Formation, function, and pathology of RNP granules.

Ribonucleoprotein (RNP) granules are diverse membrane-less organelles that form through multivalent RNA-RNA, RNA-protein, and protein-protein interactions between RNPs. RNP granules are implicated in many aspects of RNA physiology, but in most cases their functions are poorly understood. RNP granules can be described through four key principles. First, RNP granules often arise because of the large size, high localized concentrations, and multivalent interactions of RNPs. Second, cells regulate RNP granule formation by multiple mechanisms including posttranslational modifications, protein chaperones, and RNA chaperones. Third, RNP granules impact cell physiology in multiple manners. Finally, dysregulation of RNP granules contributes to human diseases. Outstanding issues in the field remain, including determining the scale and molecular mechanisms of RNP granule function and how granule dysfunction contributes to human disease.

Spatiotemporal Proteomic Analysis of Stress Granule Disassembly Using APEX Reveals Regulation by SUMOylation and Links to ALS Pathogenesis.

Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly.

Translational Repression of G3BP in Cancer and Germ Cells Suppresses Stress Granules and Enhances Stress Tolerance.

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.

ZFAND1 Recruits p97 and the 26S Proteasome to Promote the Clearance of Arsenite-Induced Stress Granules.

Stress granules (SGs) are cytoplasmic assemblies of mRNPs stalled in translation initiation. They are induced by various stress conditions, including exposure to the environmental toxin and carcinogen arsenic. While perturbed SG turnover is linked to the pathogenesis of neurodegenerative diseases, the molecular mechanisms underlying SG formation and turnover are still poorly understood. Here, we show that ZFAND1 is an evolutionarily conserved regulator of SG clearance. ZFAND1 interacts with two key factors of protein degradation, the 26S proteasome and the ubiquitin-selective segregase p97, and recruits them to arsenite-induced SGs. In the absence of ZFAND1, SGs lack the 26S proteasome and p97, accumulate defective ribosomal products, and persist after arsenite removal, indicating their transformation into aberrant, disease-linked SGs. Accordingly, ZFAND1 depletion is epistatic to the expression of pathogenic mutant p97 with respect to SG clearance, suggesting that ZFAND1 function is relevant to the multisystem degenerative disorder IBMPFD/ALS.

Stress Granules in Cancer.

The capacity of cells to organize complex biochemical reactions in intracellular space is a fundamental organizational principle of life. Key to this organization is the compartmentalization of the cytoplasm into distinct organelles, which is frequently achieved through intracellular membranes. Recent evidence, however, has added a new layer of flexibility to cellular compartmentalization. As such, in response to specific stimuli, liquid-liquid phase separations can lead to the rapid rearrangements of the cytoplasm to form membraneless organelles. Stress granules (SGs) are one such type of organelle that form specifically when cells are faced with stress stimuli, to aid cells in coping with stress. Inherently, altered SG formation has been linked to the pathogenesis of diseases associated with stress and inflammatory conditions, including cancer. Exciting discoveries have indicated an intimate link between SGs and tumorigenesis. Several pro-tumorigenic signaling molecules including the RAS oncogene, mTOR, and histone deacetylase 6 (HDAC6) have been shown to upregulate SG formation. Based on these studies, SGs have emerged as structures that can integrate oncogenic signaling and tumor-associated stress stimuli to enhance cancer cell fitness. In addition, growing evidence over the past decade suggests that SGs function not only to regulate the switch between survival and cell death, but also contribute to cancer cell proliferation, invasion, metastasis, and drug resistance. Although much remains to be learned about the role of SGs in tumorigenesis, these studies highlight SGs as a key regulatory hub in cancer and a promising therapeutic target.

Phase Separation of C9orf72 Dipeptide Repeats Perturbs Stress Granule Dynamics.

Liquid-liquid phase separation (LLPS) of RNA-binding proteins plays an important role in the formation of multiple membrane-less organelles involved in RNA metabolism, including stress granules. Defects in stress granule homeostasis constitute a cornerstone of ALS/FTLD pathogenesis. Polar residues (tyrosine and glutamine) have been previously demonstrated to be critical for phase separation of ALS-linked stress granule proteins. We now identify an active role for arginine-rich domains in these phase separations. Moreover, arginine-rich dipeptide repeats (DPRs) derived from C9orf72 hexanucleotide repeat expansions similarly undergo LLPS and induce phase separation of a large set of proteins involved in RNA and stress granule metabolism. Expression of arginine-rich DPRs in cells induced spontaneous stress granule assembly that required both eIF2α phosphorylation and G3BP. Together with recent reports showing that DPRs affect nucleocytoplasmic transport, our results point to an important role for arginine-rich DPRs in the pathogenesis of C9orf72 ALS/FTLD.

Rare Pseudo-Chediak-Higashi Inclusions in Therapy-Related Acute Myeloid Leukemia with Myelodysplasia-Related Changes.

BACKGROUND: Defined as rare large azurophilic cytoplasmic inclusions, Pseudo-Chediak-Higashi granules mimic those in granulocytes cytoplasm of Chediak-Higashi syndrome. Rare cases of hematopoietic and lymphoid tissues tumors showed Pseudo-Chediak-Higashi inclusions in cytoplasm, some of which presented with unusual morphological characteristics. METHODS: Herein, we report the first case, in which rare pseudo-Chediak-Higashi inclusions were observed in therapy-related acute myeloid leukemia with myelodysplasia-related changes (t-AML-MRC). RESULTS: The rare pseudo-Chediak-Higashi inclusions may be positive for Sudan black, and some scholars think that these rare inclusions are a kind of dysgranulopoiesis. CONCLUSIONS: The case highlights the significance of an integrated diagnostic work-up, with an interesting effect for morphology.

Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.

Eosinophil exocytosis in a poorly differentiated tubular gastric adenocarcinoma: case report.

A case of poorly differentiated tubular gastric adenocarcinoma with tumor-associated tissue eosinophilia (TATE) is studied by light and electron microscopy, focusing on membrane interactions between eosinophils and tumor cells. 29.2% of the eosinophils in contact with tumor cells showed intact granules, 28.3% exhibited piecemeal degranulation (PMD), 40% were characterized by coexistence of PMD and compound exocytosis in the same granulocyte, whereas classical exocytosis was found in 2.5% of the eosinophils with PMD. Eosinophil Sombrero Vesicles (EoSVs), important tubulovesicular carriers for delivery of cytotoxic proteins from the specific granules during PMD, were also studied at the ultrastructural level. In activated eosinophils, EoSVs and specific granules with ultrastructural signs of degranulation were polarized toward tumor cells. Ultrastructural changes in paraptosis-like cell death, such as mitochondrial swelling, dilation of the nuclear envelope, cytoplasmic vacuoles, and nuclear chromatin condensation, but without margination of the chromatin, were observed in these tumor cells. Our data support the notion that eosinophils may exert an antitumoral role in gastric cancer. Finally, the case reported provides, for the first time, ultrastructural evidence of classical and compound exocytosis of eosinophils in the tumor stroma of human adenocarcinoma.

Biomolecular condensates in neurodegeneration and cancer.

The intracellular environment is partitioned into functionally distinct compartments containing specific sets of molecules and reactions. Biomolecular condensates, also referred to as membrane-less organelles, are diverse and abundant cellular compartments that lack membranous enclosures. Molecules assemble into condensates by phase separation; multivalent weak interactions drive molecules to separate from their surroundings and concentrate in discrete locations. Biomolecular condensates exist in all eukaryotes and in some prokaryotes, and participate in various essential house-keeping, stress-response and cell type-specific processes. An increasing number of recent studies link abnormal condensate formation, composition and material properties to a number of disease states. In this review, we discuss current knowledge and models describing the regulation of condensates and how they become dysregulated in neurodegeneration and cancer. Further research on the regulation of biomolecular phase separation will help us to better understand their role in cell physiology and disease.

Structural and functional polarisation of human pancreatic beta cells in islets from organ donors with and without type 2 diabetes.

AIMS/HYPOTHESIS: We hypothesised that human beta cells are structurally and functional polarised with respect to the islet capillaries. We set out to test this using confocal microscopy to map the 3D spatial arrangement of key proteins and live-cell imaging to determine the distribution of insulin granule fusion around the cells. METHODS: Human pancreas samples were rapidly fixed and processed using the pancreatic slice technique, which maintains islet structure and architecture. Slices were stained using immunofluorescence for polarity markers (scribble, discs large [Dlg] and partitioning defective 3 homologue [Par3]) and presynaptic markers (liprin, Rab3-interacting protein [RIM2] and piccolo) and imaged using 3D confocal microscopy. Isolated human islets were dispersed and cultured on laminin-511-coated coverslips. Live 3D two-photon microscopy was used on cultured cells to image exocytic granule fusion events upon glucose stimulation. RESULTS: Assessment of the distribution of endocrine cells across human islets found that, despite distinct islet-to-islet complexity and variability, including multi-lobular islets, and intermixing of alpha and beta cells, there is still a striking enrichment of alpha cells at the islet mantle. Measures of cell position demonstrate that most beta cells contact islet capillaries. Subcellularly, beta cells consistently position polar determinants, such as Par3, Dlg and scribble, with a basal domain towards the capillaries and apical domain at the opposite face. The capillary interface/vascular face is enriched in presynaptic scaffold proteins, such as liprin, RIM2 and piccolo. Interestingly, enrichment of presynaptic scaffold proteins also occurs where the beta cells contact peri-islet capillaries, suggesting functional interactions. We also observed the same polarisation of synaptic scaffold proteins in islets from type 2 diabetic patients. Consistent with polarised function, isolated beta cells cultured onto laminin-coated coverslips target insulin granule fusion to the coverslip. CONCLUSIONS/INTERPRETATION: Structural and functional polarisation is a defining feature of human pancreatic beta cells and plays an important role in the control of insulin secretion.

In vivo stress granule misprocessing evidenced in a FUS knock-in ALS mouse model.

Many RNA-binding proteins, including TDP-43, FUS, and TIA1, are stress granule components, dysfunction of which causes amyotrophic lateral sclerosis (ALS). However, whether a mutant RNA-binding protein disrupts stress granule processing in vivo in pathogenesis is unknown. Here we establish a FUS ALS mutation, p.R521C, knock-in mouse model that carries impaired motor ability and late-onset motor neuron loss. In disease-susceptible neurons, stress induces mislocalization of mutant FUS into stress granules and upregulation of ubiquitin, two hallmarks of disease pathology. Additionally, stress aggravates motor performance decline in the mutant mouse. By using two-photon imaging in TIA1-EGFP transduced animals, we document more intensely TIA1-EGFP-positive granules formed hours but cleared weeks after stress challenge in neurons in the mutant cortex. Moreover, neurons with severe granule misprocessing die days after stress challenge. Therefore, we argue that stress granule misprocessing is pathogenic in ALS, and the model we provide here is sound for further disease mechanistic study.

The Emerging Role of Stress Granules in Hepatocellular Carcinoma.

Stress granules (SGs) are small membrane-free cytosolic liquid-phase ordered entities in which mRNAs are protected and translationally silenced during cellular adaptation to harmful conditions (e.g., hypoxia, oxidative stress). This function is achieved by structural and functional SG components such as scaffold proteins and RNA-binding proteins controlling the fate of mRNAs. Increasing evidence indicates that the capacity of cells to assemble/disassemble functional SGs may significantly impact the onset and the development of metabolic and inflammatory diseases, as well as cancers. In the liver, the abnormal expression of SG components and formation of SG occur with chronic liver diseases, hepatocellular carcinoma (HCC), and selective hepatic resistance to anti-cancer drugs. Although, the role of SG in these diseases is still debated, the modulation of SG assembly/disassembly or targeting the expression/activity of specific SG components may represent appealing strategies to treat hepatic disorders and potentially cancer. In this review, we discuss our current knowledge about pathophysiological functions of SGs in HCC as well as available molecular tools and drugs capable of modulating SG formation and functions for therapeutic purposes.

Reduced Granule Cell Proliferation and Molecular Dysregulation in the Cerebellum of Lysosomal Acid Phosphatase 2 (ACP2) Mutant Mice.

Lysosomal acid phosphatase 2 (Acp2) mutant mice (naked-ataxia, nax) have a severe cerebellar cortex defect with a striking reduction in the number of granule cells. Using a combination of in vivo and in vitro immunohistochemistry, Western blotting, BrdU assays, and RT-qPCR, we show downregulation of MYCN and dysregulation of the SHH signaling pathway in the nax cerebellum. MYCN protein expression is significantly reduced at P10, but not at the peak of proliferation at around P6 when the number of granule cells is strikingly reduced in the nax cerebellum. Despite the significant role of the SHH-MycN pathway in granule cell proliferation, our study suggests that a broader molecular pathway and additional mechanisms regulating granule cell development during the clonal expansion period are impaired in the nax cerebellum. In particular, our results indicate that downregulation of the protein synthesis machinery may contribute to the reduced number of granule cells in the nax cerebellum.

15-Deoxy-Δ12,14-prostaglandin J2 promotes phosphorylation of eukaryotic initiation factor 2α and activates the integrated stress response.

Stress granules (SGs) are cytoplasmic RNA-protein aggregates formed in response to inhibition of translation initiation. SGs contribute to the stress response and are implicated in a variety of diseases, including cancer and some forms of neurodegeneration. Neurodegenerative diseases often involve chronic phosphorylation of eukaryotic initiation factor 2α (eIF2α), with deletions of eIF2α kinases or treatment with eIF2α kinase inhibitors being protective in some animal models of disease. However, how and why the integrated stress response (ISR) is activated in different forms of neurodegeneration remains unclear. Because neuroinflammation is common to many neurodegenerative diseases, we hypothesized that inflammatory factors contribute to ISR activation in a cell-nonautonomous manner. Using fluorescence microscopy and immunoblotting, we show here that the endogenously produced product of inflammation, 15-deoxy-Δ12,14-prostaglandin J2 (15-d-PGJ2), triggers eIF2α phosphorylation, thereby activating the ISR, repressing bulk translation, and triggering SG formation. Our findings define a mechanism by which inflammation activates the ISR in a cell-nonautonomous manner and suggest that inhibition of 15-d-PGJ2 production might be a useful therapeutic strategy in some neuroinflammatory contexts.

Traumatic injury induces stress granule formation and enhances motor dysfunctions in ALS/FTD models.

Traumatic brain injury (TBI) has been predicted to be a predisposing factor for amyotrophic lateral sclerosis (ALS) and other neurological disorders. Despite the importance of TBI in ALS progression, the underlying cellular and molecular mechanisms are still an enigma. Here, we examined the contribution of TBI as an extrinsic factor and investigated whether TBI influences the susceptibility of developing neurodegenerative symptoms. To evaluate the effects of TBI in vivo, we applied mild to severe trauma to Drosophila and found that TBI leads to the induction of stress granules (SGs) in the brain. The degree of SGs induction directly correlates with the level of trauma. Furthermore, we observed that the level of mortality is directly proportional to the number of traumatic hits. Interestingly, trauma-induced SGs are ubiquitin, p62 and TDP-43 positive, and persistently remain over time suggesting that SGs might be aggregates and exert toxicity in our fly models. Intriguingly, TBI on animals expressing ALS-linked genes increased mortality and locomotion dysfunction suggesting that mild trauma might aggravate neurodegenerative symptoms associated with ALS. Furthermore, we found elevated levels of high molecular weight ubiquitinated proteins and p62 in animals expressing ALS-causing genes with TBI, suggesting that TBI may lead to the defects in protein degradation pathways. Finally, we observed that genetic and pharmacological induction of autophagy enhanced the clearance of SGs and promoted survival of flies in vivo. Together, our study demonstrates that trauma can induce SG formation in vivo and might enhance neurodegenerative phenotypes in the fly models of ALS.

CCNA2 Ablation in Aged Mice Results in Abnormal rRNA Granule Accumulation in Hippocampus.

Mounting evidence in the literature suggests that RNA-RNA binding protein aggregations can disturb neuronal homeostasis and lead to symptoms associated with normal aging as well as dementia. The specific ablation of cyclin A2 in adult neurons results in neuronal polyribosome aggregations and learning and memory deficits. Detailed histologic and ultrastructural assays of aged mice revealed that post-mitotic hippocampal pyramidal neurons maintain cyclin A2 expression and that proliferative cells in the dentate subgranular zone express cyclin A2. Cyclin A2 loss early during neural development inhibited hippocampal development through canonical/cell-cycle mechanisms, including prolonged cell cycle timing in embryonic hippocampal progenitor cells. However, in mature neurons, cyclin A2 colocalized with dendritic rRNA. Cyclin A2 ablation in adult hippocampus resulted in decreased synaptic density in the hippocampus as well as in accumulation of rRNA granules in dendrite shafts. We conclude that cyclin A2 functions in a noncanonical/non-cell cycle regulatory role to maintain adult pyramidal neuron ribostasis.

Targeting stress granules: A novel therapeutic strategy for human diseases.

Stress granules (SGs) are assemblies of mRNA and proteins that form from mRNAs stalled in translation initiation in response to stress. Chronic stress might even induce formation of cytotoxic pathological SGs. SGs participate in various biological functions including response to apoptosis, inflammation, immune modulation, and signalling pathways; moreover, SGs are involved in pathogenesis of neurodegenerative diseases, viral infection, aging, cancers and many other diseases. Emerging evidence has shown that small molecules can affect SG dynamics, including assembly, disassembly, maintenance and clearance. Thus, targeting SGs is a potential therapeutic strategy for the treatment of human diseases and the promotion of health. The established methods for detecting SGs provided ready tools for large-scale screening of agents that alter the dynamics of SGs. Here, we describe the effects of small molecules on SG assembly, disassembly, and their roles in the disease. Moreover, we provide perspective for the possible application of small molecules targeting SGs in the treatment of human diseases.