Damaged mitochondria recruit the effector NEMO to activate NF-κB signaling.

Failure to clear damaged mitochondria via mitophagy disrupts physiological function and may initiate damage signaling via inflammatory cascades, although how these pathways intersect remains unclear. We discovered that nuclear factor kappa B (NF-κB) essential regulator NF-κB effector molecule (NEMO) is recruited to damaged mitochondria in a Parkin-dependent manner in a time course similar to recruitment of the structurally related mitophagy adaptor, optineurin (OPTN). Upon recruitment, NEMO partitions into phase-separated condensates distinct from OPTN but colocalizing with p62/SQSTM1. NEMO recruitment, in turn, recruits the active catalytic inhibitor of kappa B kinase (IKK) component phospho-IKKß, initiating NF-κB signaling and the upregulation of inflammatory cytokines. Consistent with a potential neuroinflammatory role, NEMO is recruited to mitochondria in primary astrocytes upon oxidative stress. These findings suggest that damaged, ubiquitinated mitochondria serve as an intracellular platform to initiate innate immune signaling, promoting the formation of activated IKK complexes sufficient to activate NF-κB signaling. We propose that mitophagy and NF-κB signaling are initiated as parallel pathways in response to mitochondrial stress.

Autophagy adaptors mediate Parkin-dependent mitophagy by forming sheet-like liquid condensates.

During PINK1- and Parkin-mediated mitophagy, autophagy adaptors are recruited to damaged mitochondria to promote their selective degradation. Autophagy adaptors such as optineurin (OPTN) and NDP52 facilitate mitophagy by recruiting the autophagy-initiation machinery, and assisting engulfment of damaged mitochondria through binding to ubiquitinated mitochondrial proteins and autophagosomal ATG8 family proteins. Here, we demonstrate that OPTN and NDP52 form sheet-like phase-separated condensates with liquid-like properties on the surface of ubiquitinated mitochondria. The dynamic and liquid-like nature of OPTN condensates is important for mitophagy activity, because reducing the fluidity of OPTN-ubiquitin condensates suppresses the recruitment of ATG9 vesicles and impairs mitophagy. Based on these results, we propose a dynamic liquid-like, rather than a stoichiometric, model of autophagy adaptors to explain the interactions between autophagic membranes (i.e., ATG9 vesicles and isolation membranes) and mitochondrial membranes during Parkin-mediated mitophagy. This model underscores the importance of liquid-liquid phase separation in facilitating membrane-membrane contacts, likely through the generation of capillary forces.

Spatiotemporal Control of ULK1 Activation by NDP52 and TBK1 during Selective Autophagy.

Selective autophagy recycles damaged organelles and clears intracellular pathogens to prevent their aberrant accumulation. How ULK1 kinase is targeted and activated during selective autophagic events remains to be elucidated. In this study, we used chemically inducible dimerization (CID) assays in tandem with CRISPR KO lines to systematically analyze the molecular basis of selective autophagosome biogenesis. We demonstrate that ectopic placement of NDP52 on mitochondria or peroxisomes is sufficient to initiate selective autophagy by focally localizing and activating the ULK1 complex. The capability of NDP52 to induce mitophagy is dependent on its interaction with the FIP200/ULK1 complex, which is facilitated by TBK1. Ectopically tethering ULK1 to cargo bypasses the requirement for autophagy receptors and TBK1. Focal activation of ULK1 occurs independently of AMPK and mTOR. Our findings provide a parsimonious model of selective autophagy, which highlights the coordination of ULK1 complex localization by autophagy receptors and TBK1 as principal drivers of targeted autophagosome biogenesis.

A conserved core region of the scaffold NEMO is essential for signal-induced conformational change and liquid-liquid phase separation.

Scaffold proteins help mediate interactions between protein partners, often to optimize intracellular signaling. Herein, we use comparative, biochemical, biophysical, molecular, and cellular approaches to investigate how the scaffold protein NEMO contributes to signaling in the NF-κB pathway. Comparison of NEMO and the related protein optineurin from a variety of evolutionarily distant organisms revealed that a central region of NEMO, called the Intervening Domain (IVD), is conserved between NEMO and optineurin. Previous studies have shown that this central core region of the IVD is required for cytokine-induced activation of IκB kinase (IKK). We show that the analogous region of optineurin can functionally replace the core region of the NEMO IVD. We also show that an intact IVD is required for the formation of disulfide-bonded dimers of NEMO. Moreover, inactivating mutations in this core region abrogate the ability of NEMO to form ubiquitin-induced liquid-liquid phase separation droplets in vitro and signal-induced puncta in vivo. Thermal and chemical denaturation studies of truncated NEMO variants indicate that the IVD, while not intrinsically destabilizing, can reduce the stability of surrounding regions of NEMO due to the conflicting structural demands imparted on this region by flanking upstream and downstream domains. This conformational strain in the IVD mediates allosteric communication between the N- and C-terminal regions of NEMO. Overall, these results support a model in which the IVD of NEMO participates in signal-induced activation of the IKK/NF-κB pathway by acting as a mediator of conformational changes in NEMO.

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 GSK3ß 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.

Vascular Endothelial Cell-Derived Exosomal Sphingosylphosphorylcholine Attenuates Myocardial Ischemia-Reperfusion Injury through NR4A2-Mediated Mitophagy.

Cardiomyocyte survival is a critical contributing process of host adaptive responses to cardiovascular diseases (CVD). Cells of the cardiovascular endothelium have recently been reported to promote cardiomyocyte survival through exosome-loading cargos. Sphingosylphosphorylcholine (SPC), an intermediate metabolite of sphingolipids, mediates protection against myocardial infarction (MI). Nevertheless, the mechanism of SPC delivery by vascular endothelial cell (VEC)-derived exosomes (VEC-Exos) remains uncharacterized at the time of this writing. The present study utilized a mice model of ischemia/reperfusion (I/R) to demonstrate that the administration of exosomes via tail vein injection significantly diminished the severity of I/R-induced cardiac damage and prevented apoptosis of cardiomyocytes. Moreover, SPC was here identified as the primary mediator of the observed protective effects of VEC-Exos. In addition, within this investigation, in vitro experiments using cardiomyocytes showed that SPC counteracted myocardial I/R injury by activating the Parkin and nuclear receptor subfamily group A member 2/optineurin (NR4A2/OPTN) pathways, in turn resulting in increased levels of mitophagy within I/R-affected myocardium. The present study highlights the potential therapeutic effects of SPC-rich exosomes secreted by VECs on alleviating I/R-induced apoptosis in cardiomyocytes, thereby providing strong experimental evidence to support the application of SPC as a potential therapeutic target in the prevention and treatment of myocardial infarction.

Differential optineurin expression controls TGFß 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-ß (TGFß) signaling in TNBC cells by interacting with TGFß type I receptor (TßRI) and promoting its ubiquitination for degradation. Consistent with our experimental findings, the clinical TNBC samples displayed a negative correlation between OPTN mRNA expression and TGFß gene response signature and expression of proto-typic TGFß target genes. Altogether, our study demonstrates that OPTN is a negative regulator for TGFß receptor/SMAD signaling and suppresses metastasis in TNBC.

DJ-1 is an essential downstream mediator in PINK1/parkin-dependent mitophagy.

Loss-of-function mutations in the PRKN, PINK1 and PARK7 genes (encoding parkin, PINK1 and DJ-1, respectively) cause autosomal recessive forms of Parkinson's disease. PINK1 and parkin jointly mediate selective autophagy of damaged mitochondria (mitophagy), but the mechanisms by which loss of DJ-1 induces Parkinson's disease are not well understood. Here, we investigated PINK1/parkin-mediated mitophagy in cultured human fibroblasts and induced pluripotent stem cell-derived neurons with homozygous PARK7 mutations. We found that DJ-1 is essential for PINK1/parkin-mediated mitophagy. Loss of DJ-1 did not interfere with PINK1 or parkin activation after mitochondrial depolarization but blocked mitophagy further downstream by inhibiting recruitment of the selective autophagy receptor optineurin to depolarized mitochondria. By contrast, starvation-induced, non-selective autophagy was not affected by loss of DJ-1. In wild-type fibroblasts and induced pluripotent stem cell-derived dopaminergic neurons, endogenous DJ-1 translocated to depolarized mitochondria in close proximity to optineurin. DJ-1 translocation to depolarized mitochondria was dependent on PINK1 and parkin and did not require oxidation of cysteine residue 106 of DJ-1. Overexpression of DJ-1 did not rescue the mitophagy defect of PINK1- or parkin-deficient cells. These findings position DJ-1 downstream of PINK1 and parkin in the same pathway and suggest that disruption of PINK1/parkin/DJ-1-mediated mitophagy is a common pathogenic mechanism in autosomal recessive Parkinson's disease.

Functional and structural consequences of TBK1 missense variants in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.

Mutations in the Tank-binding kinase 1 (TBK1) gene were identified in 2015 in individuals with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). They account for ∼3-4% of cases. To date, over 100 distinct mutations, including missense, nonsense, deletion, insertion, duplication, and splice-site mutations have been reported. While nonsense mutations are predicted to cause disease via haploinsufficiency, the mechanisms underlying disease pathogenesis with missense mutations is not fully elucidated. TBK1 is a kinase involved in neuroinflammation, which is commonly observed in these diseases. TBK1 also phosphorylates key autophagy mediators, thereby regulating proteostasis, a pathway that is dysregulated in ALS-FTLD. Recently, several groups have characterised various missense mutations with respect to their effects on the phosphorylation of known TBK1 substrates, TBK1 homodimerization, interaction with optineurin, and the regulation of autophagy and neuroinflammatory pathways. Further, the effects of either global or conditional heterozygous knock-out of Tbk1, or the heterozygous or homozygous knock-in of ALS-FTLD associated mutations, alone or when crossed with the SOD1G93A classical ALS mouse model or a TDP-43 mouse model, have been reported. In this review we summarise the known functional effects of TBK1 missense mutations. We also present novel modelling data that predicts the structural effects of missense mutations and discuss how they correlate with the known functional effects of these mutations.

SUMOylation of optineurin is critical for inhibiting interferon ß production.

Targeting the vital kinase protein TBK1, optineurin (OPTN) performs as a suppressor involved in controlling RNA virus-induced interferon ß (IFN-ß) production. It has been determined that OPTN is altered by phosphorylation and ubiquitination, which are crucial for the generation of IFN-ß and mitophagy. In this study, endogenous OPTN was first discovered in human cells at a molecular mass of about 115 kD. The SUMOylation band was verified to be the higher molecular mass band of the OPTN. Additionally, the OPTN SUMOylation band was seen in cells from several species, including mouse, rabbit, bovine, porcine, etc., but not in cells from avian animals (chicken and duck). This finding suggests that OPTN SUMOylation is well conserved in mammals but not in avian animals. Additionally, it was determined that some lysine residues in the human OPTN had SUMOylation sites that followed the consensus motif. LPS, VSV infection, starvation, and RNA virus infection are a few of the stimuli that encourage endogens OPTN SUMOylation. OPTN SUMOylation is essential for OPTN biological activity, as evidenced by the stark differences in the cellular distribution of mutant OPTN SUMOylation sites from wild type (WT) OPTN. Additionally, we discovered that non-SUMOylated OPTN lost its ability to block both IFN-ß production. Our findings offer a preliminary understanding of how OPTN SUMOylation regulates IFN-ß production.

Optineurin in ocular herpes infection.

Herpes Simplex Virus-1 (HSV-1) is a neurotropic virus that can infect humans in the eye and travel to the trigeminal ganglion to establish latency. HSV-1 causes various disease states in both the primary and secondary sites of infection including the eye and the nervous system. This DNA virus exploits various adaptive measures to infect host cells, hijack host cell proteins, evade host immune response and spread from cell-to-cell to avoid immune detection. Recent data suggest that Optineurin (OPTN), a host protein, is a key restriction factor that prevents cell-to-cell spread of HSV-1 and guards against serious damage to the nervous system during infection. In recent years OPTN has gained increased attention because of its involvement in cellular mechanisms that promote homeostasis and prevent neurodegeneration. At the center of it all is the role OPTN plays as a receptor for selective autophagy. This review summarizes our latest understanding of the viral lifecycle, disease pathologies, and OPTN-mediated protective mechanisms during HSV-1 infection of the eye and the nervous system. We specifically highlight recent discoveries that implicate OPTN as crucial in the prevention of ocular and neurodegenerative diseases.

OPTN variants in ALS cases: a case report of a novel mutation and literature review.

INTRODUCTION: Optineurin (OPTN)-associated mutations have been implicated in the development of type 12 amyotrophic lateral sclerosis (ALS12). We reported a case of ALS with a new OPTN variant (p.D527fs) and reviewed relevant literature to better understand the phenotypes and pathophysiological mechanisms of ALS12. METHODS: We report a case of a 55-year-old female patient with a new heterozygous variant of the OPTN gene. A literature search of ALS cases associated with the OPTN gene mutations was performed in PubMed with the search criteria as [("amyotrophic lateral sclerosis") OR ("motor neuron disease")] AND ("OPTN"). RESULTS: The case of ALS with a new OPTN variant (p.D527fs) in our report manifested with bulbar involvement in onset and a rapidly progressive course. A literature review of 37 ALS patients with OPTN mutations included 20 males and 16 females with another patient whose gender was not described. The mean onset age of 37 ALS12 patients was 48 with the youngest 23 and the oldest 83 years old. Differences in onset age between male and female patients were not significant. Mean time from initiation to death was 61.8 ± 12.0 months. Patients present with either limb onset (73.5% cases) or bulbar onset (23.5% cases). CONCLUSION: Through the literature review, we summarized the clinical characteristics of ALS12. The phenotypes of the reported patients elucidate the genetic profiles and clinical phenotypes of ALS12. Clinicians should pay close attention to the role of receptor-interacting kinase 1 (RIPK1)-dependent necroptosis in the pathophysiologic development of ALS12, since necroptosis inhibitors are expected as potential therapeutic agents for treating ALS12.

Optineurin Deficiency and Insufficiency Lead to Higher Microglial TDP-43 Protein Levels.

Mutations in optineurin, a ubiquitin-binding adaptor protein, cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease of motor neurons linked to chronic inflammation and protein aggregation. The majority of ALS patients, including those carrying the optineurin mutations, exhibit cytoplasmic mislocalization, ubiquitination, and aggregation of nuclear TAR DNA-binding protein 43 kDa (TDP-43). To address the crosstalk between optineurin and TDP-43, we generated optineurin knockout (KO) neuronal and microglial cell lines using the CRISPR/Cas9 approach. Interestingly, we observed that loss of optineurin resulted in elevated TDP-43 protein expression in microglial BV2 but not neuronal Neuro 2a and NSC-34 cell lines. No changes were observed at the mRNA level, suggesting that this increase was post-translationally regulated. To confirm this observation in primary cells, we then used microglia and macrophages from an optineurin loss-of-function mouse model that lacks the C-terminal ubiquitin-binding region (Optn470T), mimicking optineurin truncations in ALS patients. As observed in the BV2 cells, we also found elevated basal levels of TDP-43 protein in Optn470T microglia and bone marrow-derived macrophages. To test if inflammation could further enhance TDP-43 accumulation in cells lacking functional optineurin, we stimulated them with lipopolysaccharide (LPS), and we observed a significant increase in TDP-43 expression following LPS treatment of WT cells. However, this was absent in both BV2 Optn KO and primary Optn470T microglia, which exhibited the same elevated TDP-43 levels as in basal conditions. Furthermore, we did not observe nuclear TDP-43 depletion or cytoplasmic aggregate formation in either Optn470T microglia or LPS-treated WT or Optn470T microglia. Taken together, our results show that optineurin deficiency and insufficiency post-translationally upregulate microglial TDP-43 protein levels and that elevated TDP-43 levels in cells lacking functional optineurin could not be further increased by an inflammatory stimulus, suggesting the presence of a plateau.

Optineurin defects cause TDP43-pathology with autophagic vacuolar formation.

We previously showed that optineurin (OPTN) mutations lead to the development of amyotrophic lateral sclerosis. The association between OPTN mutations and the pathogenesis of amyotrophic lateral sclerosis remains unclear. To investigate the mechanism underlying its pathogenesis, we generated Optn knockout mice. We evaluated histopathological observations of these mice and compared with those of OPTN- amyotrophic lateral sclerosis cases to investigate the mechanism underlying the pathogenesis of amyotrophic lateral sclerosis caused by OPTN mutations. The Optn (-/-) mice presented neuronal autophagic vacuoles immunopositive for charged multivesicular body protein 2b, one of the hallmarks of granulovacuolar degenerations, in the cytoplasm of spinal cord motor neurons at the age of 8 months and the OPTN- amyotrophic lateral sclerosis case with homozygous Q398X mutation. In addition, Optn (-/-) mice showed TAR-DNA binding protein 43/sequestosome1/p62 -positive cytoplasmic inclusions and the clearance of nuclear TAR-DNA binding protein 43. The axonal degeneration of the sciatic nerves was observed in Optn (-/-) mice. However, we could not observe significant differences in survival time, body weight, and motor functions, at 24 months. Our findings suggest that homozygous OPTN deletion or mutations might result in autophagic dysfunction and TAR-DNA binding protein 43 mislocalization, thereby leading to neurodegeneration of motor neurons. These findings indicate that the Optn (-/-) mice recapitulate both common and specific pathogenesis of amyotrophic lateral sclerosis associated with autophagic abnormalities. Optn (-/-) mice could serve as a mouse model for the development of therapeutic strategies.

Optineurin modulates ER stress-induced signaling pathways and cell death.

We have investigated the physiological role of the autophagy receptor Optineurin/Optn in endoplasmic reticulum (ER) stress response using cellular and animal models. In comparison to their normal counterparts, Optn-deficient mouse embryonic fibroblasts showed significantly higher cell death and caspase-3 activation upon treatment with tunicamycin and thapsigargin, inducers of ER stress. The transcript levels of some of the genes regulated by the IRE1-XBP1 and PERK-ATF4 pathways were upregulated in Optn-deficient cells, in comparison with normal cells, upon treatment with tunicamycin, and also in the brain cortex and liver of tunicamycin treated Optn-deficient mice. Also, the basal levels of IRE1α and PERK were higher in Optn-deficient cells. These results suggest that Optn modulates ER stress-induced signaling pathways and provides protection from ER stress-induced cell death.

Conformational stabilization of optineurin by the dynamic interaction of linear polyubiquitin.

Optineurin produces intracellular multi-functions involving autophagy, vesicular trafficking, and negative regulation of inflammation signaling through interaction with various proteins such as ATG8/LC3, Rab8, and polyubiquitin. Optineurin is a component of cytoplasmic inclusion bodies (IBs) in motor neurons from amyotrophic lateral sclerosis (ALS), and its mutation E478G, has been identified in patients with ALS. However, the mechanism by which polyubiquitin binding modulates the interaction partners of OPTN and ALS-associated IB formation is still unclear. To address this issue, we analyzed the interaction of Optineurin with Rab8 and LC3 in the absence and presence of linear polyubiquitin chains using fluorescence cross-correlation spectroscopy and IB formation efficiency of the E478G mutant of Optineurin during Rab8 depletion using fluorescence microscopy. Here, we hypothesize that linear polyubiquitin binding to Optineurin dynamically induces LC3 association and Rab8 dissociation, likely through a conformational change of Optineurin, and the dynamic conformational change may prevent the aggregate formation of mutant Optineurin.

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

BACKGROUND: Neuroinflammation 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. METHODS: BV2 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. RESULTS: As 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 Aß oligomer (Aßo) 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)-1ß in microglial cells. Moreover, RIPK1 was also found to be negatively regulated by OPTN via ubiquitin protease hydrolysis, resulting in the synthesis of IL-1ß 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. CONCLUSIONS: These 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.

Paget's Disease of Bone: Osteoimmunology and Osteoclast Pathology.

PURPOSE OF REVIEW: The purpose of this review is to recognize clinical features of Paget's disease of bone and to describe how the osteoclast, a myeloid-derived cell responsible for bone resorption, contributes to the disease. RECENT FINDINGS: Recent studies have identified several variants in SQSTM1, OPTN, and other genes that may predispose individuals to Paget's disease of bone; studies of these genes and their protein products have elucidated new roles for these proteins in bone physiology. Understanding the pathologic mechanisms in the Pagetic osteoclast may lead to the identification of future treatment targets for other inflammatory and autoimmune diseases characterized by abnormal bone erosion and/or osteoclast activation.

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.

ALS-Related Mutant SOD1 Aggregates Interfere with Mitophagy by Sequestering the Autophagy Receptor Optineurin.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive demise of motor neurons. One of the causes of familial ALS is the mutation of the gene encoding superoxide dismutase 1 (SOD1), which leads to abnormal protein aggregates. How SOD1 aggregation drives ALS is still poorly understood. Recently, ALS pathogenesis has been functionally implicated in mitophagy, specifically the clearance of damaged mitochondria. Here, to understand this mechanism, we investigated the relationship between the mitophagy receptor optineurin and SOD1 aggregates. We found that mutant SOD1 (mSOD1) proteins associate with and then sequester optineurin, which is required to form the mitophagosomes, to aggregates in N2a cells. Optineurin recruitment into mSOD1 aggregates resulted in a reduced mitophagy flux. Furthermore, we observed that an exogenous augmentation of optineurin alleviated the cellular cytotoxicity induced by mSOD1. Taken together, these studies demonstrate that ALS-linked mutations in SOD1 interfere with the mitophagy process through optineurin sequestration, suggesting that the accumulation of damaged mitochondria may play a crucial role in the pathophysiological mechanisms contributing to ALS.