Gain and loss of function of ALS-related mutations of TARDBP (TDP-43) cause motor deficits in vivo

Abstract Background: A relationship between the abundance of epicardial adipose tissue (EAT) and the risk of atrial fibrosis and atrial fibrillation (AF) in diabetes mellitus (DM) has been reported. And previous studies have shown that MicroRNA-21 (miR-21) is a regulatory factor in atrial fibrosis and AF. The aim of this study was to examine the role of different subtypes of miR-21 in EAT browning and atrial fibrosis under hyperglycemia conditions.Methods: In vivo, C57BL/6 wild type (WT) and miR-21 knockout (KO) mice were used to establish the diabetic model by intraperitoneal injection of streptozotocin (STZ). In vitro, the EAT adipocytes from miR-21 KO mice were cultured and transfected with miR-21-3p mimic or miR-21-5p mimic and co-cultured with atrial fibroblasts in both HG or LG conditions. The browning of EAT and the fibrosis of fibroblasts were assessed by western blotting, immunofluorescence, Masson staining, and ELISA. The gain- and loss-of-function experiments were used to identified fibroblast growth factor receptor 1 (FGFR1) as the target gene of miR-21-3p.Results: In patients with DM and/or AF, serum hsa-miR-21-3p, instead of hsa-miR-21-5p, was significantly up-regulated. And miR-21 KO clearly ameliorated the atrial fibrosis in the diabetic mice. miR-21-3p as a key regulator that controls EAT browning and participates in atrial fibrosis under hyperglycemia conditions. Moreover, our gain- and loss-of-function experiments showed that FGFR1, as a direct target of miR-21-3p identified a regulatory pathway in EAT adipocytes. Conclusions: MiR-21-3p regulated EAT browning and participated the process of hyperglycemia-induced atrial fibrosis by targeting FGFR1.

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Small molecule inhibition of PIKFYVE kinase rescues gain- and loss-of-function C9ORF72 ALS/FTD disease processes in vivo

10.1101/685800 ◽

2019 ◽

Cited By ~ 2

Author(s):

K. A. Staats ◽

C. Seah ◽

A. Sahimi ◽

Y. Wang ◽

N. Koutsodendris ◽

...

Keyword(s):

Small Molecule ◽

Hippocampal Neurons ◽

Neuroprotective Effect ◽

Loss Of Function ◽

Gain Of Function ◽

Small Molecule Inhibition ◽

Early Endosomes ◽

Gain And Loss ◽

Dipeptide Repeat

AbstractThe most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion (HRE) in C9ORF72 that contributes to neurodegeneration by both loss-of-function (decreased C9ORF72 protein levels) and gain-of-function (e.g. dipeptide repeat protein production) mechanisms. Although therapeutics targeting the gain-of-function mechanisms are in clinical development, it is unclear if these will be efficacious given the contribution of C9ORF72 loss-of-function processes to neurodegeneration. Moreover, there is a lack of therapeutic strategies for C9ORF72 ALS/FTD with demonstrated efficacy in vivo. Here, we show that small molecule inhibition of PIKFYVE kinase rescues both loss- and gain-of-function C9ORF72 disease mechanisms in vivo. We find that the reduction of C9ORF72 in mouse motor neurons leads to a decrease in early endosomes. In contrast, treatment with the PIKFYVE inhibitor apilimod increases the number of endosomes and lysosomes. We show that reduced C9ORF72 levels increases glutamate receptor levels in hippocampal neurons in mice, and that apilimod treatment rescues this excitotoxicity-related phenotype in vivo. Finally, we show that apilimod also alleviates the gain-of-function pathology induced by the C9ORF72 HRE by decreasing levels of dipeptide repeat proteins derived from both sense and antisense C9ORF72 transcripts in hippocampal neurons in vivo. Our data demonstrate the neuroprotective effect of PIKFYVE kinase inhibition in both gain- and loss-of-function murine models of C9ORF72 ALS/FTD.

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Hsa_circ_0004296 inhibits metastasis of prostate cancer by interacting with EIF4A3 to prevent nuclear export of ETS1 mRNA

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02138-8 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Shiyu Mao ◽

Wentao Zhang ◽

Fuhan Yang ◽

Yadong Guo ◽

Hong Wang ◽

...

Keyword(s):

Prostate Cancer ◽

Nuclear Export ◽

Rna Binding ◽

Rna Binding Proteins ◽

Transcriptional Level ◽

Loss Of Function ◽

Mesenchymal Transition ◽

Potential Biomarker ◽

Gain And Loss

Abstract Background Circular RNAs (circRNAs) have been shown to play vital biological functions in various tumors, including prostate cancer (PCa). However, the roles of circRNAs in the metastasis of PCa remain unclear. In the present study, differentially expressed circRNAs associated with PCa metastasis were screened using high-throughput RNA sequencing, from which hsa_circ_0004296 was identified. Methods Quantitative real-time PCR (qRT-PCR) was used to detect the expression of circ_0004296 in PCa tissues and adjacent normal tissues as well as in blood and urine. Gain and loss of function experiments were performed to investigate the function of circ_0004296 in PCa. Bioinformatics analyses, RNA pull-down assay, and mass spectrometry were conducted to identify RNA-binding proteins. RNA immunoprecipitation and RNA and protein nuclear-cytoplasmic fractionation were performed to investigate the underlying mechanism. A xenograft mouse model was used to analyze the effect of circ_0004296 on PCa growth and metastasis in vivo. Results The expression of circ_0004296 was decreased in PCa tissues, blood, and urine, which was negatively associated with metastasis. Furthermore, gain and loss of function experiments in vitro and in vivo showed that circ_0004296 inhibited the proliferation, migration, invasion, and epithelial-mesenchymal transition of PCa cells. Mechanistically, circ_0004296 regulated host gene ETS1 expression at the post-transcriptional level. EIF4A3 was identified and confirmed as the downstream binding protein of circ_0004296. EIF4A3 expression was significantly upregulated in PCa tissues and associated with PCa metastasis. Silencing EIF4A3 suppressed PCa cell proliferation, migration, invasion, and EMT. Conclusions Circ_0004296 overexpression efficiently inhibited ETS1 mRNA nuclear export by promoting EIF4A3 retention in the nucleus, leading to the downregulation of ETS1 expression and suppression of PCa metastasis; thus, circ_0004296 might be a potential biomarker and therapeutic target for patients with PCa.

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Circ-0005105 activates COL11A1 by targeting miR-20a-3p to promote pancreatic ductal adenocarcinoma progression

Cell Death and Disease ◽

10.1038/s41419-021-03938-8 ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Gang Ma ◽

Guichen Li ◽

Wufeng Fan ◽

Yuanhong Xu ◽

Shaowei Song ◽

...

Keyword(s):

Pancreatic Ductal Adenocarcinoma ◽

Cell Lines ◽

Poor Prognosis ◽

Cellular Proliferation ◽

Ductal Adenocarcinoma ◽

Loss Of Function ◽

Gain And Loss ◽

Proliferation And Invasion

AbstractGrowing evidence indicates that circular RNAs (circRNAs) are closely involved in tumorigenesis, but the association between circRNAs and pancreatic ductal adenocarcinoma (PDAC) is far from clear. Here, we focused on the functional investigation of circ-0005105, a newly identified circRNA, in PDAC progression. In the present study, we assessed circ-0005105 expression in PDAC tissues and cell lines with quantitative reverse transcription–polymerase chain reaction (qRT-PCR). The biological functions of circ-0005105 in cellular proliferation and invasion were identified through gain- and loss-of-function experiments in vitro and in vivo. The interaction between circ-0005105 and the microRNA (miR)-20a-3p–COL11A1 (collagen type XI alpha 1) axis was examined using luciferase reporter and RNA immunoprecipitation assays. We found that circ-0005105 expression was upregulated in both PDAC tissues and cell lines. Higher circ-0005105 expression correlated positively with the malignant clinical phenotype and poor prognosis of patients with PDAC. Gain- and loss-of-function analysis showed that circ-0005105 facilitated both in vitro and in vivo cellular proliferation and invasion. Mechanistically, circ-000510 served as a competing endogenous RNA (ceRNA) of miR-20a-3p and indirectly modulated COL11A1 expression, leading to activation of epithelial–mesenchymal transition (EMT). Rescue experiments suggested that the oncogenic activity of circ-0005105 was dependent on the modulation of the miR-20a-3p–COL11A1 axis. More importantly, COL11A1 overexpression was significantly associated with poor prognosis in PDAC, and silencing COL11A1 reduced PDAC cell tumorigenicity and metastasis. Taken together, our findings confirm for the first time that circ-0005105 has critical functions by regulating the miR-20a-3p–COL11A1 axis. In the clinic, circ-0005105 can act as a potential prognostic marker and therapeutic target in PDAC.

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In vivo and in vitro characterization of Escherichia coli protein CheZ gain- and loss-of-function mutants.

Journal of Bacteriology ◽

10.1128/jb.178.21.6275-6280.1996 ◽

1996 ◽

Vol 178 (21) ◽

pp. 6275-6280 ◽

Cited By ~ 24

Author(s):

M G Sanna ◽

M I Simon

Keyword(s):

Escherichia Coli ◽

Loss Of Function ◽

In Vitro Characterization ◽

Gain And Loss

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A novel lncRNA ROPM-mediated lipid metabolism governs breast cancer stem cell properties

Journal of Hematology & Oncology ◽

10.1186/s13045-021-01194-z ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Shuiqing Liu ◽

Yan Sun ◽

Yixuan Hou ◽

Liping Yang ◽

Xueying Wan ◽

...

Keyword(s):

Breast Cancer ◽

Stem Cells ◽

Cancer Stem Cells ◽

Phospholipid Metabolism ◽

Luciferase Reporter ◽

Breast Cancer Patients ◽

Loss Of Function ◽

Gain And Loss

Abstract Background Cancer stem cells (CSCs) are considered as the major cause to tumor initiation, recurrence, metastasis, and drug resistance, driving poor clinical outcomes in patients. Long noncoding RNAs (lncRNAs) have emerged as crucial regulators in cancer development and progression. However, limited lncRNAs involved in CSCs have been reported. Methods The novel lncROPM (a regulator of phospholipid metabolism) in breast CSCs (BCSCs) was identified by microarray and validated by qRT-PCR in BCSCs from breast cancer cells and tissues. The clinical significance of lncROPM was evaluated in two breast cancer cohorts and TANRIC database (TCGA-BRCA, RNAseq data). Gain- and loss-of-function assays were performed to examine the role of lncROPM on BCSCs both in vitro and in vivo. The regulatory mechanism of lncROPM was investigated by bioinformatics, RNA FISH, RNA pull-down, luciferase reporter assay, and actinomycin D treatment. PLA2G16-mediated phospholipid metabolism was determined by UHPLC-QTOFMS system. Cells’ chemosensitivity was assessed by CCK8 assay. Results LncROPM is highly expressed in BCSCs. The enhanced lncROPM exists in clinic breast tumors and other solid tumors and positively correlates with malignant grade/stage and poor prognosis in breast cancer patients. Gain- and loss-of-function studies show that lncROPM is required for the maintenance of BCSCs properties both in vitro and in vivo. Mechanistically, lncROPM regulates PLA2G16 expression by directly binding to 3'-UTR of PLA2G16 to increase the mRNA stability. The increased PLA2G16 significantly promotes phospholipid metabolism and the production of free fatty acid, especially arachidonic acid in BCSCs, thereby activating PI3K/AKT, Wnt/β-catenin, and Hippo/YAP signaling, thus eventually involving in the maintenance of BCSCs stemness. Importantly, lncROPM and PLA2G16 notably contribute to BCSCs chemo-resistance. Administration of BCSCs using clinic therapeutic drugs such as doxorubicin, cisplatin, or tamoxifen combined with Giripladib (an inhibitor of cytoplasmic phospholipase A2) can efficiently eliminate BCSCs and tumorigenesis. Conclusions Our study highlights that lncROPM and its target PLA2G16 play crucial roles in sustaining BCSC properties and may serve as a biomarker for BCSCs or other cancer stem cells. Targeting lncROPM-PLA2G16 signaling axis may be a novel therapeutic strategy for patients with breast cancer.

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Development of motor neurons and motor activity in zebrafish requires F-actin nucleation by Fmn2b

10.1101/2021.08.10.455777 ◽

2021 ◽

Author(s):

Dhriti Nagar ◽

Blake Carrington ◽

Shawn M Burgess ◽

Aurnab Ghose

Keyword(s):

Motor Neuron ◽

Motor Neurons ◽

Regulatory Protein ◽

Motor Deficits ◽

Axon Collateral ◽

Loss Of Function ◽

Function Model ◽

Actin Nucleation ◽

Neuronal Morphogenesis

Background: Cytoskeletal remodelling plays a pivotal role in the establishment of neuronal connectivity during development and in plasticity in adults. Mutations in the cytoskeleton regulatory protein Formin-2 (Fmn2) are associated with neurodevelopmental disorders like intellectual disability, though its function in neuronal morphogenesis has not been characterised in vivo. Results: Here we develop a loss-of-function model for fmn2b, the zebrafish orthologue of Fmn2, using CRISPR/Cas9-mediated gene editing. fmn2b mutants display motor deficits starting from the earliest motor responses in the embryo. We find that fmn2b is expressed in motor neurons and its loss reduces motor neuron innervation of the axial muscles without affecting myotome integrity. The translocation of caudal primary (CaP) motor neuron outgrowth is compromised in fmn2b mutants, while rostral primary (RoP) motor neurons have missing soma or stall at the horizontal myoseptum. Strikingly, axon collateral branching of the motor neurons is severely compromised and results in reduced synaptic coverage of the myotome. Rescue experiments identify the requirement for Fmn2-mediated actin nucleation for motor neuron outgrowth and arborisation. Conclusions: The zebrafish loss-of-function model of Fmn2 reveals the specific requirement of F-actin polymerisation by Fmn2 in neuromuscular development. It also underscores the role of Fmn2 in motor neuropathies, especially as a proportion of individuals harbouring mutations in Fmn2 present with hypotonia.

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Allosteric feed-forward activation of AP-2 via FCHO-BMP2K axis promotes clathrin-mediated endocytosis

10.1101/870899 ◽

2019 ◽

Author(s):

Shikha T. Ramesh ◽

Kolaparamba V. Navyasree ◽

Anjitha B. Ashok ◽

Nishada Qathoon ◽

Suryasikha Mohanty ◽

...

Keyword(s):

Plasma Membrane ◽

Loss Of Function ◽

Feed Forward ◽

Temporal Regulation ◽

Bona Fide ◽

Gain And Loss ◽

Spatio Temporal ◽

Functional Inactivation ◽

Zebrafish Embryogenesis

AbstractSpatio-temporal regulation of central adaptor complex, AP-2 is pivotal for clathrin-mediated endocytosis (CME). We recently discovered that FCHO proteins trigger clathrin-coated pit (CCP) formation by allosterically activating AP-2 on plasma membrane (Umasankar et al., 2014). Here, we demonstrate that this activation promotes AP-2 phosphorylation via recruitment and stabilization of BMP-2 inducible kinase (BMP2K), a bona fide AP-2 kinase leading to CCP maturation. Accordingly, BMP2K mislocalizes and degrades in FCHO knockout/ AP-2 depleted cells. Functional inactivation of kinase impairs AP-2 phosphorylation leading to altered lattice morphology and CME phenotypes reminiscent of CCP maturation defects. Reexpression of FCHO rescues AP-2 phosphorylation defects in FCHO knockout cells implying membrane activation of AP-2 is a prerequisite for kinase function. Furthermore, gain- and loss-of function phenotypes of FCHO and BMP2K are analogous and mirror altered AP-2 functions during zebrafish embryogenesis. Together, our findings reveal an in vivo allosteric feed-forward axis for operation of CME.

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