scholarly journals CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9orf72 dipeptide repeat protein toxicity

2017 ◽  
Author(s):  
Michael S. Haney ◽  
Nicholas J. Kramer ◽  
David W. Morgens ◽  
Ana Jovičić ◽  
Julien Couthouis ◽  
...  
Keyword(s):  
Er Stress ◽  
Nucleocytoplasmic Transport ◽  
Human Cells ◽  
Model Systems ◽  
Repeat Protein ◽  
Genome Wide ◽  
Primary Mouse ◽  
Repeat Expansions ◽  
Protein Toxicity ◽  
Dipeptide Repeat

AbstractHexanucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). The nucleotide repeat expansions are translated into dipeptide repeat (DPR) proteins, which are aggregation-prone and may contribute to neurodegeneration. Studies in model organisms, including yeast and flies have converged upon nucleocytoplasmic transport as one underlying pathogenic mechanism, but a comprehensive understanding of the molecular and cellular underpinnings of DPR toxicity in human cells is still lacking. We used the bacteria-derived clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to perform genome-wide gene knockout screens for suppressors and enhancers of C9orf72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. Our screens revealed genes involved in nucleocytoplasmic transport, reinforcing the previous findings from model systems. We also uncovered new potent modifiers of DPR toxicity whose gene products function in the endoplasmic reticulum (ER), proteasome, RNA processing pathways, and in chromatin modification. Since regulators of ER stress emerged prominently from the screens, we further investigated one such modifier, TMX2, which we identified as a modulator of the ER-stress signature elicited by C9orf72 DPRs in neurons. Together, this work identifies novel suppressors of DPR toxicity that represent potential therapeutic targets and demonstrates the promise of CRISPR-Cas9 screens to define mechanisms of neurodegenerative diseases.One Sentence SummaryGenome-wide CRISPR-Cas9 screens in human cells reveal mechanisms and targets for ALS-associated C9orf72 dipeptide repeat protein toxicity.

scholarly journals CRISPR–Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity

2018 ◽  
Vol 50 (4) ◽  
pp. 603-612 ◽  
Author(s):  
Nicholas J. Kramer ◽  
Michael S. Haney ◽  
David W. Morgens ◽  
Ana Jovičić ◽  
Julien Couthouis ◽  
...  
Keyword(s):  
Human Cells ◽  
Primary Neurons ◽  
Repeat Protein ◽  
Protein Toxicity ◽  
Dipeptide Repeat

scholarly journals Reduced autophagy upon C9ORF72 loss synergizes with dipeptide repeat protein toxicity in G4C2 repeat expansion disorders

2020 ◽  
Vol 39 (4) ◽  
Author(s):  
Manon Boivin ◽  
Véronique Pfister ◽  
Angeline Gaucherot ◽  
Frank Ruffenach ◽  
Luc Negroni ◽  
...  
Keyword(s):  
Repeat Expansion ◽  
Repeat Protein ◽  
Protein Toxicity ◽  
Dipeptide Repeat

scholarly journals C9orf72-generated poly-GR and poly-PR do not directly interfere with nucleocytoplasmic transport

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Joni Vanneste ◽  
Thomas Vercruysse ◽  
Steven Boeynaems ◽  
Adria Sicart ◽  
Philip Van Damme ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Motor Neurons ◽  
Hela Cells ◽  
Nuclear Import ◽  
Nucleocytoplasmic Transport ◽  
Repeat Expansions ◽  
Import And Export ◽  
Lateral Sclerosis ◽  
Dipeptide Repeat

Abstract Repeat expansions in the C9orf72 gene cause amyotrophic lateral sclerosis and frontotemporal dementia characterized by dipeptide-repeat protein (DPR) inclusions. The toxicity associated with two of these DPRs, poly-GR and poly-PR, has been associated with nucleocytoplasmic transport. To investigate the causal role of poly-GR or poly-PR on active nucleocytoplasmic transport, we measured nuclear import and export in poly-GR or poly-PR expressing Hela cells, neuronal-like SH-SY5Y cells and iPSC-derived motor neurons. Our data strongly indicate that poly-GR and poly-PR do not directly impede active nucleocytoplasmic transport.

scholarly journals Genome-wide characterization of LTR retrotransposons in the non-model deep-sea annelid Lamellibrachia luymesi

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Oluchi Aroh ◽  
Kenneth M. Halanych
Keyword(s):  
Deep Sea ◽  
Long Terminal Repeat Retrotransposons ◽  
Marine Organisms ◽  
Model Systems ◽  
Ltr Retrotransposons ◽  
Genome Wide ◽  
Lamellibrachia Luymesi ◽  
Vestimentiferan Tubeworm ◽  
Retrotransposon Activity

Abstract Background Long Terminal Repeat retrotransposons (LTR retrotransposons) are mobile genetic elements composed of a few genes between terminal repeats and, in some cases, can comprise over half of a genome’s content. Available data on LTR retrotransposons have facilitated comparative studies and provided insight on genome evolution. However, data are biased to model systems and marine organisms, including annelids, have been underrepresented in transposable elements studies. Here, we focus on genome of Lamellibrachia luymesi, a vestimentiferan tubeworm from deep-sea hydrocarbon seeps, to gain knowledge of LTR retrotransposons in a deep-sea annelid. Results We characterized LTR retrotransposons present in the genome of L. luymesi using bioinformatic approaches and found that intact LTR retrotransposons makes up about 0.1% of L. luymesi genome. Previous characterization of the genome has shown that this tubeworm hosts several known LTR-retrotransposons. Here we describe and classify LTR retrotransposons in L. luymesi as within the Gypsy, Copia and Bel-pao superfamilies. Although, many elements fell within already recognized families (e.g., Mag, CSRN1), others formed clades distinct from previously recognized families within these superfamilies. However, approximately 19% (41) of recovered elements could not be classified. Gypsy elements were the most abundant while only 2 Copia and 2 Bel-pao elements were present. In addition, analysis of insertion times indicated that several LTR-retrotransposons were recently transposed into the genome of L. luymesi, these elements had identical LTR’s raising possibility of recent or ongoing retrotransposon activity. Conclusions Our analysis contributes to knowledge on diversity of LTR-retrotransposons in marine settings and also serves as an important step to assist our understanding of the potential role of retroelements in marine organisms. We find that many LTR retrotransposons, which have been inserted in the last few million years, are similar to those found in terrestrial model species. However, several new groups of LTR retrotransposons were discovered suggesting that the representation of LTR retrotransposons may be different in marine settings. Further study would improve understanding of the diversity of retrotransposons across animal groups and environments.

scholarly journals Sex-dependent dynamics of metabolism in primary mouse hepatocytes

2021 ◽  
Author(s):  
Luise Hochmuth ◽  
Christiane Körner ◽  
Fritzi Ott ◽  
Daniela Volke ◽  
Kaja Blagotinšek Cokan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Sexual Dimorphism ◽  
New Drugs ◽  
Model Systems ◽  
Specific Gene ◽  
Primary Hepatocytes ◽  
Alcoholic Fatty Liver ◽  
Male And Female ◽  
Primary Mouse

AbstractThe liver is one of the most sexually dimorphic organs. The hepatic metabolic pathways that are subject to sexual dimorphism include xenobiotic, amino acid and lipid metabolism. Non-alcoholic fatty liver disease and hepatocellular carcinoma are among diseases with sex-dependent prevalence, progression and outcome. Although male and female livers differ in their abilities to metabolize foreign compounds, including drugs, sex-dependent treatment and pharmacological dynamics are rarely applied in all relevant cases. Therefore, it is important to consider hepatic sexual dimorphism when developing new treatment strategies and to understand the underlying mechanisms in model systems. We isolated primary hepatocytes from male and female C57BL6/N mice and examined the sex-dependent transcriptome, proteome and extracellular metabolome parameters in the course of culturing them for 96 h. The sex-specific gene expression of the general xenobiotic pathway altered and the female-specific expression of Cyp2b13 and Cyp2b9 was significantly reduced during culture. Sex-dependent differences of several signaling pathways increased, including genes related to serotonin and melatonin degradation. Furthermore, the ratios of male and female gene expression were inversed for other pathways, such as amino acid degradation, beta-oxidation, androgen signaling and hepatic steatosis. Because the primary hepatocytes were cultivated without the influence of known regulators of sexual dimorphism, these results suggest currently unknown modulatory mechanisms of sexual dimorphism in vitro. The large sex-dependent differences in the regulation and dynamics of drug metabolism observed during cultivation can have an immense influence on the evaluation of pharmacodynamic processes when conducting initial preclinical trials to investigate potential new drugs.

scholarly journals Humans and other commonly used model organisms are resistant to cycloheximide-mediated biases in ribosome profiling experiments

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Puneet Sharma ◽  
Jie Wu ◽  
Benedikt S. Nilges ◽  
Sebastian A. Leidel
Keyword(s):  
Human Cells ◽  
Ribosome Profiling ◽  
Model Organisms ◽  
Treatment Conditions ◽  
Genome Wide ◽  
Optimized Protocol ◽  
Gene Level ◽  
Translation Inhibitor ◽  
Species Specific ◽  
Conformational Restrictions

AbstractRibosome profiling measures genome-wide translation dynamics at sub-codon resolution. Cycloheximide (CHX), a widely used translation inhibitor to arrest ribosomes in these experiments, has been shown to induce biases in yeast, questioning its use. However, whether such biases are present in datasets of other organisms including humans is unknown. Here we compare different CHX-treatment conditions in human cells and yeast in parallel experiments using an optimized protocol. We find that human ribosomes are not susceptible to conformational restrictions by CHX, nor does it distort gene-level measurements of ribosome occupancy, measured decoding speed or the translational ramp. Furthermore, CHX-induced codon-specific biases on ribosome occupancy are not detectable in human cells or other model organisms. This shows that reported biases of CHX are species-specific and that CHX does not affect the outcome of ribosome profiling experiments in most settings. Our findings provide a solid framework to conduct and analyze ribosome profiling experiments.

scholarly journals Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Anayelly López-Islas ◽  
Victoria Chagoya-Hazas ◽  
Benjamin Pérez-Aguilar ◽  
Mayrel Palestino-Domínguez ◽  
Verónica Souza ◽  
...  
Keyword(s):  
Er Stress ◽  
Toxic Effect ◽  
Nuclear Translocation ◽  
Cholesterol Content ◽  
Primary Hepatocytes ◽  
Transmission Electron ◽  
Primary Mouse Hepatocytes ◽  
Primary Mouse ◽  
Mouse Hepatocytes ◽  
Stress Oxidative

Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC) diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde, while the activity of this enzyme determined in microsomes increased in the HC and in all ethanol treated hepatocytes, HC and CW. Oxidized proteins were increased in the HC cultures treated or not with the toxins. Transmission electron microscopy showed endoplasmic reticulum (ER) stress and megamitochondria in hepatocytes treated with ethanol as in HC and the ethanol HC treated hepatocytes. ER stress determined by PERK content was increased in ethanol treated hepatocytes from HC mice and CW. Nuclear translocation of ATF6 was observed in HC hepatocytes treated with ethanol, results that indicate that lipids overload and ethanol treatment favor ER stress. Oxidative stress, ER stress, and mitochondrial damage underlie potential mechanisms for increased damage in steatotic hepatocyte treated with ethanol.

scholarly journals Identification of anti-norovirus genes in mouse and human cells using genome-wide CRISPR activation screening

2018 ◽  
Author(s):  
Robert C. Orchard ◽  
Meagan E. Sullender ◽  
Bria F. Dunlap ◽  
Dale R. Balce ◽  
John G. Doench ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
World Wide ◽  
Human Cells ◽  
Gene Promoters ◽  
Murine Norovirus ◽  
Individual Gene ◽  
Genome Wide ◽  
Host Genes ◽  
Crispr Activation

AbstractNoroviruses (NoVs) are a leading cause of gastroenteritis world-wide, yet host factors that restrict NoV replication are not well understood. Here, we use a CRISPR activation (CRISPRa) genome-wide screening to identify host genes that can inhibit murine norovirus (MNoV) replication in either mouse or human cells. Our screens identified with high confidence 57 genes that can inhibit MNoV infection when overexpressed. A significant number of these genes are in interferon and immune regulation signaling networks, but surprising, the majority of the genes identified are not associated with innate or adaptive immunity nor with any antiviral activity. Confirmatory studies of eight of the genes in validate the initial screening data. Mechanistic studies on TRIM7 demonstrated a conserved role of the molecule in mouse and human cells in restricting MNoV in a step of infection after viral entry. Furthermore, we demonstrate that two isoforms of TRIM7 have differential antiviral activity. Taken together these data provide a resource for understanding norovirus biology and demonstrate a robust methodology for identifying new antiviral molecules across cell types and species.Author SummaryNorovirus is one of the leading causes of foodborne illness world-wide. Despite its prevalence, our understanding of norovirus biology is limited due to the difficulty in growing human norovirus in vitro and a lack of an animal model. Murine norovirus (MNoV) is a model norovirus system because MNoV replicates robustly in cell culture and in mice. To identify host genes that can restrict norovirus replication when overexpressed we performed genome-wide CRISPR activation (CRISPRa) screens to induce gene overexpression at the native locus through recruitment of transcriptional activators to individual gene promoters. We found 57 genes could block murine norovirus replication in either mouse or human cells. Several of these genes are associated with classical immune signaling pathways, while many of the molecules we identified have not been previously associated with antiviral activity. Our data is a resource for those studying norovirus and we provide a robust approach to identify novel antiviral genes.

scholarly journals Induction of Recurrent Break Cluster Genes in Neural Progenitor Cells Differentiated from Embryonic Stem Cells In Culture

2019 ◽  
Author(s):  
Aseda Tena ◽  
Yuxiang Zhang ◽  
Nia Kyritsis ◽  
Anne Devorak ◽  
Jeffrey Zurita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Progenitor Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Neural Progenitors ◽  
Neural Genes ◽  
Genome Wide ◽  
Primary Mouse

ABSTRACTMild replication stress enhances appearance of dozens of robust recurrent genomic break clusters, termed RDCs, in cultured primary mouse neural stem and progenitor cells (NSPCs). Robust RDCs occur within genes (“RDC-genes”) that are long and have roles in neural cell communications and/or have been implicated in neuropsychiatric diseases or cancer. We sought to develop an in vitro approach to determine whether specific RDC formation is associated with neural development. For this purpose, we adapted a system to induce neural progenitor cell (NPC) development from mouse embryonic stem cell (ESC) lines deficient for XRCC4 plus p53, a genotype that enhances DNA double-strand break (DSB) persistence to enhance detection. We tested for RDCs by our genome wide DSB identification approach that captures DSBs genome-wide via their ability to join to specific genomic Cas9/sgRNA-generated bait DSBs. In XRCC4/p53-deficient ES cells, we detected 7 RDCs, which were in genes, with two RDCs being robust. In contrast, in NPCs derived from these ES cell lines, we detected 29 RDCs, a large fraction of which were robust and associated with long, transcribed neural genes that were also robust RDC-genes in primary NSPCs. These studies suggest that many RDCs present in NSPCs are developmentally influenced to occur in this cell type and indicate that induced development of NPCs from ES cells provides an approach to rapidly elucidate mechanistic aspects of NPC RDC formation.SIGNIFICANCE STATEMENTWe previously discovered a set of long neural genes susceptible to frequent DNA breaks in primary mouse brain progenitor cells. We termed these genes RDC-genes. RDC-gene breakage during brain development might alter neural gene function and contribute to neurological diseases and brain cancer. To provide an approach to characterize the unknown mechanism of neural RDC-gene breakage, we asked whether RDC-genes appear in neural progenitors differentiated from embryonic stem cells in culture. Indeed, robust RDC-genes appeared in neural progenitors differentiated in culture and many overlapped with robust RDC-genes in primary brain progenitors. These studies indicate that in vitro development of neural progenitors provides a model system for elucidating how RDC-genes are formed.

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