Optineurin Expression Research Articles

Optineurin inhibits IBDV replication via interacting with VP1

Avibirnavirus, specifically Infectious Bursal Disease Virus (IBDV), is a highly contagious pathogen that causes significant economic losses in the poultry industry. The polymerase protein VP1 of IBDV is critical to the viral life cycle, facilitating the synthesis of viral mRNA and the genome. Previous studies have suggested that various host factors influence the regulation of IBDV polymerase activity. In this study, we identified that IBDV infection induces the expression of optineurin (OPTN), a mitophagy receptor and a protein associated with amyotrophic lateral sclerosis (ALS), as well as a negative regulator of interferon I production. The induced expression of OPTN acts as a suppressor of IBDV replication, a function dependent on its ubiquitin-binding domain (UBAN). Furthermore, we demonstrated that OPTN exerts its antiviral effects through direct interactions with VP1 and VP3, which inhibit the polymerase activity of VP1 by preventing K63-linked ubiquitination of VP1. To our knowledge, this study is the first to report that OPTN, upregulated during IBDV infection, functions as a novel antiviral host factor that limits the virus's replicative capacity, offering a potential target for anti-IBDV therapeutic strategies.

D-pinitol alleviates diabetic cardiomyopathy by inhibiting the optineurin-mediated endoplasmic reticulum stress and glycophagy signaling pathway.

Diabetic cardiomyopathy (DCM) is an important complication resulting in heart failure and death of diabetic patients. However, there is no effective drug for treatments. This study investigated the effect of D-pinitol (DP) on cardiac injury using diabetic mice and glycosylation injury of cardiomyocytes and its molecular mechanisms. We established the streptozotocin-induced SAMR1 and SAMP8 mice and DP (150 mg/kg/day) intragastrically and advanced glycation end-products (AGEs)-induced H9C2 cells. H9C2 cells were transfected with optineurin (OPTN) siRNA and overexpression plasmids. The metabolic disorder indices, cardiac dysfunction, histopathology, immunofluorescence, western blot, and immunoprecipitation were investigated. Our results showed that DP reduced the blood glucose and AGEs, and increased the expression of heart OPTN in diabetic mice and H9C2 cells, thereby inhibiting the endoplasmic reticulum stress (GRP78, CHOP) and glycophagy (STBD1, GABARAPL1), and alleviating the myocardial apoptosis and fibrosis of DCM. The expression of filamin A as an interaction protein of OPTN downregulated by AGEs decreased OPTN abundance. Moreover, OPTN siRNA increased the expression of GRP78, CHOP, STBD1, and GABARAPL1 and inhibited the expression of GAA via GSK3β phosphorylation and FoxO1. DP may be helpful to treat the onset of DCM. Targeting OPTN with DP could be translated into clinical application in the fighting against DCM.

Deciphering the mitophagy receptor network identifies a crucial role for OPTN (optineurin) in acute myeloid leukemia

ABSTRACT The selective autophagic degradation of mitochondria via mitophagy is essential for preserving mitochondrial homeostasis and, thereby, disease maintenance and progression in acute myeloid leukemia (AML). Mitophagy is orchestrated by a variety of mitophagy receptors whose interplay is not well understood. Here, we established a pairwise multiplexed CRISPR screen targeting mitophagy receptors to elucidate redundancies and gain a deeper understanding of the functional interactome governing mitophagy in AML. We identified OPTN (optineurin) as sole non-redundant mitophagy receptor and characterized its unique role in AML. Knockdown and overexpression experiments demonstrated that OPTN expression is rate-limiting for AML cell proliferation. In a MN1-driven murine transplantation model, loss of OPTN prolonged overall median survival by 7 days (+21%). Mechanistically, we found broadly impaired mitochondrial respiration and function with increased mitochondrial ROS, that most likely caused the proliferation defect. Our results decipher the intertwined network of mitophagy receptors in AML for both ubiquitin-dependent and receptor-mediated mitophagy, identify OPTN as a non-redundant tool to study mitophagy in the context of leukemia and suggest OPTN inhibition as an attractive therapeutic strategy. Abbreviations: AML: acute myeloid leukemia; CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; CTRL: control; DFP: deferiprone; GI: genetic interaction; KD: knockdown; KO: knockout; ldMBM, lineage-depleted murine bone marrow; LFC: log2 fold change; LIR: LC3-interacting region; LSC: leukemic stem cell; MAGeCK: Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout; MDIVI-1: mitochondrial division inhibitor 1; MOI: multiplicity of infection; MOM: mitochondrial outer membrane; NAC: N-acetyl-L-cysteine; OA: oligomycin-antimycin A; OCR: oxygen consumption rate; OE: overexpression; OPTN: optineurin; PINK1: PTEN induced putative kinase 1; ROS: reactive oxygen species; SEM: standard error of the mean; TCGA: The Cancer Genome Atlas; TEM: transmission electron microscopy; UBD: ubiquitin-binding domain; WT: wild type

Biphasic Dose-Response of Mn-Induced Mitochondrial Damage, PINK1/Parkin Expression, and Mitophagy in SK-N-SH Cells.

Excessive manganese (Mn) exposure produces neurotoxicity with mitochondrial damage. Mitophagy is a protective mechanism to eliminate damaged mitochondria to protect cells. The aim of this study was to determine the dose-response of Mn-induced mitochondria damage, the expression of mitophagy-mediated protein PINK1/Parkin and mitophagy in dopamine-producing SK-N-SH cells. Cells were exposed to 0, 300, 900, and 1500μM Mn2+ for 24h, and ROS production, mitochondrial damage and mitophagy were examined. The levels of dopamine were detected by ELISA and neurotoxicity and mitophagy-related proteins (α-synuclein, PINK1, Parkin, Optineurin, and LC3II/I) were detected by western blot. Mn increased intracellular ROS and apoptosis and decreased mitochondrial membrane potential in a concentration-dependent manner. However, at the low dose of 300μM Mn, autophagosome was increased 11-fold, but at the high dose of 1500μM, autophagosome was attenuated to 4-fold, together with decreased mitophagy-mediated protein PINK1/Parkin and LC3II/I ratio and increased Optineurin expression, resulting in increased α-synuclein accumulation and decreased dopamine production. Thus, Mn-induced mitophagy exhibited a novel biphasic regulation: at the low dose, mitophagy is activated to eliminate damaged mitochondria, however, at the high dose, cells gradually loss the adaptive machinery, the PINK1/Parkin-mediated mitophagy weakened, resulting in neurotoxicity.

Differential optineurin expression controls TGFβ signaling and is a key determinant for metastasis of triple negative breast cancer.

Triple-negative breast cancer (TNBC) is the most challenging breast cancer subtype to treat due to its aggressive characteristics and low response to the existing clinical therapies. Distant metastasis is the main cause of death of TNBC patients. Better understanding of the mechanisms underlying TNBC metastasis may lead to new strategies of early diagnosis and more efficient treatment. In our study, we uncovered that the autophagy receptor optineurin (OPTN) plays an unexpected role in TNBC metastasis. Data mining of publicly available data bases revealed that the mRNA level of OPTN in TNBC patients positively correlates with relapse free and distance metastasis free survival. Importantly, in vitro and in vivo models demonstrated that OPTN suppresses TNBC metastasis. Mechanistically, OPTN inhibited the pro-oncogenic transforming growth factor-β (TGFβ) signaling in TNBC cells by interacting with TGFβ type I receptor (TβRI) and promoting its ubiquitination for degradation. Consistent with our experimental findings, the clinical TNBC samples displayed a negative correlation between OPTN mRNA expression and TGFβ gene response signature and expression of proto-typic TGFβ target genes. Altogether, our study demonstrates that OPTN is a negative regulator for TGFβ receptor/SMAD signaling and suppresses metastasis in TNBC.

Optineurin-mediated mitophagy as a potential therapeutic target for intervertebral disc degeneration.

Low back pain is thought to be mainly caused by intervertebral disc degeneration (IVDD), and there is a lack of effective treatments. Cellular senescence and matrix degradation are important factors that cause disc degeneration. Mitochondrial dysfunction induced by oxidative stress is an important mechanism of cellular senescence and matrix degradation in the nucleus pulposus (NP), and mitophagy can effectively remove damaged mitochondria, restore mitochondrial homeostasis, and mitigate the damage caused by oxidative stress. Optineurin (OPTN) is a selective mitophagy receptor, and its role in intervertebral disc degeneration remains unclear. Here, we aimed to explore the effect of OPTN on H2O2-induced nucleus pulposus cell (NPCs) senescence and matrix degradation in a rat model of disc degeneration. Western blot analysis showed that OPTN expression was reduced in degenerative human and rat nucleus pulposus tissues and increased in H2O2-induced senescent NPCs. OPTN overexpression significantly inhibited H2O2-induced senescence and increased matrix-associated protein expression in NPCs, but OPTN knockdown showed the opposite effect. As previous reports have suggested that mitophagy significantly reduces mitochondrial damage and reactive oxygen species (ROS) caused by oxidative stress, and we used the mitophagy agonist CCCP, the mitophagy inhibitor cyclosporin A (CsA), and the mitochondrial ROS (mtROS) scavenger mitoTEMPO and confirmed that OPTN attenuated NPCs senescence and matrix degeneration caused by oxidative stress by promoting mitophagy to scavenge damaged mitochondria and excess reactive oxygen species, thereby slowing the progression of IVDD. In conclusion, our research suggests that OPTN is involved in IVDD and exerts beneficial effects against IVDD.

Abstract 1296: MEK inhibition overcomes chemoimmunotherapy resistance by inducing CXCL10 in cancer cells

Abstract Chemotherapy with anti PD-1/PD-L1 mAb has become the standard of care for patients with metastatic non-small cell lung cancer (NSCLC). Using lung tumor models, where pemetrexed-platinum chemotherapy (PEM/CDDP) remains unable to synergize with immune checkpoint inhibitors (ICI), we linked failure of this treatment with its inability to induce CXCL10 expression and CD8+ T cell recruitment. Using drug screening, we showed that combining a MEK inhibitor (MEKi) with PEM/CDDP triggers CXCL10 secretion by cancer cells and CD8+ T cell recruitment, and restores ICI efficacy. PEM/CDDP plus MEKi promotes optineurin (OPTN)-dependent mitophagy, resulting in CXCL10 production in a mitochondrial DNA and TLR9-dependent manner. TLR9 or autophagy/mitophagy processes genetic inactivation of abort the antitumor efficacy of PEM/CDDP plus MEKi/anti PD-L1 therapy. In human NSCLC, high OPTN, TLR9 and CXCL10 expression is associated with better response to ICI. Our results underline the role of TLR9 and OPTN-dependent mitophagy in enhancing chemoimmunotherapy efficacy. Citation Format: Emeric Limagne, Lisa Nuttin, Marion Thibaudin, Elise Jacquin, Romain Aucagne, Marjorie Bon, Elise Ballot, Solène Revy, Robby Barnestein, Caroline Truntzer, Valentin Derangère, Jean-David Fumet, Rébé Cédric, Pierre-Simon Bellaye, Coureche-Guillaume Kaderbhai, Aodrenn Spill, Bertrand Collin, Mary Callanan, Aurélie Lagrange, Laure Favier, Bruno Coudert, Laurent Arnould, Sylvain Ladoire, Bertrand Routy, Philippe Joubert, François Ghiringhelli. MEK inhibition overcomes chemoimmunotherapy resistance by inducing CXCL10 in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1296.

PDGF signaling inhibits mitophagy in glioblastoma stem cells through N6-methyladenosine.

Dysregulated growth factor receptor pathways, RNA modifications, and metabolism each promote tumor heterogeneity. Here, we demonstrate that platelet-derived growth factor (PDGF) signaling induces N6-methyladenosine (m6A) accumulation in glioblastoma (GBM) stem cells (GSCs) to regulate mitophagy. PDGF ligands stimulate early growth response 1 (EGR1) transcription to induce methyltransferase-like 3 (METTL3) to promote GSC proliferation and self-renewal. Targeting the PDGF-METTL3 axis inhibits mitophagy by regulating m6A modification of optineurin (OPTN). Forced OPTN expression phenocopies PDGF inhibition, and OPTN levels portend longer survival of GBM patients; these results suggest a tumor-suppressive role for OPTN. Pharmacologic targeting of METTL3 augments anti-tumor efficacy of PDGF receptor (PDGFR) and mitophagy inhibitors invitro and invivo. Collectively, we define PDGF signaling as an upstream regulator of oncogenic m6A regulation, driving tumor metabolism to promote cancer stem cell maintenance, highlighting PDGF-METTL3-OPTN signaling as a GBM therapeutic target.

A Unique Cooperativity between Optineurin and CCL5 Triggers Early Cell Death during Herpes Simplex Virus-1 Infection of the Cornea

Abstract Induction of cell death is considered an effective strategy to control viral infections. Rapid killing of infected cells prevents propagation of viruses and thereby, effectively reduces the infection. Herpes Simplex Virus-1 (HSV-1) is known to cause ocular and nervous system infections. While studying an autophagy receptor protein, Optineurin (OPTN), we found evidence of a unique collaboration between OPTN and CCL5 to generate signal for early cell death in HSV-1 infected cells in the cornea. OPTN knockout mice tend to die due to rapid spread of HSV-1 infection to the brain causing encephalitis. Interestingly, the OPTN knockout animals also exhibit a marked reduction in CCL5 induction. Human transcriptomic profile dataset shows a strong positive correlation between CCL5 upregulation and OPTN expression during HSV infection. Our data shows that a well-coordinated activity of both, OPTN and CCL5, is needed to trigger an early cell death signal that mimics pyroptosis to prevent viral spread. Downregulation of CCL5 and/or OPTN causes interference with this signal and a corresponding delay in cell death. Our results provide new information on an anti-HSV1 chemokine signaling pathway that participates in innate immune control of HSV-1 spread and viral transmission.

MEK inhibition overcomes chemoimmunotherapy resistance by inducing CXCL10 in cancer cells.

Chemotherapy with anti PD-1/PD-L1 antibodies has become the standard of care for patients with metastatic non-small cell lung cancer (mNSCLC). Using lung tumor models, where pemetrexed and cisplatin (PEM/CDDP) chemotherapy remains unable to synergize with immune checkpoint inhibitors (ICIs), we linked the failure of this treatment with its inability to induce CXCL10 expression and CD8+ Tcell recruitment. Using drug screening, we showed that combining a MEK inhibitor (MEKi) with PEM/CDDP triggers CXCL10 secretion by cancer cells and CD8+ Tcell recruitment, sensitizing it to ICIs. PEM/CDDP plus a MEKi promotes optineurin(OPTN)-dependent mitophagy, resulting in CXCL10 production in a mitochondrial DNA- and TLR9-dependent manner. TLR9 or autophagy/mitophagy inhibition abolishes the anti-tumor efficacy of PEM/CDDP plus MEKi/anti-PD-L1 therapy. In human NSCLCs, high OPTN, TLR9, and CXCL10 expression is associated with a better response to ICIs. Our results underline the role of TLR9- and OPTN-dependent mitophagy in enhancing chemoimmunotherapy efficacy.

Intrinsic Antiviral Activity of Optineurin Prevents Hyperproliferation of a Primary Herpes Simplex Virus Type 2 Infection.

Very little knowledge exists on virus-specific host cell intrinsic mechanisms that prevent hyperproliferation of primary HSV type 2 (HSV-2) genital infections. In this study, we provide evidence that the Nemo-related protein, optineurin (OPTN), plays a key role in restricting HSV-2 infection both in vitro and in vivo. Contrary to previous reports regarding the proviral role of OPTN during Sendai virus infection, we demonstrate that lack of OPTN in cells causes enhanced virus production. OPTN deficiency negatively affects the host autophagy response and results in a marked reduction of CCL5 induction. OPTN knockout (OPTN-/-) mice display exacerbated genital disease and dysregulated T cell frequencies in infected tissues and lymph nodes. A human transcriptomic profile dataset provides further credence that a strong positive correlation exists between CCL5 upregulation and OPTN expression during HSV-2 genital infection. Our findings underscore a previously unknown OPTN/CCL5 nexus that restricts hyperproliferative spread of primary HSV-2 infection, which may constitute an intrinsic host defense mechanism against herpesviruses in general.

Metformin promotes histone deacetylation of optineurin and suppresses tumour growth through autophagy inhibition in ocular melanoma.

ObjectiveTo explore the therapeutic potential and the underlying mechanism of metformin, an adenosine monophosphate‐activated kinase (AMPK) activator, in ocular melanoma.MethodsCCK8, transwell, and colony formation assays were performed to detect the proliferation and migration ability of ocular melanoma cells. A mouse orthotopic xenograft model was built to detect ocular tumor growth in vivo. Western blot, immunofluorescence, and electron microscopy were adopted to evaluate the autophagy levels of ocular melanoma cells, and high‐throughput proteomics and CUT & Tag assays were performed to analyze the candidate for autophagy alteration.ResultsHere, we revealed for the first time that a relatively low dose of metformin induced significant tumorspecific inhibition of the proliferation and migration of ocular melanoma cells both in vitro and in vivo. Intriguingly, we found that metformin significantly attenuated autophagic influx in ocular melanoma cells. Through high‐throughput proteomics analysis, we revealed that optineurin (OPTN), which is a key candidate for autophagosome formation and maturation, was significantly downregulated after metformin treatment. Moreover, excessive OPTN expression was associated with an unfavorable prognosis of patients. Most importantly, we found that a histone deacetylase, SIRT1, was significantly upregulated after AMPK activation, resulting in histone deacetylation in the OPTN promoter.ConclusionsOverall, we revealed for the first time that metformin significantly inhibited the progression of ocular melanoma, and verified that metformin acted as an autophagy inhibitor through histone deacetylation of OPTN. This study provides novel insights into metformin ‐ guided suppression of ocular melanoma and the potential mechanism underlying the dual role of metformin in autophagy regulation.

Downregulating expression of OPTN elevates neuroinflammation via AIM2 inflammasome- and RIPK1-activating mechanisms in APP/PS1 transgenic mice

BackgroundNeuroinflammation is thought to be a cause of Alzheimer’s disease (AD), which is partly caused by inadequate mitophagy. As a receptor of mitophagy, we aimed to reveal the regulatory roles of optineurin (OPTN) on neuroinflammation in the pathogenesis of AD.MethodsBV2 cells and APP/PS1 transgenic (Tg) mice were used as in vitro and in vivo experimental models to determine the regulatory roles of OPTN in neuroinflammation of AD. Sophisticated molecular technologies including quantitative (q) RT-PCR, western blot, enzyme linked immunosorbent assay (ELISA), co-immunoprecipitation (Co-IP) and immunofluorescence (IF) were employed to reveal the inherent mechanisms.ResultsAs a consequence, key roles of OPTN in regulating neuroinflammation were identified by depressing the activity of absent in melanoma 2 (AIM2) inflammasomes and receptor interacting serine/threonine kinase 1 (RIPK1)-mediated NF-κB inflammatory mechanisms. In detail, we found that expression of OPTN was downregulated, which resulted in activation of AIM2 inflammasomes due to a deficiency in mitophagy in APP/PS1 Tg mice. By ectopic expression, OPTN blocks the effects of Aβ oligomer (Aβo) on activating AIM2 inflammasomes by inhibiting mRNA expression of AIM2 and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), leading to a reduction in the active form of caspase-1 and interleukin (IL)-1β in microglial cells. Moreover, RIPK1 was also found to be negatively regulated by OPTN via ubiquitin protease hydrolysis, resulting in the synthesis of IL-1β by activating the transcriptional activity of NF-κB in BV2 cells. As an E3 ligase, the UBAN domain of OPTN binds to the death domain (DD) of RIPK1 to facilitate its ubiquitination. Based on these observations, ectopically expressed OPTN in APP/PS1 Tg mice deactivated microglial cells and astrocytes via the AIM2 inflammasome and RIPK-dependent NF-κB pathways, leading to reduce neuroinflammation.ConclusionsThese results suggest that OPTN can alleviate neuroinflammation through AIM2 and RIPK1 pathways, suggesting that OPTN deficiency may be a potential factor leading to the occurrence of AD.

Knockdown of optineurin controls C2C12 myoblast differentiation via regulating myogenin and MyoD expressions

Mutations in optineurin (OPTN) have been identified in a small proportion of sporadic and familial amyotrophic lateral sclerosis (ALS) cases. Recent evidences suggest that OPTN would be involved in not only the pathophysiological mechanisms of motor neuron death of ALS but also myofiber degeneration of sporadic inclusion body myositis. However, the detailed role of OPTN in muscle remains unclear. Initially, we showed that OPTN expression levels were significantly increased in the denervated muscles of mice, suggesting that OPTN may be involved in muscle homeostasis. To reveal the molecular role of OPTN in muscle atrophy, we used cultured C2C12 myotubes treated with tumor necrosis factor-like inducer of apoptosis (TWEAK) as an in vitro model of muscle atrophy. Our data showed that OPTN had no effect on the process of muscle atrophy in this model. On the other hand, we found that myogenic differentiation was affected by OPTN. Immunoblotting analysis showed that OPTN protein levels gradually decreased during C2C12 differentiation. Furthermore, OPTN knockdown inhibited C2C12 differentiation, accompanied by reduction of mRNA and protein expression levels of myogenin and MyoD. These findings suggested that OPTN may have a novel function in muscle homeostasis and play a role in the pathogenesis of neuromuscular diseases.

Suppression of optineurin impairs the progression of hepatocellular carcinoma through regulating mitophagy.

Autophagy removes damaged organelles to inhibit malignant transformation during tumor initiation. Once a cancer matures, it uses the autophagic pathway as an energy source. Optineurin (OPTN) is an autophagy adaptor protein that recruits microtubule‐associated protein 1 light chain 3, an autophagosome marker, to the autophagosome. Despite studies of the relation between cancer progression and autophagy adaptor proteins, there are no reports to our knowledge of a correlation between hepatocellular carcinoma (HCC) and OPTN. We aimed here to investigate the effects of OPTN expression on HCC progression through autophagy. Immunohistochemistry was used to measure the OPTN expression in the tissues of 141 Japanese patients with HCC. The effects of OPTN expression on HCC progression and mitophagy were assessed using an OPTN knockout (KO) cell line in vitro. We used this KO cell line to establish and exploit a mouse model of HCC to determine the effects of OPTN expression on tumor progression. Immunohistochemical analysis showed that patients with elevated expression of OPTN experienced shorter overall survival (OS) and recurrence‐free survival (RFS). OPTN KO cells proliferated relatively slower versus wild‐type (WT) cells in vitro. Western blot analysis showed that mitophagy was suppressed in OPTN KO cells, and ATP synthesis and beta‐oxidation were reduced. The mouse model of HCC showed that OPTN KO cells formed smaller tumors versus WT cells less 10 weeks after implantation. Overall, the present findings suggest that OPTN is a key mediator of mitophagy that contributes to HCC progression through mitochondrial energy production.

PDGF Signaling Promotes Mitophagy in Glioblastoma Stem Cells Through N <sup>6</sup>-Methyladenosine

Glioblastomas are lethal primary brain tumors that contain glioblastoma stem cells (GSCs) that promote tumor growth. N6-methyladenosine (m6A) is the most common internal modification of mRNA and maintains critical GSC phenotypes. Here, we interrogated upstream regulation of m6A modifications in GSCs, revealing platelet-derived growth factor (PDGF) induction of m6A levels mediated by the methyltransferase METTL3. PDGF signaling stimulated EGR1 transcription that induced METTL3 to promote GSC proliferation and self-renewal, while targeting the PDGF-METTL3 axis generated mitochondrial dysfunction via mitophagy inhibition by regulating mRNA of the mitophagy regulator optineurin (OPTN) through m6A modification. Forced OPTN expression phenocopied PDGF inhibition and OPTN levels portend longer survival of glioblastoma patients. Collectively, we describe novel upstream regulation of m6A manifesting in mitochondrial regulation to promote maintenance of GSC stemness and proliferation, highlighting PDGF-METTL3-OPTN signaling as a therapeutic target in glioblastoma.

Autophagy receptor OPTN (optineurin) regulates mesenchymal stem cell fate and bone-fat balance during aging by clearing FABP3

ABSTRACT Senile osteoporosis (OP) is often concomitant with decreased autophagic activity. OPTN (optineurin), a macroautophagy/autophagy (hereinafter referred to as autophagy) receptor, is found to play a pivotal role in selective autophagy, coupling autophagy with bone metabolism. However, its role in osteogenesis is still mysterious. Herein, we identified Optn as a critical molecule of cell fate decision for bone marrow mesenchymal stem cells (MSCs), whose expression decreased in aged mice. Aged mice revealed osteoporotic bone loss, elevated senescence of MSCs, decreased osteogenesis, and enhanced adipogenesis, as well as optn– / – mice. Importantly, restoring Optn by transplanting wild-type MSCs to optn– / – mice or infecting optn– / – mice with Optn-containing lentivirus rescued bone loss. The introduction of a loss-of-function mutant of OptnK193R failed to reestablish a bone-fat balance. We further identified FABP3 (fatty acid binding protein 3, muscle and heart) as a novel selective autophagy substrate of OPTN. FABP3 promoted adipogenesis and inhibited osteogenesis of MSCs. Knockdown of FABP3 alleviated bone loss in optn– / – mice and aged mice. Our study revealed that reduced OPTN expression during aging might lead to OP due to a lack of FABP3 degradation via selective autophagy. FABP3 accumulation impaired osteogenesis of MSCs, leading to the occurrence of OP. Thus, reactivating OPTN or inhibiting FABP3 would open a new avenue to treat senile OP. Abbreviations: ADIPOQ: adiponectin, C1Q and collagen domain containing; ALPL: alkaline phosphatase, liver/bone/kidney; BGLAP/OC/osteocalcin: bone gamma carboxyglutamate protein; BFR/BS: bone formation rate/bone surface; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CDKN1A/p21: cyclin-dependent kinase inhibitor 1A; CDKN2A/p16: cyclin dependent kinase inhibitor 2A; CDKN2B/p15: cyclin dependent kinase inhibitor 2B; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; COL1A1: collagen, type I, alpha 1; Ct. BV/TV: cortical bone volume fraction; Ct. Th: cortical thickness; Es. Pm: endocortical perimeter; FABP4/Ap2: fatty acid binding protein 4, adipocyte; H2AX: H2A.X variant histone; HE: hematoxylin and eosin; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAR: mineral apposition rate; MSCs: bone marrow mesenchymal stem cells; NBR1: NBR1, autophagy cargo receptor; OP: osteoporosis; OPTN: optineurin; PDB: Paget disease of bone; PPARG: peroxisome proliferator activated receptor gamma; Ps. Pm: periosteal perimeter; qRT-PCR: quantitative real-time PCR; γH2AX: Phosphorylation of the Serine residue of H2AX; ROS: reactive oxygen species; RUNX2: runt related transcription factor 2; SA-GLB1: senescence-associated (SA)-GLB1 (galactosidase, beta 1); SP7/Osx/Osterix: Sp7 transcription factor 7; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 (human T cell leukemia virus type I) binding protein 1; Tb. BV/TV: trabecular bone volume fraction; Tb. N: trabecular number; Tb. Sp: trabecular separation; Tb. Th: trabecular thickness; μCT: micro computed tomography.

Optineurin inhibits NLRP3 inflammasome activation by enhancing mitophagy of renal tubular cells in diabetic nephropathy.

Tubulointerstitial inflammation plays a critical role in the progression of diabetic nephropathy (DN), and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes contribute to renal interstitial inflammation in DN. Decreased expression of optineurin (OPTN) is also associated with the progression of DN. We investigated the role of OPTN in activation of the NLRP3 inflammasome in DN. We initially examined the renal biopsy tissues of 172 patients with type 2 DN and 32 nondiabetic patients with renal hamartoma. Expression of renal OPTN was significantly lower in patients with DN and negatively correlated with urinary levels of IL-1β and IL-18. Confocal microscopy analysis of the biopsies indicated no NLRP3 or IL-1β staining in OPTN-positive renal tubular epithelial cells (RTECs), indicating a negative correlation of OPTN expression with the activation of NLRP3 inflammasome. In vitro studies of murine RTECs indicated the levels of OPTN mRNA and protein decreased significantly after stimulation by a high glucose (HG) treatment. Relative to RTECs given HG, RTECs overexpressing OPTN showed significantly lower levels of NLRP3 expression, cleavage of caspase-1 and IL-1β, and release of IL-1 β and IL-18. Overexpression of OPTN in the presence of HG significantly increased the costaining of microtubule-associated protein 1A/1B-light chain 3-II and translocase of outer mitochondrial membrane 20 in RTECs, suggesting that OPTN enhances mitophagy. In addition, mitochondrial division inhibitor 1 blocked the inhibitory effect of OPTN overexpression on the activation of NLRP3 inflammasome in the presence of HG, indicating that OPTN overexpression inhibited NLRP3 inflammasome activation by enhancement of mitophagy. OPTN gene silencing significantly enhanced production of mitochondrial reactive oxygen species (mtROS) in the presence of HG. Compared with HG+OPTN small interfering RNA (siRNA)-treated RTECs, HG+OPTN siRNA+MitoTempo-treated RTECs attenuated NLRP3 inflammasome activation, suggesting that OPTN gene silencing activates the NLRP3 inflammasome by increasing mtROS in HG-treated RTECs. Taken together, our results demonstrate that OPTN inhibits the activation of NLRP3 inflammasome by enhancing mitophagy.-Chen, K., Feng, L., Hu, W., Chen, J., Wang, X., Wang, L., He, Y. Optineurin inhibits NLRP3 inflammasome activation by enhancing mitophagy of renal tubular cells in diabetic nephropathy.

Molecular effect of an OPTN common variant associated to Paget's disease of bone.

Paget’s disease of bone (PDB) is a chronic bone disorder and although genetic factors appear to play an important role in its pathogenesis, to date PDB causing mutations were identified only in the Sequestosome 1 (SQSTM1) gene at the PDB3 locus. PDB6 locus, also previously linked to PDB, contains several candidate genes for metabolic bone diseases. We focused our analysis in the most significantly associated variant with PDB, within the Optineurin (OPTN) gene, i.e. the common variant rs1561570. Although it was previously shown to be strongly associated with PDB in several populations, its contribution to PDB pathogenesis remains unclear. In this study we have shown that rs1561570 may contribute to PDB since its T allele results in the loss of a methylation site in patients’ DNA, leading to higher levels of OPTN gene expression and a corresponding increase in protein levels in patients’ osteoclasts. This increase in OPTN expression leads to higher levels of NF-κB translocation into the nucleus and increasing expression of its target genes, which may contribute to the overactivity of osteoclasts observed in PDB. We also reported a tendency for a more severe clinical phenotype in the presence of a haplotype containing the rs1561570 T allele, which appear to be re-enforced with the presence of the SQSTM1/P392L mutation. In conclusion, our work provides novel insight towards understanding the functional effects of this variant, located in OPTN intron 7, and its implication in the contribution to PDB pathogenesis.

Autophagy Disruptions Associated With Altered Optineurin Expression in Extranigral Regions in a Rotenone Model of Parkinson's Disease.

The motor features of Parkinson's disease (PD) primarily result from a lesion to the nigrostriatal dopamine system. Numerous non-motor symptoms occur in PD, many of which are postulated to stem from pathology outside of the nigrostriatal dopamine system. Perturbations to protein trafficking, disruption of mitochondrial integrity, and impaired autophagy have repeatedly been implicated in dopaminergic neuron cell death. Previously, we demonstrated that multiple markers of autophagy are disrupted in a rotenone model of PD, with alterations occurring prior to an overt lesion to the nigrostriatal dopamine system. Whether these events occur in extra-nigral nuclei in PD and when relative to a lesion in the nigrostriatal dopamine system are generally unknown. The primary goal of these studies was to determine whether autophagy disruptions, in non-dopaminergic neuronal populations occur in an environmental model of PD utilizing a mitochondrial toxin. Here, we utilized the rat rotenone PD model, with sampling time-points before and after an overt lesion to the nigrostriatal dopamine system. In analyzing autophagy changes, we focused on optineurin (OPTN) and the autophagy marker, LC3. OPTN is an autophagy cargo adapter protein genetically linked to amyotrophic lateral sclerosis and glaucoma. In the present study, we observed OPTN enrichment in all PD-relevant brain regions examined. Further, alterations in OPTN and LC3 expression and colocalized puncta suggest specific impairments to autophagy that will inform future mechanistic studies. Thus, our data suggest that autophagy disruptions may be critical to PD pathogenesis in non-dopaminergic neurons and the onset of non-motor symptoms.