scholarly journals Small molecule v-ATPase inhibitor Etidronate lowers levels of ALS protein ataxin-2

2021 ◽  
Author(s):  
Garam Kim ◽  
Lisa Nakayama ◽  
Jacob A Blum ◽  
Tetsuya Akiyama ◽  
Steven Boeynaems ◽  
...  
Keyword(s):  
Spinocerebellar Ataxia Type ◽  
Atpase Inhibitor ◽  
Protein Levels ◽  
Genome Wide ◽  
A Genome ◽  
Human Neurons ◽  
Crispr Screen ◽  
Ataxin 2 ◽  
Antisense Oligonucleotide Therapy

Antisense oligonucleotide therapy targeting ATXN2, a gene in which mutations cause neurodegenerative diseases spinocerebellar ataxia type 2 and amyotrophic lateral sclerosis, has entered clinical trials in humans. Additional methods to lower ataxin 2 levels would be beneficial not only in uncovering potentially cheaper or less invasive therapies, but also in gaining greater mechanistic insight into how ataxin 2 is normally regulated. We performed a genome-wide fluorescence activated cell sorting (FACS)-based CRISPR screen in human cells and identified multiple subunits of the lysosomal vacuolar ATPase (v ATPase) as regulators of ataxin 2 levels. We demonstrate that Etidronate, a U.S. Food and Drug Administration (FDA)-approved drug that inhibits the v ATPase, lowers ataxin 2 protein levels in mouse and human neurons. Moreover, oral administration of the drug to mice in their water supply and food is sufficient to lower ataxin-2 levels in the brain. Thus, we uncover Etidronate as a safe and inexpensive compound for lowering ataxin-2 levels and demonstrate the utility of FACS-based screens for identifying targets to modulate levels of human disease proteins.

scholarly journals Targeting RTN4/NoGo-Receptor reduces levels of ALS protein ataxin-2

2021 ◽  
Author(s):  
Caitlin M Rodriguez ◽  
Sophia C Bechek ◽  
Graham L Jones ◽  
Lisa Nakayama ◽  
Tetsuya Akiyama ◽  
...  
Keyword(s):  
Spinocerebellar Ataxia Type ◽  
Gene Encoding ◽  
Nogo Receptor ◽  
Protein Levels ◽  
Axonal Regrowth ◽  
A Genome ◽  
Human Neurons ◽  
Ataxin 2

Gene-based therapeutic strategies to lower ataxin-2 levels are emerging for neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type 2 (SCA2). To identify additional ways of reducing ataxin-2 levels, we performed a genome-wide screen in human cells for regulators of ataxin-2 and identified RTN4R, the gene encoding the RTN4/NoGo-Receptor, as a top hit. RTN4R knockdown, or treatment with a peptide inhibitor, was sufficient to lower ataxin-2 protein levels in mouse and human neurons in vitro and Rtn4r knockout mice have reduced ataxin-2 levels in vivo. Remarkably, we observed that ataxin-2 shares a role with the RTN4/NoGo-Receptor in limiting axonal regeneration. Reduction of either protein increases axonal regrowth following axotomy. These data define the RTN4/NoGo-Receptor as a novel therapeutic target for ALS and SCA2 and implicate the targeting of ataxin-2 as a potential treatment following nerve injury.

scholarly journals Genome-wide CRISPR screen identifies protein pathways modulating tau protein levels in neurons

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Carlos G. Sanchez ◽  
Christopher M. Acker ◽  
Audrey Gray ◽  
Malini Varadarajan ◽  
Cheng Song ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Tau Protein ◽  
Screening Strategy ◽  
Mtor Pathway ◽  
Protein Levels ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen ◽  
Hit Validation

AbstractAggregates of hyperphosphorylated tau protein are a pathological hallmark of more than 20 distinct neurodegenerative diseases, including Alzheimer’s disease, progressive supranuclear palsy, and frontotemporal dementia. While the exact mechanism of tau aggregation is unknown, the accumulation of aggregates correlates with disease progression. Here we report a genome-wide CRISPR screen to identify modulators of endogenous tau protein for the first time. Primary screens performed in SH-SY5Y cells, identified positive and negative regulators of tau protein levels. Hit validation of the top 43 candidate genes was performed using Ngn2-induced human cortical excitatory neurons. Using this approach, genes and pathways involved in modulation of endogenous tau levels were identified, including chromatin modifying enzymes, neddylation and ubiquitin pathway members, and components of the mTOR pathway. TSC1, a critical component of the mTOR pathway, was further validated in vivo, demonstrating the relevance of this screening strategy. These findings may have implications for treating neurodegenerative diseases in the future.

scholarly journals IPO11 mediates βcatenin nuclear import in a subset of colorectal cancers

2019 ◽  
Vol 219 (2) ◽  
Author(s):  
Monika Mis ◽  
Siobhan O’Brien ◽  
Zachary Steinhart ◽  
Sichun Lin ◽  
Traver Hart ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Gene Activation ◽  
Protein Stabilization ◽  
Nuclear Accumulation ◽  
Colorectal Cancers ◽  
Protein Levels ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen ◽  
Context Specific

Activation of Wnt signaling entails βcatenin protein stabilization and translocation to the nucleus to regulate context-specific transcriptional programs. The majority of colorectal cancers (CRCs) initiate following APC mutations, resulting in Wnt ligand—independent stabilization and nuclear accumulation of βcatenin. The mechanisms underlying βcatenin nucleocytoplasmic shuttling remain incompletely defined. Using a novel, positive selection, functional genomic strategy, DEADPOOL, we performed a genome-wide CRISPR screen and identified IPO11 as a required factor for βcatenin-mediated transcription in APC mutant CRC cells. IPO11 (Importin-11) is a nuclear import protein that shuttles cargo from the cytoplasm to the nucleus. IPO11−/− cells exhibit reduced nuclear βcatenin protein levels and decreased βcatenin target gene activation, suggesting IPO11 facilitates βcatenin nuclear import. IPO11 knockout decreased colony formation of CRC cell lines and decreased proliferation of patient-derived CRC organoids. Our findings uncover a novel nuclear import mechanism for βcatenin in cells with high Wnt activity.

scholarly journals A genome-wide CRISPR screen identifies UFMylation and TRAMP-like complexes as host factors required for hepatitis A virus infection

Cell Reports ◽  
2021 ◽  
Vol 34 (11) ◽  
pp. 108859
Author(s):  
Jessie Kulsuptrakul ◽  
Ruofan Wang ◽  
Nathan L. Meyers ◽  
Melanie Ott ◽  
Andreas S. Puschnik
Keyword(s):  
Virus Infection ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Host Factors ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

A genome wide quantitative trait linkage analysis for serum lipids in type 2 diabetes in an African population

2005 ◽  
Vol 181 (2) ◽  
pp. 389-397 ◽  
Author(s):  
Adebowale A. Adeyemo ◽  
Thomas Johnson ◽  
Joseph Acheampong ◽  
Johnnie Oli ◽  
Godfrey Okafor ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Linkage Analysis ◽  
Quantitative Trait ◽  
Serum Lipids ◽  
African Population ◽  
Quantitative Trait Linkage Analysis ◽  
Genome Wide ◽  
A Genome ◽  
Quantitative Trait Linkage

scholarly journals A Genome-Wide Linkage Scan for Diabetic Retinopathy Susceptibility Genes in Mexican Americans With Type 2 Diabetes From Starr County, Texas

Diabetes ◽  
2007 ◽  
Vol 56 (4) ◽  
pp. 1167-1173 ◽  
Author(s):  
D. M. Hallman ◽  
E. Boerwinkle ◽  
V. H. Gonzalez ◽  
B. E. K. Klein ◽  
R. Klein ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Diabetic Retinopathy ◽  
Mexican Americans ◽  
Susceptibility Genes ◽  
Genome Wide ◽  
A Genome ◽  
Starr County

scholarly journals A Genome-wide CRISPR Screen Identifies CDC25A as a Determinant of Sensitivity to ATR Inhibitors

2016 ◽  
Vol 62 (2) ◽  
pp. 307-313 ◽  
Author(s):  
Sergio Ruiz ◽  
Cristina Mayor-Ruiz ◽  
Vanesa Lafarga ◽  
Matilde Murga ◽  
Maria Vega-Sendino ◽  
...  
Keyword(s):  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

scholarly journals Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Rowena DeJesus ◽  
Francesca Moretti ◽  
Gregory McAllister ◽  
Zuncai Wang ◽  
Phil Bergman ◽  
...  
Keyword(s):  
Adaptor Protein ◽  
Er Stress Response ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen ◽  
Selection Step ◽  
A Cell ◽  
Cell Type Specific ◽  
Cellular Pathways ◽  
Mtor Signalling

SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein.

scholarly journals Crosstalk in oxygen homeostasis networks: SKN-1/NRF inhibits the HIF-1 hypoxia-inducible factor in Caenorhabditis elegans

2021 ◽  
Author(s):  
Dingxia Feng ◽  
Zhiwei Zhai ◽  
Zhiyong Shao ◽  
Yi Zhang ◽  
Jo Anne Powell-Coffman
Keyword(s):  
Oxidative Stress ◽  
Hypoxia Inducible Factor ◽  
Regulatory Sequences ◽  
Low Oxygen ◽  
Transcriptional Responses ◽  
C Elegans ◽  
Protein Levels ◽  
Oxygen Homeostasis ◽  
Genome Wide ◽  
A Genome

AbstractDuring development, homeostasis, and disease, organisms must balance responses that allow adaptation to low oxygen (hypoxia) with those that protect cells from oxidative stress. The evolutionarily conserved hypoxia-inducible factors are central to these processes, as they orchestrate transcriptional responses to oxygen deprivation. Here, we employ genetic strategies in C. elegans to identify stress-responsive genes and pathways that modulate the HIF-1 hypoxia-inducible factor and facilitate oxygen homeostasis. Through a genome-wide RNAi screen, we show that RNAi-mediated mitochondrial or proteasomal dysfunction increases the expression of hypoxia-responsive reporter Pnhr-57:GFP in C. elegans. Interestingly, only a subset of these effects requires hif-1. Of particular importance, we found that skn-1 RNAi increases the expression of hypoxia-responsive reporter Pnhr-57:GFP and elevates HIF-1 protein levels. The SKN-1/NRF transcription factor has been shown to promote oxidative stress resistance. We present evidence that the crosstalk between HIF-1 and SKN-1 is mediated by EGL-9, the prolyl hydroxylase that targets HIF-1 for oxygen-dependent degradation. Treatment that induces SKN-1, such as heat, increases expression of a Pegl-9:GFP reporter, and this effect requires skn-1 function and a putative SKN-1 binding site in egl-9 regulatory sequences. Collectively, these data support a model in which SKN-1 promotes egl-9 transcription, thereby inhibiting HIF-1. We propose that this interaction enables animals to adapt quickly to changes in cellular oxygenation and to better survive accompanying oxidative stress.

scholarly journals Genome-wide CRISPR screen identifies regulators of MAPK and MTOR pathways mediating sorafenib resistance in acute myeloid leukemia

Haematologica ◽  
2020 ◽  
Author(s):  
Alisa Damnernsawad ◽  
Daniel Bottomly ◽  
Stephen E. Kurtz ◽  
Christopher A. Eide ◽  
Shannon K. McWeeney ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Mek Inhibitors ◽  
Genome Wide ◽  
A Genome ◽  
Flt3 Mutations ◽  
Flt3 Inhibitors ◽  
Crispr Screen ◽  
Acute Myeloid

Drug resistance impedes the long-term effect of targeted therapies in acute myeloid leukemia (AML), necessitating the identification of mechanisms underlying resistance. Approximately 25% of AML patients carry FLT3 mutations and develop post-treatment insensitivity to FLT3 inhibitors, including sorafenib. Using a genome-wide CRISPR screen, we identified LZTR1, NF1, TSC1 or TSC2, negative regulators of the MAPK and MTOR pathways, as mediators of sorafenib resistance. Analyses of ex vivo drug sensitivity assays in FLT3-ITD AML patient samples revealed lower expression of LZTR1, NF1, and TSC2 correlated with sorafenib sensitivity. Importantly, MAPK and/or MTOR complex1 (MTORC1) activity were upregulated in AML cells made resistant to several FLT3 inhibitors, including crenolanib, quizartinib, or sorafenib. These cells were sensitive to MEK inhibitors, and the combination of FLT3 and MEK inhibitors showed enhanced efficacy, suggesting its effectiveness in AML patients with FLT3 mutations and those with resistance to FLT3 inhibitors.

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