TDP-43 Pathology in Alzheimer’s Disease

Transactive response DNA binding protein of 43 kDa (TDP-43) is an intranuclear protein encoded by the TARDBP gene that is involved in RNA splicing, trafficking, stabilization, and thus, the regulation of gene expression. Cytoplasmic inclusion bodies containing phosphorylated and truncated forms of TDP-43 are hallmarks of amyotrophic lateral sclerosis (ALS) and a subset of frontotemporal lobar degeneration (FTLD). Additionally, TDP-43 inclusions have been found in up to 57% of Alzheimer’s disease (AD) cases, most often in a limbic distribution, with or without hippocampal sclerosis. In some cases, TDP-43 deposits are also found in neurons with neurofibrillary tangles. AD patients with TDP-43 pathology have increased severity of cognitive impairment compared to those without TDP-43 pathology. Furthermore, the most common genetic risk factor for AD, apolipoprotein E4 (APOE4), is associated with increased frequency of TDP-43 pathology. These findings provide strong evidence that TDP-43 pathology is an integral part of multiple neurodegenerative conditions, including AD. Here, we review the biology and pathobiology of TDP-43 with a focus on its role in AD. We emphasize the need for studies on the mechanisms that lead to TDP-43 pathology, especially in the setting of age-related disorders such as AD.

A subpopulation of arenavirus nucleoprotein localizes to mitochondria

Viruses need cells for their replication and, therefore, ways to hijack cellular functions. Mitochondria play fundamental roles within the cell in metabolism, immunity and regulation of homeostasis due to which some viruses aim to alter mitochondrial functions. Herein we show that the nucleoprotein (NP) of arenaviruses enters the mitochondria of infected cells, affecting the mitochondrial morphology. Reptarenaviruses cause boid inclusion body disease (BIBD) that is characterized, especially in boas, by the formation of cytoplasmic inclusion bodies (IBs) comprising reptarenavirus NP within the infected cells. We initiated this study after observing electron-dense material reminiscent of IBs within the mitochondria of reptarenavirus infected boid cell cultures in an ultrastructural study. We employed immuno-electron microscopy to confirm that the mitochondrial inclusions indeed contain reptarenavirus NP. Mutations to a putative N-terminal mitochondrial targeting signal (MTS), identified via software predictions in both mamm- and reptarenavirus NPs, did not affect the mitochondrial localization of NP, suggesting that it occurs independently of MTS. In support of MTS-independent translocation, we did not detect cleavage of the putative MTSs of arenavirus NPs in reptilian or mammalian cells. Furthermore, in vitro translated NPs could not enter isolated mitochondria, suggesting that the translocation requires cellular factors or conditions. Our findings suggest that MTS-independent mitochondrial translocation of NP is a shared feature among arenaviruses. We speculate that by targeting the mitochondria arenaviruses aim to alter mitochondrial metabolism and homeostasis or affect the cellular defense.

Characterization of the interaction domains between the phosphoprotein and the nucleocapsid of human Metapneumovirus

Human metapneumovirus (HMPV) causes severe respiratory diseases in young children. The HMPV RNA genome is encapsidated by the viral nucleoprotein, forming an RNA-N complex (NNuc), which serves as template for genome replication and mRNA transcription by the RNA-dependent RNA polymerase (RdRp). The RdRp is formed by the association of the large polymerase subunit (L), which has RNA polymerase, capping and methyltransferase activities, and the tetrameric phosphoprotein (P). P plays a central role in the RdRp complex by binding to NNuc and L, allowing the attachment of the L polymerase to the nucleocapsid template. During infection these proteins concentrate in cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. By analogy to the closely related pneumovirus respiratory syncytial virus (RSV), it is likely that the formation of IBs depends on the interaction between P and NNuc. However, the HMPV P-NNuc interaction still remains to characterize. Here, we finely characterized the binding domains involved in HMPV P and NNuc interaction by studying binding between recombinant proteins, combined with the use of a functional assay of the polymerase complex activity and the study of the recruitment of these proteins to IBs by immunofluorescence. We show that the last 6 C-terminal residues of HMPV P are necessary and sufficient for binding to NNuc, that P binds the N-terminal domain of N (NNTD), and identified conserved N residues critical for the interaction. Our results allowed to propose a structural model of the HMPV P-NNuc interaction.

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

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.

Supplementary data on virus-like particles in the brainstem of Parkinson’s disease patients and controls

In this study, we present 84 transmission electron microscopy (TEM) images of human brainstem tissue from 11 cases of late onset Parkinson’s disease (PD). The tissues were fixed, embedded, sectioned, and stained for TEM application. In addition, we present 14 images from autopsy specimens of 1 case of human poliomyelitis infection as positive controls and 14 images from 8 cases of autopsy specimens of other conditions as negative controls. In the TEM images of the PD cases there were cytoplasmic inclusion bodies consisting of virus-like particles (VLP) 30 nm in diameter that were associated with endoplasmic reticulum membranes. In the nuclei of the PD neurons there were VLP ranging from 40 nm to 50 nm in diameter. In the poliomyelitis cases, similar particles as were observed in PD which were interpreted to be poliomyelitis virus particles. In the negative controls one case was identified which showed similar VLP (Figure 1, controls). A Lewy body was found in this “control” case (Figure 10) suggesting that this was an undiagnosed case of PD. Cytoplasmic ribosomes measuring approximately 17 nm were observed in the control neurons.

Peste Des Petits Ruminants Virus and Goatpox Virus Co-Infection in Goats

Infection of small ruminants with peste des petits ruminants virus (PPRV) and goatpox virus (GTPV) are endemic and can have devastating economic consequences in Asia and Africa. Co-infection with these viruses have recently been reported in goats and sheep in Nigeria. In this study, we evaluated samples from the lips of a red Sokoto goat, and describe co-infection of keratinocytes with PPRV and GTPV using histopathology and transmission electron microscopy. Eosinophilic cytoplasmic inclusion bodies were identified histologically, and ultrastructural analysis revealed numerous large cytoplasmic viral factories containing poxvirus particles and varying sizes of smaller cytoplasmic inclusions composed of PPRV nucleocapsids. These histopathological and ultrastructural findings show concurrent infection with the 2 viruses for the first time as well as the detection of PPRV particles in epithelial cells of the mucocutaneous junction of the lip.

Localization of immunoreactive, dystonia-associated protein torsinA near the Golgi apparatus of cultured rodent astrocytes

ABSTRACTAn in-frame deletion of a single glutamic acid codon in the TOR1A gene causes the neurological disorder DYT1 dystonia, but the cellular pathophysiology of this disorder remains elusive. A current model postulates that the wild-type (WT) torsinA protein is mainly localized to the endoplasmic reticulum (ER), but that the mutant form (ΔE-torsinA) is diverted to the nuclear envelope and cytoplasmic inclusion bodies. This mis-localization has been observed by overexpressing the proteins in neuronal and non-neuronal cells. However, it is not clear whether this model is valid for the astrocytic glial cells that support and modify neuronal functions. Here we report, using rodent astrocytes in primary culture, that the overexpressed torsinA proteins were distributed as predicted by the mis-localization model. We also found by immunocytochemistry that the cultured astrocytes express torsinA endogenously. Most of the signals from endogenous protein, whether the WT or ΔE form, were localized near a cis-Golgi marker GM130. Such localization of endogenous proteins was found in glial cells from several sources: the hippocampus of WT rats, the hippocampus and striatum of WT mice, and the hippocampus and striatum of ΔE-torsinA knock-in mice, a model of DYT1 dystonia. These results show that the mis-localization model is applicable to overexpressed torsinA proteins, but not applicable to those expressed at endogenous levels, at least in cultured rodent astrocytes. These discrepancies in the distribution of overexpressed versus endogenous torsinA proteins highlight the potential need for caution in interpreting the results of overexpression studies.

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

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.

Rotavirus Induces Formation of Remodeled Stress Granules and P Bodies and Their Sequestration in Viroplasms To Promote Progeny Virus Production

ABSTRACTRotavirus replicates in unique virus-induced cytoplasmic inclusion bodies called viroplasms (VMs), the composition and structure of which have yet to be understood. Based on the analysis of a few proteins, earlier studies reported that rotavirus infection inhibits stress granule (SG) formation and disrupts P bodies (PBs). However, the recent demonstration that rotavirus infection induces cytoplasmic relocalization and colocalization with VMs of several nuclear hnRNPs and AU-rich element-binding proteins (ARE-BPs), which are known components of SGs and PBs, suggested the possibility of rotavirus-induced remodeling of SGs and PBs, prompting us to analyze a large number of the SG and PB components to understand the status of SGs and PBs in rotavirus-infected cells. Here we demonstrate that rotavirus infection induces molecular triage by selective exclusion of a few proteins of SGs (G3BP1 and ZBP1) and PBs (DDX6, EDC4, and Pan3) and sequestration of the remodeled/atypical cellular organelles, containing the majority of their components, in the VM. The punctate SG and PB structures are seen at about 4 h postinfection (hpi), coinciding with the appearance of small VMs, many of which fuse to form mature large VMs with progression of infection. By use of small interfering RNA (siRNA)-mediated knockdown and/or ectopic overexpression, the majority of the SG and PB components, except for ADAR1, were observed to inhibit viral protein expression and virus growth. In conclusion, this study demonstrates that VMs are highly complex supramolecular structures and that rotavirus employs a novel strategy of sequestration in the VM and harnessing of the remodeled cellular RNA recycling bins to promote its growth.IMPORTANCERotavirus is known to replicate in specialized virus-induced cytoplasmic inclusion bodies called viroplasms (VMs), but the composition and structure of VMs are not yet understood. Here we demonstrate that rotavirus interferes with normal SG and PB assembly but promotes formation of atypical SG-PB structures by selective exclusion of a few components and employs a novel strategy of sequestration of the remodeled SG-PB granules in the VMs to promote virus growth by modulating their negative influence on virus infection. Rotavirus VMs appear to be complex supramolecular structures formed by the union of the triad of viral replication complexes and remodeled SGs and PBs, as well as other host factors, and designed to promote productive virus infection. These observations have implications for the planning of future research with the aim of understanding the structure of the VM, the mechanism of morphogenesis of the virus, and the detailed roles of host proteins in rotavirus biology.