Induction Of Mitophagy Research Articles

Engineered inhalable nanocatalytic therapeutics for Parkinson's disease by inducing mitochondrial autophagy

Reactive oxygen species (ROS) - induced oxidative stress damage of dopaminergic neurons is the principal etiology of Parkinson's disease (PD). While most nanoenzymes can catalyze the breakdown of ROS present in the brain, they cannot eradicate the source of ROS production attributed to damaged mitochondria. Herein, we introduce a Co-doped Prussian blue (PB/Co) nanozyme that demonstrates multi-enzyme-like coordinated activity for scavenging present ROS and triggers mitophagy to remove damaged mitochondria. To further augment the brain entry efficiency of PB/Co nanozyme, we encapsulated it in 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 2,3-bis (palmitoyloxy)propyl-2-(trimethylammonio)ethylphosphate (DPPC) cationic liposomes and developed a non-invasive inhalable nanospray ((PB/Co)@DD) that permeates the brain via the olfactory bulb. In the PD mice model, (PB/Co)@DD nanospray induced mitophagy in the striatum to eliminate the ROS production source, preventing excessive ROS-induced sustained damage to dopaminergic neurons and averting the buildup of α-synuclein deposits, thereby ultimately restoring motor function in PD mice. Our research lays the foundation for catalytic therapy to eliminate abnormal mitochondria via the induction of mitophagy in PD mice models and highlights the potential of inhalable nanoenzymes as a non-invasive therapeutic strategy for the treatment of neuroinflammatory diseases.

TNIP1 inhibits selective autophagy via bipartite interaction with LC3/GABARAP and TAX1BP1

Mitophagy is a form of selective autophagy that disposes of superfluous and potentially damage-inducing organelles in a tightly controlled manner. While the machinery involved in mitophagy induction is well known, the regulation of the components is less clear. Here, we demonstrate that TNIP1 knockout in HeLa cells accelerates mitophagy rates and that ectopic TNIP1 negatively regulates the rate of mitophagy. These functions of TNIP1 depend on an evolutionarily conserved LIR motif as well as an AHD3 domain, which are required for binding to the LC3/GABARAP family of proteins and the autophagy receptor TAX1BP1, respectively. We further show that phosphorylation appears to regulate its association with the ULK1 complex member FIP200, allowing TNIP1 to compete with autophagy receptors, which provides a molecular rationale for its inhibitory function during mitophagy. Taken together, our findings describe TNIP1 as a negative regulator of mitophagy that acts at the early steps of autophagosome biogenesis.

Staphylococcus aureus induces mitophagy to promote its survival within bovine mammary epithelial cells

Mitophagy occurs in a variety of pathogenic infections. However, the role of mitophagy in the intracellular survival of Staphylococcus aureus (S.aureus) within bovine mammary epithelial cells (BMECs) and which molecules specifically mediate the induction of mitophagy remains unclear. Therefore, this study aims to investigate the role and mechanism of mitophagy in the intracellular survival of S.aureus. Here, we reported that S.aureus induced complete mitophagy to promote its survival within BMECs. The further mechanistic study showed that S. aureus induced mitophagy by activating the p38-PINK1-Parkin signaling pathway. These findings expand our knowledge of the intracellular survival mechanism of S.aureus in the host and provide a desirable therapeutic strategy against S.aureus and other intracellular infections.

Nrf2/PINK1-mediated mitophagy induction alleviates sodium fluoride-induced hepatic injury by improving mitochondrial function, oxidative stress, and inflammation

Mitophagy has distinct functions, which can lead to either protection or damage of tissues. Though current evidence indicated that NaF triggers mitophagy, the role and regulation of mitophagy in sodium fluoride (NaF)-induced liver injury still remain unclear. Therefore, we exployed the cell and mouse models and confirmed that NaF treatment activates mitophagy. Knocking down PTEN-induced putative kinase protein 1 (PINK1) expression attenuated mitophagy and increased the degree of mitochondrial impairment, oxidative stress, and apoptosis in NaF-treated HepG2 cells. In vivo experiments indicated that PINK1 deficiency weakened NaF-induced mitophagy. Moreover, PINK1-deficient mices aggravated NaF-induced hepatic mitochondrial injury, oxidative stress, and inflammation in livers, evidenced by the increased number of abnormal mitochondria, decreased adenosine triphosphate (ATP) and glutathione (GSH) levels, elevated reactive oxygen species (ROS) and malondialdehyde (MDA) content, enhanced hepatic macrophage infiltration and inflammatory cytokine levels. Notably, NaF exposure activated Nrf2 signaling both in vitro and in vivo. Nrf2 siRNA transfection blocked the upregulation of PINK1 expression and the induction of mitophagy in NaF-treated HepG2 cells. Also, ML385 (Nrf2 inhibitor) partially blocked the upregulation of PINK1 expression caused by NaF in mice livers. To sum up, the present study provided the demonstration that Nrf2/PINK1-mediated mitophagy activation offers a hepatoprotective effect by inhibiting NaF-induced mitochondrial dysfunction, oxidative stress, and inflammation.

Neuroprotective effects of melatonin-mediated mitophagy through nucleotide-binding oligomerization domain and leucine-rich repeat-containing protein X1 in neonatal hypoxic-ischemic brain damage.

Hypoxia-ischemia (HI) is a major cause of brain damage in neonates. Mitochondrial dysfunction acts as a hub for a broad spectrum of signaling events, culminating in cell death triggered by HI. A neuroprotective role of melatonin (MT) has been proposed, and mitophagy regulation seems to be important for cell survival. However, the molecular mechanisms underlying MT-mediated mitophagy during HI treatment are poorly defined. Nucleotide-binding oligomerization domain and leucine-rich repeat-containing protein X1 (NLRX1) has emerged as a critical regulator of mitochondrial dynamics and neuronal death that participates in the pathology of diverse diseases. This study aimed to clarify whether NLRX1 participates in the regulation of mitophagy during MT treatment for hypoxic-ischemic brain damage (HIBD). We demonstrated that MT protected neonates from HIBD through NLRX1-mediated mitophagy in vitro and in vivo. Meanwhile, MT upregulated the expression of NLRX1, Beclin-1, and autophagy-related 7 (ATG7) but decreased the expression of the mammalian target of rapamycin (mTOR) and translocase of the inner membrane of mitochondrion 23 (TIM23). Moreover, the neuroprotective effects of MT were abolished by silencing NLRX1 after oxygen-glucose deprivation (OGD). In addition, the downregulation of mTOR and upregulation of Beclin-1 and ATG7 by MT were inhibited after silencing NLRX1 under OGD. In summary, MT modulates mitophagy induction through NLRX1 and plays a protective role in HIBD, providing insight into potential therapeutic targets for MT to exert neuroprotection.

TRIM72 Alleviates Muscle Inflammation in mdx Mice via Promoting Mitophagy-Mediated NLRP3 Inflammasome Inactivation.

Chronic muscle inflammation exacerbates the pathogenesis of Duchenne muscular dystrophy (DMD), which is characterized by progressive muscle degeneration and weakness. NLRP3 (nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3) inflammasome plays a key role in the inflammatory process, and its abnormal activation leads to a variety of inflammatory or immune diseases. TRIM72 (MG53) is a protective myokine for tissue repair and regeneration. However, little is known about the potential impact of TRIM72 in the crosstalk between mitophagy and inflammatory process of DMD. Here, 10-week-old male mdx mice were injected intramuscularly with adeno-associated virus (AAV-TRIM72) to overexpress TRIM72 protein for 6 weeks. Then, skeletal muscle samples were collected, and relevant parameters were measured by histopathological analysis and molecular biology techniques. C2C12 cell line was transfected with lentivirus (LV-TRIM72) to overexpress or siRNA (si-TRIM72) to suppress the TRIM72 expression for the following experiment. Our data firstly showed that the TRIM72 expression was decreased in skeletal muscles of mdx mice. Then, we observed the increased NLRP3 inflammasome and impaired mitophagy in mdx mice compared with wild type mice. In mdx mice, administration of AAV-TRIM72 alleviated the accumulation of NLRP3 inflammasome and the consequent IL-18 and IL1β maturation by inducing autophagy, while this protective effect was reversed by chloroquine. Mitochondrial reactive oxygen species (mtROS), as a recognized activator for NLRP3 inflammasome, was attenuated by TRIM72 through the induction of mitophagy in C2C12 cells. Additionally, we proposed that the TRIM72 overexpression might promote mitophagy through both the early stage by PI3K-AKT pathway and the late stage by autolysosome fusion. In conclusion, the current study suggests that TRIM72 prevents DMD inflammation via decreasing NLRP3 inflammasomes and enhancing mitophagy. Collectively, our study provides insight into TRIM72 as a promising target for therapeutic intervention for DMD.

P53 Regulates Mitochondrial Dynamics in Vascular Smooth Muscle Cell Calcification.

Arterial calcification is an important characteristic of cardiovascular disease. It has key parallels with skeletal mineralization; however, the underlying cellular mechanisms responsible are not fully understood. Mitochondrial dynamics regulate both bone and vascular function. In this study, we therefore examined mitochondrial function in vascular smooth muscle cell (VSMC) calcification. Phosphate (Pi)-induced VSMC calcification was associated with elongated mitochondria (1.6-fold increase, p < 0.001), increased mitochondrial reactive oxygen species (ROS) production (1.83-fold increase, p < 0.001) and reduced mitophagy (9.6-fold decrease, p < 0.01). An increase in protein expression of optic atrophy protein 1 (OPA1; 2.1-fold increase, p < 0.05) and a converse decrease in expression of dynamin-related protein 1 (DRP1; 1.5-fold decrease, p < 0.05), two crucial proteins required for the mitochondrial fusion and fission process, respectively, were noted. Furthermore, the phosphorylation of DRP1 Ser637 was increased in the cytoplasm of calcified VSMCs (5.50-fold increase), suppressing mitochondrial translocation of DRP1. Additionally, calcified VSMCs showed enhanced expression of p53 (2.5-fold increase, p < 0.05) and β-galactosidase activity (1.8-fold increase, p < 0.001), the cellular senescence markers. siRNA-mediated p53 knockdown reduced calcium deposition (8.1-fold decrease, p < 0.01), mitochondrial length (3.0-fold decrease, p < 0.001) and β-galactosidase activity (2.6-fold decrease, p < 0.001), with concomitant mitophagy induction (3.1-fold increase, p < 0.05). Reduced OPA1 (4.1-fold decrease, p < 0.05) and increased DRP1 protein expression (2.6-fold increase, p < 0.05) with decreased phosphorylation of DRP1 Ser637 (3.20-fold decrease, p < 0.001) was also observed upon p53 knockdown in calcifying VSMCs. In summary, we demonstrate that VSMC calcification promotes notable mitochondrial elongation and cellular senescence via DRP1 phosphorylation. Furthermore, our work indicates that p53-induced mitochondrial fusion underpins cellular senescence by reducing mitochondrial function.

PS-B01-4: ANGIOTENSIN II TYPE1 RECEPTOR INHIBITION IMPROVES VASCULAR SENESCENCE THROUGH IMPROVEMENT OF MITOCHONDRIAL QUALITY CONTROL VIA CRAF/MEK/ERK PATHWAY

Introduction: Prevention of atherosclerotic cardiovascular disease has a problem of residual risk that is insufficient even if lipids are properly lowered by drugs such as statin. It has been reported that lectin-like oxidized low-density lipoprotein scavenger receptor-1 (LOX-1) associated with Angiotensin II Type 1 Receptor (AT1R). Mitochondria play crucial role in vascular senescence and atherosclerosis. We hypothesized receptor association with LOX-1 and AT1R might be a breakthrough to solve residual risk in terms of mitochondrial quality control. Methods: We performed in vitro and in vivo experiments using vascular smooth muscle cell (VSMC), C57BL6 (WT) and apolipoprotein E deficient (ApoE KO) mice. Results: Administration of ox-LDL to VSMC induced mitochondrial dysfunction and cellular senescence accompanied by excessive mitochondrial fission through Drp1 activation. This Drp1 activation was derived from the activation of CRAF/MEK/ERK pathway, which is a downstream of AT1R. AT1R inhibition inactivated ox-LDL-mediated Drp1 activation and retarded mitochondrial dysfunction, excessive mitochondrial fission and cellular senescence, indicating that LDL receptor LOX-1 associates with AT1R and activates CRAF/MEK/ERK/Drp1, leading to induction of mitochondrial fission and resulting in facilitating cellular senescence. AT1R inhibition also induced mitophagy assessed by electron microscopy and immunohistochemistry of LAMP2 and TOMM20. AT1R inhibition-induced mitophagy was not affected by Atg7 knockdown, whereas it was diminished by Rab9 knockdown. Immunohistochemistry showed TOMM20 dots were co-localized to LAMP2 and Rab9 but not LC3. These results suggest that AT1R signal induces mitophagy derived from Rab9-dependent alternative autophagy. Moreover, inhibition of either CRAF or MEK also decreased excessive mitochondrial fission and induced mitophagy, suggesting that AT1R signal followed by CRAF/MEK pathway modulates both mitochondrial dynamics and mitophagy. Taken together, inhibition of AT1R induces dual modification of mitochondrial dynamics and mitophagy through inactivation of CRAF/MEK/ERK axis. Finally, we confirmed that the degree of vascular senescence and the number of fragmented mitochondria were higher in ApoE KO than those of WT mice, and treatment with Candesartan to ApoE KO inhibited vascular senescence through induction of mitophagy and imbalance of mitochondrial dynamics. Conclusion: Receptor association of LOX-1 with AT1R plays a crucial role in cellular/vascular senescence induced by ox-LDL, and AT1R inhibition improves mitochondrial quality control through regulation of mitochondrial dynamics and mitophagy derived from Rab9-dependent alternative autophagy.

THE ROLE OF AUTOPHAGY IN THE REGULATION OF NEUROINFLAMMATION IN ACUTE ISCHEMIC STROKE (REVIEW OF LITERATURE)

Postischemic neuroinflammation is a critical pathophysiological process within the entire scheme of cerebral ischemia, covering early damage and the period of tissue repair. It is characterized by microglial and astroglial activation with increased expression of inflammatory mediators and is accompanied by impaired innate and adaptive immune responses. In acute ischemic stroke (IS), neuroinflammation is caused by the response of resident immune cells of microglia and peripheral immunocompetent cells infiltrating the brain tissue, which penetrate the blood-brain barrier (BBB) into the lesion. Recent studies have shown the important role of the NLRP3-mediated inflammation in the death of neurons and glial cells in acute IS. The review presents the main mechanisms of activation of NLRP3-mediated inflammation in acute IS, leading to the caspase-1 formation and the IL-1β and IL-18 release, which are involved in the initiation and progression of inflammation in the brain parenchyma. The literature data on the role of autophagy in the inhibition of postischemic neuroinflammation are summarized. Autophagy can suppress neuroinflammation through a wide range of the autophagy - related proteins. The role of autophagy as a negative regulator of NLRP3-mediated inflammation in acute IS is analyzed. Data on the participation of autophagic proteins Beclin-1, LC3, and p62 in the suppression of NLRP3 inflammation due to the induction of basic mitophagy are presented. Prospects for modulating autophagy aimed at suppressing postischemic neuroinflammation, including the inhibition of NLRP3-inflammasome, have been noted. The review was based on sources from international and national data bases: Scopus, Web of Science, Springer, RINC.

722 NATURAL ACTIVATORS OF AUTOPHAGY PROTECT THE HEART FROM DOX-INDUCED CARDIOMYOPATHY

Abstract Introduction Heart failure is a frequent death cause for oncological patients who received treatment with doxorubicin (DOX). DOX administration inhibits autophagy in cardiomyocytes and causes myocardial damage. Natural activators of autophagy (NAA) such as trehalose, a natural disaccharide, and spermidine, a putrescine-derived polyamine initially isolated from semen, were previously found to be promising candidates to recover the autophagic flux and treat cardiovascular diseases in mice. Hypothesis The administration of natural activators of autophagy protects the heart from DOX-induced myocardial injury. Methods C57BL/6J WT mice were fed ad libitum with trehalose or spermidine in drinking water and received 3 weekly injections of DOX, reaching a final cumulative dose of 15 mg/kg. Echocardiographic, histological and biochemical analyses were performed 6 weeks after the first administration of DOX. Also, in vitro experimentation was performed on neonatal rat primary cardiomyocytes. Results Mice treated with DOX had reduced systolic function (FS: 44±1.05% vs. 34.1±2.33%, n=6-8), but trehalose administration preserved left ventricular fractional shortening (FS: 34.1 [Office1]±2.33% vs. 45.1±0.75%, n=6-8). Autophagy-deficient Beclin1+/- mice had reduced FS and were not protected by trehalose administration (35.4±2.48% vs. 45.1±0.75%, n=7-8). Mice treated with DOX developed fibrosis (0.1±0.08% vs. 8.6±3.5%, n=5), but this effect was reduced in trehalose-fed mice that received DOX (8.6±3.5% vs. 2±0.68%, n=4-5). DOX administration increased the levels of damaged-mitochondria disposal (2.6±0.74 vs. 9.4±1.7 mitophagic bodies per TEM field, n=13) and trehalose administration further boosted this effect (9.4±1.7 vs. 24.1±1.45, n=13). The mRFP-eGFP-Lc3-dots assay was used for evaluating the autophagic flux in neonatal primary cardiomyocytes and a reduction of autophagy was observed in cells treated with DOX (51.9±4.57 vs. 29.9±3.52 red dots per cell, n=34-51), while trehalose recovered the autophagic flux (29.9±3.52 vs. 121.5±8.02 red dots per cell, n=33-34). Mitochondrial biogenesis reporter ‘MitoTimer’ mice that received DOX showed increased levels of biogenesis (0.8±0.38 vs. 3.3±0.9, n=5-6), while mice receiving DOX and trehalose did not (3.3±0.9 vs. 0.3±0.12, n=5-6). Similarly, spermidine preserved systolic function (FS: 29.4±2.03 vs. 39±3.22, n=5-6) and increased myocardial autophagy. Conclusion NAA-mediated mitophagy induction protects the heart from developing DOX-induced cardiomyopathy by disposing of damaged mitochondria. [Office1]Le virgole diventano punti

Heme-dependent induction of mitophagy program during differentiation of murine erythroid cells

Although establishment and maintenance of mitochondria are essential for the production of massive amounts of heme in erythroblasts, mitochondria must be degraded upon terminal differentiation to red blood cells (RBCs), thus creating a biphasic regulatory process. Previously, we reported that iron deficiency in mice promotes mitochondrial retention in RBCs, suggesting that a proper amount of iron and/or heme is necessary for the degradation of mitochondria during erythroblast maturation. Because the transcription factor GATA1 regulates autophagy in erythroid cells, which involves mitochondrial clearance (mitophagy), we investigated the relationship between iron or heme and mitophagy by analyzing the expression of genes related to GATA1 and autophagy and the impact of iron or heme restriction on the amount of mitochondria. We found that heme promotes the expression of GATA1-regulated mitophagy-related genes and the induction of mitophagy. GATA1 might induce the expression of the autophagy-related genes Atg4d and Stk11 for mitophagy through a heme-dependent mechanism in murine erythroleukemia (MEL) cells and a genetic rescue system with G1E-ER-GATA1 erythroblast cells derived from Gata1-null murine embryonic stem cells. These results provide evidence for a biphasic mechanism in which mitochondria are essential for heme generation, and the heme generated during differentiation promotes mitophagy and mitochondrial disposal. This mechanism provides a molecular framework for understanding this fundamentally important cell biological process.

Autophagy and retinal ganglion cell death in glaucoma: Friend or foe?

AbstractGlaucoma, a leading cause of irreversible blindness worldwide, is an age‐related neurodegenerative disease characterized by progressive alterations of the optic nerve head and degeneration of retinal ganglion cells (RGCs). Although elevated intraocular pressure (IOP) has been identified as the main and the only modifiable risk factor, the pathogenesis of the disease is still unclear. Indeed, lowering IOP is often insufficient to prevent the damage progression leaving unmet the therapeutic need of IOP‐independent pharmacological targets.Autophagy, and the more selective mitophagy, are responsible for breaking down cellular components, preserving cellular homeostasis and preventing the accumulation of altered and aggregated proteins and damaged organelles.Several evidence have shown that modulation of autophagy is a recurrent and functional response of RGCs to IOP‐dependent or independent glaucoma‐related insults. However, in RGCs, autophagy has been found to take part to either neuroprotection as well as neuronal death, depending on the experimental setting (i.e. animal model, timing of the experiments, drugs/doses used to modulate the pathway).Here we describe the dynamic changes of autophagy flux and the modulation of mitophagy related proteins in a model of acute glaucoma induced in C57BL/6J mice by transient elevation of intraocular pressure (IOP). We also report and discuss experimental evidence supporting the pro‐survival role of autophagy and mitophagy induction in mice subjected to ocular hypertension suggesting the possibility to targeting autophagy to achieve retinal neuroprotection in glaucoma.

ATP13A2 modifies mitochondrial localization of overexpressed TOM20 to autolysosomal pathway.

Mutations in ATP13A2 cause Kufor-Rakeb Syndrome (KRS), a juvenile form of Parkinson's Disease (PD). The gene product belongs to a diverse family of ion pumps and mediates polyamine influx from lysosomal lumen. While the biochemical and structural studies highlight its unique mechanics, how PD pathology is linked to ATP13A2 function remains unclear. Here we report that localization of overexpressed TOM20, a mitochondrial outer-membrane protein, is significantly altered upon ATP13A2 expression to partially merge with lysosome. Using Halo-fused version of ATP13A2, ATP13A2 was identified in lysosome and autophagosome. Upon ATP13A2 co-expression, overexpressed TOM20 was found not only in mitochondria but also within ATP13A2-containing autolysosome. This modification of TOM20 localization was inhibited by adding 1-methyl-4-phenylpyridinium (MPP+) and not accompanied with mitophagy induction. We suggest that ATP13A2 may participate in the control of overexpressed proteins targeted to mitochondrial outer-membrane.

Vaspin Attenuates Atrial Abnormalities by Promoting ULK1/FUNDC1-Mediated Mitophagy.

The worldwide incidence and prevalence of atrial fibrillation (AF) are increasing, making it a life-threatening condition due to the higher numbers of people suffering from obesity. Vaspin, an adipokine derived from epicardial adipose tissue, has been reported to reduce inflammation, inhibit apoptosis, and induce autophagy; however, its role in the pathogenesis of AF is not known. In this study, we investigated the role of vaspin in patients with AF and explored the molecular mechanisms using atrial myocytes in vitro. Our data showed that vaspin levels were significantly reduced in the plasma of patients with AF. Lower plasma levels of vaspin were also associated with a higher risk of AF in patients with obesity. Vaspin treatment in vitro alleviated cardiomyocyte injury, atrial fibrosis, atrial myocyte apoptosis, and mitochondrial injury in atrial myocytes following Ang-II stress. Moreover, our results demonstrated that vaspin protected against Ang-II-induced atrial myocyte dysfunction by inducing mitophagy. We also observed that vaspin treatment enhanced the phosphorylation of Fun14 domain-containing protein 1 (FUNDC1) at Ser17 by unc-51 like autophagy activating kinase 1 (ULK1), resulting in the induction of mitophagy. These positive effects of vaspin were reversed by ULK1 silencing in Ang-II-stimulated HL-1 cells. Our study is the first to propose that vaspin plays a vital role in AF pathogenesis via ULK1/FUNDC1-regulated mitophagy and could be a novel therapeutic target for AF.

Bnip3lb Regulated Mitophagy Maintains the Embryonic Pool of Hematopoietic Stem Cells By Protecting Them from ROS Induced Apoptosis

We have previously shown that Reactive Oxygen Species (ROS) promote Hematopoietic Stem and Progenitor Cell (HSPC) specification in the dorsal aorta, however, in the adult marrow it is established that HSPCs must maintain low levels of ROS to protect the stem cell pool against excessive cycling, differentiation, loss of stemness and apoptosis. We aim to elucidate the developmental timing and the mechanism by which HSCs transition from their initial dependence on ROS, to their precise regulation of it in the adult marrow. Since adult HSPCs regulate ROS by maintaining high levels of mitophagy, a process in which damaged mitochondria, which disproportionately generate ROS, are removed from the cell and recycled, we sought to define the initiation, regulation and impact of mitophagy in embryonic HSPC development. Our recent data shows that oxidative stress limits HSPC numbers as early as their colonization of the secondary hematopoietic niche, the Caudal Hematopoietic Tissue (CHT). This is the zebrafish analog of the mammalian fetal liver in that it is the tissue to which the HSPCs migrate after emerging from the hemogenic endothelium of the dorsal aorta. Once in the CHT they mature and proliferate before migrating again to their terminal niche in the marrow. In contrast to published data showing that ROS promotes HSPC specification in the dorsal aorta, when ROS levels were increased in the embryo using a morpholino against PRDX1 we saw a decrease in runx1/c-myb expression in the CHT. Conversely, when ROS was decreased using the antioxidant N-acetyl cystine (NAC), there was an increase in HSPC marker expression. By live imaging of zebrafish embryos expressing a recently developed fluorescent transgenic mitophagy-reporter, Tg(ubi:mitoGR), we observed that mitophagy is low in the hemogenic endothelial tissue which gives rise to HSPCs, but is upregulated as HSPCs emerge and begin to migrate to the CHT, and is occurring at high levels at the developmental stage when ROS begins to become damaging to the HSPC population. Our scRNAseq analysis and in situ-hybridization experiments allowed us to examine which genes and pathways were regulating the induction of mitophagy in these cells. These data show that the increase in mitophagy induction in the HSPC population at this timepoint coincides precisely with the initialization of expression of the bnip3lb mitophagy receptor gene. Fluorescent whole-mount in situ hybridization experiments demonstrated that bnip3lb is co-expressed with runx1 (a HSPC marker), in the CHT (figure 1), and, by examining our Tg(ubi:mitoGR) mitophagy reporter line after morpholino knock down of bnip3lb we confirmed that it is this gene which is driving mitophagy in HSPCs at this developmental stage. NIX, the murine homolog of bnip3lb, is responsible for developmentally regulated mitophagy in reticulocytes, lens fibers and macrophages, but has, thus far, had no reported role in maintaining HSPCs. Functionally, mitophagy appears to be essential for the survival of the HSPC population in the CHT. Reducing mitophagy via morpholino directed knockdown of bnip3lb has no effect on the initial specification of HSPCs in the dorsal aorta, but significantly decreases HSPC marker expression in the CHT, as measured by runx1/c-myb in-situ hybridization, and quantified by CD41:GFP HSPC reporter expression. In contrast upregulating mitophagy, which can be done via a novel heat-shock driven ΔOTC transgenic line or via small molecule mitophagy induction, elevates HSPC numbers in the CHT. Mechanistically, we found that bnip3lb knockdown increases detection of CellROX, a fluorescent dye which reports ROS levels, and increases the number of acridine orange+ apoptotic runx1+ HSPCs. Further, reducing ROS with antioxidant NAC reduces apoptosis and rescues the HSPC population (figure 2). We therefore propose that developmentally programmed mitophagy directed by bnip3lb is responsible for protecting proliferative embryonic HSPCs from the harmful effects of oxidative stress while the HSPC pool is expanding to seed lifelong hematopoiesis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Platelet activation and mitophagy induction by thymic stromal lymphopoietin (TSLP) is associated with thrombosis in Kawasaki disease.

Kawasaki disease (KD) is an acute vasculitis of early childhood with unknown cause, which can be complicated by coronary artery lesions resulting in thrombosis. Fu and coworkers identify thymic stromal lymphopoietin (TSLP) to be increased in KD patients with thrombosis. They demonstrate that TSLP can activate platelets, induce mitophagy and trigger thrombus formation in vitro, suggesting that TSLP may have potential as a therapeutic target against KD-associated thrombosis. Commentary on: Fu L, et al. TSLP induces platelet mitophagy and promotes thrombosis in Kawasaki disease. Br J Haematol. 2023; 200:776-791.

Cobalt chloride has beneficial effects across species through a hormetic mechanism.

Severe oxygen and iron deficiencies have evolutionarily conserved detrimental effects, leading to pathologies in mammals and developmental arrest as well as neuromuscular degeneration in the nematode Caenorhabditis elegans. Yet, similar to the beneficial effects of mild hypoxia, non-toxic levels of iron depletion, achieved with the iron chelator bipyridine or through frataxin silencing, extend C. elegans lifespan through hypoxia-like induction of mitophagy. While the positive health outcomes of hypoxia preconditioning are evident, its practical application is rather challenging. Here, we thus test the potential beneficial effects of non-toxic, preconditioning interventions acting on iron instead of oxygen availability. We find that limiting iron availability through the iron competing agent cobalt chloride has evolutionarily conserved dose-dependent beneficial effects: while high doses of cobalt chloride have toxic effects in mammalian cells, iPS-derived neurospheres, and in C. elegans, sub-lethal doses protect against hypoxia- or cobalt chloride-induced death in mammalian cells and extend lifespan and delay age-associated neuromuscular alterations in C. elegans. The beneficial effects of cobalt chloride are accompanied by the activation of protective mitochondrial stress response pathways.

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.

Mitochondrial Localization of SARM1 in Acrylamide Intoxication Induces Mitophagy and Limits Neuropathy.

Sterile α and toll/interleukin 1 receptor motif-containing protein 1 (SARM1) is the defining molecule and central executioner of programmed axon death, also known as Wallerian degeneration. SARM1 has a mitochondrial targeting sequence, and it can bind to and stabilize PTEN-induced putative kinase 1 (PINK1) for mitophagy induction, but the deletion of the mitochondrial localization sequence is found to disrupt the mitochondrial localization of SARM1 in neurons without altering its ability to promote axon degeneration after axotomy. The biological significance of SARM1 mitochondrial localization remains elusive. In this study, we observed that the pro-degeneration factor, SARM1, was upregulated in acrylamide (ACR) neuropathy, a slow, Wallerian-like, programmed axonal death process. The upregulated SARM1 accumulated on mitochondria, interfered with mitochondrial dynamics, and activated PINK1-mediated mitophagy. Importantly, rapamycin (RAPA) intervention eliminated mitochondrial accumulation of SARM1 and partly attenuated ACR neuropathy. Thus, mitochondrial localization of SARM1 may contribute to its clearance through the SARM1-PINK1 mitophagy pathway, which inhibits axonal degeneration through a negative feedback loop. The mitochondrial localization of SARM1 complements the coordinated activity of the pro-survival factor, nicotinamide mononucleotide adenyltransferase 2 (NMNAT2), and SARM1 and is part of the self-limiting molecular mechanisms underpinning programmed axon death in ACR neuropathy. Mitophagy clearance of SARM1 is complementary to the coordinated activity of NMNAT2 and SARM1 in ACR neuropathy.

Azulene hydrazide-hydrazones for selective targeting of pancreatic cancer cells

Dysregulation of iron homeostasis is one of the important processes in the development of many oncological diseases, such as pancreatic cancer. Targeting it with specific agents, such as an iron chelator, are promising therapeutic methods. In this study, we tested the cytotoxicity of novel azulene hydrazide-hydrazone-based chelators against pancreatic cancer cell lines (MIA PaCa-2, PANC-1, AsPC-1). All prepared chelators (compounds 4-6) showed strong cytotoxicity against pancreatic cancer cell lines and high selectivity for cancer cell lines compared to the healthy line. Their cytotoxicity is lower than thiosemicarbazone-based chelators Dp44mT and DpC, but significantly higher than hydroxamic acid-based chelator DFO. The chelator tested showed mitochondrial and lysosomal co-localization and its mechanism of action was based on the induction of hypoxia-inducible factor-1-alpha (HIF-1α), N-myc downstream-regulated gene-1 (NDRG1) and transferrin receptor 1 (TfR1). This strongly implies that the cytotoxic effect of tested chelators could be associated with mitophagy induction. Lipinski's rule of five analyses was performed to determine whether the prepared compounds had properties ensuring their bioavailability. In addition, the drug-likeness and drug-score were calculated and discussed.