Abstract 4: Macrophage p62/SQSTM1 Ameliorates Atherosclerosis by Sequestering Inclusion Bodies and Mediating Mitophagy

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Ismail Sergin ◽  
Somashubhra Bhattacharya ◽  
Carl J Stokes ◽  
John A Curci ◽  
Babak Razani
Keyword(s):  
Inclusion Bodies ◽  
Cytoplasmic Inclusion ◽  
Protein Aggregates ◽  
Whole Body ◽  
Plaque Burden ◽  
Protein Accumulation ◽  
Inflammasome Activation ◽  
Cytoplasmic Inclusions ◽  
Cytoplasmic Inclusion Bodies

Protein and organelle turnover is critical for cellular homeostasis and is prominently mediated by autophagy. Disruptions in autophagy lead to accumulation of protein aggregates and dysfunctional organelles such as mitochondria. Recent evidence suggests that the chaperone protein p62 is a critical link for targeting polyubiquitinated protein aggregates/damaged mitochondria to autophagosomes for degradation. Herein we describe a p62-centric mechanism of handling protein aggregates and dysfunctional mitochondria in atherosclerosis. Macrophages deficient in autophagy (ATG5-/-) or rendered deficient by incubation with atherogenic lipids have significantly increased levels of p62. This coincides with 1) the accumulation of polyubiquitinated proteins co-localizing with p62 and present as cytoplasmic inclusion bodies, and 2) p62 co-localization with mitochondrial markers. Aortas from atherosclerotic (ApoE-/-) mice also have progressive and marked elevations in p62, polyubiquitinated proteins, and mitochondrial reactive oxygen species that predominantly co-localize with plaque macrophages, a process further exacerbated in the autophagy-deficient setting. The formation of cytoplasmic inclusions and maintenance of adequate mitochondrial function appears to be dependent on p62. Lipid-loaded p62-null macrophages show polyubiquitinated protein accumulation present in a diffuse/disrupted cytoplasmic pattern. These macrophages also develop larger dysmorphic mitochondria with increased polarization and decreased oxidative phosphorylation capacity. As a result, p62-null macrophages display apoptotic susceptibility to atherogenic lipids and increased IL-1β secretion likely through mitochondrial-dependent inflammasome activation. Consistent with our in vitro observations, mice with either whole-body p62-deficiency or transplanted with p62-deficient bone marrow show significantly increased atherosclerotic plaque burden and lesion complexity with increased apoptosis and necrotic cores. Taken together, these data demonstrate a previously unrecognized atheroprotective role for macrophage p62 by facilitating the formation of inclusion bodies and maintaining healthy mitochondria.

scholarly journals Minimal Elements Required for the Formation of Respiratory Syncytial Virus Cytoplasmic Inclusion Bodies In Vivo and In Vitro

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Marie Galloux ◽  
Jennifer Risso-Ballester ◽  
Charles-Adrien Richard ◽  
Jenna Fix ◽  
Marie-Anne Rameix-Welti ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Liquid Phase ◽  
Inclusion Bodies ◽  
Cytoplasmic Inclusion ◽  
Cytoplasmic Inclusion Bodies ◽  
Syncytial Virus ◽  
P Proteins ◽  
Nucleoprotein N ◽  
Liquid Liquid Phase Separation

ABSTRACT Infection of host cells by the respiratory syncytial virus (RSV) is characterized by the formation of spherical cytoplasmic inclusion bodies (IBs). These structures, which concentrate all the proteins of the polymerase complex as well as some cellular proteins, were initially considered aggresomes formed by viral dead-end products. However, recent studies revealed that IBs are viral factories where viral RNA synthesis, i.e., replication and transcription, occurs. The analysis of IBs by electron microscopy revealed that they are membrane-less structures, and accumulated data on their structure, organization, and kinetics of formation revealed that IBs share the characteristics of cellular organelles, such as P-bodies or stress granules, suggesting that their morphogenesis depends on a liquid-liquid phase separation mechanism. It was previously shown that expression of the RSV nucleoprotein N and phosphoprotein P of the polymerase complex is sufficient to induce the formation of pseudo-IBs. Here, using a series of truncated P proteins, we identified the domains of P required for IB formation and show that the oligomeric state of N, provided it can interact with RNA, is critical for their morphogenesis. We also show that pseudo-IBs can form in vitro when recombinant N and P proteins are mixed. Finally, using fluorescence recovery after photobleaching approaches, we reveal that in cellula and in vitro IBs are liquid organelles. Our results strongly support the liquid-liquid phase separation nature of IBs and pave the way for further characterization of their dynamics. IMPORTANCE Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants, elderly, and immunocompromised people. No vaccine or efficient antiviral treatment is available against this virus. The replication and transcription steps of the viral genome are appealing mechanisms to target for the development of new antiviral strategies. These activities take place within cytoplasmic inclusion bodies (IBs) that assemble during infection. Although expression of both the viral nucleoprotein (N) and phosphoprotein (P) allows induction of the formation of these IBs, the mechanism sustaining their assembly remains poorly characterized. Here, we identified key elements of N and P required for the scaffolding of IBs and managed for the first time to reconstitute RSV pseudo-IBs in vitro by coincubating recombinant N and P proteins. Our results provide strong evidence that the biogenesis of RSV IBs occurs through liquid-liquid phase transition mediated by N-P interactions.

scholarly journals Morphogenesis of Light and Electron Microscopic Lesions in Porcine GM2-Gangliosidosis

1979 ◽  
Vol 16 (1) ◽  
pp. 6-17 ◽  
Author(s):  
S. D. Kosanke ◽  
K. R. Pierce ◽  
W. K. Read
Keyword(s):  
Glial Cells ◽  
Inclusion Bodies ◽  
Cytoplasmic Inclusion ◽  
Particulate Material ◽  
Electron Microscopic ◽  
Cytoplasmic Body ◽  
Cytoplasmic Inclusions ◽  
Neuronal Inclusions ◽  
Cytoplasmic Inclusion Bodies ◽  
Cytoplasmic Structures

The neurons and glial cells of 1- to 140-day-old pigs with GM2-gangliosidosis had membranous cytoplasmic inclusion bodies. These bodies appeared as small vacuolated cytoplasmic structures in paraffin-embedded, hematoxylin and eosin-stained sections and as solid, dark, round granules in 1-micrometer sections embedded in plastic and stained with toluidine blue. Ultrastructurally, the cytoplasmic inclusion bodies appeared as round, dense structures from 0.6 to 1.2 micrometers in diameter, that were filled with various amounts of small to large arrays of membranous lamellae. The cortical neuronal inclusions were seen initially as lysosomes containing a small amount of particulate material. The appearance of these inclusions changed as they progressed through different configurational stages. Inclusions resembled the granulomembranous body, the zebra body, possibly other intermediate forms and, finally, the classical membranous cytoplasmic body. The cytoplasmic inclusions in glial cells resembled membranovesicular bodies and, although also of apparent lysosomal origin, were morphologically different from the neuronal inclusions. The morphologic lesions in the neurons and glial cells of the affected pigs were similar to those described for human gangliosidoses.

Abstract 4: Enhancing the Ability of Macrophages to Orchestrate Selective Autophagy and Lysosomal Biogenesis Protects Against Atherosclerosis

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Ismail Sergin ◽  
Somashubhra Bhattacharya ◽  
Xiangyu Zhang ◽  
Trent D Evans ◽  
Babak Dehestani ◽  
...  
Keyword(s):  
Therapeutic Strategy ◽  
Membrane Integrity ◽  
Protein Aggregates ◽  
Plaque Burden ◽  
Inflammasome Activation ◽  
Lysosomal Degradation ◽  
Cytoplasmic Inclusions ◽  
Selective Autophagy ◽  
Mechanistic Basis

The autophagy-lysosome system is a catabolic cellular mechanism that degrades dysfunctional proteins and organelles. The pro-atherogenic phenotype of mice with macrophage-specific autophagy deficiency (ATG5-/-) confirm the importance of this degradation system in the pathogenesis of atherosclerosis. The mechanistic basis appears to involve a two-step process in macrophages: the development of lysosomal dysfunction/membrane integrity by atherogenic lipids followed by an inability of lysosomes to handle and degrade cargo supplied by autophagy. A prominent sequelae of such blockage is the accumulation of cytoplasmic inclusions composed of polyubiquitinated protein aggregates and organelles which are normally targeted for selective autophagy by the protein chaperone p62. In order to stimulate the degradative capacity of macrophages, we developed mice with macrophage-specific overexpression of TFEB, a master transcriptional activator of both autophagy and lysosomal biogenesis. Macrophage TFEB ameliorated several deleterious effects of atherogenic lipids, namely the blunting of inflammasome activation, enhancing cholesterol efflux, accelerating the degradation of protein aggregates, and decreasing apoptosis. In vivo, macrophage TFEB overexpression reduced both plaque burden and plaque complexity in pro-atherogenic ApoE-/- mice fed a Western diet. Interestingly, TFEB’s atheroprotective effects were not only abrogated in the background of macrophage autophagy deficiency (ATG5-/-) but also in the background of p62-deficiency (p62-/-) suggesting the critical importance of selective autophagy and degradation of p62-enriched protein aggregates. Taken together, these data support the induction of a holistic pro-degradative response in macrophages (i.e. selective autophagy followed by lysosomal degradation) as a viable therapeutic strategy in atherosclerosis.

scholarly journals Targeting lysosome function causes selective cytotoxicity in VHL-inactivated renal cell carcinomas

2019 ◽  
Vol 41 (6) ◽  
pp. 828-840 ◽  
Author(s):  
Nadia Bouhamdani ◽  
Dominique Comeau ◽  
Alexandre Coholan ◽  
Kevin Cormier ◽  
Sandra Turcotte
Keyword(s):  
Renal Cell ◽  
Cytoplasmic Inclusion ◽  
Bioactive Compound ◽  
Suppressor Gene ◽  
Primary Tumors ◽  
Von Hippel Lindau ◽  
Chromosome 3P ◽  
Open Platform ◽  
Cytoplasmic Inclusion Bodies

Abstract The inactivation of the tumor suppressor gene, von Hippel-Lindau (VHL), has been identified as the earliest event in renal cell carcinoma (RCC) development. The loss of heterogeneity by chromosome 3p deletion followed by inactivating mutations on the second VHL copy are events present in close to 90% of patients. Our study illustrates a lysosomal vulnerability in VHL-inactivated RCC in vitro. By investigating the mechanism of action of the previously identified STF-62247, a small bioactive compound known for its selective cytotoxic properties towards VHL-defective models, we present the promising approach of targeting truncal-driven VHL inactivation through lysosome disruption. Furthermore, by analyzing the open platform for exploring cancer genomic data (cbioportal), we uncover the high alteration frequency of essential lysosomal and autophagic genes in sequenced biopsies from clear cell RCC patient primary tumors. By investigating lysosome physiology, we also identify VHL-inactivated cells’ inability to maintain their lysosomes at the perinuclear localization in response to STF-62247-induced stress and accumulate cytoplasmic inclusion bodies in response to an inefficient lysosomal degradative capacity. Finally, by testing other known lysosomal-disrupting agents (LDAs), we show that these are selectively cytotoxic to cells lacking VHL functions. Our study builds a strong platform that could specifically link genetic clonal ccRCC evolution to lysosomal and trafficking vulnerabilities.

scholarly journals Searching for the cause of Kawasaki disease — cytoplasmic inclusion bodies provide new insight

2008 ◽  
Vol 6 (5) ◽  
pp. 394-401 ◽  
Author(s):  
Anne H. Rowley ◽  
Susan C. Baker ◽  
Jan M. Orenstein ◽  
Stanford T. Shulman
Keyword(s):  
Kawasaki Disease ◽  
Inclusion Bodies ◽  
Cytoplasmic Inclusion ◽  
Cytoplasmic Inclusion Bodies
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