TFEB activation triggers pexophagy for functional adaptation during oxidative stress under calcium deficient-conditions.

BACKGROUND: Calcium is a ubiquitous intracellular messenger that regulates the expression of various genes involved in cell proliferation, differentiation, and motility. The involvement of calcium in diverse metabolic pathways has been suggested. However, the effect of calcium in peroxisomes, which are involved in fatty acid oxidation and scavenges the result reactive oxygen species (ROS), remains elusive. In addition, impaired peroxisomal ROS inhibit the mammalian target of rapamycin complex 1 (mTORC1) and promote autophagy. Under stress, autophagy serves as a protective mechanism to avoid cell death. In response to oxidative stress, lysosomal calcium mediates transcription factor EB (TFEB) activation. However, the impact of calcium on peroxisome function and the mechanisms governing cellular homeostasis to prevent diseases caused by calcium deficiency are currently unknown. METHODS: To investigate the significance of calcium in peroxisomes and their roles in preserving cellular homeostasis, we established an in-vitro scenario of calcium depletion. RESULTS: This study demonstrated that calcium deficiency reduces catalase activity, resulting in increased ROS accumulation in peroxisomes. This, in turn, inhibits mTORC1 and induces pexophagy through TFEB activation. However, treatment with the antioxidant N-acetyl-l-cysteine (NAC) and the autophagy inhibitor chloroquine impeded the nuclear translocation of TFEB and attenuated peroxisome degradation. CONCLUSIONS: Collectively, our study revealed that ROS-mediated TFEB activation triggers pexophagy during calcium deficiency, primarily because of attenuated catalase activity. We posit that calcium plays a significant role in the proper functioning of peroxisomes, critical for fatty-acid oxidation and ROS scavenging in maintaining cellular homeostasis. These findings have important implications for signaling mechanisms in various pathologies, including Zellweger's syndrome and ageing.

Catalase-deficient mice induce aging faster through lysosomal dysfunction.

BACKGROUND: Lysosomes are a central hub for cellular metabolism and are involved in the regulation of cell homeostasis through the degradation or recycling of unwanted or dysfunctional organelles through the autophagy pathway. Catalase, a peroxisomal enzyme, plays an important role in cellular antioxidant defense by decomposing hydrogen peroxide into water and oxygen. In accordance with pleiotropic significance, both impaired lysosomes and catalase have been linked to many age-related pathologies with a decline in lifespan. Aging is characterized by progressive accumulation of macromolecular damage and the production of high levels of reactive oxygen species. Although lysosomes degrade the most long-lived proteins and organelles via the autophagic pathway, the role of lysosomes and their effect on catalase during aging is not known. The present study investigated the role of catalase and lysosomal function in catalase-knockout (KO) mice. METHODS: We performed experiments on WT and catalase KO younger (9 weeks) and mature adult (53 weeks) male mice and Mouse embryonic fibroblasts isolated from WT and KO mice from E13.5 embryos as in vivo and in ex-vivo respectively. Mouse phenotyping studies were performed with controls, and a minimum of two independent experiments were performed with more than five mice in each group. RESULTS: We found that at the age of 53 weeks (mature adult), catalase-KO mice exhibited an aging phenotype faster than wild-type (WT) mice. We also found that mature adult catalase-KO mice induced leaky lysosome by progressive accumulation of lysosomal content, such as cathespin D, into the cytosol. Leaky lysosomes inhibited autophagosome formation and triggered impaired autophagy. The dysregulation of autophagy triggered mTORC1 (mechanistic target of rapamycin complex 1) activation. However, the antioxidant N-acetyl-L-cysteine and mTORC1 inhibitor rapamycin rescued leaky lysosomes and aging phenotypes in catalase-deficient mature adult mice. CONCLUSIONS: This study unveils the new role of catalase and its role in lysosomal function during aging. Video abstract.

Intracellular cholesterol transport inhibition Impairs autophagy flux by decreasing autophagosome-lysosome fusion.

BACKGROUND: Autophagy is an intracellular degradation process crucial for homeostasis. During autophagy, a double-membrane autophagosome fuses with lysosome through SNARE machinery STX17 to form autolysosome for degradation of damaged organelle. Whereas defective autophagy enhances cholesterol accumulation in the lysosome and impaired autophagic flux that results Niemann-Pick type C1 (NPC1) disease. However, exact interconnection between NPC1 and autophagic flux remain obscure due to the existence of controversial reports. RESULTS: This study aimed at a comparison of the effects of three autophagic inhibitor drugs, including chloroquine, U18666A, and bafilomycin A1, on the intracellular cholesterol transport and autophagy flux. Chloroquine, an autophagic flux inhibitor; U1866A, a NPC1 inhibitor, and bafilomycin A, a lysosomotropic agent are well known to inhibit autophagy by different mechanism. Here we showed that treatment with U1866A and bafilomycin A induces lysosomal cholesterol accumulation that prevented autophagic flux by decreasing autophagosome-lysosome fusion. We also demonstrated that accumulation of cholesterol within the lysosome did not affect lysosomal pH. Although the clearance of accumulated cholesterol by cyclodextrin restored the defective autophagosome-lysosome fusion, the autophagy flux restoration was possible only when lysosomal acidification was not altered. In addition, a failure of STX17 trafficking to autophagosomes plays a key role in prevention of autophagy flux caused by intracellular cholesterol transport inhibitors. CONCLUSIONS: Our data provide a new insight that the impaired autophagy flux does not necessarily result in lysosomal cholesterol accumulation even though it prevents autophagosome-lysosome fusion. Video abstract.

Catalase deficiency facilitates the shuttling of free fatty acid to brown adipose tissue through lipolysis mediated by ROS during sustained fasting.

BACKGROUND: Fatty acids (FA) derived from adipose tissue and liver serve as the main fuel in thermogenesis of brown adipose tissue (BAT). Catalase, a peroxisomal enzyme, plays an important role in maintaining intracellular redox homeostasis by decomposing hydrogen peroxide to either water or oxygen that oxidize and provide fuel for cellular metabolism. Although the antioxidant enzymatic activity of catalase is well known, its role in the metabolism and maintenance of energy homeostasis has not yet been revealed. The present study investigated the role of catalase in lipid metabolism and thermogenesis during nutrient deprivation in catalase-knockout (KO) mice. RESULTS: We found that hepatic triglyceride accumulation in KO mice decreased during sustained fasting due to lipolysis through reactive oxygen species (ROS) generation in adipocytes. Furthermore, the free FA released from lipolysis were shuttled to BAT through the activation of CD36 and catabolized by lipoprotein lipase in KO mice during sustained fasting. Although the exact mechanism for the activation of the FA receptor enzyme, CD36 in BAT is still unclear, we found that ROS generation in adipocytes mediated the shuttling of FA to BAT. CONCLUSIONS: Taken together, our findings uncover the novel role of catalase in lipid metabolism and thermogenesis in BAT, which may be useful in understanding metabolic dysfunction.

Knockdown of PEX16 Induces Autophagic Degradation of Peroxisomes.

Peroxisome abundance is regulated by homeostasis between the peroxisomal biogenesis and degradation processes. Peroxin 16 (PEX16) is a peroxisomal protein involved in trafficking membrane proteins for de novo peroxisome biogenesis. The present study demonstrates that PEX16 also modulates peroxisome abundance through pexophagic degradation. PEX16 knockdown in human retinal pigment epithelial-1 cells decreased peroxisome abundance and function, represented by reductions in the expression of peroxisome membrane protein ABCD3 and the levels of cholesterol and plasmalogens, respectively. The activation of pexophagy under PEX16 knockdown was shown by (i) abrogated peroxisome loss under PEX16 knockdown in autophagy-deficient ATG5 knockout cell lines, and (ii) increased autophagy flux and co-localization of p62-an autophagy adaptor protein-with ABCD3 in the presence of the autophagy inhibitor chloroquine. However, the levels of cholesterol and plasmalogens did not recover despite the restoration of peroxisome abundance following chloroquine treatment. Thus, PEX16 is indispensable for maintaining peroxisome homeostasis by regulating not only the commonly known biogenesis pathway but also the autophagic degradation of peroxisomes.

Catalase deficiency induces reactive oxygen species mediated pexophagy and cell death in the liver during prolonged fasting.

Peroxisomes are dynamic organelles that participate in a diverse array of cellular processes, including ß-oxidation, which produces a considerable amount of reactive oxygen species (ROS). Although we showed that catalase depletion induces ROS-mediated pexophagy in cells, the effect of catalase deficiency during conditions that favor ROS generation remains elusive in mice. In this study, we reported that prolonged fasting in catalase-knockout (KO) mice drastically increased ROS production, which induced liver-specific pexophagy, an autophagic degradation of peroxisomes. In addition, increased ROS generation induced the production of pro-inflammatory cytokines in the liver tissues of catalase-KO mice. Furthermore, there was a significant increase in the levels of aspartate transaminase and alanine transaminase as well as apparent cell death in the liver of catalase-KO mice during prolonged fasting. However, an intra-peritoneal injection of the antioxidant N-acetyl-l-cysteine (NAC) and autophagy inhibitor chloroquine inhibited the inflammatory response, liver damage, and pexophagy in the liver of catalase-KO mice during prolonged fasting. Consistently, genetic ablation of autophagy, Atg5 led to suppression of pexophagy during catalase inhibition by 3-aminotriazole (3AT). Moreover, treatment with chloroquine also ameliorated the inflammatory response and cell death in embryonic fibroblast cells from catalase-KO mice. Taken together, our data suggest that ROS-mediated liver-specific pexophagy observed during prolonged fasting in catalase-KO mice may be responsible for the process associated with hepatic cell death.

TMEM135 regulates primary ciliogenesis through modulation of intracellular cholesterol distribution.

Recent evidence has linked the lysosomal cholesterol accumulation in Niemann-Pick type C1 with anomalies associated with primary ciliogenesis. Here, we report that perturbed intracellular cholesterol distribution imposed by lysosomal cholesterol accumulation during TMEM135 depletion is closely associated with impaired ciliogenesis. TMEM135 depletion does not affect the formation of the basal body and the ciliary transition zone. TMEM135 depletion severely blunts Rab8 trafficking to the centrioles without affecting the centriolar localization of Rab11 and Rabin8, the upstream regulators of Rab8 activation. Although TMEM135 depletion prevents enhanced IFT20 localization at the centrioles, ciliary vesicle formation is not affected. Furthermore, enhanced IFT20 localization at the centrioles is dependent on Rab8 activation. Supplementation of cholesterol in complex with cyclodextrin rescues Rab8 trafficking to the centrioles and Rab8 activation, thereby recovering primary ciliogenesis in TMEM135-depleted cells. Taken together, our data suggest that TMEM135 depletion prevents ciliary vesicle elongation, a characteristic of impaired Rab8 function. Our study thus reveals a previously uncharacterized effect of erroneous intracellular cholesterol distribution on impairing Rab8 function and primary ciliogenesis.

Dimethyloxaloylglycine induces pexophagy in a HIF-2α dependent manner involving autophagy receptor p62.

Peroxisomes are metabolically active oxygen demanding organelles with a high abundance of oxidases making it vulnerable to low oxygen levels such as hypoxic conditions. However, the exact mechanism of peroxisome degradation in hypoxic condition remains elusive. In order to study the mechanism of peroxisome degradation in hypoxic condition, we use Dimethyloxaloylglycine (DMOG), a cell-permeable prolyl-4-hydroxylase inhibitor, which mimics hypoxic condition by stabilizing hypoxia-inducible factors. Here we report that DMOG degraded peroxisomes by selectively activating pexophagy in a HIF-2α dependent manner involving autophagy receptor p62. Furthermore, DMOG not only increased peroxisome turnover by pexophagy but also reduced HIF-2α dependent peroxisome proliferation at the transcriptional level. Taken together, our data suggest that hypoxic condition is a negative regulator for peroxisome abundance through increasing pexophagy and decreasing peroxisome proliferation in HIF-2α dependent manner.

Ciliogenesis is reciprocally regulated by PPARA and NR1H4/FXR through controlling autophagy in vitro and in vivo.

The primary cilia are evolutionarily conserved microtubule-based cellular organelles that perceive metabolic status and thus link the sensory system to cellular signaling pathways. Therefore, ciliogenesis is thought to be tightly linked to autophagy, which is also regulated by nutrient-sensing transcription factors, such as PPARA (peroxisome proliferator activated receptor alpha) and NR1H4/FXR (nuclear receptor subfamily 1, group H, member 4). However, the relationship between these factors and ciliogenesis has not been clearly demonstrated. Here, we present direct evidence for the involvement of macroautophagic/autophagic regulators in controlling ciliogenesis. We showed that activation of PPARA facilitated ciliogenesis independently of cellular nutritional states. Importantly, PPARA-induced ciliogenesis was mediated by controlling autophagy, since either pharmacological or genetic inactivation of autophagy significantly repressed ciliogenesis. Moreover, we showed that pharmacological activator of autophagy, rapamycin, recovered repressed ciliogenesis in ppara-/- cells. Conversely, activation of NR1H4 repressed cilia formation, while knockdown of NR1H4 enhanced ciliogenesis by inducing autophagy. The reciprocal activities of PPARA and NR1H4 in regulating ciliogenesis were highlighted in a condition where de-repressed ciliogenesis by NR1H4 knockdown was further enhanced by PPARA activation. The in vivo roles of PPARA and NR1H4 in regulating ciliogenesis were examined in greater detail in ppara-/- mice. In response to starvation, ciliogenesis was facilitated in wild-type mice via enhanced autophagy in kidney, while ppara-/- mice displayed impaired autophagy and kidney damage resembling ciliopathy. Furthermore, an NR1H4 agonist exacerbated kidney damage associated with starvation in ppara-/- mice. These findings indicate a previously unknown role for PPARA and NR1H4 in regulating the autophagy-ciliogenesis axis in vivo.

Catalase inhibition induces pexophagy through ROS accumulation.

Peroxisomes are dynamic and multifunctional organelles involved in various cellular metabolic processes, and their numbers are tightly regulated by pexophagy, a selective degradation of peroxisomes through autophagy to maintain peroxisome homeostasis in cells. Catalase, a major peroxisome protein, plays a critical role in removing peroxisome-generated reactive oxygen species (ROS) produced by peroxisome enzymes, but the contribution of catalase to pexophagy has not been reported. Here, we investigated the role of catalase in peroxisome degradation during nutrient deprivation. Both short interfering RNA-mediated silencing of catalase and pharmacological inhibition by 3-aminotriazole (3AT) decreased the number of peroxisomes and resulted in the downregulation of peroxisomal proteins, such as PMP70 and PEX14 under serum starvation. In addition, treatment with 3AT induced NBR1-dependent autophagy and PEX5 ubiquitination in the absence of serum, which was accompanied by accumulation of ROS. Co-treatment with antioxidant agent N-acetyl-l-cysteine (NAC) prevented ROS accumulation and pexophagy by modulating peroxisome protein levels and the association of NBR1, a pexophagy receptor with peroxisomes. Taken together, these findings demonstrate that catalase plays an important role in pexophagy during nutrient deprivation.

Autophagy alteration prevents primary cilium disassembly in RPE1 cells.

Primary cilium is a microtubule structure that emanates from the surface of most human cells. Primary cilia assemble during the resting stage (G0 phase) and disassemble with cell cycle progression. Defects associated with the control of the assembly or disassembly of the primary cilium have been implicated in various human diseases, including ciliopathy and cancer. Although studies have suggested the interplay between activation of autophagy and ciliogenesis, any direct mechanism between autophagy abatement and disassembly of primary cilium remains elusive. In this study, we found that the gradual abatement in autophagy during serum-restimulation was a dynamic process and significantly correlated with the disassembly of primary cilium in human retinal pigmented epithelial (RPE1) cells. Although autophagy activity was gradually decreased during serum-restimulation, the alteration in autophagy under the same condition prevented the disassembly of the primary cilium. Autophagy inhibitors such as chloroquine, U18666A and 3-methyladenine (3-MA) retained both the number of ciliated cells and cilium length. In contrast, rapamycin treatment during serum-restimulation maintained the number of ciliated cells with shortened cilia. Taken together, alteration in autophagy during serum-restimulation prevent the disassembly of the primary cilium, and autophagy modulators may serve as useful compounds for studying mechanistic details related to the disassembly of the primary cilium and ciliopathy.

3-Aminotriazole protects from CoCl2-induced ototoxicity by inhibiting the generation of reactive oxygen species and proinflammatory cytokines in mice.

Cobalt is an essential heavy metal that is necessary for the formation of vitamin B12 (hydroxocobalamin). However, exposure to excess cobalt for a prolonged period can harm the human body, causing pulmonary fibrosis, blindness, deafness, and peripheral neuropathy. 3-Aminotriazole (3-AT) is a catalase inhibitor that is often used to investigate the physiological effects of catalase. The present study found that injection of 3-AT in mice significantly reduced CoCl2-induced hearing impairment. In cultured organ of Corti explants from rats, 3-AT treatment protected hair cells from CoCl2-induced cytotoxicity. To determine the mechanism by which 3-AT protected from CoCl2-induced ototoxicity, we used the HEI-OC1 auditory cell line. Pretreatment with 10 mM 3-AT attenuated CoCl2-induced accumulation of ROS and induction of proinflammatory cytokine expression. Interestingly, these protective effects of 3-AT did not require catalase activity, as demonstrated by a series of experiments using RNA interference-mediated catalase knockdown in HEI-OC1 cells and using catalase-deficient mouse embryonic fibroblasts. Our results demonstrated the mechanisms of CoCl2-induced ototoxicity that may provide better ways to prevent the ototoxic effect of cobalt exposure.

Fenofibrate, a peroxisome proliferator-activated receptor α ligand, prevents abnormal liver function induced by a fasting-refeeding process.

Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, is an anti-hyperlipidemic agent that has been widely used in the treatment of dyslipidemia. In this study, we examined the effect of fenofibrate on liver damage caused by refeeding a high-fat diet (HFD) in mice after 24h fasting. Here, we showed that refeeding HFD after fasting causes liver damage in mice determined by liver morphology and liver cell death. A detailed analysis revealed that hepatic lipid droplet formation is enhanced and triglyceride levels in liver are increased by refeeding HFD after starvation for 24h. Also, NF-κB is activated and consequently induces the expression of TNF-α, IL1-ß, COX-2, and NOS2. However, treating with fenofibrate attenuates the liver damage and triglyceride accumulation caused by the fasting-refeeding HFD process. Fenofibrate reduces the expression of NF-κB target genes but induces genes for peroxisomal fatty acid oxidation, peroxisome biogenesis and mitochondrial fatty acid oxidation. These results strongly suggest that the treatment of fenofibrate ameliorates the liver damage induced by fasting-refeeding HFD, possibly through the activation of fatty acid oxidation.