Mitophagy In Response Research Articles

Blunted Cardiac Mitophagy in Response to Metabolic Stress Contributes to HFpEF.

Metabolic remodeling and mitochondrial dysfunction are hallmarks of heart failure with reduced ejection fraction. However, their role in the pathogenesis of HF with preserved ejection fraction (HFpEF) is poorly understood. In a mouse model of HFpEF, induced by high-fat diet and Nω-nitrol-arginine methyl ester, cardiac energetics was measured by 31P NMR spectroscopy and substrate oxidation profile was assessed by 13C-isotopmer analysis. Mitochondrial functions were assessed in the heart tissue and human induced pluripotent stem cell-derived cardiomyocytes. HFpEF hearts presented a lower phosphocreatine content and a reduced phosphocreatine/ATP ratio, similar to that in heart failure with reduced ejection fraction. Decreased respiratory function and increased reactive oxygen species production were observed in mitochondria isolated from HFpEF hearts suggesting mitochondrial dysfunction. Cardiac substrate oxidation profile showed a high dependency on fatty acid oxidation in HFpEF hearts, which is the opposite of heart failure with reduced ejection fraction but similar to that in high-fat diet hearts. However, phosphocreatine/ATP ratio and mitochondrial function were sustained in the high-fat diet hearts. We found that mitophagy was activated in the high-fat diet heart but not in HFpEF hearts despite similar extent of obesity suggesting that mitochondrial quality control response was impaired in HFpEF hearts. Using a human induced pluripotent stem cell-derived cardiomyocyte mitophagy reporter, we found that fatty acid loading stimulated mitophagy, which was obliterated by inhibiting fatty acid oxidation. Enhancing fatty acid oxidation by deleting ACC2 (acetyl-CoA carboxylase 2) in the heart stimulated mitophagy and improved HFpEF phenotypes. Maladaptation to metabolic stress in HFpEF hearts impairs mitochondrial quality control and contributed to the pathogenesis, which can be improved by stimulating fatty acid oxidation.

CgPHB2 involved in the haemocyte mitophagy in response to Vibrio splendidus stimulation in Pacific oyster Crassostrea gigas

Prohibitin2 (PHB2) is recently identified as a novel inner membrane mitophagy receptor to mediate mitophagy. In the present study, the function of CgPHB2 in mediating mitophagy in response to Vibrio splendidus stimulation was investigated in Crassostrea gigas. CgPHB2 protein was mainly distributed in the cytoplasm of three subpopulations of haemocytes. After V. splendidus stimulation, the expressions of CgPHB2 mRNA in haemocytes were up-regulated significantly at 6, 12 and 24 h, and the abundance of CgPHB2 protein was also enhanced at 12–24 h compared to control group. Furthermore, the green signals of CgPHB2 were colocalized respectively with the red signals of mitochondria and CgLC3 in the haemocytes at 12 h after V. splendidus stimulation, and the co-localization value of CgPHB2 and mtphagy Dye was significantly increased. The direct interaction between CgPHB2 and CgLC3 was simulated by molecular docking. In PHB2-inhibitor Fluorizoline-treated oysters, the mRNA expressions of mitophagy-related genes and the ratio of mitophagy were significantly decreased in haemocytes of oysters after V. splendidus stimulation. All the results collectively suggested that CgPHB2 participated in mediating the haemocyte mitophagy in the antibacterial immune response of oysters.

Abstract 5265: Targeting mtDNA dynamics enhances immunogenicity and sensitizes KRAS mutant cancers to PD-1 blockade

Abstract Mitochondrial DNA (mtDNA) can stimulate an innate immune response that potentiates the efficacy of cancer immunotherapy. However, mtDNA-targeting therapy has not been developed to enhance this innate immune system in specific cancer subtypes efficiently. Here we discover that mtDNA is enriched in KRAS mutant tumors where the DNA sensor machinery cGAS-STING signaling is primed for innate immune activation. Intriguingly, MEK inhibitor induces cytosolic mtDNA release to potently activate the cGAS-STING signaling, which in turn induces PRKN-mediated mitophagy in response to mitochondrial stress in KRAS mutant cancer cells. During the process of mitophagy, PRKN, as an E3 ubiquitin ligase, degrades BAX protein and attenuates mitochondrial pore formation, which is essential for mtDNA efflux upon MEK inhibition. Combined MEK and mitophagy inhibitors efficiently stimulate type I IFNs in both mtDNA- and STING-dependent manners, and this combination enhances the efficacy of PD-1 blockade in Kras mutant murine syngeneic tumors. In the clinic, PRKN is associated with a ‘cold tumor’ phenotype among KRAS mutant tumors. Together, targeting mtDNA dynamics by co-inhibition of MEK and mitophagy represents a promising strategy to convert ‘cold to hot tumor’ in mtDNA-enriched KRAS mutant cancers. Citation Format: Kosuke Tanaka, Yasuki Adachi, Miyuki Yoshiya, Takuya Owari, Tomoko Yamamori-Morita, Akihiro Ohashi, Susumu Kobayashi, Shohei Koyama, Hiroyoshi Nishikawa. Targeting mtDNA dynamics enhances immunogenicity and sensitizes KRAS mutant cancers to PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5265.

The HRI branch of the integrated stress response selectively triggers mitophagy

To maintain mitochondrial homeostasis, damaged or excessive mitochondria are culled in coordination with the physiological state of the cell. The integrated stress response (ISR) is a signaling network that recognizes diverse cellular stresses, including mitochondrial dysfunction. Because the four ISR branches converge to common outputs, it is unclear whether mitochondrial stress detected by this network can regulate mitophagy, the autophagic degradation of mitochondria. Using a whole-genome screen, we show that the heme-regulated inhibitor (HRI) branch of the ISR selectively induces mitophagy. Activation of the HRI branch results in mitochondrial localization of phosphorylated eukaryotic initiation factor 2, which we show is sufficient to induce mitophagy. The HRI mitophagy pathway operates in parallel with the mitophagy pathway controlled by the Parkinson's disease related genes PINK1 and PARKIN and is mechanistically distinct. Therefore, HRI repurposes machinery that is normally used for translational initiation to trigger mitophagy in response to mitochondrial damage.

HILPDA-mediated lipidomic remodelling promotes radiotherapy resistance in nasopharyngeal carcinoma by accelerating mitophagy.

Radiotherapy resistance is a major obstacle to nasopharyngeal carcinoma (NPC) therapy and contributes to tumour recurrence and metastasis. Lipid metabolism is a key regulatory mechanism in cancer biology; however, its role in NPC radiotherapy resistance remains unclear. In this study, we identified hypoxia-inducible lipid droplet-associated protein (HILPDA) as a newly discovered regulator of radioresistance that induces not only lipid droplet (LD) formation but also intracellular lipid remodelling, notably changing mitochondrial cardiolipin (CL) levels. Additionally, we found that the upregulation of CL promotes mitophagy in response to irradiation exposure. Mechanistically, HILPDA inhibits PINK1-mediated CLS1 ubiquitination and degradation. The combination of a mitophagy inhibitor and irradiation significantly increases the radiosensitivity of NPC cells. Human cancer-derived data confirmed that the HILPDA-CLS1 pathway promotes NPC radioresistance. Collectively, these findings suggest that HILPDA plays a critical role in promoting NPC radioresistance and might be targeted to overcome radiotherapeutic resistance in NPC patients in the clinic.

The role of CK2 in the regulation of mitochondrial autophagy induced by rotenone

CK2 regulates receptor-mediated mitophagy that removes damaged mitochondria. The PINK1/Parkin pathways also involve mitochondrial clearance through mitophagy. However, it is not clear whether CK2 regulates PINK1/Parkin-dependent mitophagy in response to stress. Rotenone treatment showed a decrease of FUNDC1 expression in the mitochondrial fraction of SH-SY5Y and HeLa cells, but an increase of PINK1/Parkin expression only in SH-SY5Y cells. Interestingly, CK2 inhibition increased mitochondrial LC3II expression in rotenone-treated HeLa cells, whereas it decreased in SH-SY5Y cells, indicating that CK2 mediates rotenone-induced mitophagy in dopaminergic neurons. Furthermore, FUNDC1 expression increased in rotenone-treated SH-SY5Y cells by CK2 inhibition, whereas it decreased in HeLa cells. CK2 inhibition also blocked the increase of Drp1, PINK1 and Parkin translocation into mitochondria and decrease of PGAM5 expression in rotenone-treated SH-SY5Y cells. As expected, rotenone treatment in PGAM5-knockdown cells reduced the expression of PINK1 and Parkin and decrease of LC3II expression. Interestingly, we observed that knockdown of CK2α or PGAM5 induced a further increase in caspase-3 expression. These results suggest that PINK1/Parkin-dependent mitophagy was dominant over FUNDC1 receptor-mediated mitophagy. Collectively, our findings suggest that CK2 can positively induce PINK1/Parkin-dependent mitophagy, and that mitophagy regulates cytoprotective effects by CK2 signaling in dopaminergic neurons. Data availability statementAll data generated or analyzed during this study are available upon request.

Mitochonic acid 5 promotes the migration of mouse microglial BV‑2 cells in the presence of LPS‑induced inflammation via Mfn2‑associated mitophagy.

Effective strategies are needed to prevent the development of neuroinflammation, which is associated with nervous system disease,\\r\\nin patients. A previous study indicated that mitochonic acid 5 (MA‑5) may promote the survival of microglial cells via mitofusin 2 (Mfn2)‑associated mitophagy in response to lipopolysaccharide (LPS)‑induced inflammation. The current study investigated the role and underlying mechanisms of MA‑5 in the migration of BV‑2 cells following LPS‑mediated inflammation. The results of the present study revealed that MA‑5 promoted migration and upregulated the expression of F‑actin, C‑X‑C motif chemokine receptor (CXCR) 4 and CXCR7 in BV‑2 cells in response to LPS‑induced inflammation. The results also indicate that MA‑5 did not promote migration or upregulate the expression of F‑actin, CXCR4 or CXCR7 following the inhibition of Mfn2. Overall, the results of the present study suggest that MA‑5 may promote the migration of microglial cells via Mfn2‑associated mitophagy following LPS‑induced inflammation.

2D MoS2 and BN Nanosheets Damage Mitochondria through Membrane Penetration.

With the progression of nanotechnology, a growing number of nanomaterials have been created and incorporated into organisms and ecosystems, which raises significant concern about potential hazards of these materials on human health, wildlife, and the environment. Two-dimensional (2D) nanomaterials are one type of nanomaterials with thicknesses ranging from that of a single atom or of several atoms and have been proposed for a variety of biomedical applications such as drug delivery and gene therapy, but the toxicity thereof on subcellular organelles remains to be studied. In this work, we studied the impact of two typical 2D nanomaterials, MoS2 and BN nanosheets, on mitochondria, which are a type of membranous subcellular organelle that provides energy to cells. While 2D nanomaterials at a low dose exhibited a negligible cell mortality rate, significant mitochondrial fragmentation and partially reduced mitochondrial functions occurred; cells initiate mitophagy in response to mitochondrial damages, which cleans damaged mitochondria to avoid damage accumulation. Moreover, the molecular dynamics simulation results revealed that both MoS2 and BN nanosheets can spontaneously penetrate the mitochondrial lipid membrane through the hydrophobic interaction. The membrane penetration induced heterogeneous lipid packing resulting in damages. Our results demonstrate that even at a low dose 2D nanomaterials can physically damage mitochondria by penetrating the membrane, which draws attention to carefully evaluating the cytotoxicity of 2D nanomaterials for the potential biomedical application.

Inner mitochondrial membrane protein Prohibitin 1 mediates Nix-induced, Parkin-independent mitophagy

Autophagy of damaged mitochondria, called mitophagy, is an important organelle quality control process involved in the pathogenesis of inflammation, cancer, aging, and age-associated diseases. Many of these disorders are associated with altered expression of the inner mitochondrial membrane (IMM) protein Prohibitin 1. The mechanisms whereby dysfunction occurring internally at the IMM and matrix activate events at the outer mitochondrial membrane (OMM) to induce mitophagy are not fully elucidated. Using the gastrointestinal epithelium as a model system highly susceptible to autophagy inhibition, we reveal a specific role of Prohibitin-induced mitophagy in maintaining intestinal homeostasis. We demonstrate that Prohibitin 1 induces mitophagy in response to increased mitochondrial reactive oxygen species (ROS) through binding to mitophagy receptor Nix/Bnip3L and independently of Parkin. Prohibitin 1 is required for ROS-induced Nix localization to mitochondria and maintaining homeostasis of epithelial cells highly susceptible to mitochondrial dysfunction.

Sensing local energetics to acutely regulate mitophagy in skeletal muscle.

The energetic requirements of skeletal muscle to sustain movement, as during exercise, is met largely by mitochondria, which form an intricate, interconnected reticulum. Maintenance of a healthy mitochondrial reticulum is essential for skeletal muscle function, suggesting quality control pathways are spatially governed. Mitophagy, the process by which damaged and/or dysfunctional regions of the mitochondrial reticulum are removed and degraded, has emerged as an integral part of the molecular response to exercise. Upregulation of mitophagy in response to acute exercise is directly connected to energetic sensing mechanisms through AMPK. In this review, we discuss the connection of mitophagy to muscle energetics and how AMPK may spatially control mitophagy through multiple potential means.

Isolation, Characterization, and Autophagy Function of BECN1-Splicing Isoforms in Cancer Cells.

Alternative splicing allows the synthesis of different protein variants starting from a single gene. Human Beclin 1 (BECN1) is a key autophagy regulator that acts as haploinsufficient tumor suppressor since its decreased expression correlates with tumorigenesis and poor prognosis in cancer patients. Recent studies show that BECN1 mRNA undergoes alternative splicing. Here, we report on the isolation and molecular and functional characterization of three BECN1 transcript variants (named BECN1-α, -β and -γ) in human cancer cells. In ovarian cancer NIHOVCAR3, these splicing variants were found along with the canonical wild-type. BECN1-α lacks 143 nucleotides at its C-terminus and corresponds to a variant previously described. BECN1-β and -γ lack the BCL2 homology 3 domain and other regions at their C-termini. Following overexpression in breast cancer cells MDA-MB231, we found that BECN1-α stimulates autophagy. Specifically, BECN1-α binds to Parkin and stimulates mitophagy. On the contrary, BECN1-β reduces autophagy with a dominant negative effect over the endogenous wild-type isoform. BECN1-γ maintains its ability to interact with the vacuolar protein sorting 34 and only has a slight effect on autophagy. It is possible that cancer cells utilize the alternative splicing of BECN1 for modulating autophagy and mitophagy in response to environmental stresses.

Role of TFE3 in Mitochondrial Adaptations to Skeletal Muscle Disuse

Prolonged muscle disuse is accompanied by a phenotypic shift characterized by declines in mitochondrial content and function within skeletal muscle. This loss in mitochondrial content can be partially attributed to elevations in various catabolic processes that occur with atrophy. One of these is termed mitophagy, a selective form of cellular recycling (autophagy) whereby dysfunctional mitochondria are degraded via lysosomes. TFEB and TFE3 are key transcription factors that regulate lysosomes by activating the expression of various lysosomal genes. Loss of TFE3 has been associated with depressed levels of autophagy and with mitochondrial dysfunction. It is speculated that these functional impairments are due to diminished clearance via the lysosomes, however to date this has not been examined. We hypothesized that the loss of TFE3 would amplify the mitochondrial dysfunction associated with muscle disuse, while paradoxically preserving muscle mass and mitochondrial content. Using a severe model of muscle disuse, sciatic denervation, we observed a 10% loss in hindlimb muscle mass within 7 days of denervation. In the absence of TFE3, a trend for muscle preservation was observed, as these animals lost 20% less muscle mass than WT counterparts. Reduced rates of respiration supported by complex I and II were observed irrespective of genotype, concurrent with elevated ROS emissions, suggesting an impaired oxidative capacity following 7 days of denervation. Surprisingly, TFE3 KO animals did exhibit lower levels of oxidative stress basally, but this too increased following 7 days of denervation, and taken relative to baseline the fold induction of ROS emission was 3 times greater than WT animals. Higher levels of mitophagy flux were observed in the absence of TFE3 basally, which supports the observed 35% decline in mitochondrial content as measured by COX activity, as well as the lower levels of ROS emissions. Following only 1 day of denervation, increases in mitophagy flux were observed in WT animals, while the response in KO animals was clearly attenuated. In the WT animals, denervation led to a 30% reduction in mitochondrial content, however no change was observed in the absence of TFE3. Finally, increases in a number of autophagy‐related markers such Beclin‐1 and ATG7 were observed following denervation irrespective of genotype. However, the mature form of Cathepsin B, a hydrolytic enzyme, was markedly reduced by 55% in the absence of TFE3 and did not increase to the same extent as WT following 7 days of denervation suggesting an impairment in lysosomal function. Together, our results suggest that TFE3 exerts multiple roles in skeletal muscle plasticity, as a partial mediator of muscle mass, and in the control of lysosomal function and mitophagy in response to the acute stress of muscle disuse.

Ulk1-dependent alternative mitophagy plays a protective role during pressure overload in the heart.

Well-controlled mitochondrial homeostasis, including a mitochondria-specific form of autophagy (hereafter referred to as mitophagy), is essential for maintaining cardiac function. The molecular mechanism mediating mitophagy during pressure overload (PO) is poorly understood. We have shown previously that mitophagy in the heart is mediated primarily by Atg5/Atg7-independent mechanisms, including Unc-51-like kinase 1 (Ulk1)-dependent alternative mitophagy, during myocardial ischaemia. Here, we investigated the role of alternative mitophagy in the heart during PO-induced hypertrophy. Mitophagy was observed in the heart in response to transverse aortic constriction (TAC), peaking at 3-5 days. Whereas mitophagy is transiently up-regulated by TAC through an Atg7-dependent mechanism in the heart, peaking at 1 day, it is also activated more strongly and with a delayed time course through an Ulk1-dependent mechanism. TAC induced more severe cardiac dysfunction, hypertrophy, and fibrosis in ulk1 cardiac-specific knock-out (cKO) mice than in wild-type mice. Delayed activation of mitophagy was characterized by the co-localization of Rab9 dots and mitochondria and phosphorylation of Rab9 at Ser179, major features of alternative mitophagy. Furthermore, TAC-induced decreases in the mitochondrial aspect ratio were abolished and the irregularity of mitochondrial cristae was exacerbated, suggesting that mitochondrial quality control mechanisms are impaired in ulk1 cKO mice in response to TAC. TAT-Beclin 1 activates mitophagy even in Ulk1-deficient conditions. TAT-Beclin 1 treatment rescued mitochondrial dysfunction and cardiac dysfunction in ulk1 cKO mice during PO. Ulk1-mediated alternative mitophagy is a major mechanism mediating mitophagy in response to PO and plays an important role in mediating mitochondrial quality control mechanisms and protecting the heart against cardiac dysfunction.

Mitochonic acid 5 regulates mitofusin 2 to protect microglia

Microglial apoptosis is associated with neuroinflammation and no effective strategies are currently available to protect microglia against inflammation-induced apoptosis. Mouse microglial BV-2 cells (5 × 106) were incubated with 10 μg/mL lipopolysaccharides for 12 hours to mimic an inflammatory environment. Then the cells were co-cultured with mitochonic acid 5 (MA-5) for another 12 hours. MA-5 improved the survival of lipopolysaccharide-exposed cells. MA-5 decreased the activity of caspase-3, which is associated with apoptosis. MA-5 reduced the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells, and increased adenosine triphosphate levels in cells. MA-5 decreased the open state of the mitochondrial permeability transition pore and reduced calcium overload and diffusion of second mitochondria-derived activator of caspase (Smac). MA-5 decreased the expression of apoptosis-related proteins (mitochondrial Smac, cytoplasmic Smac, pro-caspase-3, cleaved-caspase-3, and caspase-9), and increased the levels of anti-apoptotic proteins (Bcl2 and X-linked inhibitor of apoptosis protein), mitochondria-related proteins (mitochondrial fusion protein 2, mitochondrial microtubule-associated proteins 1A/1B light chain 3B II), and autophagy-related proteins (Beclin1, p62 and autophagy related 5). However, MA-5 did not promote mitochondrial homeostasis or decrease microglial apoptosis when Mitofusin 2 expression was silenced. This shows that MA-5 increased Mitofusin 2-related mitophagy, reversed cellular energy production and maintained energy metabolism in BV-2 cells in response to lipopolysaccharide-induced inflammation. These findings indicate that MA-5 may promote the survival of microglial cells via Mitofusin 2-related mitophagy in response to lipopolysaccharide-induced inflammation.

Mitophagy protects β cells from inflammatory damage in diabetes.

Inflammatory damage contributes to β cell failure in type 1 and 2 diabetes (T1D and T2D, respectively). Mitochondria are damaged by inflammatory signaling in β cells, resulting in impaired bioenergetics and initiation of proapoptotic machinery. Hence, the identification of protective responses to inflammation could lead to new therapeutic targets. Here, we report that mitophagy serves as a protective response to inflammatory stress in both human and rodent β cells. Utilizing in vivo mitophagy reporters, we observed that diabetogenic proinflammatory cytokines induced mitophagy in response to nitrosative/oxidative mitochondrial damage. Mitophagy-deficient β cells were sensitized to inflammatory stress, leading to the accumulation of fragmented dysfunctional mitochondria, increased β cell death, and hyperglycemia. Overexpression of CLEC16A, a T1D gene and mitophagy regulator whose expression in islets is protective against T1D, ameliorated cytokine-induced human β cell apoptosis. Thus, mitophagy promotes β cell survival and prevents diabetes by countering inflammatory injury. Targeting this pathway has the potential to prevent β cell failure in diabetes and may be beneficial in other inflammatory conditions.

MAPK1/3 kinase-dependent ULK1 degradation attenuates mitophagy and promotes breast cancer bone metastasis

ABSTRACT The function of mitophagy in cancer is controversial. ULK1 is critical for induction of macroautophagy/autophagy and has a more specific role in mitophagy in response to hypoxia. Here, we show that ULK1 deficiency induces an invasive phenotype of breast cancer cells under hypoxia and increases osteolytic bone metastasis. Mechanistically, ULK1 depletion attenuates mitophagy ability during hypoxia. As a result, the accumulation of damaged, ROS-generating mitochondria leads to activation of the NLRP3 inflammasome, which induces abnormal soluble cytokines secretion, then promotes the differentiation and maturation of osteoclasts, and ultimately results in bone metastasis. Notably, phosphorylation of ULK1 by MAPK1/ERK2-MAPK3/ERK1 kinase triggers its interaction with BTRC and subsequent K48-linked ubiquitination and proteasome degradation. Also, a clearly negative correlation between the expression levels of ULK1 and p-MAPK1/3 was observed in human breast cancer tissues. The MAP2K/MEK inhibitor trametinib is sufficient to restore mitophagy function via upregulation of ULK1, leading to inhibition of NLRP3 inflammasome activation, thereby reduces bone metastasis. These results indicate that ULK1 knockout-mediated mitophagy defect promotes breast cancer bone metastasis and provide evidence to explore MAP2K/MEK- MAPK1/3 pathway inhibitors for therapy, especially in cancers displaying low levels of ULK1. Abbreviations: ATG: autophagy-related; Baf A1: bafilomycin A1; BTRC/β-TrCP: beta-transducin repeat containing E3 ubiquitin protein ligase; CHX: cycloheximide; CM: conditioned media; FBXW7/FBW7: F-box and WD repeat domain containing 7; MAPK1: mitogen-activated protein kinase 1; MTDR: MitoTracker Deep Red; mtROS: mitochondrial reactive oxygen species; microCT: micro-computed tomography; mtROS: mitochondrial reactive oxygen species; OCR: oxygen consumption rate; SQSTM1: sequestosome 1; ACP5/TRAP: acid phosphatase, tartrate resistant; ULK1: unc-51 like autophagy activating kinase 1

Tauopathy-associated tau modifications selectively impact neurodegeneration and mitophagy in a novel C. elegans single-copy transgenic model

BackgroundA defining pathological hallmark of the progressive neurodegenerative disorder Alzheimer’s disease (AD) is the accumulation of misfolded tau with abnormal post-translational modifications (PTMs). These include phosphorylation at Threonine 231 (T231) and acetylation at Lysine 274 (K274) and at Lysine 281 (K281). Although tau is recognized to play a central role in pathogenesis of AD, the precise mechanisms by which these abnormal PTMs contribute to the neural toxicity of tau is unclear.MethodsHuman 0N4R tau (wild type) was expressed in touch receptor neurons of the genetic model organism C. elegans through single-copy gene insertion. Defined mutations were then introduced into the single-copy tau transgene through CRISPR-Cas9 genome editing. These mutations included T231E, to mimic phosphorylation of a commonly observed pathological epitope, and K274/281Q, to mimic disease-associated lysine acetylation – collectively referred as “PTM-mimetics” – as well as a T231A phosphoablation mutant. Stereotypical touch response assays were used to assess behavioral defects in the transgenic strains as a function of age. Genetically-encoded fluorescent biosensors were expressed in touch neurons and used to measure neuronal morphology, mitochondrial morphology, mitophagy, and macro autophagy.ResultsUnlike existing tau overexpression models, C. elegans single-copy expression of tau did not elicit overt pathological phenotypes at baseline. However, strains expressing disease associated PTM-mimetics (T231E and K274/281Q) exhibited reduced touch sensation and neuronal morphological abnormalities that increased with age. In addition, the PTM-mimetic mutants lacked the ability to engage neuronal mitophagy in response to mitochondrial stress.ConclusionsLimiting the expression of tau results in a genetic model where modifications that mimic pathologic tauopathy-associated PTMs contribute to cryptic, stress-inducible phenotypes that evolve with age. These findings and their relationship to mitochondrial stress provides a new perspective into the pathogenic mechanisms underlying AD.

Inhibition of proteasome reveals basal mitochondrial ubiquitination

Strict quality control for mitochondrial proteins is necessary to ensure cell homeostasis. Two cellular pathways–Ubiquitin Proteasome System (UPS) and autophagy–contribute to mitochondrial homeostasis under stressful conditions. Here, we investigate changes to the mitochondria proteome and to the ubiquitin landscape at mitochondria in response to proteasome inhibition. Treatment of HeLa cells devoid of Parkin, the primary E3 ligase responsible for mitophagy, with proteasome inhibitor MG132 for a few hours caused mitochondrial oxidative stress and fragmentation, reduced energy output, and increased mitochondrial ubiquitination without inducing mitophagy. Overexpression of Parkin did not show any induction of mitophagy in response to MG132 treatment. Analysis of ubiquitin chains on isolated mitochondria revealed predominance of K48, K29 and K63-linked polyubiquitin. Interestingly, of all ubiquitinated mitochondrial proteins detected in response to MG132 treatment, a majority (≥90%) were intramitochondrial irrespective of Parkin expression. However, overall levels of these ubiquitinated mitochondrial proteins did not change significantly upon proteasome inhibition when evaluated by quantitative proteomics (LFQ and SILAC), suggesting that only a small portion are ubiquitinated under basal conditions. Another aspect of proteasome inhibition is significant enrichment of UPS, lysosomal and phagosomal components, and other heat shock proteins associated with isolated mitochondria. Taken together, our study highlights a critical role of UPS for ubiquitinating and removing imported proteins as part of a basal mitochondrial quality control system independent of Parkin. SignificanceAs centers of cellular bioenergetics, numerous metabolic pathways and signaling cascades, the health of mitochondria is of utmost importance for ensuring cell survival. Due to their unique physiology, mitochondria are constantly subjected to damaging oxidative radicals (ROS) and protein import-related stress due to buildup of unfolded aggregate-prone proteins. Thus, for quality control purposes, mitochondria are constantly under surveillance by Autophagy and the Ubiquitin Proteasome System (UPS), both of which share ubiquitin as a common signal. The ubiquitin landscape of mitochondria has been studied in detail under stressful conditions, however, little is known about basal mitochondrial ubiquitination. Our study reveals that the extent of ubiquitination at mitochondria greatly increases upon proteasome inhibition, pointing to a large number of potential substrates for proteasomal degradation. Interestingly, most of the ubiquitination occurs on intramitochondrial proteins, components of the electron transport chain (ETC) and matrix-resident metabolic enzymes in particular. Moreover, numerous cytosolic UPS components, chaperones and autophagy-lysosomal proteins were recruited to mitochondria upon proteasome inhibition. Taken together, this suggests that the levels and functions of mitochondrial proteins are constantly regulated through ubiquitin-dependent proteasomal degradation even under basal conditions. Unclogging mitochondrial import channels may provide a mechanism to alleviate stress associated with mitochondrial protein import or to adapt cells according to their metabolic needs. Therefore, targeting the mitochondrial ubiquitination/deubiquitination machinery, such as improving the therapeutic potency of proteasome inhibitors, may provide an additional therapeutic arsenal against tumors.

Mitochondrial phosphatase PGAM5 modulates cellular senescence by regulating mitochondrial dynamics

Mitochondria undergo dynamic fusion/fission, biogenesis and mitophagy in response to stimuli or stresses. Disruption of mitochondrial homeostasis could lead to cell senescence, although the underlying mechanism remains unclear. We show that deletion of mitochondrial phosphatase PGAM5 leads to accelerated retinal pigment epithelial (RPE) senescence in vitro and in vivo. Mechanistically, PGAM5 is required for mitochondrial fission through dephosphorylating DRP1. PGAM5 deletion leads to increased mitochondrial fusion and decreased mitochondrial turnover. As results, cellular ATP and reactive oxygen species (ROS) levels are elevated, mTOR and IRF/IFN-β signaling pathways are enhanced, leading to cellular senescence. Overexpression of Drp1 K38A or S637A mutant phenocopies or rescues mTOR activation and senescence in PGAM5−/− cells, respectively. Young but not aging Pgam5−/− mice are resistant to sodium iodate-induced RPE cell death. Our studies establish a link between defective mitochondrial fission, cellular senescence and age-dependent oxidative stress response, which have implications in age-related diseases.

Increasing fatty acid oxidation prevents high fat diet induced cardiomyopathy through regulating mitophagy activity

Under obese condition, an imbalance between fatty acid uptake and fatty acid oxidation (FAO) promotes the development of cardiac lipotoxicity. We have previously demonstrated that enhancing FAO via Acetyl-CoA carboxylase 2 (ACC2) deletion prevents high fat diet (HFD) induced cardiac dysfunction and attenuates pathological remodeling in obese mice. In this study, we examined the effect of enhancing FAO in regulating mitochondrial function in obese mouse heart. ACC2 flox/flox (Con) and ACC2 flox/flox-MerCreMer+ (iKO) were injected with tamoxifen and subjected to HFD feeding for 24 weeks. Mitochondria oxygen consumption rate in Con HFD-fed hearts were reduced, which was maintained in iKO mice. Electron microscopy analysis revealed that ACC2 iKO mice prevented HFD induced accumulation of damaged mitochondria (3 ± 0.24% (CON) vs. 1.27 ± 0.36% (iKO), p < .05, n = 5). In vitro, palmitate (PA) induced mitochondria fragmation and increased reactive oxggen species production was partially attenuated in cardiomyocytes when ACC2 was knocked down. Knocking down of ACC2 also prevented PA induced cell death (65.2 ± 3.1% vs. 85.3 ± 3.9%, p < .05, n = 3). These data suggest that enhancing FAO prevents HFD induced mitochondria dysfunction. Moreover, HFD treatment reduced the expression of LC3 II and p62 in mitochondria fraction. EM analysis revealed that the number of mitochondria in autophagosome was less in Con HFD-fed heart compared with Con chow-fed group in response to starvation and CCCP treatment. Mt-Keima signal, indicative of mitophagy activity in vivo, decreased in mice with HFD feeding, suggesting that HFD treatment impairs mitophagy activity. However, ACC2 iKO mice partially restored mitophagy in response to HFD feeding. Therefore, the beneficial effect for enhancing FAO in HFD induced obesity model mediates by maintenance of mitochondria function through regulating mitophagy. Our findings suggest that promoting FAO is an effective strategy to resist the development of cardiac lipotoxicity during diet-induced obesity.