Membrane remodeling and trafficking piloted by SARS-CoV-2.

The molecular mechanisms underlying SARS-CoV-2 host cell invasion and life cycle have been studied extensively in recent years, with a primary focus on viral entry and internalization with the aim of identifying antiviral therapies. By contrast, our understanding of the molecular mechanisms involved in the later steps of the coronavirus life cycle is relatively limited. In this review, we describe what is known about the host factors and viral proteins involved in the replication, assembly, and egress phases of SARS-CoV-2, which induce significant host membrane rearrangements. We also discuss the limits of the current approaches and the knowledge gaps still to be addressed.

Role of trafficking protein particle complex 2 in medaka development.

The skeletal dysplasia spondyloepiphyseal dysplasia tarda (SEDT) is caused by mutations in the TRAPPC2 gene, which encodes Sedlin, a component of the trafficking protein particle (TRAPP) complex that we have shown previously to be required for the export of type II collagen (Col2) from the endoplasmic reticulum. No vertebrate model for SEDT has been generated thus far. To address this gap, we generated a Sedlin knockout animal by mutating the orthologous TRAPPC2 gene (olSedl) of Oryzias latipes (medaka) fish. OlSedl deficiency leads to embryonic defects, short size, diminished skeletal ossification and altered Col2 production and secretion, resembling human defects observed in SEDT patients. Moreover, SEDT knock-out animals display photoreceptor degeneration and gut morphogenesis defects, suggesting a key role for Sedlin in the development of these organs. Thus, by studying Sedlin function in vivo, we provide evidence for a mechanistic link between TRAPPC2-mediated membrane trafficking, Col2 export, and developmental disorders.

Dynamic tandem proximity-based proteomics-Protein trafficking at the proteome-scale.

TransitID is a new methodology based on proximity labeling allowing for the study of protein trafficking a the proteome scale.

Loss of the batten disease protein CLN3 leads to mis-trafficking of M6PR and defective autophagic-lysosomal reformation.

Batten disease, one of the most devastating types of neurodegenerative lysosomal storage disorders, is caused by mutations in CLN3. Here, we show that CLN3 is a vesicular trafficking hub connecting the Golgi and lysosome compartments. Proteomic analysis reveals that CLN3 interacts with several endo-lysosomal trafficking proteins, including the cation-independent mannose 6 phosphate receptor (CI-M6PR), which coordinates the targeting of lysosomal enzymes to lysosomes. CLN3 depletion results in mis-trafficking of CI-M6PR, mis-sorting of lysosomal enzymes, and defective autophagic lysosomal reformation. Conversely, CLN3 overexpression promotes the formation of multiple lysosomal tubules, which are autophagy and CI-M6PR-dependent, generating newly formed proto-lysosomes. Together, our findings reveal that CLN3 functions as a link between the M6P-dependent trafficking of lysosomal enzymes and lysosomal reformation pathway, explaining the global impairment of lysosomal function in Batten disease.

Distinct changes in endosomal composition promote NLRP3 inflammasome activation.

Inflammasome complexes are pivotal in the innate immune response. The NLR family pyrin domain containing protein 3 (NLRP3) inflammasome is activated in response to a broad variety of cellular stressors. However, a primary and converging sensing mechanism by the NLRP3 receptor initiating inflammasome assembly remains ill defined. Here, we demonstrate that NLRP3 inflammasome activators primarily converge on disruption of endoplasmic reticulum-endosome membrane contact sites (EECS). This defect causes endosomal accumulation of phosphatidylinositol 4-phosphate (PI4P) and a consequent impairment of endosome-to-trans-Golgi network trafficking (ETT), necessary steps for endosomal recruitment of NLRP3 and subsequent inflammasome activation. Lowering endosomal PI4P levels prevents endosomal association of NLRP3 and inhibits inflammasome activation. Disruption of EECS or ETT is sufficient to enhance endosomal PI4P levels, to recruit NLRP3 to endosomes and to potentiate NLRP3 inflammasome activation. Mice with defects in ETT in the myeloid compartment are more susceptible to lipopolysaccharide-induced sepsis. Our study thus identifies a distinct cellular mechanism leading to endosomal NLRP3 recruitment and inflammasome activation.

Oculocerebrorenal syndrome of Lowe protein controls cytoskeletal reorganisation during human platelet spreading.

Lowe syndrome (LS) is a rare, X-linked disorder characterised by numerous symptoms affecting the brain, the eyes, and the kidneys. It is caused by mutations in the oculocerebrorenal syndrome of Lowe (OCRL) protein, a 5-phosphatase localised in different cellular compartments that dephosphorylates phosphatidylinositol-4,5-bisphosphate into phosphatidylinositol-4-monophosphate. Some patients with LS also have bleeding disorders, with normal to low platelet (PLT) count and impaired PLT function. However, the mechanism of PLT dysfunction in patients with LS is not completely understood. The main function of PLTs is to activate upon vessel wall injury and stop the bleeding by clot formation. PLT activation is accompanied by a shape change that is a result of massive cytoskeletal rearrangements. Here, we show that OCRL-inhibited human PLTs do not fully spread, form mostly filopodia, and accumulate actin nodules. These nodules co-localise with ARP2/3 subunit p34, vinculin, and sorting nexin 9. Furthermore, OCRL-inhibited PLTs have a retained microtubular coil with high levels of acetylated tubulin. Also, myosin light chain phosphorylation is decreased upon OCRL inhibition, without impaired degranulation or integrin activation. Taken together, these results suggest that OCRL contributes to cytoskeletal rearrangements during PLT activation that could explain mild bleeding problems in patients with LS.

ER exit sites take the strain.

Cells are able to adapt their growth to external mechanical strain. A recent study by Phuyal et al (2022) has shown that these responses depend on the heterodimerization of two small GTPases.

The role of NSP6 in the biogenesis of the SARS-CoV-2 replication organelle.

SARS-CoV-2, like other coronaviruses, builds a membrane-bound replication organelle to enable RNA replication1. The SARS-CoV-2 replication organelle is composed of double-membrane vesicles (DMVs) that are tethered to the endoplasmic reticulum (ER) by thin membrane connectors2, but the viral proteins and the host factors involved remain unknown. Here we identify the viral non-structural proteins (NSPs) that generate the SARS-CoV-2 replication organelle. NSP3 and NSP4 generate the DMVs, whereas NSP6, through oligomerization and an amphipathic helix, zippers ER membranes and establishes the connectors. The NSP6(ΔSGF) mutant, which arose independently in the Alpha, Beta, Gamma, Eta, Iota and Lambda variants of SARS-CoV-2, behaves as a gain-of-function mutant with a higher ER-zippering activity. We identified three main roles for NSP6: first, to act as a filter in communication between the replication organelle and the ER, by allowing lipid flow but restricting the access of ER luminal proteins to the DMVs; second, to position and organize DMV clusters; and third, to mediate contact with lipid droplets (LDs) through the LD-tethering complex DFCP1-RAB18. NSP6 thus acts as an organizer of DMV clusters and can provide a selective means of refurbishing them with LD-derived lipids. Notably, both properly formed NSP6 connectors and LDs are required for the replication of SARS-CoV-2. Our findings provide insight into the biological activity of NSP6 of SARS-CoV-2 and of other coronaviruses, and have the potential to fuel the search for broad antiviral agents.

Drug Repurposing in Rare Diseases: An Integrative Study of Drug Screening and Transcriptomic Analysis in Nephropathic Cystinosis.

Diagnosis and cure for rare diseases represent a great challenge for the scientific community who often comes up against the complexity and heterogeneity of clinical picture associated to a high cost and time-consuming drug development processes. Here we show a drug repurposing strategy applied to nephropathic cystinosis, a rare inherited disorder belonging to the lysosomal storage diseases. This approach consists in combining mechanism-based and cell-based screenings, coupled with an affordable computational analysis, which could result very useful to predict therapeutic responses at both molecular and system levels. Then, we identified potential drugs and metabolic pathways relevant for the pathophysiology of nephropathic cystinosis by comparing gene-expression signature of drugs that share common mechanisms of action or that involve similar pathways with the disease gene-expression signature achieved with RNA-seq.

Correction of oxidative stress enhances enzyme replacement therapy in Pompe disease.

Pompe disease is a metabolic myopathy due to acid alpha-glucosidase deficiency. In addition to glycogen storage, secondary dysregulation of cellular functions, such as autophagy and oxidative stress, contributes to the disease pathophysiology. We have tested whether oxidative stress impacts on enzyme replacement therapy with recombinant human alpha-glucosidase (rhGAA), currently the standard of care for Pompe disease patients, and whether correction of oxidative stress may be beneficial for rhGAA therapy. We found elevated oxidative stress levels in tissues from the Pompe disease murine model and in patients' cells. In cells, stress levels inversely correlated with the ability of rhGAA to correct the enzymatic deficiency. Antioxidants (N-acetylcysteine, idebenone, resveratrol, edaravone) improved alpha-glucosidase activity in rhGAA-treated cells, enhanced enzyme processing, and improved mannose-6-phosphate receptor localization. When co-administered with rhGAA, antioxidants improved alpha-glucosidase activity in tissues from the Pompe disease mouse model. These results indicate that oxidative stress impacts on the efficacy of enzyme replacement therapy in Pompe disease and that manipulation of secondary abnormalities may represent a strategy to improve the efficacy of therapies for this disorder.

RNA, a new member in the glycan-club that gets exposed at the cell surface.

In this article we discuss implications of the recent discovery of glycoRNAs found to be present at the cell surface of mammalian cells which was reported by Flynn et al. Cell 2021.

Repurposing of tamoxifen ameliorates CLN3 and CLN7 disease phenotype.

Batten diseases (BDs) are a group of lysosomal storage disorders characterized by seizure, visual loss, and cognitive and motor deterioration. We discovered increased levels of globotriaosylceramide (Gb3) in cellular and murine models of CLN3 and CLN7 diseases and used fluorescent-conjugated bacterial toxins to label Gb3 to develop a cell-based high content imaging (HCI) screening assay for the repurposing of FDA-approved compounds able to reduce this accumulation within BD cells. We found that tamoxifen reduced the lysosomal accumulation of Gb3 in CLN3 and CLN7 cell models, including neuronal progenitor cells (NPCs) from CLN7 patient-derived induced pluripotent stem cells (iPSC). Here, tamoxifen exerts its action through a mechanism that involves activation of the transcription factor EB (TFEB), a master gene of lysosomal function and autophagy. In vivo administration of tamoxifen to the CLN7Δex2 mouse model reduced the accumulation of Gb3 and SCMAS, decreased neuroinflammation, and improved motor coordination. These data strongly suggest that tamoxifen may be a suitable drug to treat some types of Batten disease.

Regulation and physiology of membrane contact sites.

Membrane contact sites (MCSs) in addition to impacting the functions of membrane-limited organelles also have a role in the spatial and functional organization of cells, tissues and whole organisms. MCSs have been identified between all organelles and the identification of their molecular composition has progressed significantly in recent years. Equally important is how MCSs respond dynamically to physiological stimuli, how this is regulated, and the physiological roles of MCSs in tissues and at the organismal level, an area that still remains relatively unexplored. In the present review, we focus on the regulation of MCSs, considerations of their function at the organismal level, and how mutations of MCS components linked to genetic diseases might inform us about their physiological relevance.

Deregulation of phosphatidylinositol-4-phosphate in the development of amyotrophic lateral sclerosis 8.

Amyotrophic lateral sclerosis 8 (ALS8) is one of a heterogeneous group of progressive neurodegenerative disorders characterized by the death of motor neurons. ALS8 is caused by mutations in VAPB, a protein that acts at multiple membrane contact sites between the endoplasmic reticulum (ER) and almost all other organelles and thus affects functions at diverse cellular locations. One prominent function mediated by VAPB at these sites is lipid exchange, and a recurrent phenotype observed in all models investigating knockout or knockdown of VAPs is a significant increase in the levels of phosphatidylinositol-4-phosphate (PI4P). Here we consider the relevance of this PI4P deregulation in the development of ALS8 that might represent a potential target for therapeutic intervention.

Memory Deficits in Children with Developmental Dyslexia: A Reading-Level and Chronological-Age Matched Design.

Developmental Dyslexia (DD) is considered a multifactorial deficit. Among the neurocognitive impairments identified in DD, it has been found that memory plays a particularly important role in reading and learning. The present study aims to investigate whether short-term memory (STM) and long-term memory (LTM) deficits could be related to poor reading experience or could be causal factors in DD. To verify that memory deficits in DD did not simply reflect differences in reading experience, 16 children with DD were not only compared to 16 chronological age-matched children (CA) but also to 16 reading level-matched children (RL) in verbal, visual-object, and visual-spatial STM and LTM tasks. Children with DD performed as well as RL, but worse than CA in all STM tasks. Considering LTM, the three groups did not differ in Visual-Object and Visual-Spatial Learning tasks. In the Verbal LTM task, DD recalled significantly fewer words than CA but not RL, while CA and RL showed a similar performance. The present results suggest that when reading experience was equated, children with DD and typical readers did not differ in STM and LTM, especially in the verbal modality, weakening claims that memory has a causal effect in reading impairments.

Homicide and concealment of the corpse. Autopsy case series and review of the literature.

INTRODUCTION: The concealment of the body following a homicide undermines different moments of the forensic and medico-legal investigations. The aim of the present study is to provide an overview of the literature and the forensic casuistry of the Institute of Legal Medicine of Padova for analyzing and discussing diverse methodological approaches for the forensic pathologist dealing with covered-up homicides. MATERIAL AND METHODS: A literature review, updated until September 2019, was performed, and a literature pool of forensic cases was built. In-house cases were included by conducting a retrospective analysis of the forensic caseworks of Padova of the last 20 years. Data regarding epidemiology, methodology of assessment, methods of concealment, and answers to medico-legal issues were extracted for both data sets. RESULTS AND DISCUSSION: Seventy-eight papers were included in the literature review (78.2% being case reports or case series, 17% retrospective studies, and 6% experimental studies or reviews). Literature and in-house data sets consisted of 145 and 13 cases, respectively. Death scene investigation, radiology, toxicology, and additional analyses were performed in 20-54% of literature and 62-77% of in-house cases. Cover-up by multiple methods prevailed. Death was caused by head trauma in about 40% of cases (both data sets), strangulation in 21% of literature, and 7% of in-house cases, and was undetermined in 17% of literature and 7% of in-house cases. CONCLUSIONS: The methodology of ascertainment should be case-specific and based on a multidisciplinary and multimodal evaluation of all data, including those gained through novel radiological and/or analytical techniques.