scholarly journals Knockout of the non-essential gene SUGCT creates diet-linked, age-related microbiome disbalance with a diabetes-like metabolic syndrome phenotype

2019 ◽  
Vol 77 (17) ◽  
pp. 3423-3439 ◽  
Author(s):  
Joanna Niska-Blakie ◽  
Lakshmi Gopinathan ◽  
Kia Ngee Low ◽  
Yang Lay Kien ◽  
Christine M. F. Goh ◽  
...  
Keyword(s):  
Essential Gene ◽  
Glutaric Aciduria Type ◽  
Loss Of Function ◽  
Age Related ◽  
Related Phenotype ◽  
Type 3 ◽  
Kidney Liver ◽  
Gut Microbiota Dysbiosis ◽  
Lysine Catabolism

Abstract SUGCT (C7orf10) is a mitochondrial enzyme that synthesizes glutaryl-CoA from glutarate in tryptophan and lysine catabolism, but it has not been studied in vivo. Although mutations in Sugct lead to Glutaric Aciduria Type 3 disease in humans, patients remain largely asymptomatic despite high levels of glutarate in the urine. To study the disease mechanism, we generated SugctKO mice and uncovered imbalanced lipid and acylcarnitine metabolism in kidney in addition to changes in the gut microbiome. After SugctKO mice were treated with antibiotics, metabolites were comparable to WT, indicating that the microbiome affects metabolism in SugctKO mice. SUGCT loss of function contributes to gut microbiota dysbiosis, leading to age-dependent pathological changes in kidney, liver, and adipose tissue. This is associated with an obesity-related phenotype that is accompanied by lipid accumulation in kidney and liver, as well as “crown-like” structures in adipocytes. Furthermore, we show that the SugctKO kidney pathology is accelerated and exacerbated by a high-lysine diet. Our study highlights the importance of non-essential genes with no readily detectable early phenotype, but with substantial contributions to the development of age-related pathologies, which result from an interplay between genetic background, microbiome, and diet in the health of mammals.

scholarly journals Transient non-integrative nuclear reprogramming promotes multifaceted reversal of aging in human cells

2019 ◽  
Author(s):  
Tapash Jay Sarkar ◽  
Marco Quarta ◽  
Shravani Mukherjee ◽  
Alex Colville ◽  
Patrick Paine ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Human Fibroblasts ◽  
Human Cells ◽  
Cellular Aging ◽  
Epigenetic Reprogramming ◽  
Epigenetic Clock ◽  
Nuclear Reprogramming ◽  
Loss Of Function ◽  
Age Related

SummaryAging is characterized by a gradual loss of function occurring at the molecular, cellular, tissue and organismal levels1-3. At the chromatin level, aging is associated with the progressive accumulation of epigenetic errors that eventually lead to aberrant gene regulation, stem cell exhaustion, senescence, and deregulated cell/tissue homeostasis3. The technology of nuclear reprogramming to pluripotency, through over-expression of a small number of transcription factors, can revert both the age and the identity of any cell to that of an embryonic cell by driving epigenetic reprogramming2,4,5. Recent evidence has shown that transient transgenic reprogramming can ameliorate age-associated hallmarks and extend lifespan in progeroid mice6. However, it is unknown how this form of ‘epigenetic rejuvenation’ would apply to physiologically aged cells and, importantly, how it might translate to human cells. Here we show that transient reprogramming, mediated by transient expression of mRNAs, promotes a rapid reversal of both cellular aging and of epigenetic clock in human fibroblasts and endothelial cells, reduces the inflammatory profile in human chondrocytes, and restores youthful regenerative response to aged, human muscle stem cells, in each case without abolishing cellular identity. Our method, that we named Epigenetic Reprogramming of Aging (ERA), paves the way to a novel, potentially translatable strategy for ex vivo cell rejuvenation treatment. In addition, ERA holds promise for in vivo tissue rejuvenation therapies to reverse the physiological manifestations of aging and the risk for the development of age-related diseases.

scholarly journals Engineering gene overlaps to sustain genetic constructs in vivo

2021 ◽  
Vol 17 (10) ◽  
pp. e1009475
Author(s):  
Antoine L. Decrulle ◽  
Antoine Frénoy ◽  
Thomas A. Meiller-Legrand ◽  
Aude Bernheim ◽  
Chantal Lotton ◽  
...  
Keyword(s):  
Essential Gene ◽  
Loss Of Function ◽  
Evolutionary Potential ◽  
Reading Frame ◽  
E Coli ◽  
Overlapping Reading Frames ◽  
The Stability ◽  
Genetic Constructs ◽  
Selection Of

Evolution is often an obstacle to the engineering of stable biological systems due to the selection of mutations inactivating costly gene circuits. Gene overlaps induce important constraints on sequences and their evolution. We show that these constraints can be harnessed to increase the stability of costly genes by purging loss-of-function mutations. We combine computational and synthetic biology approaches to rationally design an overlapping reading frame expressing an essential gene within an existing gene to protect. Our algorithm succeeded in creating overlapping reading frames in 80% of E. coli genes. Experimentally, scoring mutations in both genes of such overlapping construct, we found that a significant fraction of mutations impacting the gene to protect have a deleterious effect on the essential gene. Such an overlap thus protects a costly gene from removal by natural selection by associating the benefit of this removal with a larger or even lethal cost. In our synthetic constructs, the overlap converts many of the possible mutants into evolutionary dead-ends, reducing the evolutionary potential of the system and thus increasing its stability over time.

scholarly journals Manipulation of EGFR-Induced Signaling for the Recruitment of Quiescent Neural Stem Cells in the Adult Mouse Forebrain

2021 ◽  
Vol 15 ◽  
Author(s):  
Loïc M. Cochard ◽  
Louis-Charles Levros ◽  
Sandra E. Joppé ◽  
Federico Pratesi ◽  
Anne Aumont ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Signaling Pathways ◽  
Intracellular Signaling ◽  
Signal Pathways ◽  
Loss Of Function ◽  
Egfr Signaling ◽  
Extrinsic Factors ◽  
Age Related

The ventricular-subventricular zone (V-SVZ) is the principal neurogenic niche in the adult mammalian forebrain. Neural stem/progenitor cell (NSPC) activity within the V-SVZ is controlled by numerous of extrinsic factors, whose downstream effects on NSPC proliferation, survival and differentiation are transduced via a limited number of intracellular signaling pathways. Here, we investigated the relationship between age-related changes in NSPC output and activity of signaling pathways downstream of the epidermal growth factor receptor (EGFR), a major regulator of NSPC activity. Biochemical experiments indicated that age-related decline of NSPC activity in vivo is accompanied by selective deficits amongst various EGFR-induced signal pathways within the V-SVZ niche. Pharmacological loss-of-function signaling experiments with cultured NSPCs revealed both overlap and selectivity in the biological functions modulated by the EGFR-induced PI3K/AKT, MEK/ERK and mTOR signaling modules. Specifically, while all three modules promoted EGFR-mediated NSPC proliferation, only mTOR contributed to NSPC survival and only MEK/ERK repressed NSPC differentiation. Using a gain-of-function in vivo genetic approach, we electroporated a constitutively active EGFR construct into a subpopulation of quiescent, EGFR-negative neural stem cells (qNSCs); this ectopic activation of EGFR signaling enabled qNSCs to divide in 3-month-old early adult mice, but not in mice at middle-age or carrying familial Alzheimer disease mutations. Thus, (i) individual EGFR-induced signaling pathways have dissociable effects on NSPC proliferation, survival, and differentiation, (ii) activation of EGFR signaling is sufficient to stimulate qNSC cell cycle entry during early adulthood, and (iii) the proliferative effects of EGFR-induced signaling are dominantly overridden by anti-proliferative signals associated with aging and Alzheimer’s disease.

scholarly journals LncRNA CRNED Hinders the Progression of Osteoarthritis by Epigenetic Regulation of DACT1

2021 ◽  
Author(s):  
Ziqi Zhang ◽  
Pei Yang ◽  
Chunsheng Wang ◽  
Run Tian
Keyword(s):  
Rat Model ◽  
Epigenetic Modification ◽  
Colorectal Neoplasia ◽  
Cruciate Ligament ◽  
Cartilage Damage ◽  
Loss Of Function ◽  
Cell Functions ◽  
Age Related

Abstract Background: Osteoarthritis (OA) is usually characterized by articular cartilage degeneration, synovial fibrosis and inflammation. LncRNA CRNED (colorectal neoplasia differentially expressed) has been reported to be down-regulated in age-related OA, but its role in injury-induced OA needs to be further explored.Methods: An OA rat model was established by using anterior cruciate ligament transection, and the adenovirus-mediated CRNED overexpression (Ad-CRNED) or DACT1 (dapper antagonist of catenin-1) interference (sh-DACT1) vectors were injected into the rat model through tail vein. ATDC5 cells were treated by IL-1β (10 ng/mL) to simulate OA conditions in vitro. Histological staining was performed to evaluate knee cartilage damage and synovitis. Gain-and loss-of-function assays analyzed the effects of CRNED and DACT1 on cell functions and Wnt/β-catenin pathway activity in chondrocytes. Bioinformatic analysis, RNA immunoprecipitation and chromatin immunoprecipitation were used to assess the regulatory interaction of CRNED, p300 and DACT1.Results: Overexpression of CRNED alleviated cartilage damage and synovitis in OA rats, and suppressed IL-1β-induced apoptosis, inflammation, and extracellular matrix (ECM) degradation in DACT5 cells, while silencing DACT1 effectively antagonized the protective effect of CRNED both in vitro and in vivo. Mechanism studies found that DACT1 could act as a downstream target of CRNED. By recruiting p300, CRNED promoted the enrichment of H3K27ac in the DACT1 promoter, thus promoting DACT1 transcription. In addition, CRNED hindered the activation of the Wnt pathway in IL-1β-stimulated chondrocytes by inducing DACT1 expression.Conclusion: CRNED promoted DACT1 expression through epigenetic modification and restrained the activation of Wnt/β-catenin signaling to impede the progression of OA.

scholarly journals Renal medullary carcinomas depend upon SMARCB1 loss and are sensitive to proteasome inhibition

2018 ◽  
Author(s):  
Andrew L. Hong ◽  
Yuen-Yi Tseng ◽  
Jeremiah Wala ◽  
Won Jun Kim ◽  
Bryan D. Kynnap ◽  
...  
Keyword(s):  
Sickle Cell Trait ◽  
Essential Gene ◽  
Functional Characterization ◽  
Proteasome Inhibition ◽  
Medullary Carcinoma ◽  
Cyclin B1 ◽  
Loss Of Function ◽  
Rhabdoid Tumors

AbstractRenal medullary carcinoma (RMC) is a rare and deadly kidney cancer in patients of African descent with sickle cell trait. Through direct-to-patient outreach, we developed genomically faithful patient-derived models of RMC. Using whole genome sequencing, we identified intronic fusion events in one SMARCB1 allele with concurrent loss of the other allele, confirming that SMARCB1 loss occurs in RMC. Biochemical and functional characterization of these RMC models revealed that RMC depends on the loss of SMARCB1 for survival and functionally resemble other cancers that harbor loss of SMARCB1, such as malignant rhabdoid tumors or atypical teratoid rhabdoid tumors. We performed RNAi and CRISPR-Cas9 loss of function genetic screens and a small-molecule screen and identified UBE2C as an essential gene in SMARCB1 deficient cancers. We found that the ubiquitin-proteasome pathway was essential for the survival of SMARCB1 deficient cancers in vitro and in vivo. Genetic or pharmacologic inhibition of this pathway leads to G2/M arrest due to constitutive accumulation of cyclin B1. These observations identify a synthetic lethal relationship that may serve as a therapeutic approach for patients with SMARCB1 deficient cancers.

scholarly journals Loss of Frrs1l disrupts synaptic AMPA receptor function, and results in neurodevelopmental, motor, cognitive and electrographical abnormalities

2018 ◽  
Author(s):  
Michelle Stewart ◽  
Petrina Lau ◽  
Gareth Banks ◽  
Rasneer Sonia Bains ◽  
Enrico Castroflorio ◽  
...  
Keyword(s):  
Ampa Receptor ◽  
Ex Vivo ◽  
Postsynaptic Membrane ◽  
Motor Deficits ◽  
Loss Of Function ◽  
Test Environment ◽  
Receptor Associated Protein ◽  
Age Related ◽  
Severe Motor

Summary statementIn this study, we show that the loss of the epilepsy-related gene Frrs1l in mice causes a dramatic reduction in AMPA receptor levels at the synapse. This change elicits severe motor and coordination disabilities, hyperactivity, cognitive defects, behavioural seizures and abnormal electroencephalographic (EEG) patterns.AbstractLoss of function mutations in the human AMPA receptor-associated protein, ferric chelate reductase 1-like (FRRS1L), are associated with a devastating neurological condition incorporating choreoathetosis, cognitive deficits and epileptic encephalopathies. Furthermore, evidence from overexpression and ex vivo studies have implicated FRRS1L in AMPA receptor biogenesis and assembly, suggesting that changes in glutamatergic signalling might underlie the disorder. Here, we investigated the neurological and neurobehavioural correlates of the disorder using a mouse Frrs1l null mutant. The study revealed several neurological defects that mirrored those seen in human patients. We established that mice lacking Frrs1l suffered from a broad spectrum of early-onset motor deficits with no progressive, age-related deterioration. Moreover, Frrs1l-/- mice were hyperactive irrespective of test environment, exhibited working memory deficits and displayed significant sleep fragmentation. Longitudinal electroencephalographic recordings also revealed abnormal EEG in Frrs1l-/- mice. Parallel investigations into disease aetiology identified a specific deficiency in AMPA receptor levels in the brain of Frrs1l-/- mice, while the general levels of several other synaptic components remained unchanged with no obvious alterations in the number of synapses. Furthermore, we established that Frrsl1 deletion results in glycosylation deficits in GLUA2 and GLUA4 AMPA receptor proteins, leading to cytoplasmic retention and a reduction of those specific AMPA receptor levels in the postsynaptic membrane. Overall, this study determines, for the first time in vivo, how loss of FRRS1L function can affect glutamatergic signalling and provides mechanistic insight into the development and progression of a human hyperkinetic disorder.

scholarly journals Engineering gene overlaps to sustain genetic constructs in vivo

2019 ◽  
Author(s):  
Antoine L. Decrulle ◽  
Antoine Frenoy ◽  
Thomas A. Meiller-Legrand ◽  
Aude Bernheim ◽  
Chantal Lotton ◽  
...  
Keyword(s):  
Fitness Landscape ◽  
Essential Gene ◽  
Loss Of Function ◽  
Evolutionary Potential ◽  
Reading Frame ◽  
E Coli ◽  
The Stability ◽  
Genetic Constructs ◽  
Selection Of

AbstractEvolution is often an obstacle to the engineering of stable biological systems due to the selection of mutations inactivating costly gene circuits. Gene overlaps induce important constraints on sequences and their evolution. We show that these constraints can be harnessed to increase the stability of synthetic circuits by purging loss-of-function mutations. We combine computational and synthetic biology approaches to rationally design an overlapping reading frame expressing an essential gene within an existing gene to protect. Our algorithm succeeded in creating overlapping reading frames in 80% of E. coli genes. Experimentally, scoring mutations in both genes of such overlapping construct, we found that a significant fraction of mutations impacting the gene to protect have a deleterious effect on the essential gene. Such an overlap thus protects a costly gene from removal by natural selection by associating the benefit of this removal with a larger or even lethal cost. In our synthetic constructs, the overlap converts many of the possible mutants into evolutionary dead-ends, effectively changing the fitness landscape and reducing the evolutionary potential of the system.

scholarly journals Gemin3 Is an Essential Gene Required for Larval Motor Function and Pupation in Drosophila

2009 ◽  
Vol 20 (1) ◽  
pp. 90-101 ◽  
Author(s):  
Karl B. Shpargel ◽  
Kavita Praveen ◽  
T. K. Rajendra ◽  
A. Gregory Matera
Keyword(s):  
Protein Function ◽  
Muscular Atrophy ◽  
Essential Gene ◽  
Survival Motor Neuron ◽  
Loss Of Function ◽  
Transposon Insertion ◽  
Associated Proteins ◽  
Smn Protein

The assembly of metazoan Sm-class small nuclear ribonucleoproteins (snRNPs) is an elaborate, step-wise process that takes place in multiple subcellular compartments. The initial steps, including formation of the core RNP, are mediated by the survival motor neuron (SMN) protein complex. Loss-of-function mutations in human SMN1 result in a neuromuscular disease called spinal muscular atrophy. The SMN complex is comprised of SMN and a number of tightly associated proteins, collectively called Gemins. In this report, we identify and characterize the fruitfly ortholog of the DEAD box protein, Gemin3. Drosophila Gemin3 (dGem3) colocalizes and interacts with dSMN in vitro and in vivo. RNA interference for dGem3 codepletes dSMN and inhibits efficient Sm core assembly in vitro. Transposon insertion mutations in Gemin3 are larval lethals and also codeplete dSMN. Transgenic overexpression of dGem3 rescues lethality, but overexpression of dSMN does not, indicating that loss of dSMN is not the primary cause of death. Gemin3 mutant larvae exhibit motor defects similar to previously characterized Smn alleles. Remarkably, appreciable numbers of Gemin3 mutants (along with one previously undescribed Smn allele) survive as larvae for several weeks without pupating. Our results demonstrate the conservation of Gemin3 protein function in metazoan snRNP assembly and reveal that loss of either Smn or Gemin3 can contribute to neuromuscular dysfunction.

scholarly journals The PNUTS-PP1 axis regulates endothelial aging and barrier function via SEMA3B suppression

2020 ◽  
Author(s):  
Noelia Lozano-Vidal ◽  
Laura Stanicek ◽  
Diewertje I. Bink ◽  
Veerle Kremer ◽  
Alyson W. MacInnes ◽  
...  
Keyword(s):  
Barrier Function ◽  
Health Care Systems ◽  
Multiorgan Failure ◽  
Cell Aging ◽  
Binding Motif ◽  
Loss Of Function ◽  
Nuclear Targeting ◽  
Age Related ◽  
Care Systems

ABSTRACTAge-related diseases pose great challenges to health care systems worldwide. During aging, endothelial senescence increases the risk for cardiovascular disease. Recently, it was described that Phosphatase 1 Nuclear Targeting Subunit (PNUTS) has a central role in cardiomyocyte aging and homeostasis. Here, we determined the role of PNUTS in endothelial cell aging. We confirmed that PNUTS is repressed in senescent endothelial cells (ECs). Moreover, PNUTS silencing elicits several of the hallmarks of endothelial aging: senescence, reduced angiogenesis and loss of barrier function. To validate our findings in vivo, we generated an endothelial-specific inducible PNUTS-deficient mouse line (Cdh5-CreERT2;PNUTSfl/fl), termed PNUTSEC-KO. Two weeks after PNUTS deletion, PNUTSEC-KO mice presented severe multiorgan failure and vascular leakage. We showed that the PNUTS binding motif for protein phosphatase 1 (PP1) is essential to maintain endothelial barrier function. Transcriptomic analysis of PNUTS-silenced HUVECs and lungs of PNUTSEC-KO mice revealed that the PNUTS-PP1 axis tightly regulates the expression of semaphorin 3B (SEMA3B). Indeed, silencing of SEMA3B completely restored barrier function after PNUTS loss-of-function. These results reveal a pivotal role for PNUTS in endothelial homeostasis through a PP1-SEMA3B downstream pathway that provides a potential target against the effects of aging in ECs.

scholarly journals microRNA-186 in extracellular vesicles from bone marrow mesenchymal stem cells alleviates idiopathic pulmonary fibrosis via interaction with SOX4 and DKK1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jing Zhou ◽  
Yang Lin ◽  
Xiuhua Kang ◽  
Zhicheng Liu ◽  
Wei Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Extracellular Vesicles ◽  
Luciferase Assay ◽  
Therapeutic Effects ◽  
Loss Of Function ◽  
Fibroblast Activation ◽  
Promising Therapeutic Approach

Abstract Background Previous reports have identified that human bone marrow mesenchymal stem cell-derived extracellular vesicles (BMSC-EVs) with their cargo microRNAs (miRNAs) are a promising therapeutic approach for the treatment of idiopathic pulmonary fibrosis (IPF). Therefore, we explored whether delivery of microRNA-186 (miR-186), a downregulated miRNA in IPF, by BMSC EVs could interfere with the progression of IPF in a murine model. Methods In a co-culture system, we assessed whether BMSC-EVs modulated the activation of fibroblasts. We established a mouse model of PF to evaluate the in vivo therapeutic effects of BMSC-EVs and determined miR-186 expression in BMSC-EVs by polymerase chain reaction. Using a loss-of-function approach, we examined how miR-186 delivered by BMSC-EVs affected fibroblasts. The putative relationship between miR-186 and SRY-related HMG box transcription factor 4 (SOX4) was tested using luciferase assay. Next, we investigated whether EV-miR-186 affected fibroblast activation and PF by targeting SOX4 and its downstream gene, Dickkopf-1 (DKK1). Results BMSC-EVs suppressed lung fibroblast activation and delayed IPF progression in mice. miR-186 was downregulated in IPF but enriched in the BMSC-EVs. miR-186 delivered by BMSC-EVs could suppress fibroblast activation. Furthermore, miR-186 reduced the expression of SOX4, a target gene of miR-186, and hence suppressed the expression of DKK1. Finally, EV-delivered miR-186 impaired fibroblast activation and alleviated PF via downregulation of SOX4 and DKK1. Conclusion In conclusion, miR-186 delivered by BMSC-EVs suppressed SOX4 and DKK1 expression, thereby blocking fibroblast activation and ameliorating IPF, thus presenting a novel therapeutic target for IPF.

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