[Optineurin and mitochondrial dysfunction in neurodegeneration]. / L'optineurine et les dysfonctionnements mitochondriaux dans la neurodégénérescence.

Optineurin (OPTN) is a multifunctional protein playing a crucial role as a receptor in selective autophagy. OPTN gene mutations are linked to diseases such as normal-tension glaucoma and amyotrophic lateral sclerosis. Recognized as a critical receptor for mitophagy, OPTN is pivotal in selectively degrading damaged mitochondria. This process is essential to prevent their accumulation, the generation of reactive oxygen species, and the release of pro-apoptotic factors. Mitophagy's quality control is governed by the PINK1 kinase and the cytosolic ubiquitin ligase Parkin, whose mutations are associated with Parkinson's disease. This review highlights recent insights emphasizing OPTN's role in mitophagy and its potential involvement in neurodegenerative diseases.

Title: L'optineurine et les dysfonctionnements mitochondriaux dans la neurodégénérescence. Abstract: L'optineurine (OPTN) est une protéine multifonctionnelle jouant un rôle crucial en tant que récepteur dans l'autophagie sélective. Les mutations du gène OPTN sont liées à des maladies telles que le glaucome à tension normale et la sclérose latérale amyotrophique. L'OPTN exerce une fonction essentielle dans la dégradation sélective des mitochondries endommagées. Ce processus est requis pour empêcher leur accumulation, la production d'espèces réactives de l'oxygène et la libération de facteurs pro-apoptotiques. Le contrôle de la qualité de la mitophagie est orchestré par la kinase PINK1 et la ligase de l'ubiquitine cytosolique Parkin, dont les mutations sont associées à la maladie de Parkinson. Cette revue met en lumière des perspectives récentes soulignant le rôle de l'OPTN dans la mitophagie et son implication potentielle dans les maladies neurodégénératives.

Analysis of ATG8 Family Members Using LC3-Interacting Regions (LIR)-Based Molecular Traps.

The ATG8 family of proteins regulates the autophagy process from the autophagosome maturation and cargo recruitment up to degradation. Autophagy dysfunction is involved in the development of multiple diseases. The LC3 interacting region (LIR)-based molecular traps have been designed to isolate endogenous ATG8 proteins and their interactors in order to facilitate the study of selective autophagy events. Here, we summarize protocols describing LC3 traps and sample preparation as well as adaptations for the analysis of ATG8 proteins in different biological models. This protocol was optimized to prepare affinity columns, reduce background, and improve the protein recovery to be analyzed by immunodetection with antibodies recognizing proteins of interest.

Exploring selective autophagy events in multiple biologic models using LC3-interacting regions (LIR)-based molecular traps.

Autophagy is an essential cellular pathway that ensures degradation of a wide range of substrates including damaged organelles or large protein aggregates. Understanding how this proteolytic pathway is regulated would increase our comprehension on its role in cellular physiology and contribute to identify biomarkers or potential drug targets to develop more specific treatments for disease in which autophagy is dysregulated. Here, we report the development of molecular traps based in the tandem disposition of LC3-interacting regions (LIR). The estimated affinity of LC3-traps for distinct recombinant LC3/GABARAP proteins is in the low nanomolar range and allows the capture of these proteins from distinct mammalian cell lines, S. cerevisiae and C. elegans. LC3-traps show preferences for GABARAP/LGG1 or LC3/LGG2 and pull-down substrates targeted to proteaphagy and mitophagy. Therefore, LC3-traps are versatile tools that can be adapted to multiple applications to monitor selective autophagy events in distinct physiologic and pathologic circumstances.

Role of Optineurin in the Mitochondrial Dysfunction: Potential Implications in Neurodegenerative Diseases and Cancer.

Optineurin (Optn) is a 577 aa protein encoded by the Optn gene. Mutations of Optn are associated with normal tension glaucoma and amyotrophic lateral sclerosis, and its gene has also been linked to the development of Paget's disease of bone and Crohn's disease. Optn is involved in diverse cellular functions, including NF-κB regulation, membrane trafficking, exocytosis, vesicle transport, reorganization of actin and microtubules, cell cycle control, and autophagy. Besides its role in xenophagy and autophagy of aggregates, Optn has been identified as a primary autophagy receptor, among the five adaptors that translocate to mitochondria during mitophagy. Mitophagy is a selective macroautophagy process during which irreparable mitochondria are degraded, preventing accumulation of defective mitochondria and limiting the release of reactive oxygen species and proapoptotic factors. Mitochondrial quality control via mitophagy is central to the health of cells. One of the important surveillance pathways of mitochondrial health is the recently defined signal transduction pathway involving the mitochondrial PTEN-induced putative kinase 1 (PINK1) protein and the cytosolic RING-between-RING ubiquitin ligase Parkin. Both of these proteins, when mutated, have been identified in certain forms of Parkinson's disease. By targeting ubiquitinated mitochondria to autophagosomes through its association with autophagy related proteins, Optn is responsible for a critical step in mitophagy. This review reports recent discoveries on the role of Optn in mitophagy and provides insight into its link with neurodegenerative diseases. We will also discuss the involvement of Optn in other pathologies in which mitophagy dysfunctions are involved including cancer.

Zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells.

The cytopathic effects of Zika virus (ZIKV) are poorly characterized. Innate immunity controls ZIKV infection and disease in most infected patients through mechanisms that remain to be understood. Here, we studied the morphological cellular changes induced by ZIKV and addressed the role of interferon-induced transmembrane proteins (IFITM), a family of broad-spectrum antiviral factors, during viral replication. We report that ZIKV induces massive vacuolization followed by "implosive" cell death in human epithelial cells, primary skin fibroblasts and astrocytes, a phenomenon which is exacerbated when IFITM3 levels are low. It is reminiscent of paraptosis, a caspase-independent, non-apoptotic form of cell death associated with the formation of large cytoplasmic vacuoles. We further show that ZIKV-induced vacuoles are derived from the endoplasmic reticulum (ER) and dependent on the PI3K/Akt signaling axis. Inhibiting the Sec61 ER translocon in ZIKV-infected cells blocked vacuole formation and viral production. Our results provide mechanistic insight behind the ZIKV-induced cytopathic effect and indicate that IFITM3, by acting as a gatekeeper for incoming virus, restricts virus takeover of the ER and subsequent cell death.

Regulation of TBK1 activity by Optineurin contributes to cell cycle-dependent expression of the interferon pathway.

The innate immune system has evolved to detect and neutralize viral invasions. Triggering of this defense mechanism relies on the production and secretion of soluble factors that stimulate intracellular antiviral defense mechanisms. The Tank Binding Kinase 1 (TBK1) is a serine/threonine kinase in the innate immune signaling pathways including the antiviral response and the host defense against cytosolic infection by bacteries. Given the critical roles of TBK1, important regulatory mechanisms are required to regulate its activity. Among these, Optineurin (Optn) was shown to negatively regulate the interferon response, in addition to its important role in membrane trafficking, protein secretion, autophagy and cell division. As Optn does not carry any enzymatic activity, its functions depend on its precise subcellular localization and its interaction with other proteins, especially with components of the innate immune pathway. This review highlights advances in our understanding of Optn mechanisms of action with focus on the relationships between Optn and TBK1 and their implication in host defense against pathogens. Specifically, how the antiviral immune system is controlled during the cell cycle by the Optn/TBK1 axis and the physiological consequences of this regulatory mechanism are described. This review may serve to a better understanding of the relationships between the different functions of Optn, including those related to immune responses and its associated pathologies such as primary open-angle glaucoma, amyotrophic lateral sclerosis and Paget's disease of bone.

Optineurin regulates the interferon response in a cell cycle-dependent manner.

Viral invasion into a host is initially recognized by the innate immune system, mainly through activation of the intracellular cytosolic signaling pathway and coordinated activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB) transcription factors that promote type I interferon gene induction. The TANK-binding Kinase 1 (TBK1) phosphorylates and activates IRF3. Here, we show that Optineurin (Optn) dampens the antiviral innate immune response by targeting the deubiquitinating enzyme CYLD to TBK1 in order to inhibit its enzymatic activity. Importantly, we found that this regulatory mechanism is abolished at the G2/M phase as a consequence of the nuclear translocation of CYLD and Optn. As a result, we observed, at this cell division stage, an increased activity and phosphorylation of TBK1 that lead to its relocalization to mitochondria and to enhanced interferon production, suggesting that this process, which relies on Optn function, might be of major importance to mount a preventive antiviral response during mitosis.

Toward an integrative view of Optineurin functions.

This review highlights recent advances in our understanding of the mechanisms of Optineurin (Optn) action and its implication in diseases. Optn has emerged as a key player regulating various physiological processes, including membrane trafficking, protein secretion, cell division and host defense against pathogens. Furthermore, there is growing evidence for an association of Optn mutations with human diseases such as primary open-angle glaucoma, amyotrophic lateral sclerosis and Paget's disease of bone. Optn functions depend on its precise subcellular localization and its interaction with other proteins. Here, we review the mechanisms that allow Optn to ensure a timely and spatially coordinated integration of different physiological processes and discuss how their deregulation may lead to different pathologies.

Recruitment of histone deacetylase 3 to the interferon-A gene promoters attenuates interferon expression.

BACKGROUND: Induction of Type I Interferon (IFN) genes constitutes an essential step leading to innate immune responses during virus infection. Sendai virus (SeV) infection of B lymphoid Namalwa cells transiently induces the transcriptional expression of multiple IFN-A genes. Although transcriptional activation of IFN-A genes has been extensively studied, the mechanism responsible for the attenuation of their expression remains to be determined. PRINCIPAL FINDINGS: In this study, we demonstrate that virus infection of Namalwa cells induces transient recruitment of HDAC3 (histone deacetylase 3) to IFN-A promoters. Analysis of chromatin-protein association by Chip-QPCR demonstrated that recruitment of interferon regulatory factor (IRF)3 and IRF7, as well as TBP correlated with enhanced histone H3K9 and H3K14 acetylation, whereas recruitment of HDAC3 correlated with inhibition of histone H3K9/K14 acetylation, removal of IRF7 and TATA-binding protein (TBP) from IFN-A promoters and inhibition of virus-induced IFN-A gene transcription. Additionally, HDAC3 overexpression reduced, and HDAC3 depletion by siRNA enhanced IFN-A gene expression. Furthermore, activation of IRF7 enhanced histone H3K9/K14 acetylation and IFN-A gene expression, whereas activation of both IRF7 and IRF3 led to recruitment of HDAC3 to the IFN-A gene promoters, resulting in impaired histone H3K9 acetylation and attenuation of IFN-A gene transcription. CONCLUSION: Altogether these data indicate that reversal of histone H3K9/K14 acetylation by HDAC3 is required for attenuation of IFN-A gene transcription during viral infection.

Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors.

The therapeutic efficacy of anticancer chemotherapies may depend on dendritic cells (DCs), which present antigens from dying cancer cells to prime tumor-specific interferon-gamma (IFN-gamma)-producing T lymphocytes. Here we show that dying tumor cells release ATP, which then acts on P2X(7) purinergic receptors from DCs and triggers the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)-dependent caspase-1 activation complex ('inflammasome'), allowing for the secretion of interleukin-1beta (IL-1beta). The priming of IFN-gamma-producing CD8+ T cells by dying tumor cells fails in the absence of a functional IL-1 receptor 1 and in Nlpr3-deficient (Nlrp3(-/-)) or caspase-1-deficient (Casp-1(-/-)) mice unless exogenous IL-1beta is provided. Accordingly, anticancer chemotherapy turned out to be inefficient against tumors established in purinergic receptor P2rx7(-/-) or Nlrp3(-/-) or Casp1(-/-) hosts. Anthracycline-treated individuals with breast cancer carrying a loss-of-function allele of P2RX7 developed metastatic disease more rapidly than individuals bearing the normal allele. These results indicate that the NLRP3 inflammasome links the innate and adaptive immune responses against dying tumor cells.

The role of differential expression of human interferon--a genes in antiviral immunity.

Immune recognition of virus-associated molecules by Toll-like receptors (TLRs) and/or RIG-I-like receptors (RLRs) triggers intracellular signaling cascades that converge on the activation of interferon regulatory factors - particularly IRF3 and IRF7, leading to the transcriptional induction of type 1 interferon genes. This review summarizes new data describing how these factors regulate the temporal and quantitative differences in the expression of the multigenic IFN-A family. The distinctive DNA-binding features of IRF3 and IRF7 affect the selectivity and affinity of these factors for IFN-A promoters; modification of the ratio of promoter-bound IRF3 and IRF7 during virus infection may influence both transcriptional activation and repression of IFN-A genes. This review also summarizes the structural differences between IFN-beta and different IFN-alpha subtypes, their interaction with their common receptor IFNAR, and their potency to elicit antiviral, antiproliferative and antitumoral responses. Taken together, this information enhances our understanding of the selective advantage of the multiplicity of IFN-alpha subtypes in the regulation of innate and adaptive immunity.

RIG-I-like receptors: sensing and responding to RNA virus infection.

Viral and microbial pathogens contain specific motifs or pathogen-associated molecular patterns (PAMPs) that are recognized by cell surface- and endosome-associated Toll-like receptors (TLRs). RNA virus infection is also detected through TLR-independent mechanisms. Early viral replicative intermediates are detected by two recently characterized cystolic viral RNA receptors-RIG-I and MDA-5. Both are DExDH/box RNA helicases, and RIG-I specifically recognizes 5'-triphosphate containing viral RNA and transmits signals that induce type I interferon-mediated host immunity against virus infection. In this review, we will focus on RIG-I-like receptor (RLR) signal transduction and the regulatory mechanisms - ubiquitination, deubiquitination, ISGylation - underlying this important host response.

Differential regulation of human interferon A gene expression by interferon regulatory factors 3 and 7.

Differential expression of the human interferon A (IFN-A) gene cluster is modulated following paramyxovirus infection by the relative amounts of active interferon regulatory factor 3 (IRF-3) and IRF-7. IRF-3 expression activates predominantly IFN-A1 and IFN-B, while IRF-7 expression induces multiple IFN-A genes. IFN-A1 gene expression is dependent on three promoter proximal IRF elements (B, C, and D modules, located at positions -98 to -45 relative to the mRNA start site). IRF-3 binds the C module of IFN-A1, while other IFN-A gene promoters are responsive to the binding of IRF-7 to the B and D modules. Maximal expression of IFN-A1 is observed with complete occupancy of the three modules in the presence of IRF-7. Nucleotide substitutions in the C modules of other IFN-A genes disrupt IRF-3-mediated transcription, whereas a G/A substitution in the D modules enhances IRF7-mediated expression. IRF-3 exerts dual effects on IFN-A gene expression, as follows: a synergistic effect with IRF-7 on IFN-A1 expression and an inhibitory effect on other IFN-A gene promoters. Chromatin immunoprecipitation experiments reveal that transient binding of both IRF-3 and IRF-7, accompanied by CBP/p300 recruitment to the endogenous IFN-A gene promoters, is associated with transcriptional activation, whereas a biphasic recruitment of IRF-3 and CBP/p300 represses IFN-A gene expression. This regulatory mechanism contributes to differential expression of IFN-A genes and may be critical for alpha interferon production in different cell types by RIG-I-dependent signals, leading to innate antiviral immune responses.

Distinct functions of IRF-3 and IRF-7 in IFN-alpha gene regulation and control of anti-tumor activity in primary macrophages.

Type I IFN (IFN-alpha/beta) have important biological functions ranging from immune cell development and activation, to tumor cell killing and most importantly inhibition of virus replication. Following viral infection or activation of Toll-like receptors (TLRs) via distinct ligands, IFN-alpha/beta are produced. Two members of the interferon regulatory factor (IRF) family - IRF-3 and IRF-7 - are the major modulators of IFN gene expression. Activation of IRF-3 and IRF-7 by TBK1/IKKvarepsilon mediated phosphorylation promotes IFN gene expression and potentiates the production of IFN responsive genes important to the development of an effective antiviral immune response. IFN treatment can augment anti-tumor properties and they are potentially key players in cancer therapy. For example, adoptive transfer of IFN-gamma-activated macrophages can mediate tumor cell killing via direct cell-cell contact, as well as release of soluble cytotoxic pro-inflammatory molecules. A recent study investigated whether IRF-3 and IRF-7 could mediate the acquisition of new anti-tumor effector functions in macrophages. Adenovirus mediated transduction of the active form of IRF-7 into primary macrophages resulted in the production of type I IFN, upregulation of target genes including TRAIL and increased tumoricidal activity of macrophages; in contrast, the active form of IRF-3 led to induction of cell death. These studies indicate that IRF-7 transduced macrophages may be an attractive candidate for in vivo adoptive therapy of cancer.

Promoter organization of the interferon-A genes differentially affects virus-induced expression and responsiveness to TBK1 and IKKepsilon.

Virus-induced expression of interferon (IFN)-A genes is regulated by two members of the IFN regulatory factor (IRF) family, IRF-3 and IRF-7, which are activated by phosphorylation during viral infection by the IKK-related serine/threonine kinases TBK1 and IkappaB kinase epsilon (IKKepsilon). In this study, we demonstrate that three IRF-binding sites located in the virus-responsive element mediate the transcriptional activation of the IFN-A4 promoter by IRF-3. The precise arrangement of these IRF elements is required for synergistic activation of the IFN-A4 promoter following Newcastle disease virus infection or activation by TBK1 or IKKepsilon. The ordered assembly of IRF-3 multimers on the promoter also determines cooperative recruitment of IRF-3 and CREB-binding protein and differential virus-induced expression of IFN-A4 gene promoter compared with IFN-A11. Naturally occurring nucleotide substitutions disrupt two of the IRF elements in the IFN-A11 gene promoter, leading to a dramatic decrease in IRF-3 and CREB-binding protein recruitment and in IRF-3-dependent transcription. Transcription of the IFN-A4 promoter by IRF-7 is mediated by two IRF elements; promoter mutants that carry a reversed IRF element retain the ability to respond to IKKepsilon or TBK1 expression in the presence of IRF-7 but lose the capacity to respond to virus or kinase-induced IRF-3. Interestingly, IKKepsilon or TBK1 stimulates the IRF-7-mediated transcription of IFN-A11, although at a lesser extent compared with IFN-A4. Our data indicate that virus-induced expression of IFN-A genes is dictated by the organization of IRF elements within the IFN-A promoters and that the differential IFN-A gene expression, based on the IRF-3 responsiveness, is partially compensated in the presence of IRF-7 when both factors are activated by IKKepsilon or TBK1.

Impairment of interferon-induced IRF-7 gene expression due to inhibition of ISGF3 formation by trichostatin A.

Two members of the signal transducer and activator of transcription family, STAT1 and STAT2, form, together with interferon regulatory factor 9 (IRF-9), the ISGF3 complex that activates the expression of the interferon-stimulated genes (ISG). The ISGF3 complex also participates in the virus-induced alpha/beta interferon (IFN-alpha/beta) gene amplification cascade by up-regulating IRF-7 gene expression. Here, we show that treatment of cells with trichostatin A (TSA), a deacetylase inhibitor, inhibits the virus-induced activation of IFN-alpha/beta promoters and dramatically reduces the ability of different ISG promoters to respond to IFN stimulation. Impairment of IFN-alpha/beta and ISG expression by TSA in infected cells is due to the blockage of interferon-stimulated ISGF3 complex formation, which leads to the abolition of IRF-7 gene expression. We also show that the TSA-dependent inhibition of ISGF3 is related to impaired nuclear accumulation of STAT2. Our data suggest that an acetylation/deacetylation mechanism participates in the regulation of cellular distribution and function of STAT2 in IFN-alpha/beta signaling.

Positive and negative control of virus-induced interferon-A gene expression.

Transcriptional regulation is a consequence of the combination of both activation and repression for establishing specific patterns of eukaryotic gene expression. The regulation of the expression of type I interferon (IFN-A and -B) multigene family is controlled primarily at the transcriptional level and has been widely studied as a model to understand the mechanisms of stable repression, transient expression and postinduction repression of genes. The positive and negative regulatory elements required for this on/off switch have been defined within a complex 5' upstream region of their transcription start site. The differential expression pattern of IFN-A genes is thought to involve both substitutions in the virus responsive element (VRE-A) and presence or absence of the distal negative regulatory element (DNRE) which is delimited upstream of the VRE-A. The interferon regulatory factors (IRF)-3 and -7 binding to the VRE-A and interacting as homodimers or heterodimers participate in the virus-induced transcriptional activation of IFN-A family. This data and the presence of homeodomain protein pituitary homeobox 1 (Pitx1) binding to the distal DNRE, negatively regulating the IRF-3 and IRF-7 activities and interacting physically with IRF-3 and IRF-7 contribute to our understanding of the complex differential transcriptional activation and repression of the IFN-A genes.

Regulation of virus-induced interferon-A genes.

Different members of the interferon regulatory factor (IRF) family are early activated by viral infection of eukaryotic cells. The IRFs participate in the virus-induced transcriptional regulation of different genes, including the multigenic interferon-A (IFN-A) family, members of which are involved in the establishment of an antiviral state, cell growth inhibition or apoptosis. This study presents the recent progress in the field of virus-induced transactivation and repression of IFN-A gene promoters. Data presented on the modular organization of IFN-A gene promoters and their transactivation dependent on IRF-3 and IRF-7 provide a new insight on the cooperativity mechanisms among the different IRF family members. Data on the transcriptional repression of virus-induced interferon-A promoters by the homeodomain protein Pitx1 contribute to our understanding of the complex differential transcriptional activation, repression and antirepression of the IFN-A genes.