Autophagic Receptor Research Articles

Activation of PINK1-Parkin-dependent mitophagy in Tri-ortho-cresyl phosphate-treated Neuro2a cells

Tri-ortho-cresyl phosphate (TOCP) is a typical organophosphorus compound that can cause organophosphate-induced delayed neuropathy (OPIDN), which is pathologically characterized by axonal degeneration. Nowadays, mitochondrial dysfunction is regarded as a potential mechanism contributing to OPIDN progress. Mitophagy, a selective type of autophagy, is required to segregate damaged mitochondria from healthy mitochondrial networks and deliver them to lysosome for degradation. This research was designed to investigate the role of mitophagy in axon degeneration following TOCP administration in an in vitro model. Differentiated neuro2a (N2a) cells were divided into four groups and treated with 0, 5, 10, and 20 μM TOCP for 24 h, respectively. The critical proteins in PINK1-Parkin-dependent mitophagy including LC3, P62, PINK1, Parkin, mitochondrial proteins, and autophagic receptors were detected by immunoblotting and immunofluorescence. After TOCP treatment, increased level of ROS in N2a cells revealed a significant mitochondria damage. Meanwhile, it was observed that much more PINK1, Parkin, and LC3-II were translocated to the mitochondria. Furthermore, immunofluorescence analysis demonstrated that the co-localization of Parkin and LC3 was significantly increased. These results suggested that PINK1-Parkin dependent mitophagy pathway in N2a cells was activated by TOCP treatment. In addition, P62, a major autophagic receptor, was markedly accumulated on the mitochondria, which indicated that P62 might play a critical role in facilitating mitophagy under TOCP-induced axonal degeneration. Taken together, our results suggest that TOCP exposure resulted in the activation of PINK1-Parkin-dependent mitophagy in N2a cells. Mitophagy may act as a positively reactive mode in eliminating dysfunctional mitochondria and therefore protect neurons against TOCP neurotoxicity.

BAG3 and SYNPO (synaptopodin) facilitate phospho-MAPT/Tau degradation via autophagy in neuronal processes

ABSTRACTA major cellular catabolic pathway in neurons is macroautophagy/autophagy, through which misfolded or aggregation-prone proteins are sequestered into autophagosomes that fuse with lysosomes, and are degraded. MAPT (microtubule-associated protein tau) is one of the protein clients of autophagy. Given that accumulation of hyperphosphorylated MAPT contributes to the pathogenesis of Alzheimer disease and other tauopathies, decreasing endogenous MAPT levels has been shown to be beneficial to neuronal health in models of these diseases. A previous study demonstrated that the HSPA/HSP70 co-chaperone BAG3 (BCL2-associated athanogene 3) facilitates endogenous MAPT clearance through autophagy. These findings prompted us to further investigate the mechanisms underlying BAG3-mediated autophagy in the degradation of endogenous MAPT. Here we demonstrate for the first time that BAG3 plays an important role in autophagic flux in the neurites of mature neurons (20–24 days in vitro [DIV]) through interaction with the post-synaptic cytoskeleton protein SYNPO (synaptopodin). Loss of either BAG3 or SYNPO impeded the fusion of autophagosomes and lysosomes predominantly in the post-synaptic compartment. A block of autophagy leads to accumulation of the autophagic receptor protein SQSTM1/p62 (sequestosome 1) as well as MAPT phosphorylated at Ser262 (p-Ser262). Furthermore, p-Ser262 appears to accumulate in autophagosomes at post-synaptic densities. Overall these data provide evidence of a novel role for the co-chaperone BAG3 in synapses. In cooperation with SYNPO, it functions as part of a surveillance complex that facilitates the autophagic clearance of MAPT p-Ser262, and possibly other MAPT species at the post-synapse. This appears to be crucial for the maintenance of a healthy, functional synapse.Abbreviations: aa: amino acids; ACTB: actin beta; BafA1: bafilomycin A1; BAG3: BCL2 associated athanogene 3; CQ chloroquine; CTSL: cathepsin L; DIV: days in vitro; DLG4/PSD95: discs large MAGUK scaffold protein 4; HSPA/HSP70: heat shock protein family A (Hsp70); MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP2: microtubule associated protein 2; MAPT: microtubule associated protein tau; p-Ser262: MAPT phosphorylated at serine 262; p-Ser396/404: MAPT phosphorylated at serines 396 and 404; p-Thr231: MAPT phosphorylated at threonine 231; PBS: phosphate buffered saline; PK: proteinase K; scr: scrambled; shRNA: short hairpin RNA; SQSTM1/p62 sequestosome 1; SYN1: synapsin I; SYNPO synaptopodin; SYNPO2/myopodin: synaptopodin 2; VPS: vacuolar protein sorting

DEAD Box Protein 5 Inhibits Liver Tumorigenesis by Stimulating Autophagy via Interaction with p62/SQSTM1.

In hepatocellular carcinoma (HCC), dysregulated expression of DDX5 (DEAD box protein 5) and impaired autophagy have been reported separately. However, the relationship between them has not been explored. Here we present evidence to show that, by interacting with autophagic receptor p62, DDX5 promotes autophagy and suppresses tumorigenesis. DDX5 inversely correlated with p62/sequestosome 1 (SQSTM1) expression in hepatitis B virus (HBV)-associated and non-HBV-associated HCCs. Patients with low DDX5 expression showed poor prognosis after tumor resection. We found that DDX5 overexpression induced, while DDX5 knockdown attenuated, autophagic flux in HepG2 and Huh7 cells. DDX5 promoted p62 degradation and markedly reduced the half-life of p62. Moreover, DDX5 overexpression dramatically reduced, while DDX5 knockdown promoted, cancer cell growth and tumorigenesis in vitro and in vivo. We found that DDX5 bound to p62 and interfered with p62/TRAF6 (tumor necrosis factor receptor-associated factor 6) interaction. Further findings revealed that the N-terminal domain of DDX5, involved in the interaction with p62, was sufficient to induce autophagy independent of its RNA binding and helicase activity. DDX5 overexpression decreased p62/TRAF6-mediated lysine 63-linked ubiquitination of mammalian target of rapamycin (mTOR) and subsequently inhibited the mTOR signaling pathway. Knockdown of TRAF6 blocked DDX5-induced autophagy. Furthermore, we showed that miR-17-5p downregulated DDX5 and impaired autophagy. Inhibition of miR-17-5p promoted autophagic flux and suppressed tumor growth in HCC xenograft models. Conclusion: Our findings define a noncanonical pathway that links miR-17-5p, DDX5, p62/TRAF6, autophagy, and HCC. These findings open an avenue for the treatment of HCC.

Acetylation modulates LC3 stability and cargo recognition.

LC3 is a key autophagy-related protein involved in both autophagosome formation and autophagy cargo recruitment. Despite these functions being exerted by deacetylated LC3, this protein is more abundantly distributed in its acetylated form. Here, we reveal that the stability and cargo recognition ability of LC3 are highly dependent on its acetylation. Through detecting the diffusion rate of soluble LC3 by fluorescence recovery after photobleaching (FRAP), we found that nutrient-state-related acetylation inhibited LC3 complex formation. Acetylation blocked LC3's interaction with p62, the autophagic cargo receptor, preventing the mis-targeting of p62 to nonautophagic LC3 and thus permitting the efficient degradation of autophagic cargoes. Acetylation also inhibited LC3 proteasome-dependent degradation, thus maintaining LC3 as a long-lived protein that could serve as a reserve. Altogether, acetylated LC3, the nonactivated form, is suitable for storage and avoids inopportune interactions with other proteins, assuring autophagic degradation.

The Roles of Ubiquitin-Binding Protein Shuttles in the Degradative Fate of Ubiquitinated Proteins in the Ubiquitin-Proteasome System and Autophagy.

The ubiquitin-proteasome system (UPS) and autophagy are the two major intracellular protein quality control (PQC) pathways that are responsible for cellular proteostasis (homeostasis of the proteome) by ensuring the timely degradation of misfolded, damaged, and unwanted proteins. Ubiquitination serves as the degradation signal in both these systems, but substrates are precisely targeted to one or the other pathway. Determining how and when cells target specific proteins to these two alternative PQC pathways and control the crosstalk between them are topics of considerable interest. The ubiquitin (Ub) recognition code based on the type of Ub-linked chains on substrate proteins was believed to play a pivotal role in this process, but an increasing body of evidence indicates that the PQC pathway choice is also made based on other criteria. These include the oligomeric state of the Ub-binding protein shuttles, their conformation, protein modifications, and the presence of motifs that interact with ATG8/LC3/GABARAP (autophagy-related protein 8/microtubule-associated protein 1A/1B-light chain 3/GABA type A receptor-associated protein) protein family members. In this review, we summarize the current knowledge regarding the Ub recognition code that is bound by Ub-binding proteasomal and autophagic receptors. We also discuss how cells can modify substrate fate by modulating the structure, conformation, and physical properties of these receptors to affect their shuttling between both degradation pathways.

Influenza A Virus NS1 Protein Suppresses JNK1-Dependent Autophagosome Formation Mediated by Rab11a Recycling Endosomes.

Autophagy is an essential process for cellular metabolism and homeostasis, but also functions as one of innate immune responses against pathogen infection. However, in contrast to cellular metabolism and homeostasis pathways, less is known about how virus infection leads to autophagosome formation. Here, we showed that influenza A virus NS1 protein inhibits the formation of autophagosomes. The autophagosome formation was induced by infection with NS1 mutant virus lacking the dsRNA-binding activity for inhibition of innate immune responses (R38AK41A) or the activation of PI3K-Akt signaling pathway (Y89F). R38AK41A mutant infection induced phosphorylation of JNK1 and up-regulated the expression of autophagy-related genes which are downstream of JNK1 signaling pathway. We also found that the amount of phosphorylated TSC2, which activates mTOR, increased in wild type-infected cells but not in Y89F mutant-infected cells. These findings suggest that NS1 inhibits the autophagosome formation through both the inhibition of JNK1 and the activation of PI3K-Akt-mTOR pathway. Further, viral ribonucleoprotein (vRNP) complexes were selectively sequestered into autophagosomes, and knockdown of Rab11a, which is responsible for the apical transport of vRNP complexes, impaired not only engulfment of vRNP complexes by autophagosomes but also the formation of autophagosomes in R38AK41A mutant-infected cells. This indicates that Rab11a-positive recycling endosomes function as a donor membrane for the phagophore elongation and an autophagic receptor for the selective engulfment of viral RNP complexes. Based on these results, we propose that NS1 inhibits JNK1-mediated autophagy induction and the sequestration of vRNP complexes into autophagosomes.

Tryptophan Deprivation Promotes an Adaptive Response and Contributes to Bioenergetics in Multiple Myeloma

Background and Objective:There is an increasing interest about the role of amino acid degrading enzymes in cancer immunotherapy.In multiple myeloma (MM) disease progression depends on the ability of malignant plasma cells (PC) to subvert the local microenvironment and reshape host immunity to support tumor growth.Our previous work showed that i) MM cells have metabolic plasticity due to availability of external nutrients; ii) aminoacid shortage due to the tryptohan (trp) degrading enzyme IDO-1 was associated to clinical MM progression. Thus, we tested bio-energetic changes in MM upon trp deprivation.Experimental design:We evaluated the trascriptome changes in the adaptive response in four human MM cell lines (MM1.s, H929, U266, OPM2), chosen for their cytogenetic alterations and previously characterized for differential expression of CD38 and c-myc and primary BM samples of MGUS and MM to trp shortage.The pathway was confirmed looked at the expression levels of key metabolic genes in a large series of highly purified BM PC samples from healthy donors (4N), 129 MM, 24 primary plasma cell leukemia (pPCL), 12 secondary PCL (sPCL) cases from a proprietary dataset (GSE66293).ResultsTrp deprivation induced an adaptive response through increased expression, time and dose-dependent, of ATF4-ASNS-CHOP-GADD34, part of GCN2 signaling, associated to increased expression of p62 (the main autophagic receptor in MM) followed by autophagy flux induction, as shown by flow cytometry and immunofluorescence evaluations.In MM cell lines, trp deprivation altered the cellular dependence on mitochondrial ATP generation via oxidative phosphorylation, inducing in vitro increase of lactate dehydrogenase (LDH) and intracellular lactate and NAD/NADH with down-regulation of surface expression of the NAD-ase CD38. Similarly, short-term trp deprivation in CD138+ plasma cells isolated by magnetic sorting from MM patients, downregulated surface expression of CD38 and intra-cellular amount of c-myc, as detected by flow cytometry.This phenomenon was associated to increased isocitrate dehydrogenase (IDH-1), enolase-1 (ENO-1), phosphoglycerate kinase 1 (PGK-1), and dihydrolipoamide dehydrogenase (DLD), suggesting that branched chain amino acids with tolerogenic meaning like trp are used in MM to sustain NADH availability and energy production.In the GSE66293 MM progression was associated to increased expression of ENO-1, PGK-1, and DLD, suggesting that changes in transcription of genes involved in bioenergetics could be clinically relevant.ConclusionOverall, our study reveals that trp deprivation promotes an adaptive response of MM offering new putative targets in the bio-energetic pathway for synthetic lethality. DisclosuresPalumbo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Di Raimondo:Takeda: Honoraria, Research Funding; Celgene: Honoraria.

ZZ-dependent regulation of p62/SQSTM1 in autophagy

Autophagic receptor p62 is a critical mediator of cell detoxification, stress response, and metabolic programs and is commonly deregulated in human diseases. The diverse functions of p62 arise from its ability to interact with a large set of ligands, such as arginylated (Nt-R) substrates. Here, we describe the structural mechanism for selective recognition of Nt-R by the ZZ domain of p62 (p62ZZ). We show that binding of p62ZZ to Nt-R substrates stimulates p62 aggregation and macroautophagy and is required for autophagic targeting of p62. p62 is essential for mTORC1 activation in response to arginine, but it is not a direct sensor of free arginine in the mTORC1 pathway. We identified a regulatory linker (RL) region in p62 that binds p62ZZ in vitro and may modulate p62 function. Our findings shed new light on the mechanistic and functional significance of the major cytosolic adaptor protein p62 in two fundamental signaling pathways.

NDP52 interacts with mitochondrial RNA poly(A) polymerase to promote mitophagy.

Parkin-mediated mitophagy is a quality control pathway that selectively removes damaged mitochondria via the autophagic machinery. Autophagic receptors, which interact with ubiquitin and Atg8 family proteins, contribute to the recognition of damaged mitochondria by autophagosomes. NDP52, an autophagy receptor, is required for autophagic engulfment of damaged mitochondria during mitochondrial uncoupler treatment. The N-terminal SKICH domain and C-terminal zinc finger motif of NDP52 are both required for its function in mitophagy. While the zinc finger motif contributes to poly-ubiquitin binding, the function of the SKICH domain remains unclear. Here, we show that NDP52 interacts with mitochondrial RNA poly(A) polymerase (MTPAP) via the SKICH domain. During mitophagy, NDP52 invades depolarized mitochondria and interacts with MTPAP dependent on the proteasome but independent of ubiquitin binding. Loss of MTPAP reduces NDP52-mediated mitophagy, and the NDP52-MTPAP complex attracts more LC3 than NDP52 alone. These results indicate that NDP52 and MTPAP form an autophagy receptor complex, which enhances autophagic elimination of damaged mitochondria.

PARK7 modulates autophagic proteolysis through binding to the N-terminally arginylated form of the molecular chaperone HSPA5

ABSTRACT Macroautophagy is induced under various stresses to remove cytotoxic materials, including misfolded proteins and their aggregates. These protein cargoes are collected by specific autophagic receptors such as SQSTM1/p62 (sequestosome 1) and delivered to phagophores for lysosomal degradation. To date, little is known about how cells sense and react to diverse stresses by inducing the activity of SQSTM1. Here, we show that the peroxiredoxin-like redox sensor PARK7/DJ-1 modulates the activity of SQSTM1 and the targeting of ubiquitin (Ub)-conjugated proteins to macroautophagy under oxidative stress caused by TNFSF10/TRAIL (tumor necrosis factor [ligand] superfamily, member 10). In this mechanism, TNFSF10 induces the N-terminal arginylation (Nt-arginylation) of the endoplasmic reticulum (ER)-residing molecular chaperone HSPA5/BiP/GRP78, leading to cytosolic accumulation of Nt-arginylated HSPA5 (R-HSPA5). In parallel, TNFSF10 induces the oxidation of PARK7. Oxidized PARK7 acts as a co-chaperone-like protein that binds the ER-derived chaperone R-HSPA5, a member of the HSPA/HSP70 family. By forming a complex with PARK7 (and possibly misfolded protein cargoes), R-HSPA5 binds SQSTM1 through its Nt-Arg, facilitating self-polymerization of SQSTM1 and the targeting of SQSTM1-cargo complexes to phagophores. The 3-way interaction among PARK7, R-HSPA5, and SQSTM1 is stabilized by the Nt-Arg residue of R-HSPA5. PARK7-deficient cells are impaired in the targeting of R-HSPA5 and SQSTM1 to phagophores and the removal of Ub-conjugated cargoes. Our results suggest that PARK7 functions as a co-chaperone for R-HSPA5 to modulate autophagic removal of misfolded protein cargoes generated by oxidative stress.

P36. 4-hydroxy(phenyl) retinamide (4HPR) worsens the pathological features of ALS-related TBK1-mutant motor neurons by further disrupting the autophagic flux at the elongation phase

TANK-binding kinase 1 (TBK1) is a protein playing a critical role in the activation of pro-survival pathways, by phosphorylating Akt, and in autophagy, as its phosphorylation and activation of the autophagic receptors p62 and optineurin is fundamental for the correct cargo recruitment at the forming autophagosome. Moreover, mutations in TBK1 gene have been recently linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In our study we aimed to uncover the molecular pathological mechanisms characterizing ALS patients carrying mutations in TBK1 gene. For this scope, we used as a model human induced Pluripotent Stem Cells (hiPSCs), and differentiated them into spinal motor neurons (MNs). TBK1-mutant MNs display reduced TBK1 protein levels up to 50%, supporting the theory that haploinsufficiency might be the pathogenic mechanism leading to ALS in these specific cases. Interestingly, as observed in post-mortem tissues, TBK1 MNs are characterized by decreased viability during long-term culture, and accumulate aberrant cytosolic aggresomes. In order to elucidate the impairment affecting the degrative pathway leading to aggresomes accumulation, and eventually circumvent the pathological phenotype, we performed a small molecule screening, which identified 4-hydroxy(phenyl) retinamide (4HPR) as a potent autophagy modifier. This synthetic retinoid failed in reverting the pathological phenotype in mutant motor neurons:in fact, TBK1-mutant cells accumulated immature phagophores when treated with 4HPR. This highlighted an impaired elongation phase in autophagy, further confirmed by the down regulation of several genes involved in this specific step of the degradative pathway. Notably, over expression of Atg10, a protein directly involved in the autophagic elongation phase, rescued the detrimental effect exerted by the retinoid. Finally, we demonstrated that 4HPR leads to a strong and faster accumulation of disease markers also in human C9ORF72-mutant motor neurons and pre-synthomatic SOD1G93A mice. Our results bring novel mechanistic insights into the autophagy impairment occurring in ALS, confirming that induction of the degratative pathway worsens the disease progression if the levels of the autophagy interplayers are not properly re-established.

Optineurin: A Coordinator of Membrane-Associated Cargo Trafficking and Autophagy.

Optineurin is a multifunctional adaptor protein intimately involved in various vesicular trafficking pathways. Through interactions with an array of proteins, such as myosin VI, huntingtin, Rab8, and Tank-binding kinase 1, as well as via its oligomerisation, optineurin has the ability to act as an adaptor, scaffold, or signal regulator to coordinate many cellular processes associated with the trafficking of membrane-delivered cargo. Due to its diverse interactions and its distinct functions, optineurin is an essential component in a number of homeostatic pathways, such as protein trafficking and organelle maintenance. Through the binding of polyubiquitinated cargoes via its ubiquitin-binding domain, optineurin also serves as a selective autophagic receptor for the removal of a wide range of substrates. Alternatively, it can act in an ubiquitin-independent manner to mediate the clearance of protein aggregates. Regarding its disease associations, mutations in the optineurin gene are associated with glaucoma and have more recently been found to correlate with Paget’s disease of bone and amyotrophic lateral sclerosis (ALS). Indeed, ALS-associated mutations in optineurin result in defects in neuronal vesicular localisation, autophagosome–lysosome fusion, and secretory pathway function. More recent molecular and functional analysis has shown that it also plays a role in mitophagy, thus linking it to a number of other neurodegenerative conditions, such as Parkinson’s. Here, we review the role of optineurin in intracellular membrane trafficking, with a focus on autophagy, and describe how upstream signalling cascades are critical to its regulation. Current data and contradicting reports would suggest that optineurin is an important and selective autophagy receptor under specific conditions, whereby interplay, synergy, and functional redundancy with other receptors occurs. We will also discuss how dysfunction in optineurin-mediated pathways may lead to perturbation of critical cellular processes, which can drive the pathologies of number of diseases. Therefore, further understanding of optineurin function, its target specificity, and its mechanism of action will be critical in fully delineating its role in human disease.

The flux of iron through ferritin in erythrocyte development.

Terminal differentiation of erythropoietic progenitors requires the rapid accumulation of large amounts of iron, which is transported to the mitochondria, where it is incorporated into heme. Ferritin is the sole site of iron storage present in the cytosol. Yet the role of iron accumulation into ferritin in the context of red cell development had not been clearly defined. Early studies indicated that at the onset of terminal differentiation, iron initially accumulates in ferritin and precedes heme synthesis. Whether this accumulation is physiologically important for red cell development was unclear until recent studies defined an obligatory pathway of iron flux through ferritin. The iron chaperone functions of poly rC-binding protein 1 (PCBP1) and the autophagic cargo receptor for ferritin, nuclear co-activator 4 (NCOA4) are required for the flux of iron through ferritin in developing red cells. In the absence of these functions, iron delivery to mitochondria for heme synthesis is impaired. The regulated trafficking of iron through ferritin is important for maintaining a consistent flow of iron to mitochondria without releasing potentially damaging redox-active species in the cell. Other components of the iron trafficking machinery are likely to be important in red cell development.

SKP2 attenuates NF-κB signaling by mediating IKKβ degradation through autophagy.

NF-κB signaling controls a large set of physiological processes ranging from inflammatory responses to cell death. Its activation is tightly regulated through controlling the activity and stability of multiple signaling components. Here, we identify that NF-κB activation is suppressed by an F-box protein, S-phase kinase associated protein 2 (SKP2). SKP2 deficiency enhanced NF-κB activation as well as the production of inflammatory cytokines. In addition, SKP2 potently blocked the NF-κB activation at the IκB kinase (IKK) level. Mechanistic study further revealed that SKP2 functions as an adaptor to promote an interaction between active IKKβ and the autophagic cargo receptor p62 to mediate IKKβ degradation via selective autophagy. These findings identify a previously unrecognized role of SKP2 in NF-κB activation by which SKP2 acts as a secondary receptor to assist IKKβ delivery to autophagosomes for degradation in a p62-dependent manner.

Characterizing the Spatial Variance of Autophagic Cargo Receptor, OPTN

The mitochondria are required for various cellular processes spanning from cellular energy generation to signal transduction, growth and survival. Not surprisingly, cumulative mitochondrial damage is tightly linked to the development of multiple human diseases including various neurodegenerative diseases such as Parkinson's and Huntington's disease. In order to maintain functional mitochondria, cells have coped with multiple mitochondrial quality control pathways such as mitochondrial Unfolded Protein Response (mt‐UPR), the ubiquitin proteasome system, and mitochondrial autophagy (mitophagy). The most well‐characterized mitophagy is the PINK1‐PARKIN pathway, where damaged mitochondria are selectively marked with ubiquitin chains by the PINK1 kinase and PARKIN ubiquitin E3 ligase, engulfed by autophagosome, and cleared by the lysosome. While we now have a better understanding on this pathway, it is still unclear how autophagic cargo adaptor proteins, such as optineurin (OPTN), get recruited to damaged mitochondria, synthesized or recycled throughout the process. We hypothesized that the availability of OPTN would be the rate‐limiting factor to achieve complete removal of damaged mitochondria and this would require synthesis of new OPTN molecule. To test this hypothesis, we generated a cell line containing a genomic Halo tag at OPTN N‐terminus by utilizing CRISPR/cas9, where differentially fluorescent labeled Halo tag can distinguish older versus newer OPTN populations over time. After successfully generating a CRISPR/Cas9 knock‐in cell line for OPTN, single cell imaging was performed to determine the spatial variance and emergence of OPTN recruited to mitochondria. We were able to conclude that OPTN localizes to a fraction of damaged mitochondria despite the fact that the entire mitochondria are coated with ubiquitin chains, suggesting specificity of this recruitment. Additionally, our preliminary results indicated that newly synthesized OPTN is recruited to mitochondria for the continuation of mitophagy. Together, these observations suggest that OPTN is the rate‐limiting factor and that in order to completely remove all dysfunctional mitochondria, cells need to synthesize new OPTN. In the future, we would like to further explore the underlying mechanism of OPTN's binding specificity and determine what drives this selective recruitment of OPTN to mitochondria.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Age-related pathology after adenoviral overexpression of the leucine-rich repeat kinase 2 in the mouse striatum

Mutations in leucine-rich repeat kinase 2 (LRRK2) age-dependently cause Parkinson's disease and are associated with several inflammatory diseases. So far, the potential role of LRRK2 expression in glial cells as mediators of neuroinflammation and the influence of aging have not been investigated in viral vector-based LRRK2 animal models. In this study, we compared the effect of striatal injection of high-capacity adenoviral vectors expressing either a kinase-overactive LRRK2 with the familial G2019S mutation or a kinase-inactive LRRK2 variant in young and old C57BL/6J mice. The intrinsic adenovirus tropism guided preferentially glial transduction, and the vector design led to stable expression for at least 6 months. In histopathological analysis, young mice expressing either LRRK2 variant presented with transient vacuolization of striatal white fiber tracts accompanied by accumulation of microglial cells and astrogliosis, but inflammation resolved without permanent damage. Old mice had a stronger and prolonged inflammatory reaction and experienced permanent damage in form of partial neuron loss after 3 months exclusively in case of LRRK2_G2019S expression. The autophagic receptor p62 accumulated in cells with high levels of either LRRK2 variant, even more so in old mice. We conclude that the aging mouse brain is more susceptible to LRRK2-associated pathology, and in this model, glial LRRK2 expression significantly contributed to neuroinflammation, ultimately causing neurodegeneration.

LRRC25 inhibits type I IFN signaling by targeting ISG15-associated RIG-I for autophagic degradation.

The RIG-I-like receptors (RLRs) are critical for protection against RNA virus infection, and their activities must be stringently controlled to maintain immune homeostasis. Here, we report that leucine-rich repeat containing protein 25 (LRRC25) is a key negative regulator of RLR-mediated type I interferon (IFN) signaling. Upon RNA virus infection, LRRC25 specifically binds to ISG15-associated RIG-I to promote interaction between RIG-I and the autophagic cargo receptor p62 and to mediate RIG-I degradation via selective autophagy. Depletion of either LRRC25 or ISG15 abrogates RIG-I-p62 interaction as well as the autophagic degradation of RIG-I. Collectively, our findings identify a previously unrecognized role of LRRC25 in type I IFN signaling activation by which LRRC25 acts as a secondary receptor to assist RIG-I delivery to autophagosomes for degradation in a p62-dependent manner.

Ferritin iron regulators, PCBP1 and NCOA4, respond to cellular iron status in developing red cells

Developing red blood cells exhibit multiple, redundant systems for regulating and coordinating the uptake of iron, the synthesis of heme, and the formation of hemoglobin during terminal differentiation. We recently described the roles of poly rC-binding protein (PCBP1) and nuclear coactivator 4 (NCOA4) in mediating the flux of iron through ferritin in developing erythroid cells, with PCBP1, an iron chaperone, delivering iron to ferritin and NCOA4, an autophagic cargo receptor, directing ferritin to the lysosome for degradation and iron release. Ferritin iron flux is critical, as mice lacking these factors develop microcytic anemia. Here we report that these processes are regulated by cellular iron levels in a murine model of ex vivo terminal differentiation. PCBP1 delivers iron to ferritin via a direct protein-protein interaction. This interaction is developmentally regulated, enhanced by iron deprivation, and inhibited by iron excess, both in developing cells and in vitro. NCOA4 activity also exhibited developmental regulation and regulation by cellular iron levels. Excess iron uptake during differentiation triggered lysosomal degradation of NCOA4, which was dependent on the E3 ubiquitin ligase HERC2. Thus, developing red blood cells express a series of proteins that both mediate and regulate the flux of iron to the mitochondria.

N-3 PUFAs induce inflammatory tolerance by formation of KEAP1-containing SQSTM1/p62-bodies and activation of NFE2L2

ABSTRACTInflammation is crucial in the defense against infections but must be tightly controlled to limit detrimental hyperactivation. Our diet influences inflammatory processes and omega-3 polyunsaturated fatty acids (n-3 PUFAs) have known anti-inflammatory effects. The balance of pro- and anti-inflammatory processes is coordinated by macrophages and macroautophagy/autophagy has recently emerged as a cellular process that dampens inflammation. Here we report that the n-3 PUFA docosahexaenoic acid (DHA) transiently induces cytosolic speckles of the autophagic receptor SQSTM1/p62 (sequestosome 1) (described as SQSTM1/p62-bodies) in macrophages. We suggest that the formation of SQSTM1/p62-bodies represents a fast mechanism of NFE2L2/Nrf2 (nuclear factor, erythroid 2 like 2) activation by recruitment of KEAP1 (kelch like ECH associated protein 1). Further, the autophagy receptor TAX1BP1 (Tax1 binding protein 1) and ubiquitin-editing enzyme TNFAIP3/A20 (TNF α induced protein 3) could be identified in DHA-induced SQSTM1/p62-bodies. Simultaneously, DHA strongly dampened the induction of pro-inflammatory genes including CXCL10 (C-X-C motif chemokine ligand 10) and we suggest that formation of SQSTM1/p62-bodies and activation of NFE2L2 leads to tolerance towards selective inflammatory stimuli. Finally, reduced CXCL10 levels were related to the improved clinical outcome in n-3 PUFA-supplemented heart-transplant patients and we propose CXCL10 as a robust marker for the clinical benefits mobilized by n-3 PUFA supplementation.

Modulating cancer cell survival by targeting intracellular cholesterol transport

Background:Demand for cholesterol is high in certain cancers making them potentially sensitive to therapeutic strategies targeting cellular cholesterol homoeostasis. A potential approach involves disruption of intracellular cholesterol transport, which occurs in Niemann–Pick disease as a result of acid sphingomyelinase (ASM) deficiency. Hence, a class of lysosomotropic compounds that were identified as functional ASM inhibitors (FIASMAs) might exhibit chemotherapeutic activity by disrupting cancer cell cholesterol homoeostasis.Methods:Here, the chemotherapeutic utility of ASM inhibition was investigated. The effect of FIASMAs on intracellular cholesterol levels, cholesterol homoeostasis, cellular endocytosis and signalling cascades were investigated. The in vivo efficacy of ASM inhibition was demonstrated using melanoma xenografts and a nanoparticle formulation was developed to overcome dose-limiting CNS-associated side effects of certain FIASMAs.Results:Functional ASM inhibitors inhibited intracellular cholesterol transport leading to disruption of autophagic flux, cellular endocytosis and receptor tyrosine kinase signalling. Consequently, major oncogenic signalling cascades on which cancer cells were reliant for survival were inhibited. Two tested ASM inhibitors, perphenazine and fluphenazine that are also clinically used as antipsychotics, were effective in inhibiting xenografted tumour growth. Nanoliposomal encapsulation of the perphenazine enhanced its chemotherapeutic efficacy while decreasing CNS-associated side effects.Conclusions:This study suggests that disruption of intracellular cholesterol transport by targeting ASM could be utilised as a potential chemotherapeutic approach for treating cancer.