Exon-specific U1 snRNAs improve ELP1 exon 20 definition and rescue ELP1 protein expression in a familial dysautonomia mouse model

Familial dysautonomia (FD) is an autosomal recessive neurodegenerative disease caused by a splicing mutation in the gene encoding Elongator complex protein 1 (ELP1, also known as IKBKAP). This mutation results in tissue-specific skipping of exon 20 with a corresponding reduction of ELP1 protein, predominantly in the central and peripheral nervous system. Although FD patients have a complex neurological phenotype caused by continuous depletion of sensory and autonomic neurons, progressive visual decline leading to blindness is one of the most problematic aspect of the disease, as it severely affects their quality of life. To better understand the disease mechanism as well as to test the in vivo efficacy of targeted therapies for FD, we have recently generated a novel phenotypic mouse model, TgFD9; Elp1Δ20/flox. This mouse exhibits most of the clinical features of the disease and accurately recapitulates the tissue-specific splicing defect observed in FD patients. Driven by the dire need to develop therapies targeting retinal degeneration in FD, herein, we comprehensively characterized the progression of the retinal phenotype in this mouse, and we demonstrated that it is possible to correct ELP1 splicing defect in the retina using the splicing modulator compound (SMC) BPN-15477.

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TMOD-18. TARGETING THE PI3K/AKT PATHWAY IN MYCN AMPLIFIED HIGH GRADE GLIOMAS

Neuro-Oncology ◽

10.1093/neuonc/noaa215.969 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii231-ii232

Author(s):

Katharine Halligan ◽

Ann-Catherine Stanton ◽

Matthew Halbert ◽

Brian Golbourn ◽

Stephen Mack ◽

...

Keyword(s):

Stem Cells ◽

Tumor Suppressor ◽

Mouse Model ◽

Protein Expression ◽

Neural Stem Cells ◽

Tumor Suppressor Genes ◽

Akt Pathway ◽

High Grade ◽

Suppressor Genes ◽

High Grade Gliomas

Abstract Pediatric glioblastoma (pGBM) are incurable brain tumors with overall poor prognosis and response to treatments due to molecular and epigenetic heterogeneity. In particular, the MYCN subtype of pGBM are a highly aggressive form of GBM with a dismal median survival of only 14 months. Furthermore, this subtype is enriched with loss of the tumor suppressor genes TP53 and PTEN, leading to aberrantly active PI3K-AKT signaling pathway and DNA-checkpoint abnormalities. Here, we report the generation of a novel syngeneic mouse model that recapitulates the features of the MYCN subtype of pGBM. We isolated Sox2-Cre neural stem cells from C57BL/6 mice and transduced inverted retroviral-cassettes of the murine Mycn oncogene simultaneously with shRNA targeting tumor suppressor genes p53 and Pten. Retroviral-cassettes are flanked by tandem LoxP sites arranged so that Cre recombinase expression inverts the cassettes in frame allowing for MYCN protein expression and loss of the P53/PTEN proteins. Transgene activation is accompanied with selectable cell surface markers and fluorescent tags enabling for fluorescent activated cell sorting (FACS) of the desired cell populations. Neural stem cells with MYCN protein expression and concurrent silencing of P53 and PTEN protein (NPP cells) result in significantly increased proliferation and activation of PI3K-AKT pathway as compared to control neural stem cells and have. Injection of NPP cells into the forebrain of immune competent C57BL/6 mice result in the formation of invasive high-grade gliomas with a lethal phenotype at ~50 days post injection. Using several next generation brain penetrant small molecule inhibitors of the PI3K-AKT pathway, we show inhibition of tumorigenesis in vitro. Moreover, we have identified several novel mechanisms of PI3KAKT treatment resistance and are currently identifying therapies that may overcome this resistance through RNA seq analysis. In summary, well defined genetic drivers of GBM can lead to informed mouse model generation to test promising therapies.

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Therapeutic manipulation of IKBKAP mis-splicing with a small molecule to cure familial dysautonomia

Nature Communications ◽

10.1038/s41467-021-24705-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Masahiko Ajiro ◽

Tomonari Awaya ◽

Young Jin Kim ◽

Kei Iida ◽

Masatsugu Denawa ◽

...

Keyword(s):

Small Molecule ◽

Genetic Disease ◽

Therapeutic Strategy ◽

Therapeutic Potential ◽

Familial Dysautonomia ◽

Therapeutic Modality ◽

Aberrant Splicing ◽

Intronic Splicing Enhancer ◽

Exon 20 ◽

Splicing Enhancer

AbstractApproximately half of genetic disease-associated mutations cause aberrant splicing. However, a widely applicable therapeutic strategy to splicing diseases is yet to be developed. Here, we analyze the mechanism whereby IKBKAP-familial dysautonomia (FD) exon 20 inclusion is specifically promoted by a small molecule splice modulator, RECTAS, even though IKBKAP-FD exon 20 has a suboptimal 5′ splice site due to the IVS20 + 6 T > C mutation. Knockdown experiments reveal that exon 20 inclusion is suppressed in the absence of serine/arginine-rich splicing factor 6 (SRSF6) binding to an intronic splicing enhancer in intron 20. We show that RECTAS directly interacts with CDC-like kinases (CLKs) and enhances SRSF6 phosphorylation. Consistently, exon 20 splicing is bidirectionally manipulated by targeting cellular CLK activity with RECTAS versus CLK inhibitors. The therapeutic potential of RECTAS is validated in multiple FD disease models. Our study indicates that small synthetic molecules affecting phosphorylation state of SRSFs is available as a new therapeutic modality for mechanism-oriented precision medicine of splicing diseases.

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Bone biomechanical properties and tissue-scale bone quality in a genetic mouse model of familial dysautonomia

Osteoporosis International ◽

10.1007/s00198-021-06006-1 ◽

2021 ◽

Author(s):

G. Vahidi ◽

H. Flook ◽

V. Sherk ◽

M. Mergy ◽

F. Lefcort ◽

...

Keyword(s):

Mouse Model ◽

Bone Quality ◽

Biomechanical Properties ◽

Familial Dysautonomia ◽

Genetic Mouse Model

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Effects of Shenqi Fuzheng injection on Fas/FasL protein expression levels in the cardiomyocytes of a mouse model of viral myocarditis

Experimental and Therapeutic Medicine ◽

10.3892/etm.2016.3165 ◽

2016 ◽

Vol 11 (5) ◽

pp. 1839-1846 ◽

Cited By ~ 2

Author(s):

TIANMIN WU ◽

JINSHUI CHEN ◽

LIUFANG FAN ◽

WENYAN XIE ◽

CHANGSHENG XU ◽

...

Keyword(s):

Mouse Model ◽

Protein Expression ◽

Viral Myocarditis ◽

Expression Levels ◽

Shenqi Fuzheng Injection

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Gene and Protein Expression Profiles in a Mouse Model of Collagen-Induced Arthritis

International Journal of Medical Sciences ◽

10.7150/ijms.22345 ◽

2018 ◽

Vol 15 (1) ◽

pp. 77-85 ◽

Cited By ~ 5

Author(s):

Sun-Yeong Gwon ◽

Ki-Jong Rhee ◽

Ho Joong Sung

Keyword(s):

Mouse Model ◽

Protein Expression ◽

Expression Profiles ◽

Collagen Induced Arthritis ◽

Gene And Protein Expression ◽

Protein Expression Profiles

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α-Hederin Inhibits the Growth of Lung Cancer A549 Cells in vitro and in vivo by Increasing SIRT6 Dependent Glycolysis

10.21203/rs.3.rs-36418/v1 ◽

2020 ◽

Author(s):

cong fang ◽

Yahui Liu ◽

Lanying Chen ◽

Yingying Luo ◽

Yaru Cui ◽

...

Keyword(s):

Lung Cancer ◽

Mouse Model ◽

Protein Expression ◽

Glucose Uptake ◽

A549 Cells ◽

Lactate Production ◽

Tumor Xenograft ◽

Xenograft Mouse Model ◽

Atp Generation

Abstract Background: α-hederin an effective component of Pulsatilla chinensis (Bunge) Regel, Studies showed that α-hederin exert many pharmacological activities, However, the effect of α-hederin on metabolism is still unclear. This study aimed to illuminate the role of α-hederin in glucose metabolism in lung cancer cells and investigate the molecular mechanism of α-hederin. Methods: CCK8 and colony formation assays were employed to assess the anti-proliferative effects induced by α-hederin. Glucose uptake, ATP generation, and reduced lactate production were measured using kits, and an A549 tumor xenograft mouse model of lung cancer was used to assess the in vivo antitumor effect of α-hederin (5, 10 mg/kg). Glycolytic-related key enzymes hexokinase 2 (HK2), glucose transporters 1 (GLUT1), pyruvate kinase M2 (PKM2), lactate dehydrogenase A (LDHA), monocarboxylate transporter (MCT4), c-Myc, Hypoxia inducible factor-1α (HIF-1α) and Sirtuin 6 (SIRT6) protein expression were detected by western blotting and immunohistochemical staining and SIRT6 inhibitors was verified in A549 cells. Results: Our results showed that cell proliferation was significantly inhibited by α-hederin in a dose-dependent manner and that α-hederin inhibited glucose uptake and ATP generation and reduced lactate production. Furthermore, α-hederin remarkably inhibited HK2, GLUT1, PKM2, LDHA, MCT4, c-Myc, HIF-1α and activated SIRT6 protein expression. Using inhibitors, we proved that α-hederin inhibits glycolysis by activating SIRT6. Moreover, a tumor xenograft mouse model of lung cancer further confirmed that α-hederin inhibits lung cancer growth via inhibiting glycolysis in vivo. Conclusions: α-hederin inhibits the growth of non-small cell lung cancer A549 cells by inhibiting glycolysis. The mechanism of glycolysis inhibition includes α-hederin activating the expression of the glycolytic related protein SIRT6.

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The Therapeutic Effects and Possible Mechanism of Pranoprofen in Mouse Model of Corneal Alkali Burns

Journal of Ophthalmology ◽

10.1155/2020/7485912 ◽

2020 ◽

Vol 2020 ◽

pp. 1-9

Author(s):

Minting Chen ◽

Abdirahman Abdinasir Gureeye ◽

Yacouba Cissé ◽

Lang Bai

Keyword(s):

Mouse Model ◽

Protein Expression ◽

Western Blot ◽

Saline Group ◽

Therapeutic Effects ◽

Eye Drops ◽

Corneal Tissue ◽

Corneal Epithelial ◽

Mrna And Protein Expression ◽

Corneal Structure

Objective. To investigate therapeutic effects and possible mechanism of pranoprofen in a mouse model of corneal alkali burns and provide new evidence for the clinical treatment of corneal alkali burns. Methods. A unilateral alkali burn was created in the central cornea by placing a piece of 2 mm diameter filter paper soaked in 1N NaOH on the right eye for 30 seconds. After the model was performed, C57BL/6J mice received topical treatment with saline eye drops or pranoprofen eye drops and were, respectively, categorized as saline group and pranoprofen group, whereas the remaining normal mice that were not subjected to alkali burns served as control, each group containing 15 mice (n = 45). On the 5th day after model establishment, the corneal fluorescein sodium staining score was evaluated in order to assess corneal epithelial damage. Tissue HE stain was used to observe the pathological changes of corneal tissue in each group. Real-time RT-PCR and western blot were also performed to detect the mRNA and protein expression of NLRP3, IL-1β/p17, and matrix metallopeptidase MMP-13. Results. 5 days after burns, microscopic observations of the pranoprofen group showed less corneal opacity and neovascularization development than the saline group. Sodium fluorescein staining showed obvious corneal structure disorders, poor corneal epithelium continuity, and a larger corneal epithelial defect area in the saline group (10.33±+−0.57) as opposed to the pranoprofen group (8.33 ± 0.57） (p<0.05). HE stain results showed the saline group had obvious corneal structure disorder and the corneal epithelial layer was incomplete as opposed to the pranoprofen group. PCR and western blot results suggested that the pranoprofen group expressed less NLRP3, IL-1β, and MMP-13 mRNA and protein expression in corneal tissue than the saline group (p<0.05). Conclusion. Pranoprofen may alleviate inflammatory response by inhibiting the expression levels of NLRP3 and IL-1β at the early stage of corneal alkali injury, lowering the expression of MMP-13 and ultimately reducing corneal epithelial damage.

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