS in both Jeko-1 and Mino cells while Granta-519 cells maintained intermediate to higher membrane potential with relatively higher total or mROS levels compared to controls. Cell fractionation analysis revealed that a fraction of resident ATM protein is located inside the mitochondrial fraction and phosphorylated upon CCCP/FCCP treatment in both Jeko-1 and Mino cells but not in Granta-519 leading to elimination of depolarized mitochondria as revealed by their basal COX4 protein levels. Immuno-precipitation analysis revealed that CCCP/FCCP treatment in both Jeko-1 and Mino cells resulted in ATM cleavage more specifically in the mitochondrial fraction. Interestingly, ATM status is unrelated to global autophagy as judged by their respective LC3I/II processing in these cell lines. Stable lentiviral knockdown of ATM in both Jeko-1 and Mino cells resulted in defective mitophagy with relatively higher COX4 expression and mROS levels. Neocarzinostatin induced DNA damage revealed loss of ATMser1981, Kap1ser824 and p53ser15 phosphorylation compared with control shRNA reconfirmed the loss of ATM function in these cells. Human primary MCL subjects with known ATM translocation [t(11;14) n=4] retain relatively higher mitochondrial mass and mROS, and are resistant to CCCP/FCCP induced mitophagy compared with ATM proficient primary MCL cells. These data underscore the possibility that ATM plays a decisive role in modulating mitophagy and contributes to the cancer-prone phenotype observed in MCL. Citation Format: Aloke Kumar Sarkar, Kumudha Balakrishnan, Mary Ayres, Sattva S. Neelapu, Varsha Gandhi. Essential role of ataxia telangiectasia in mitochondrial autophagy in mantle cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 313. doi:10.1158/1538-7445.AM2014-313

The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy.

Cells maintain healthy mitochondria by degrading damaged mitochondria through mitophagy; defective mitophagy is linked to Parkinson's disease. Here we report that USP30, a deubiquitinase localized to mitochondria, antagonizes mitophagy driven by the ubiquitin ligase parkin (also known as PARK2) and protein kinase PINK1, which are encoded by two genes associated with Parkinson's disease. Parkin ubiquitinates and tags damaged mitochondria for clearance. Overexpression of USP30 removes ubiquitin attached by parkin onto damaged mitochondria and blocks parkin's ability to drive mitophagy, whereas reducing USP30 activity enhances mitochondrial degradation in neurons. Global ubiquitination site profiling identified multiple mitochondrial substrates oppositely regulated by parkin and USP30. Knockdown of USP30 rescues the defective mitophagy caused by pathogenic mutations in parkin and improves mitochondrial integrity in parkin- or PINK1-deficient flies. Knockdown of USP30 in dopaminergic neurons protects flies against paraquat toxicity in vivo, ameliorating defects in dopamine levels, motor function and organismal survival. Thus USP30 inhibition is potentially beneficial for Parkinson's disease by promoting mitochondrial clearance and quality control.

The deubiquitinase USP15 antagonizes Parkin-mediated mitochondrial ubiquitination and mitophagy.

Loss-of-function mutations in PARK2, the gene encoding the E3 ubiquitin ligase Parkin, are the most frequent cause of recessive Parkinson's disease (PD). Parkin translocates from the cytosol to depolarized mitochondria, ubiquitinates outer mitochondrial membrane proteins and induces selective autophagy of the damaged mitochondria (mitophagy). Here, we show that ubiquitin-specific protease 15 (USP15), a deubiquitinating enzyme (DUB) widely expressed in brain and other organs, opposes Parkin-mediated mitophagy, while a panel of other DUBs and a catalytically inactive version of USP15 do not. Moreover, knockdown of USP15 rescues the mitophagy defect of PD patient fibroblasts with PARK2 mutations and decreased Parkin levels. USP15 does not affect the ubiquitination status of Parkin or Parkin translocation to mitochondria, but counteracts Parkin-mediated mitochondrial ubiquitination. Knockdown of the DUB CG8334, the closest homolog of USP15 in Drosophila, largely rescues the mitochondrial and behavioral defects of parkin RNAi flies. These data identify USP15 as an antagonist of Parkin and suggest that USP15 inhibition could be a therapeutic strategy for PD cases caused by reduced Parkin levels.

Defective Mitophagy in XPA via PARP-1 Hyperactivation and NAD+/SIRT1 Reduction

Mitochondrial dysfunction is a common feature in neurodegeneration and aging. We identify mitochondrial dysfunction in xeroderma pigmentosum group A (XPA), a nucleotide excision DNA repair disorder with severe neurodegeneration, in silico and in vivo. XPA-deficient cells show defective mitophagy with excessive cleavage of PINK1 and increased mitochondrial membrane potential. The mitochondrial abnormalities appear to be caused by decreased activation of the NAD(+)-SIRT1-PGC-1α axis triggered by hyperactivation of the DNA damage sensor PARP-1. This phenotype is rescued by PARP-1 inhibition or by supplementation with NAD(+) precursors that also rescue the lifespan defect in xpa-1 nematodes. Importantly, this pathogenesis appears common to ataxia-telangiectasia and Cockayne syndrome, two other DNA repair disorders with neurodegeneration, but absent in XPC, a DNA repair disorder without neurodegeneration. Our findings reveal a nuclear-mitochondrial crosstalk that is critical for the maintenance of mitochondrial health.

A role for mitochondrial dynamics in the segregation of mitochondrial matrix proteins during stationary phase mitophagy (557.1)

Mitophagy, the autophagic degradation of mitochondria, is an important housekeeping function in eukaryotic cells. Defects in mitophagy occur in degenerative disorders such as Parkinson’s disease, and decreases in the levels of mitophagy correlate with aging phenomena. An important goal is to achieve an understanding of the quality control mechanism(s) that specifically cull defective mitochondrial components or compartments, during mitophagy. Specifically, it is not known whether a small cohort of defective molecules will cause the ablation of an entire compartment, or whether an active process segregates defective molecules from functional ones and allows a more efficient culling mechanism. In this study we have combined a proteomic study with a molecular genetic and cell biology approach to determine whether such a segregation process occurs in yeast mitochondria. We report that different mitochondrial matrix proteins undergo mitophagic degradation at distinctly different rates, supporting the active segregation hypothesis. These differential degradation rates depend on mitochondrial dynamics, suggesting a mechanism that couples a weak physical segregation with mitochondrial fission and fusion to achieve a distillation‐like effect. In agreement with this interpretation, the differential rates of mitophagy that we observed for different mitochondrial matrix proteins strongly correlate with the degree of physical segregation observed for these proteins, within the matrix.Grant Funding Source: Supported by the Israel Science Foundation

Defects in GABA metabolism affect selective autophagy pathways and are alleviated by mTOR inhibition

In addition to key roles in embryonic neurogenesis and myelinogenesis, γ-aminobutyric acid (GABA) serves as the primary inhibitory mammalian neurotransmitter. In yeast, we have identified a new role for GABA that augments activity of the pivotal kinase, Tor1. GABA inhibits the selective autophagy pathways, mitophagy and pexophagy, through Sch9, the homolog of the mammalian kinase, S6K1, leading to oxidative stress, all of which can be mitigated by the Tor1 inhibitor, rapamycin. To confirm these processes in mammals, we examined the succinic semialdehyde dehydrogenase (SSADH)-deficient mouse model that accumulates supraphysiological GABA in the central nervous system and other tissues. Mutant mice displayed increased mitochondrial numbers in the brain and liver, expected with a defect in mitophagy, and morphologically abnormal mitochondria. Administration of rapamycin to these mice reduced mTOR activity, reduced the elevated mitochondrial numbers, and normalized aberrant antioxidant levels. These results confirm a novel role for GABA in cell signaling and highlight potential pathomechanisms and treatments in various human pathologies, including SSADH deficiency, as well as other diseases characterized by elevated levels of GABA.

Defective mitophagy driven by dysregulation of rheb and KIF5B contributes to mitochondrial reactive oxygen species (ROS)-induced nod-like receptor 3 (NLRP3) dependent proinflammatory response and aggravates lipotoxicity

High-fat diet (HFD) and inflammation are the key contributors to insulin resistance and type 2 diabetes (T2D). Previous study shows fatty acid-induced accumulation of damaged, reactive oxygen species (ROS)-generating mitochondria, and this in turn activates the NLRP3 inflammasome interference with insulin signaling. Our previous research shows NLRP3 inflammasome activation signal originates from defects in autophagy. Yet how the fatty acid related to mitophagy alteration leads to the activation of NLRP3-ASC inflammasome has not been considered. Here we demonstrated that palmitate (PA) induced mitophagy deficiency, leading to damaged mitochondrion as characterized by mito-ROS production and loss of membrane potential. Antioxidant APDC or Ca2+ signaling inhibitor Nifedipine blocked PA-induced NLRP3 inflammasome activation. Further, we provided evidences that PA reduced the expression of Ras homolog enriched in brain (Rheb) and disrupted Rheb recruitment to the mitochondrial outer membrane. In addition, sustained PA caused disassociation of kinesin family member 5B (KIF5B) from binding with mitochondria via Ca2+-dependent effects. Disruption of Rheb and KIF5B interaction with mitochondria blocked mitochondrial degradation along with IL-1β dependent insulin resistance, which was majorly attenuated by Rheb/KIF5B overexpression. In a consequence, defective mitophagy led to the accumulation of damaged-ROS-generating mitochondria, down pathway of NLRP3-ASC-Caspase 1 activation, and subsequently, insulin resistance. These findings provide insights into the association of inflammation, mitophagy and T2D.

Involvement of mitochondrial dynamics in the segregation of mitochondrial matrix proteins during stationary phase mitophagy

Mitophagy, the autophagic degradation of mitochondria, is an important housekeeping function in eukaryotic cells and defects in mitophagy correlate with ageing phenomena and with several neurodegenerative disorders. A central mechanistic question regarding mitophagy is whether mitochondria are consumed en masse, or whether an active process segregates defective molecules from functional ones within the mitochondrial network, thus allowing a more efficient culling mechanism. Here, we combine a proteomic study with a molecular genetic and cell biology approach to determine whether such a segregation process occurs in yeast mitochondria. We find that different mitochondrial matrix proteins undergo mitophagic degradation at distinctly different rates, supporting the active segregation hypothesis. These differential degradation rates depend on mitochondrial dynamics, suggesting a mechanism coupling weak physical segregation with mitochondrial dynamics to achieve a distillation-like effect. In agreement, the rates of mitophagic degradation strongly correlate with the degree of physical segregation of specific matrix proteins.