Induction Of Mitophagy Research Articles

Abstract 3271: The casein kinase 2/IKZF1 axis attenuates ceramide accumulation through up-regulation of acid ceramidase: Implications for use of CK2 inhibitors in high-risk pediatric B-cell acute lymphoid leukemia

Abstract Casein Kinase II (CK2), a growth-promoting non-oncogene, is frequently over-expressed in pediatric high-risk B-cell acute lymphoblastic leukemia (B-ALL) leading to proliferation and survival. CK2 phosphorylates the tumor suppressor IKZF1 (IKAROS) resulting in a loss-of DNA-binding activity. Loss of IKZF1 function is associated with poor prognosis in high-risk B-ALL. As such, inhibition of CK2 using silmitasertib (CX-4945), to restore IKZF1 function, is being therapeutically investigated in high-risk B-ALL. CK2 also phosphorylates/activates the mammalian ceramide synthases to increase production of the pro-apoptotic sphingolipid, ceramide (Cer). Therefore, we hypothesized that inhibition of CK2 with CX-4945 would exert both pro-death (restoration of IKAROS function) and pro-survival (decreased Cer production) effects on B-ALL cells. Moreover, this suggests that targeting the sphingolipid metabolic pathway in tandem with CX-4945 could be a novel strategy for treatment of high-risk B-ALL. To this end, we examined the effects of CX-4945 on sphingolipid levels and the enzymes of the sphingolipid metabolic pathway. Unexpectedly, we determined that CX-4945 dose-dependently increased expression of ceramide synthase 1 (CERS1) and acid ceramidase (AC) proteins, in an IKZF1 dependent manner. Increased CERS1 expression/production of C18:0 Cer has been linked to enhanced mitophagy (i.e., lysosomal clearance of damaged mitochondria) in head and neck squamous cell carcinoma. Induction of mitophagy by CX-4945 was confirmed by western blot analysis of mitofusin-1 and Drp-1 levels. Up-regulation of AC, a lysosomal protein, metabolizes C18:0 Cer to sphingosine for recycling and prevents Cer-induced lysosomal membrane permeabilization. Thus, induction of mitophagy and metabolic breakdown of Cer could potentially reduce the apoptotic efficacy of CX-4945 in B-ALL cell lines. We, therefore, tested whether inhibition of AC would synergize with CX-4945 to enhance B-ALL cell death. Indeed, synergy was observed with the AC inhibitor, SACLAC, in multiple cell lines. Consistent with the observation of synergy between CX-4945 and SACLAC, the combination also enhanced cleavage of PARP indicating an enhancement of cell death. We are currently testing the efficacy of a combination of CX-4945 and AC inhibitors in cell-line derived xenograft models of human B-ALL. Together, these findings indicate that simultaneous targeting of CK2 and AC represents a rationally selected combinatorial therapeutic strategy that overcomes the ability of cancer cells to adapt to single agent targeted therapies by altering pro- and/or anti-apoptotic pathways. Citation Format: Jeremy A. Hengst, Diwakar Bastihalli Tukaramrao, Todd Fox, Arati Sharma, Dhimant Desai, Sinisa Dovat. The casein kinase 2/IKZF1 axis attenuates ceramide accumulation through up-regulation of acid ceramidase: Implications for use of CK2 inhibitors in high-risk pediatric B-cell acute lymphoid leukemia [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 3271.

Mitophagy defect mediates the aging-associated hallmarks in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal disease manifested by premature aging and aging-related phenotypes, making it a disease model for aging. The cellular machinery mediating age-associated phenotypes in HGPS remains largely unknown, resulting in limited therapeutic targets for HGPS. In this study, we showed that mitophagy defects impaired mitochondrial function and contributed to cellular markers associated with aging in mesenchymal stem cells derived from HGPS patients (HGPS-MSCs). Mechanistically, we discovered that mitophagy affected the aging-associated phenotypes of HGPS-MSCs by inhibiting the STING-NF-ĸB pathway and the downstream transcription of senescence-associated secretory phenotypes (SASPs). Furthermore, by utilizing UMI-77, an effective mitophagy inducer, we showed that mitophagy induction alleviated aging-associated phenotypes in HGPS and naturally aged mice. Collectively, our results uncovered that mitophagy defects mediated the aging-associated markers in HGPS, highlighted the function of mitochondrial homeostasis in HGPS progression, and suggested mitophagy as an intervention target for HGPS and aging.

Non-cytopathic bovine viral diarrhea virus (BVDV) inhibits innate immune responses via induction of mitophagy

Bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus within the family Flaviviridae. Mitophagy plays important roles in virus-host interactions. Here, we provide evidence that non-cytopathic (NCP) BVDV shifts the balance of mitochondrial dynamics toward fission and induces mitophagy to inhibit innate immune responses. Mechanistically, NCP BVDV triggers the translocation of dynamin-related protein (Drp1) to mitochondria and stimulates its phosphorylation at Ser616, leading to mitochondrial fission. In parallel, NCP BVDV-induced complete mitophagy via Parkin-dependent pathway contributes to eliminating damaged mitochondria to inhibit MAVS- and mtDNA-cGAS-mediated innate immunity responses, mtROS-mediated inflammatory responses and apoptosis initiation. Importantly, we demonstrate that the LIR motif of ERNS is essential for mitophagy induction. In conclusion, this study is the first to show that NCP BVDV-induced mitophagy plays a central role in promoting cell survival and inhibiting innate immune responses in vitro.

PsAF5 functions as an essential adapter for PsPHB2-mediated mitophagy under ROS stress in Phytophthora sojae

Host-derived reactive oxygen species (ROS) are an important defense means to protect against pathogens. Although mitochondria are the main intracellular targets of ROS, how pathogens regulate mitochondrial physiology in response to oxidative stress remains elusive. Prohibitin 2 (PHB2) is an inner mitochondrial membrane (IMM) protein, recognized as a mitophagy receptor in animals and fungi. Here, we find that an ANK and FYVE domain-containing protein PsAF5, is an adapter of PsPHB2, interacting with PsATG8 under ROS stress. Unlike animal PHB2 that can recruit ATG8 directly to mitochondria, PsPHB2 in Phytophthora sojae cannot recruit PsATG8 to stressed mitochondria without PsAF5. PsAF5 deletion impairs mitophagy under ROS stress and increases the pathogen’s sensitivity to H2O2, resulting in the attenuation of P. sojae virulence. This discovery of a PsPHB2-PsATG8 adapter (PsAF5) in plant-pathogenic oomycetes reveals that mitophagy induction by IMM proteins is conserved in eukaryotes, but with differences in the details of ATG8 recruitment.

The Mst1/2-BNIP3 axis is required for mitophagy induction and neuronal viability under mitochondrial stress

Mitophagy induction upon mitochondrial stress is critical for maintaining mitochondrial homeostasis and cellular function. Here, we found that Mst1/2 (Stk3/4), key regulators of the Hippo pathway, are required for the induction of mitophagy under various mitochondrial stress conditions. Knockdown of Mst1/2 or pharmacological inhibition by XMU-MP-1 treatment led to impaired mitophagy induction upon CCCP and DFP treatment. Mechanistically, Mst1/2 induces mitophagy independently of the PINK1-Parkin pathway and the canonical Hippo pathway. Moreover, our results suggest the essential involvement of BNIP3 in Mst1/2-mediated mitophagy induction upon mitochondrial stress. Notably, Mst1/2 knockdown diminishes mitophagy induction, exacerbates mitochondrial dysfunction, and reduces cellular survival upon neurotoxic stress in both SH-SY5Y cells and Drosophila models. Conversely, Mst1 and Mst2 expression enhances mitophagy induction and cell survival. In addition, AAV-mediated Mst1 expression reduced the loss of TH-positive neurons, ameliorated behavioral deficits, and improved mitochondrial function in an MPTP-induced Parkinson’s disease mouse model. Our findings reveal the Mst1/2-BNIP3 regulatory axis as a novel mediator of mitophagy induction under conditions of mitochondrial stress and suggest that Mst1/2 play a pivotal role in maintaining mitochondrial function and neuronal viability in response to neurotoxic treatment.

MoAti1 mediates mitophagy by facilitating recruitment of MoAtg8 to promote invasive growth in Magnaporthe oryzae.

Mitophagy is a selective autophagy for the degradation of damaged or excessive mitochondria to maintain intracellular homeostasis. In Magnaporthe oryzae, a filamentous ascomycetous fungus that causes rice blast, the most devastating disease of rice, mitophagy occurs in the invasive hyphae to promote infection. To date, only a few proteins are known to participate in mitophagy and the mechanisms of mitophagy are largely unknown in pathogenic fungi. Here, by a yeast two-hybrid screen with the core autophagy-related protein MoAtg8 as a bait, we obtained a MoAtg8 interactor MoAti1 (MoAtg8-interacting protein 1). Fluorescent observations and protease digestion analyses revealed that MoAti1 is primarily localized to the peripheral mitochondrialouter membrane and is responsible for recruiting MoAtg8 to mitochondria under mitophagy induction conditions. MoAti1 is specifically required for mitophagy, but not for macroautophagy and pexophagy. Infection assays suggested that MoAti1 is required for mitophagy in invasive hyphae during pathogenesis. Notably, no homologues of MoAti1 were found in rice and human protein databases, indicating that MoAti1 may be used as a potential target to control rice blast. By the host-induced gene silencing (HIGS) strategy, transgenic rice plants targeted to silencing MoATI1 showed enhanced resistance against M. oryzae with unchanged agronomic traits. Our results suggest that MoATI1 is required for mitophagy and pathogenicity in M. oryzae and can be used as a target for reducing rice blast.

A novel MTORC2-AKT-ROS axis triggers mitofission and mitophagy-associated execution of colorectal cancer cells upon drug-induced activation of mutant KRAS

ABSTRACT RAS is one of the most commonly mutated oncogenes associated with multiple cancer hallmarks. Notably, RAS activation induces intracellular reactive oxygen species (ROS) generation, which we previously demonstrated as a trigger for autophagy-associated execution of mutant KRAS-expressing cancer cells. Here we report that drug (merodantoin; C1)-induced activation of mutant KRAS promotes phospho-AKT S473-dependent ROS-mediated S616 phosphorylation and mitochondrial localization of DNM1L/DRP1 (dynamin 1 like) and cleavage of the fusion-associated protein OPA1 (OPA1 mitochondrial dynamin like GTPase). Interestingly, accumulation of the outer mitochondrial membrane protein VDAC1 (voltage dependent anion channel 1) is observed in mutant KRAS-expressing cells upon exposure to C1. Conversely, silencing VDAC1 abolishes C1-induced mitophagy, and gene knockdown of either KRAS, AKT or DNM1L rescues ROS-dependent VDAC1 accumulation and stability, thus suggesting an axis of mutant active KRAS-phospho-AKT S473-ROS-DNM1L-VDAC1 in mitochondrial morphology change and cancer cell execution. Importantly, we identified MTOR (mechanistic target of rapamycin kinsase) complex 2 (MTORC2) as the upstream mediator of AKT phosphorylation at S473 in our model. Pharmacological or genetic inhibition of MTORC2 abrogated C1-induced phosphorylation of AKT S473, ROS generation and mitophagy induction, as well as rescued tumor colony forming ability and migratory capacity. Finally, increase in thermal stability of KRAS, AKT and DNM1L were observed upon exposure to C1 only in mutant KRAS-expressing cells. Taken together, our work has unraveled a novel mechanism of selective targeting of mutant KRAS-expressing cancers via MTORC2-mediated AKT activation and ROS-dependent mitofission, which could have potential therapeutic implications given the relative lack of direct RAS-targeting strategies in cancer. Abbreviations: ACTB/ß-actin: actin beta; AKT: AKT serine/threonine kinase; C1/merodantoin: 1,3-dibutyl-2-thiooxo-imidazoldine-4,5-dione; CAT: catalase; CETSA: cellular thermal shift assay; CHX: cycloheximide; DKO: double knockout; DNM1L/DRP1: dynamin 1 like; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H2O2: hydrogen peroxide; HSPA1A/HSP70-1: heat shock protein family A (Hsp70) member 1A; HSP90AA1/HSP90: heat shock protein 90 alpha family class A member 1; KRAS: KRAS proto-oncogene, GTPase; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; LC3B-I: unlipidated form of LC3B; LC3B-II: phosphatidylethanolamine-conjugated form of LC3B; MAPKAP1/SIN1: MAPK associated protein 1; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; MFI: mean fluorescence intensity; MiNA: Mitochondrial Network Analysis; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; MTORC2: mechanistic target of rapamycin kinase complex 2; O2.−: superoxide; OMA1: OMA1 zinc metallopeptidase; OPA1: OPA1 mitochondrial dynamin like GTPase; RICTOR: RPTOR independent companion of MTOR complex 2; ROS: reactive oxygen species; RPTOR/raptor: regulatory associated protein of MTOR complex 1; SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2; SQSTM1/p62: sequestosome 1; VDAC1: voltage dependent anion channel 1; VDAC2: voltage dependent anion channel 2.

Gonococcal OMV-delivered PorB induces epithelial cell mitophagy

The bacterial pathogen Neisseria gonorrhoeae is able to invade epithelial cells and survive intracellularly. During this process, it secretes outer membrane vesicles (OMVs), however, the mechanistic details for interactions between gonococcal OMVs and epithelial cells and their impact on intracellular survival are currently not established. Here, we show that gonococcal OMVs induce epithelial cell mitophagy to reduce mitochondrial secretion of reactive oxygen species (ROS) and enhance intracellular survival. We demonstrate that OMVs deliver PorB to mitochondria to dissipate the mitochondrial membrane potential, resulting in mitophagy induction through a conventional PINK1 and OPTN/NDP52 mechanism. Furthermore, PorB directly recruits the E3 ubiquitin ligase RNF213, which decorates PorB lysine residue 171 with K63-linked polyubiquitin to induce mitophagy in a p62-dependent manner. These results demonstrate a mechanism in which polyubiquitination of a bacterial virulence factor that targets mitochondria directs mitophagy processes to this organelle to prevent its secretion of deleterious ROS.

Asprosin contributes to pathogenesis of obesity by adipocyte mitophagy induction to inhibit white adipose browning in mice.

Asprosin (ASP) is a newly discovered adipokine secreted by white adipose tissue (WAT), which can regulate the homeostasis of glucose and lipid metabolism. However, it is not clear whether it can regulate the browning of WAT and mitophagy during the browning process. Accordingly, this study aims to investigate the effects and possible mechanisms of ASP on the browning of WAT and mitophagy in vivo and in vitro. In in vivo experiments, some mouse models were used including adipose tissue ASP-specific deficiency (ASP-/-), high fat diet (HFD)-induced obesity and white adipose browning; in in vitro experiments, some cell models were also established and used, including ASP-deficient 3T3-L1 preadipocyte (ASP-/-) and CL-316243 (CL, 1 µM)-induced browning. Based on these models, the browning of WAT and mitophagy were evaluated by morphology, functionality and molecular markers. Our in vivo data show that adipose tissue-specific deletion of ASP contributes to weight loss in mice; supplementation of ASP inhibits the expressions of browning-related proteins including UCP1, PRDM16 and PGC1ɑ during the cold exposure-induced browning, and promotes the expressions of mitophagy-related proteins including PINK1 and Parkin under the conditions of whether normal diet (ND) or HFD. Similarly, our in vitro data also show that the deletion of ASP in 3T3-L1 cells significantly increases the expressions of the browning-related proteins and decreases the expressions of the mitophagy-related proteins. These data demonstrate that ASP deletion can facilitate the browning and inhibit mitophagy in WAT. The findings will lay an experimental foundation for the development of new drugs targeting ASP and the clinical treatment of metabolic diseases related to obesity.

Mitophagy curtails cytosolic mtDNA-dependent activation of cGAS/STING inflammation during aging

Macroautophagy decreases with age, and this change is considered a hallmark of the aging process. It remains unknown whether mitophagy, the essential selective autophagic degradation of mitochondria, also decreases with age. In our analysis of mitophagy in multiple organs in the mito-QC reporter mouse, mitophagy is either increased or unchanged in old versus young mice. Transcriptomic analysis shows marked upregulation of the type I interferon response in the retina of old mice, which correlates with increased levels of cytosolic mtDNA and activation of the cGAS/STING pathway. Crucially, these same alterations are replicated in primary human fibroblasts from elderly donors. In old mice, pharmacological induction of mitophagy with urolithin A attenuates cGAS/STING activation and ameliorates deterioration of neurological function. These findings point to mitophagy induction as a strategy to decrease age-associated inflammation and increase healthspan.

Icaritin with autophagy/mitophagy inhibitors synergistically enhances anticancer efficacy and apoptotic effects through PINK1/Parkin-mediated mitophagy in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is among the deadliest malignancies worldwide and still a pressing clinical problem. Icaritin, a natural compound obtained from the Epimedium genus plant, has garnered significant attention as a potential therapeutic drug for HCC therapies. Mitophagy plays a crucial role in mitochondrial quality control through efficiently eliminating damaged mitochondria. However, the specific mechanisms of the interplay between mitophagy and apoptosis in HCC is still unclear. We aimed to explore the cross-talk between icaritin-induced mitophagy and apoptosis in HCC cells and investigate its potential mechanisms. Firstly, we confirmed that icaritin inhibits proliferation and migration while inducing mitochondrial damage and reactive oxygen species (ROS) production in HCC cells. Secondly, based on proteomics analysis, we discovered that icaritin inhibits the growth of tumor cells and disrupts their mitochondrial homeostasis through the regulation of both mitophagy and apoptosis. Thirdly, icaritin causes mitophagy mediated by PINK1-Parkin signaling via regulating feedforward loop. Furthermore, knockdown of PINK1/Parkin leads to inhibition of mitophagy, which promotes cell death induced by icaritin in HCC cells. Finally, autophagy/mitophagy inhibitors remarkably enhance icaritin-induced cell death and anticancer efficacy. Collectively, our findings reveal that icaritin suppresses growth, proliferation and migration of HCC cell through induction of mitophagy and apoptosis, while inhibition of mitophagy significantly increased the anti-cancer and pro-apoptotic effects of icaritin, indicating that targeting autophagy or mitophagy is a novel approach to overcome drug resistance and enhance anticancer therapies.

5-ALA localises to the autophagy compartment and increases its fluorescence upon autophagy enhancement through caloric restriction and spermidine treatment in human glioblastoma

Glioblastoma Multiforme (GBM) is the most invasive and prevalent Central Nervous System (CNS) malignancy. It is characterised by diffuse infiltrative growth and metabolic dysregulation that impairs the extent of surgical resection (EoR), contributing to its poor prognosis. 5-Aminolevulinic acid (5-ALA) fluorescence-guided surgical resection (FGR) takes advantage of the preferential generation of 5-ALA-derived fluorescence signal in glioma cells, thereby improving visualisation and enhancing the EoR. However, despite 5-ALA FGR is a widely used technique in the surgical management of malignant gliomas, the infiltrative tumour margins usually show only vague or no visible fluorescence and thus a significant amount of residual tumour tissue may hence remain in the resection cavity, subsequently driving tumour recurrence. To investigate the molecular mechanisms that govern the preferential accumulation of 5-ALA in glioma cells, we investigated the precise subcellular localisation of 5-ALA signal using Correlative Light and Electron Microscopy (CLEM) and colocalisation analyses in U118MG glioma cells. Our results revealed strong 5-ALA signal localisation in the autophagy compartment – specifically autolysosomes and lysosomes. Flow cytometry was employed to investigate whether autophagy enhancement through spermidine treatment (SPD) or nutrient deprivation/caloric restriction (CR) would enhance 5-ALA fluorescence signal generation. Indeed, SPD, CR and a combination of SPD/CR treatment significantly increased 5-ALA signal intensity, with a most robust increase in signal intensity observed in the combination treatment of SPD/CR. When using 3-D glioma spheroids to assess the effect of 5-ALA on cellular ultrastructure, we demonstrate that 5-ALA exposure leads to cytoplasmic disruption, vacuolarisation and large-scale mitophagy induction. These findings not only suggest a critical role for the autophagy compartment in 5-ALA engagement and signal generation but also point towards a novel and practically feasible approach to enhance 5-ALA fluorescence signal intensity. The findings may highlight that indeed autophagy control may serve as a promising avenue to promote an improved resection and GBM prognosis.

ADP-Ribosylation Factor-Interacting Protein 2 Acts as a Novel Regulator of Mitophagy and Autophagy in Podocytes in Diabetic Nephropathy.

(1) Background: Differentiated podocytes are particularly vulnerable to oxidative stress and cellular waste products. The disease-related loss of postmitotic podocytes is a direct indicator of renal disease progression and aging. Podocytes use highly specific regulated networks of autophagy and endocytosis that counteract the increasing number of damaged protein aggregates and help maintain cellular homeostasis. Here, we demonstrate that ARFIP2 is a regulator of autophagy and mitophagy in podocytes both in vitro and in vivo. (2) Methods: In a recent molecular regulatory network analysis of mouse glomeruli, we identified ADP-ribosylation factor-interacting protein 2 (Arfip2), a cytoskeletal regulator and cofactor of ATG9-mediated autophagosome formation, to be differentially expressed with age. We generated an Arfip2-deficient immortalized podocyte cell line using the CRISPR/Cas technique to investigate the significance of Arfip2 for renal homeostasis in vitro. For the in vivo analyses of Arfip2 deficiency, we used a mouse model of Streptozotozin-induced type I diabetes and investigated physiological data and (patho)histological (ultra)structural modifications. (3) Results: ARFIP2 deficiency in immortalized human podocytes impedes autophagy. Beyond this, ARFIP2 deficiency in human podocytes interferes with ATG9A trafficking and the PINK1-Parkin pathway, leading to the compromised fission of mitochondria and short-term increase in mitochondrial respiration and induction of mitophagy. In diabetic mice, Arfip2 deficiency deteriorates autophagy and leads to foot process effacement, histopathological changes, and early albuminuria. (4) Conclusions: In summary, we show that ARFIP2 is a novel regulator of autophagy and mitochondrial homeostasis in podocytes by facilitating ATG9A trafficking during PINK1/Parkin-regulated mitophagy.

Varicella zoster virus glycoprotein E facilitates PINK1/Parkin-mediated mitophagy to evade STING and MAVS-mediated antiviral innate immunity

Viruses have evolved to control mitochondrial quality and content to facilitate viral replication. Mitophagy is a selective autophagy, in which the damaged or unnecessary mitochondria are removed, and thus considered an essential mechanism for mitochondrial quality control. Although mitophagy manipulation by several RNA viruses has recently been reported, the effect of mitophagy regulation by varicella zoster virus (VZV) remains to be fully determined. In this study, we showed that dynamin-related protein-1 (DRP1)-mediated mitochondrial fission and subsequent PINK1/Parkin-dependent mitophagy were triggered during VZV infection, facilitating VZV replication. In addition, VZV glycoprotein E (gE) promoted PINK1/Parkin-mediated mitophagy by interacting with LC3 and upregulating mitochondrial reactive oxygen species. Importantly, VZV gE inhibited MAVS oligomerization and STING translocation to disrupt MAVS- and STING-mediated interferon (IFN) responses, and PINK1/Parkin-mediated mitophagy was required for VZV gE-mediated inhibition of IFN production. Similarly, carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-mediated mitophagy induction led to increased VZV replication but attenuated IFN production in a three-dimensional human skin organ culture model. Our results provide new insights into the immune evasion mechanism of VZV gE via PINK1/Parkin-dependent mitophagy.

Fluorescence Methods to Measure Pexophagy.

We outline our approach for studying the selective autophagy of peroxisomes (pexophagy), using fluorescence microscopy in tissue cell culture models. Ratiometric reporters, which specifically localize to peroxisomes, allow a quantitative assessment of pexophagy in fixed and live cells, as well as whole organisms. We discuss chemical and physiological inducers of pexophagy and any overlap with the induction of mitophagy.

Selective induction of Rab9-dependent alternative mitophagy using a synthetic derivative of isoquinoline alleviates mitochondrial dysfunction and cognitive deficits in Alzheimer's disease models.

Rationale: Promotion of mitophagy is considered a promising strategy for the treatment of neurodegenerative diseases including Alzheimer's disease (AD). The development of mitophagy-specific inducers with low toxicity and defined molecular mechanisms is essential for the clinical application of mitophagy-based therapy. The aim of this study was to investigate the potential of a novel small-molecule mitophagy inducer, ALT001, as a treatment for AD. Methods: ALT001 was developed through chemical optimization of an isoquinolium scaffold, which was identified from a chemical library screening using a mitophagy reporter system. In vitro and in vivo experiments were conducted to evaluate the potential of ALT001 as a mitophagy-targeting therapeutic agent and to investigate the molecular mechanisms underlying ALT001-induced mitophagy. The therapeutic effect of ALT001 was assessed in SH-SY5Y cells expressing mutant APP and mouse models of AD (5×FAD and PS2APP) by analyzing mitochondrial dysfunction and cognitive defects. Results: ALT001 specifically induces mitophagy both in vitro and in vivo but is nontoxic to mitochondria. Interestingly, we found that ALT001 induces mitophagy through the ULK1-Rab9-dependent alternative mitophagy pathway independent of canonical mitophagy pathway regulators such as ATG7 and PINK1. Importantly, ALT001 reverses mitochondrial dysfunction in SH-SY5Y cells expressing mutant APP in a mitophagy-dependent manner. ALT001 induces alternative mitophagy in mice and restores the decreased mitophagy level in a 5×FAD AD model mouse. In addition, ALT001 reverses mitochondrial dysfunction and cognitive defects in the PS2APP and 5×FAD AD mouse models. AAV-mediated silencing of Rab9 in the hippocampus further confirmed that ALT001 exerts its therapeutic effect through alternative mitophagy. Conclusion: Our results highlight the therapeutic potential of ALT001 for AD via alleviation of mitochondrial dysfunction and indicate the usefulness of the ULK1-Rab9 alternative mitophagy pathway as a therapeutic target.

Mutant NOTCH3ECD Triggers Defects in Mitochondrial Function and Mitophagy in CADASIL Cell Models.

Cerebral autosomal-dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL) is an inherited small-vessel disease that affects the white matter of the brain. Recent studies have confirmed that the deposition of NOTCH3ECD is the main pathological basis of CADASIL; however, whether different mutations present the same pathological characteristics remains to be further studied. Some studies have found that mitochondrial dysfunction is related to CADASIL; however, the specific effects of NOTCH3ECD on mitochondrial remain to be determined. We aimed to explore the role of mitochondrial dysfunction in CADASIL. We established transgenic human embryonic kidney-293T cell models (involving alterations in cysteine and non-cysteine residues) via lentiviral transfection. Mitochondrial function and structure were assessed using flow cytometry and transmission electron microscopy, respectively. Mitophagy was assessed using western blotting and immunofluorescence. We demonstrated that NOTCH3ECD deposition affects mitochondrial morphology and function, and that its protein levels are significantly correlated with mitochondrial quality and can directly bind to mitochondria. Moreover, NOTCH3ECD deposition promoted the induction of autophagy and mitophagy. However, these processes were impaired, leading to abnormal mitochondrial accumulation. This study revealed a common pathological feature of NOTCH3ECD deposition caused by different NOTCH3 mutations and provided new insights into the role of NOTCH3ECD in mitochondrial dysfunction and mitophagy.

Synergistic celecoxib and dimethyl-celecoxib combinations block cervix cancer growth through multiple mechanisms.

The synergistic inhibitory effect of celecoxib (CXB) and dimethyl-celecoxib (DMC) plus paclitaxel (PA) or cisplatin (CP) on human cervix HeLa and SiHa cells was assessed at multiple cellular levels in order to elucidate the biochemical mechanisms triggered by the synergistic drug combinations. The effect of CXB (5 μM)/CP (2 μM) or CXB (5 μM)/PA (15 μM) and DMC (15 μM)/CP (5 μM) or DMC (15 μM)/PA (20 μM) for 24 h was assayed on cancer cell proliferation, energy metabolism, mitophagy, ROS production, glycoprotein-P activity, DNA stability and apoptosis/necrosis. Drug combinations synergistically decreased HeLa and SiHa cell proliferation (>75%) and arrested cellular cycle by decreasing S and G2/M phases as well as the Ki67 content (HeLa) by 7.5-30 times. Cell viability was preserved (>90%) and no apparent effects on non-cancer cell growth were observed. Mitochondrial and glycolytic protein contents (44-95%) and ΔΨm (45-50%) in HeLa cells and oxidative phosphorylation and glycolysis fluxes (70-90%) in HeLa and SiHa cells were severely decreased, which in turn promoted a drastic fall in the ATP supply (85-88%). High levels of mitophagy proteins in HeLa cells and active mitochondrial digestion in HeLa and SiHa cells was observed. Mitochondrial fission and microtubule proteins were also affected. Intracellular ROS content (2-2.3-fold) and ROS production was stimulated (2.3-4 times), whereas content and activity of glycoprotein-P (45-85%) were diminished. DNA fragmentation was not observed and apoptosis/necrosis was not detected suggesting that cell death could be mainly associated to mitophagy induction. CXB or DMC combination with canonical chemotherapy may be a promising chemotherapy strategy against cervical cancer growth, because it can selectively block multiple cell processes including inhibition of energy pathways and in consequence ATP-dependent processes such as cell proliferation, glycoprotein-P activity, ROS production and mitophagy, with no apparent effects on non-cancer cells.

Cranberry extracts and cranberry polyphenols induce mitophagy in human fibroblast cells

Mitophagy, a process that removes damaged mitochondria, plays a key role in healthy cellular homeostasis. Decreased mitophagy contributes to the aging process and induction of mitophagy has been identified as a novel anti-aging mechanism for a few polyphenols. Cranberries have numerous health benefits which have been ascribed to their rich polyphenol content. Cranberries and cranberry polyphenols may also have the ability to induce mitophagy. To evaluate this hypothesis, the mitophagy inducing abilities of cranberry extracts and a few abundant cranberry polyphenols were examined in normal human fibroblasts, monitoring via confocal microscopy and Western blots. A cranberry extract (polyphenol content 120–125 mg/g) induced mitophagy with a maximum effective concentration of ∼4 μg/mL. Cranberry polyphenol subfractions, flavonols (0.4 μg/mL) and anthocyanins (5 μg/mL), induced mitophagy at low concentrations, while proanthocyanidins required a significantly higher concentration (50 μg/mL) to achieve efficacy. When tested separately, the most abundant cranberry flavonols, quercetin, myricetin and kaempferol each induced mitophagy in a dose-dependent manner but were less efficient than the flavonol subfraction. A mixture of quercetin, myricetin and kaempferol at their most effective concentrations induced mitophagy comparable to the flavonol subfraction. These results suggest cranberry polyphenols induce mitophagy with flavonols requiring the lowest concentration to effectively induce mitophagy due to complementary or synergistic effects, shedding new light on the bioactivity of cranberries.

Generation of inducible mitophagy mice.

Mitophagy is programmed selective autophagy of mitochondria and is important for mitochondrial quality control and cellular homeostasis. Mitochondrial dysfunction and impaired mitophagy are closely associated with various diseases, including heart failure and diabetes. To better understand the pathophysiological role of mitophagy, we generated doxycycline-inducible mitophagy mice using a synthetic mitophagy adaptor protein consisting of an outer mitochondrial membrane targeting sequence and an engineered LIR. To evaluate the activation of mitophagy upon doxycycline treatment, we also generated mitophagy reporter mito-QC mice in which mitochondria tandemly express mCherry and GFP, and only GFP signals are lost in acidic lysosomes subjected to mitophagy. With the ROSA26 promoter-driven rtTA, mitophagy was observed at least in heart, liver, and skeletal muscle. We investigated the relationship between mitophagy activation and pressure overload heart failure or high fat diet-induced obesity. Unexpectedly, we were unable to confirm the protective effect of mitophagy in these two pathological models. Further titration of the level of mitophagy induction is required to demonstrate the potency of the protective effects of mitophagy in disease models.