Optineurin Research Articles

UBQLN2 Promotes the Production of Type I Interferon via the TBK1-IRF3 Pathway.

Mutations of Ubiquilin 2 (UBQLN2) or TANK-binding kinase 1 (TBK1) are associated with amyotrophic lateral sclerosis and frontotemporal degeneration (ALS/FTD). However, the mechanisms whereby UBQLN2 or TBK1 mutations lead to ALS and FTD remain unclear. Here, we explored the effect of UBQLN2 on TBK1 in HEK-293T cells or in CRISPR–Cas9-mediated IRF3 and IRF7 knockout (KO) cells. We found an interaction between TBK1 and UBQLN2, which was affected by ALS/FTD-linked mutations in TBK1 or UBQLN2. Co-expression of UBQLN2 with TBK1 elevated the protein level of TBK1 as well as the phosphorylation of TBK1 and IRF3 in a UBQLN2 dose-dependent manner, and this phosphorylation was reduced by mutant UBQLN2. In addition, the cellular production of IFN1 and related pro-inflammatory cytokines was substantially elevated when UBQLN2 and TBK1 were co-expressed, which was also decreased by mutant UBQLN2. Functional assay revealed that mutant UBQLN2 significantly reduced the binding affinity of TBK1 for its partners, including IRF3, (SQSTM1)/p62 and optineurin (OPTN). Moreover, complete loss of IRF3 abolished the induction of IFN1 and related pro-inflammatory cytokines enhanced by UBQLN2 in HEK-293T cells, whereas no significant change in IRF7 knockout cells was observed. Thus, our findings suggest that UBQLN2 promotes IRF3 phosphorylation via TBK1, leading to enhanced IFN1 induction, and also imply that the dysregulated TBK1-IRF3 pathway may play a role in UBQLN2-related neurodegeneration.

Noncoding RNA MaIL1 is an integral component of the TLR4–TRIF pathway

RNA has been proposed as an important scaffolding factor in the nucleus, aiding protein complex assembly in the dense intracellular milieu. Architectural contributions of RNA to cytosolic signaling pathways, however, remain largely unknown. Here, we devised a multidimensional gradient approach, which systematically locates RNA components within cellular protein networks. Among a subset of noncoding RNAs (ncRNAs) cosedimenting with the ubiquitin-proteasome system, our approach unveiled ncRNA MaIL1 as a critical structural component of the Toll-like receptor 4 (TLR4) immune signal transduction pathway. RNA affinity antisense purification-mass spectrometry (RAP-MS) revealed MaIL1 binding to optineurin (OPTN), a ubiquitin-adapter platforming TBK1 kinase. MaIL1 binding stabilized OPTN, and consequently, loss of MaIL1 blunted OPTN aggregation, TBK1-dependent IRF3 phosphorylation, and type I interferon (IFN) gene transcription downstream of TLR4. MaIL1 expression was elevated in patients with active pulmonary infection and was highly correlated with IFN levels in bronchoalveolar lavage fluid. Our study uncovers MaIL1 as an integral RNA component of the TLR4-TRIF pathway and predicts further RNAs to be required for assembly and progression of cytosolic signaling networks in mammalian cells.

Solution Structural Behavior of UBAN Domains Revealed by Coarse‐Grained Computer Simulations

Proteins containing a Ubiquitin‐binding domain in ABIN and NEMO (UBAN) have a broad range of biological roles. Much of the control of ubiquitin recognition by individual UBAN‐domain containing proteins is based on protein structure. Ubiquitin recognition is further modulated by post‐translational modifications. X‐ray crystal structures of UBAN domains display coiled‐coil dimers in the absence or presence of M1‐linked ubiquitin. However, the solution structural behavior of these UBAN domains is not well understood. Extensive coarse‐grained molecular dynamics simulations of UBAN domains from NEMO, optineurin (OPTN), CEP55, and ABIN1 using a predictive energy landscape based model reveal unique signatures of solution structural conformational heterogeneity in NEMO and ABIN1 but a much more homogeneous distribution of highly extended OPTN conformations. Simulating phosphorylation of OPTN S473, a known post‐translational modification that increases M1‐linked ubiquitin affinity for OPTN, greatly increases OPTN conformational heterogeneity. Furthermore, phosphorylation of ABIN1 S442, a likely post‐translational modification in vivo, shifts the ABIN1 conformational distribution. These results indicate the conformational distribution of UBAN domains provides a means to tailor cellular response in specific biological roles, and post‐translational modifications in combination with conformational heterogeneity provide a rheostat‐like means to control cellular signaling pathways.Support or Funding InformationThis work was supported by internal funding from the University of Connecticut to James Halpert.

Motor Neuron Pathology Leads to Respiratory Deficiency in Optn−/− Mice

Amyotrophic Lateral Sclerosis (ALS) is a devastating and fatal neurodegenerative disease with no current cure. Respiratory failure is the leading cause of death in ALS. Death occurs3–5 years after diagnosis when patients with ALS ultimately succumb to inadequate ventilation, hypoxia, and respiratory failure. Several genes are associated with ALS. One of these genes encodes optineurin (OPTN) which is associated with neurodegeneration in both ALS and glaucoma. Optineurin has multiple roles in various biochemical pathways such as regulation of inflammation and autophagy. However, the exact mechanism by which loss of OPTN results in progressive neural degeneration and respiratory failure in ALS is still unclear.In order to understand the impact of OPTN deficiency on respiratory function, our goal was to study the respiratory pathophysiology in the Optn−/− mouse. The hypothesis driving this work is that the Optn−/− mouse has respiratory insufficiency due to degeneration of the respiratory motor unit – motor neuron, nerve, neuromuscular junction and muscle. Physiological, histological and molecular outcome measures were used to assess the impact of OPTN on the respiratory system. We used whole body plethysmography (WBP) to assess breathing at baseline and during a respiratory challenge with hypercapnic and hypoxic conditions (FiCO2: 0.07, FiO2: 0.10; nitrogen balance). During the respiratory challenge, compared to the WT mice, Optn−/− mice had significantly lower tidal volume, minute ventilation, peak inspiratory flow, and peak expiratory flow starting at 6 months of age indicating weakened muscle strength. Furthermore, throughout the challenge period Optn−/− mice spent a greater amount of time in apnea indicating pathology in the respiratory control centers. The weakness and pathology in control of breathing during the respiratory challenge progresses as the mice age. At 1 year of age, Optn−/− mice have stunted growth accompanied by reduced diaphragm size compared to WT mice. Postmortem immunohistochemical studies of the medulla reveal Optn−/− mice have fewer hypoglossal motor neurons. Finally, the hypoglossal (XII) nerves of Optn−/− mice had significantly reduced g‐ratio which indicates pathology and decompaction of myelin sheaths. Within the XII nerve, there are an increase in both the number of mitochondria and area of mitochondria. These along with elevated levels of LC3‐II to LC3‐I indicate reduced mitophagy. In conclusion, the Optn−/− ALS mouse model displays motor neuron pathology, increased nerve demyelination and aberrant control of breathing leading to respiratory insufficiency.

Optineurin regulates osteoblastogenesis through STAT1

A sophisticated and delicate balance between bone resorption by osteoclasts and bone formation by osteoblasts regulates bone metabolism. Optineurin (OPTN) is a gene involved in primary open-angle glaucoma and amyotrophic lateral sclerosis. Although its function has been widely studied in ophthalmology and neurology, recent reports have shown its possible involvement in bone metabolism through negative regulation of osteoclast differentiation. However, little is known about the role of OPTN in osteoblast function. Here, we demonstrated that OPTN controls not only osteoclast but also osteoblast differentiation. Different parameters involved in osteoblastogenesis and osteoclastogenesis were assessed in Optn−/- mice. The results showed that osteoblasts from Optn−/- mice had impaired alkaline phosphatase activity, defective mineralized nodules, and inability to support osteoclast differentiation. Moreover, OPTN could bind to signal transducer and activator of transcription 1 (STAT1) and regulate runt-related transcription factor 2 (RUNX2) nuclear localization by modulating STAT1 levels in osteoblasts. These data suggest that OPTN is involved in bone metabolism not only by regulating osteoclast function but also by regulating osteoblast function by mediating RUNX2 nuclear translocation via STAT1.

Genome-wide siRNA screening reveals that DCAF4-mediated ubiquitination of optineurin stimulates autophagic degradation of Cu,Zn-superoxide dismutase

Cu, Zn superoxide dismutase (SOD1) is one of the genes implicated in the devastating neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Although the precise mechanisms of SOD1 mutant (SOD1mut)-induced motoneuron toxicity are still unclear, defects in SOD1 proteostasis are known to have a critical role in ALS pathogenesis. We previously reported that the SOD1mut adopts a conformation that exposes a Derlin-1-binding region (DBR) and that DBR-exposed SOD1 interacts with Derlin-1, leading to motoneuron death. We also found that an environmental change, i.e. zinc depletion, induces a conformational change in WT SOD1 (SOD1WT) to the DBR-exposed conformation, suggesting the presence of an equilibrium state between the DBR-masked and DBR-exposed states even with SOD1WT Here, we conducted a high-throughput screening based on time-resolved FRET to further investigate the SOD1WT conformational change, and we used a genome-wide siRNA screen to search for regulators of SOD1 proteostasis. This screen yielded 30 candidate genes that maintained an absence of the DBR-exposed SOD1WT conformation. Among these genes was one encoding DDB1- and CUL4-associated factor 4 (DCAF4), a substrate receptor of the E3 ubiquitin-protein ligase complex. Of note, we found that DCAF4 mediates the ubiquitination of an ALS-associated protein and autophagy receptor, optineurin (OPTN), and facilitates autophagic degradation of DBR-exposed SOD1. In summary, our screen identifies DCAF4 as being required for proper proteostasis of DBR-exposed SOD1, which may have potential relevance for the development of therapies for managing ALS.

Sorting Rare ALS Genetic Variants by Targeted Re-Sequencing Panel in Italian Patients: OPTN, VCP, and SQSTM1 Variants Account for 3% of Rare Genetic Forms.

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disease due to motor neuron loss variably associated with frontotemporal dementia (FTD). Next generation sequencing technology revealed an increasing number of rare and novel genetic variants and interpretation of their pathogenicity represents a major challange in the diagnosis of ALS. We selected 213 consecutive patients with sporadic or familial (16%) ALS, tested negative for SOD1, FUS, TARDBP, and C9orf72 mutations. To reveal rare forms of genetic ALS, we performed a comprehensive multi-gene panel screening including 46 genes associated with ALS, hereditary motor neuronopathies, spastic paraplegia, and FTD. Our study allowed the identification of pathogenic or likely pathogenic variants in 4.2% of patients. The genes with the highest percentage of pathogenic variants were OPTN (1%), VCP (1%) SQSTM1(1%), SETX (0.4%), FIG4 (0.4%), and GARS1 (0.4%) genes. We also found 49 novel or rare gene variants of unknown significance in 30 patients (14%), 44 unlikely pathogenic variants (39%), and 48 variants in ALS susceptibility genes. The results of our study suggest the screening of OPTN, VCP, and SQSTM1 genes in routine diagnostic investigations for both sporadic and familial cases, and confirm the importance of diagnosis and couselling for patients and their relative family members.

Neurofilament Proteins as Prognostic Biomarkers in Neurological Disorders.

Neurofilaments: light, medium, and heavy (abbreviated as NF-L, NF-M, and NF-H, respectively), which belong to Type IV intermediate filament family (IF), are neuron-specific cytoskeletal components. Neurofilaments are axonal structural components and integral components of synapses, which are important for neuronal electric signal transmissions along the axons and post-translational modification. Abnormal assembly of neurofilaments is found in several human neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), infantile spinal muscular atrophy (SMA), and hereditary sensory-motor neuropathy (HSMN). In addition, those pathological neurofilament accumulations are known in α-synuclein in Parkinson's disease (PD), Aβ and tau in Alzheimer's disease (AD), polyglutamine in CAG trinucleotide repeat disorders, superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TDP43), neuronal FUS proteins, optineurin (OPTN), ubiquilin 2 (UBQLN2), and dipeptide repeat protein (DRP) in amyotrophic lateral sclerosis (ALS). When axon damage occurs in central nervous disorders, neurofilament proteins are released and delivered into cerebrospinal fluid (CSF), which are then circulated into blood. New quantitative analyses and assay techniques are well-developed for the detection of neurofilament proteins, particularly NF-L and the phosphorylated NF-H (pNF-H) in CSF and serum. This review discusses the potential of using peripheral blood NF quantities and evaluating the severity of damage in the nervous system. Intermediate filaments could be promising biomarkers for evaluating disease progression in different nervous system disorders.

Lactobacillus johnsonii L531 ameliorates enteritis via elimination of damaged mitochondria and suppression of SQSTM1-dependent mitophagy in a Salmonella infantis model of piglet diarrhea.

Newly weaned piglets challenged with Salmonella infantis were particularly susceptible, whereas oral preadministration of Lactobacillus johnsonii L531 alleviated enteritis and promoted intestinal secretory IgA production. Salmonella infantis-induced activation of NLRC4 and NLRP3 inflammasomes and (nuclear factor kappa B) NF-κB signaling in the small intestine was also inhibited by L. johnsonii L531 pretreatment, thus limiting inflammation. An IPEC-J2 cell model of S. infantis infection yielded similar results. Salmonella infantis infection also resulted in mitochondrial damage and impaired mitophagy in the ileum and IPEC-J2 cells, as demonstrated by immunofluorescence colocalization of mitochondria with microtubule-binding protein light chain 3 (LC3) and high expression of autophagy-related proteins PTEN-induced putative kinase 1 (PINK1), sequestosome 1 (SQSTM1/p62), optineurin (OPTN), and LC3 by Western blotting analysis. However, L. johnsonii L531 pretreatment reduced both the extent of mitochondrial damage and autophagy-related protein expression. Our findings suggest that the amelioration of S. infantis-associated enteritis by L. johnsonii L531 is associated with regulation of NLRC4 and NLRP3 inflammasomes and NF-κB signaling pathway activation and suppression of mitochondrial damage. Amelioration of impaired mitophagy by L. johnsonii L531 could involve eliminating damaged mitochondria and regulating S. infantis-induced activation of the NF-κB-SQSTM1mitophagy signaling pathway in host cells to prevent the further mitochondrial damage and S. infantis dissemination.

Novel mutation in optineurin causing aggressive ALS+/−frontotemporal dementia

ObjectiveMutations in optineurin (OPTN) have been identified in familial and sporadic amyotrophic lateral sclerosis (ALS). We screened a cohort of Chinese patients for mutations in optineurin. We also performed an extensive literatures review of all mutations in optineurin identified previously to detect genotype–phenotype associations.MethodsAll 16 exons of the OPTN gene in a cohort of 15 familial ALS indexes and 275 sporadic ALS patients of Chinese origin were sequenced by targeted next generation sequencing.ResultsTwo known heterozygous missense mutations in the OPTN, c.1481T> G (p.L494W), and c.1546G> C (p.E516Q), as well as one novel heterozygous missense mutation c.1690G> C (p.D564H) were each detected in one sporadic ALS patient. The patient carrying the p.E516Q mutation developed clinical features of ALS‐frontotemporal dementia (FTD) and the patient carrying the p.D564H mutation showed a phenotype of ALS. They both had an aggressive course, with a survival of 18 and 14 months respectively. Literature review showed that the clinical phenotypes in OPTN mutated ALS were not homogeneous, although some individuals showed a relatively slow progression and a long duration, some mutations carriers developed an aggressive progression and a short survival.Interpretation OPTN mutations contribute to ALS in Chinese population and account for 0.8% of sporadic ALS patients and 1.5% of familial ALS in the pooled Chinese ALS cohorts. Mutations in optineurin can cause aggressive ALS+/−FTD.

SQSTM1/p62 variants in 486 patients with familial ALS from Germany and Sweden

Several studies reported amyotrophic lateral sclerosis (ALS)-linked mutations in TBK1, OPTN, VCP, UBQLN2, and SQSTM1 genes encoding proteins involved in autophagy. SQSTM1 was originally identified by a candidate gene approach because it encodes p62, a multifunctional protein involved in protein degradation both through proteasomal regulation and autophagy. Both p62 and optineurin (encoded by OPTN) are direct interaction partners and substrates of TBK1, and these 3 proteins form the core of a genetic and functional network that may connect autophagy with ALS. Considering the molecular and conceptual relevance of the TBK1/OPTN/SQSTM1 “triangle,” we here performed a targeted screen for SQSTM1 variants in 486 patients with familial ALS from Germany and Sweden by analyzing whole-exome sequencing data. We report 9 novel and 5 previously reported rare variants in SQSTM1 and discuss the current evidence for SQSTM1 as a primary disease gene for ALS. We conclude that the evidence for causality remains vague for SQSTM1 and is weaker than for the other autophagy genes, for example, TBK1 and OPTN.

The ICP0 Protein of Herpes Simplex Virus 1 (HSV-1) Downregulates Major Autophagy Adaptor Proteins Sequestosome 1 and Optineurin during the Early Stages of HSV-1 Infection.

Herpes simplex virus 1 (HSV-1) infects mucosal epithelial cells and establishes lifelong infections in sensory neurons. Following reactivation, the virus is transferred anterograde to the initial site of infection or to sites innervated by infected neurons, causing vesicular lesions. Upon immunosuppression, frequent HSV-1 reactivation can cause severe diseases, such as blindness and encephalitis. Autophagy is a process whereby cell components are recycled, but it also serves as a defense mechanism against pathogens. HSV-1 is known to combat autophagy through the functions of the γ134.5 protein, which prevents formation of the autophagophore by binding to Beclin 1, a key factor involved in the elongation of the isolation membrane, and by redirecting the protein phosphatase 1α (PP1α) to dephosphorylate the translation initiation factor 2α (eIF2α) to prevent host translational shutoff. Other viral proteins that counteract innate immunity negatively impact autophagy. Here, we present a novel strategy of HSV-1 to evade the host through the downregulation of the autophagy adaptor protein sequestosome (p62/SQSTM1) and of the mitophagy adaptor optineurin (OPTN). This down-modulation occurs during the early steps of the infection. We also found that infected cell protein 0 (ICP0) of the virus mediates the down-modulation of the two autophagy adaptors in a mechanism independent of its E3 ubiquitin ligase activity. Cells depleted of either p62 or OPTN were able to mount greater antiviral responses, whereas cells expressing exogenous p62 displayed decreased virus yields. We conclude that downregulation of p62/SQSTM1 and OPTN is a viral strategy to counteract the host.IMPORTANCE Autophagy is a homeostatic mechanism of cells to recycle components, as well as a defense mechanism to get rid of pathogens. Strategies that HSV-1 has developed to counteract autophagy have been described and involve inhibition of autophagosome formation or indirect mechanisms. Here, we present a novel mechanism that involves downregulation of two major autophagy adaptor proteins, sequestosome 1 (p62/SQSTM1) and optineurin (OPTN). These findings generate the question of why the virus targets two major autophagy adaptors if it has mechanisms to block autophagosome formation. P62/SQSTM1 and OPTN proteins have pleiotropic functions, including regulation of innate immunity, inflammation, protein sorting, and chromatin remodeling. The decrease in virus yields in the presence of exogenous p62/SQSTM1 suggests that these adaptors have an antiviral function. Thus, HSV-1 may have developed multiple strategies to incapacitate autophagy to ensure replication. Alternatively, the virus may target another antiviral function of these proteins.

Glaucoma-Associated Mutations in the Optineurin Gene Have Limited Impact on Parkin-Dependent Mitophagy

Glaucoma results in progressive degeneration of the optic nerve and irreversible vision loss. Several mutations in the gene encoding optineurin (OPTN), the receptor for Parkin-dependent mitochondrial autophagy (mitophagy), are associated with glaucoma and amyotrophic lateral sclerosis (ALS). ALS mutations in the ubiquitin-binding domain of OPTN impair Parkin-dependent mitophagy. However, the effects of glaucoma mutations in this region remain unknown. We examined the impact of glaucoma-associated OPTN mutations on Parkin-dependent mitophagy. The mitochondria-localized, pH-sensitive fluorescent protein mito-Keima was used to monitor mitophagy. HeLa cells expressing Parkin were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or oligomycin/antimycin A (O/A) to induce Parkin-dependent mitophagy. Two complementary mitophagy receptors, OPTN and NDP52, were deleted in HeLa cells expressing mito-Keima and Parkin (DKO_HeLa). The mutant OPTN genes were re-introduced into DKO_HeLa cells using retroviruses or through transfection. Mitophagy activity and OPTN localization were evaluated via microscopic analyses. OPTN binding to ubiquitin was examined using an immunoprecipitation assay. Parkin-dependent mitophagy was inhibited in DKO_HeLa cells. Introduction of two glaucoma mutations in the ubiquitin-interacting region of OPTN restored mitophagy in CCCP-treated DKO_HeLa cells, whereas the two ALS mutations failed to replicate this effect. Under treatment with CCCP, the two glaucoma-mutant OPTN proteins normally translocated to mitochondria and bound to ubiquitinated proteins. Furthermore, five additional glaucoma-mutant OPTN proteins restored CCCP-induced mitophagy. Moreover, treatment with O/A exhibited similar results. Glaucoma-mutant OPTN proteins retain their normal properties as mitophagy receptors, suggesting that mutations in the OPTN gene cause glaucoma through a mechanism independent of mitophagy defects.

Optineurin downregulation induces endoplasmic reticulum stress, chaperone-mediated autophagy, and apoptosis in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) shows a high level of basal autophagy. Here we investigated the role of optineurin (OPTN) in PDAC cell lines, which is a prominent member of the autophagy system. To that purpose, mining of publically available databases showed that OPTN is highly expressed in PDAC and that high levels of expression are related to reduced survival. Therefore, the role of OPTN on proliferation, migration, and colony formation was investigated by transient knockdown in Miapaca, BXPC3, and Suit2-007 human PDAC cells. Furthermore, gene expression modulation in response to OPTN knockdown was assessed by microarray. The influence on cell cycle distribution and cell death signaling cascades was followed by FACS, assays for apoptosis, RT-PCR, and western blot. Finally, autophagy and ROS induction were screened by acridine orange and DCFH-DA fluorescent staining respectively. OPTN knockdown caused significant inhibition of colony formation, increased migration and no significant effect on proliferation in Miapaca, BXPC3 and Suit2-007 cells. The microarray showed modulation of 293 genes in Miapaca versus 302 in Suit2-007 cells, of which 52 genes overlapped. Activated common pathways included the ER stress response and chaperone-mediated autophagy, which was confirmed at mRNA and protein levels. Apoptosis was activated as shown by increased levels of cleaved PARP, Annexin V binding and nuclear fragmentation. OPTN knockdown caused no increased vacuole formation as assessed by acridine orange. Also, there was only marginally increased ROS production. Combination of OPTN knockdown with the autophagy inducer erufosine or LY294002, an inhibitor of autophagy, showed additive effects, which led us to hypothesize that they address different pathways. In conclusion, OPTN knockdown was related to activation of ER stress response and chaperone-mediated autophagy, which tend to confine the damage caused by OPTN knockdown and thus question its value for PDAC therapy.

Somatic and germline mutations in the tumor suppressor gene PARK2 impair PINK1/Parkin-mediated mitophagy in lung cancer cells.

PARK2, which encodes Parkin, is a disease-causing gene for both neurodegenerative disorders and cancer. Parkin can function as a neuroprotector that plays a crucial role in the regulation of mitophagy, and germline mutations in PARK2 are associated with Parkinson’s disease (PD). Intriguingly, recent studies suggest that Parkin can also function as a tumor suppressor and that somatic and germline mutations in PARK2 are associated with various human cancers, including lung cancer. However, it is presently unknown how the tumor suppressor activity of Parkin is affected by these mutations and whether it is associated with mitophagy. Herein, we show that wild-type (WT) Parkin can rapidly translocate onto mitochondria following mitochondrial damage and that Parkin promotes mitophagic clearance of mitochondria in lung cancer cells. However, lung cancer-linked mutations inhibit the mitochondrial translocation and ubiquitin-associated activity of Parkin. Among all lung cancer-linked mutants that we tested, A46T Parkin failed to translocate onto mitochondria and could not recruit downstream mitophagic regulators, including optineurin (OPTN) and TFEB, whereas N254S and R275W Parkin displayed slower mitochondrial translocation than WT Parkin. Moreover, we found that deferiprone (DFP), an iron chelator that can induce mitophagy, greatly increased the death of A46T Parkin-expressing lung cancer cells. Taken together, our results reveal a novel mitophagic mechanism in lung cancer, suggesting that lung cancer-linked mutations in PARK2 are associated with impaired mitophagy and identifying DFP as a novel therapeutic agent for PARK2-linked lung cancer and possibly other types of cancers driven by mitophagic dysregulation.

PHB2 (prohibitin 2) promotes PINK1-PRKN/Parkin-dependent mitophagy by the PARL-PGAM5-PINK1 axis

ABSTRACT Mitophagy, which is a conserved cellular process for selectively removing damaged or unwanted mitochondria, is critical for mitochondrial quality control and the maintenance of normal cellular physiology. However, the precise mechanisms underlying mitophagy remain largely unknown. Prior studies on mitophagy focused on the events in the mitochondrial outer membrane. PHB2 (prohibitin 2), which is a highly conserved membrane scaffold protein, was recently identified as a novel inner membrane mitophagy receptor that mediates mitophagy. Here, we report a new signaling pathway for PHB2-mediated mitophagy. Upon mitochondrial membrane depolarization or misfolded protein aggregation, PHB2 depletion destabilizes PINK1 in the mitochondria, which blocks the mitochondrial recruitment of PRKN/Parkin, ubiquitin and OPTN (optineurin), leading to an inhibition of mitophagy. In addition, PHB2 overexpression directly induces PRKN recruitment to the mitochondria. Moreover, PHB2-mediated mitophagy is dependent on the mitochondrial inner membrane protease PARL, which interacts with PHB2 and is activated upon PHB2 depletion. Furthermore, PGAM5, which is processed by PARL, participates in PHB2-mediated PINK1 stabilization. Finally, a ligand of PHB proteins that we synthesized, called FL3, was found to strongly inhibit PHB2-mediated mitophagy and to effectively block cancer cell growth and energy production at nanomolar concentrations. Thus, our findings reveal that the PHB2-PARL-PGAM5-PINK1 axis is a novel pathway of PHB2-mediated mitophagy and that targeting PHB2 with the chemical compound FL3 is a promising strategy for cancer therapy. Abbreviations AIFM1: apoptosis inducing factor mitochondria associated 1; ATP5F1A/ATP5A1: ATP synthase F1 subunit alpha; BAF: bafilomycin A1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: chemical reagent carbonyl cyanide m-chlorophenyl hydrazine; FL3: flavaglines compound 3; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; LC3B/MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryo fibroblasts; MPP: mitochondrial-processing peptidase; MT-CO2/COX2: mitochondrially encoded cytochrome c oxidase II; MTS: mitochondrial targeting sequence; OA: oligomycin and antimycin A; OPTN: optineurin; OTC: ornithine carbamoyltransferase; PARL: presenilin associated rhomboid like; PBS: phosphate-buffered saline; PGAM5: PGAM family member 5, mitochondrial serine/threonine protein phosphatase; PHB: prohibitin; PHB2: prohibitin 2; PINK1: PTEN induced kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; Roc-A: rocaglamide A; TOMM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin beta class I.

Global deletion of Optineurin results in altered type I IFN signaling and abnormal bone remodeling in a model of Paget's disease.

Genome-wide association studies (GWAS) have identified Optineurin (OPTN) as genetically linked to Paget's disease of the bone (PDB), a chronic debilitating bone remodeling disorder characterized by localized areas of increased bone resorption and abnormal bone remodeling. However, only ~10% of mouse models with a mutation in Optn develop PDB, thus hindering the mechanistic understanding of the OPTN-PDB axis. Here, we reveal that 100% of aged Optn global knockout (Optn-/-) mice recapitulate the key clinical features observed in PDB patients, including polyostotic osteolytic lesions, mixed-phase lesions, and increased serum levels of alkaline phosphatase (ALP). Differentiation of primary osteoclasts ex vivo revealed that the absence of Optn resulted in an increased osteoclastogenesis. Mechanistically, Optn-deficient osteoclasts displayed a significantly decreased type I interferon (IFN) signature, resulting from both defective production of IFNβ and impaired signaling via the IFNα/βR, which acts as a negative feedback loop for osteoclastogenesis and survival. These data highlight the dual roles of OPTN in the type I IFN response to restrain osteoclast activation and bone resorption, offering a novel therapeutic target for PDB. Therefore, our study describes a novel and essential mouse model for PDB and define a key role for OPTN in osteoclast differentiation.

Coding Region Mutation Screening in Optineurin in Chinese Normal-Tension Glaucoma Patients.

Purpose To study the roles of sequence alterations in the optineurin (OPTN) gene-coding region in normal-tension glaucoma (NTG) among Chinese patients. Methods Genomic DNA was extracted from 190 NTG patients and 201 control subjects. The thirteen exons of OPTN were amplified by polymerase chain reaction and analyzed by direct sequencing. Detected sequence changes were compared between NTG patients and control subjects. Results Seven sequence changes in OPTN were identified in both NTG patients and control subjects. Among them, c.464G>A (T34 T), c.509C>T (T49T), c.806G>A (V148V), and c.959T>C (P199P) were synonymous codon changes, whilst c.655T>A (M98K), c.1996G>A (R545Q), and c.1582T>C (I407T) were missense changes. Two previously reported heterozygous mutations, c.458G>A (E50K) in exon 4 and c.691_692insAG in exon 6, were not found in this study. Out of these seven OPTN sequence variants, c.464G>A (T34T) was significantly associated with NTG in both the allelic and genotypic association analyses (allelic association: p = 0.0001, OR = 2.20, 95% CI: 1.46-3.31; genotypic association: p = 0.0001), whereas the association of other variants with NTG did not reach statistical significance (p > 0.05). Variants c.1582 T>C (I407T) and c.806G>A (V148V) were identified in one and two NTG patients, respectively, but not in the control subjects. Conclusions This study confirmed the association of the OPTN T34T variant with NTG, suggesting that OPTN is a susceptibility gene for NTG in Chinese. Moreover, a variant with amino acid change (I407T) was identified in NTG but not in controls. Further studies are warranted to assess whether this variant is a causative mutation for NTG.

Acteoside inhibits autophagic apoptosis of retinal ganglion cells to rescue glaucoma-induced optic atrophy.

BackgroundGlaucoma is the world's second biggest cause of blindness, and patients progressively lose their eyesight. The current clinical treatment for glaucoma involves controlling intraocular pressure with drugs or surgery; however, some patients still progressively lose their eyesight. This treatment is also similar to the treatment of traumatic optic neuropathy. Thus, saving retinal ganglion cells (RGCs) from apoptosis is essential.MethodsThe role of Acteoside on autophagy modulation in the 661 W cell line.ResultsIn this study, we first find that Acteoside inhibits autophagy, Rapamycin alleviates this inhibition and the PI3K inhibitor, 3‐MA or LY294002, synergistically promotes it. In a mechanistic study, we find that Optineurin (OPTN) mediates Acteoside regulation of autophagy. OPTN overexpression or knockdown activates or inhibits autophagy, respectively. OPTN is inhibited by autophagy inhibitors, such as Acteoside and 3‐MA and is promoted by the autophagy activator, Rapamycin. Meanwhile, PI3K and AKT are elevated by Acteoside and 3‐MA and inhibited by Rapamycin. Finally, we find that Acteoside inhibits apoptosis in parallel to autophagy and that this inhibition is also mediated by OPTN.ConclusionIn summary, we conclude that Acteoside inhibits autophagy‐induced apoptosis in RGCs through the OPTN and PI3K/AKT/mTOR pathway, and glaucoma patients may benefit from Acteoside treatment alone or in combination with other autophagy inhibitors.

LRRK2 mutations impair depolarization-induced mitophagy through inhibition of mitochondrial accumulation of RAB10

ABSTRACTParkinson disease (PD) is a disabling, incurable disorder with increasing prevalence in the western world. In rare cases PD is caused by mutations in the genes for PINK1 (PTEN induced kinase 1) or PRKN (parkin RBR E3 ubiquitin protein ligase), which impair the selective autophagic elimination of damaged mitochondria (mitophagy). Mutations in the gene encoding LRRK2 (leucine rich repeat kinase 2) are the most common monogenic cause of PD. Here, we report that the LRRK2 kinase substrate RAB10 accumulates on depolarized mitochondria in a PINK1- and PRKN-dependent manner. RAB10 binds the autophagy receptor OPTN (optineurin), promotes OPTN accumulation on depolarized mitochondria and facilitates mitophagy. In PD patients with the two most common LRRK2 mutations (G2019S and R1441C), RAB10 phosphorylation at threonine 73 is enhanced, while RAB10 interaction with OPTN, accumulation of RAB10 and OPTN on depolarized mitochondria, depolarization-induced mitophagy and mitochondrial function are all impaired. These defects in LRRK2 mutant patient cells are rescued by LRRK2 knockdown and LRRK2 kinase inhibition. A phosphomimetic RAB10 mutant showed less OPTN interaction and less translocation to depolarized mitochondria than wild-type RAB10, and failed to rescue mitophagy in LRRK2 mutant cells. These data connect LRRK2 with PINK1- and PRKN-mediated mitophagy via its substrate RAB10, and indicate that the pathogenic effects of mutations in LRRK2, PINK1 and PRKN may converge on a common pathway.Abbreviations : ACTB: actin beta; ATP5F1B: ATP synthase F1 subunit beta; CALCOCO2: calcium binding and coiled-coil domain 2; CCCP: carbonyl cyanide m-chlorophenylhydrazone; Co-IP: co-immunoprecipitation; EBSS: Earle’s balanced salt solution; GFP: green fluorescent protein; HSPD1: heat shock protein family D (Hsp60) member 1; LAMP1: lysosomal associated membrane protein 1; LRRK2: leucine rich repeat kinase 2; IF: immunofluorescence; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MFN2: mitofusin 2; OMM: outer mitochondrial membrane; OPTN: optineurin; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RHOT1: ras homolog family member T1; ROS: reactive oxygen species; TBK1: TANK binding kinase 1; WB: western blot.