scholarly journals A novel and translational role for autophagy in antisense oligonucleotide trafficking and activity

2019 ◽  
Vol 47 (21) ◽  
pp. 11284-11303 ◽  
Author(s):  
Joseph Ochaba ◽  
Andrew F Powers ◽  
Kaitlyn A Tremble ◽  
Sarah Greenlee ◽  
Noah M Post ◽  
...  
Keyword(s):  
Ketogenic Diet ◽  
Antisense Oligonucleotides ◽  
Endocytic Pathway ◽  
Serum Starvation ◽  
Endosomal Trafficking ◽  
Chemical Methods ◽  
Mtor Inhibition ◽  
Molecular Machinery

Abstract Endocytosis is a mechanism by which cells sense their environment and internalize various nutrients, growth factors and signaling molecules. This process initiates at the plasma membrane, converges with autophagy, and terminates at the lysosome. It is well-established that cellular uptake of antisense oligonucleotides (ASOs) proceeds through the endocytic pathway; however, only a small fraction escapes endosomal trafficking while the majority are rendered inactive in the lysosome. Since these pathways converge and share common molecular machinery, it is unclear if autophagy-related trafficking participates in ASO uptake or whether modulation of autophagy affects ASO activity and localization. To address these questions, we investigated the effects of autophagy modulation on ASO activity in cells and mice. We found that enhancing autophagy through small-molecule mTOR inhibition, serum-starvation/fasting, and ketogenic diet, increased ASO-mediated target reduction in vitro and in vivo. Additionally, autophagy activation enhanced the localization of ASOs into autophagosomes without altering intracellular concentrations or trafficking to other compartments. These results support a novel role for autophagy and the autophagosome as a previously unidentified compartment that participates in and contributes to enhanced ASO activity. Further, we demonstrate non-chemical methods to enhance autophagy and subsequent ASO activity using translatable approaches such as fasting or ketogenic diet.

scholarly journals Inhibition of Kirsten-Ras reduces fibrosis and protects against renal dysfunction in a mouse model of chronic folic acid nephropathy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Lucy J. Newbury ◽  
Jui-Hui Wang ◽  
Gene Hung ◽  
Bruce M. Hendry ◽  
Claire C. Sharpe
Keyword(s):  
Folic Acid ◽  
Kidney Disease ◽  
Mouse Model ◽  
Renal Dysfunction ◽  
Antisense Oligonucleotides ◽  
Underlying Mechanism ◽  
Therapeutic Benefit ◽  
End Stage

Abstract Chronic Kidney Disease is a growing problem across the world and can lead to end-stage kidney disease and cardiovascular disease. Fibrosis is the underlying mechanism that leads to organ dysfunction, but as yet we have no therapeutics that can influence this process. Ras monomeric GTPases are master regulators that direct many of the cytokines known to drive fibrosis to downstream effector cascades. We have previously shown that K-Ras is a key isoform that drives fibrosis in the kidney. Here we demonstrate that K-Ras expression and activation are increased in rodent models of CKD. By knocking down expression of K-Ras using antisense oligonucleotides in a mouse model of chronic folic acid nephropathy we can reduce fibrosis by 50% and prevent the loss of renal function over 3 months. In addition, we have demonstrated in vitro and in vivo that reduction of K-Ras expression is associated with a reduction in Jag1 expression; we hypothesise this is the mechanism by which targeting K-Ras has therapeutic benefit. In conclusion, targeting K-Ras expression with antisense oligonucleotides in a mouse model of CKD prevents fibrosis and protects against renal dysfunction.

scholarly journals TBC-2 Regulates RAB-5/RAB-7-mediated Endosomal Trafficking inCaenorhabditis elegans

2010 ◽  
Vol 21 (13) ◽  
pp. 2285-2296 ◽  
Author(s):  
Laëtitia Chotard ◽  
Ashwini K. Mishra ◽  
Marc-André Sylvain ◽  
Simon Tuck ◽  
David G. Lambright ◽  
...  
Keyword(s):  
Rab Gtpase ◽  
Endosomal Trafficking ◽  
Dependent Manner ◽  
Nucleotide Exchange ◽  
Late Endosomes ◽  
Arginine Finger ◽  
Endosome Maturation ◽  
Hops Complex

During endosome maturation the early endosomal Rab5 GTPase is replaced with the late endosomal Rab7 GTPase. It has been proposed that active Rab5 can recruit and activate Rab7, which in turn could inactivate and remove Rab5. However, many of the Rab5 and Rab7 regulators that mediate endosome maturation are not known. Here, we identify Caenorhabditis elegans TBC-2, a conserved putative Rab GTPase-activating protein (GAP), as a regulator of endosome to lysosome trafficking in several tissues. We show that tbc-2 mutant animals accumulate enormous RAB-7–positive late endosomes in the intestine containing refractile material. RAB-5, RAB-7, and components of the homotypic fusion and vacuole protein sorting (HOPS) complex, a RAB-7 effector/putative guanine nucleotide exchange factor (GEF), are required for the tbc-2(−) intestinal phenotype. Expression of activated RAB-5 Q78L in the intestine phenocopies the tbc-2(−) large late endosome phenotype in a RAB-7 and HOPS complex-dependent manner. TBC-2 requires the catalytic arginine-finger for function in vivo and displays the strongest GAP activity on RAB-5 in vitro. However, TBC-2 colocalizes primarily with RAB-7 on late endosomes and requires RAB-7 for membrane localization. Our data suggest that TBC-2 functions on late endosomes to inactivate RAB-5 during endosome maturation.

Cytoskeletal changes induced by GRAF, the GTPase regulator associated with focal adhesion kinase, are mediated by Rho

1999 ◽  
Vol 112 (2) ◽  
pp. 231-242 ◽  
Author(s):  
J.M. Taylor ◽  
M.M. Macklem ◽  
J.T. Parsons
Keyword(s):  
Focal Adhesion Kinase ◽  
Pc12 Cells ◽  
Focal Adhesion ◽  
Fiber Formation ◽  
Serum Starvation ◽  
3T3 Cells ◽  
Neurite Retraction ◽  
Swiss 3T3

Graf, the GTPase regulator associated with focal adhesion kinase was previously shown to have GAP activity for Ρ A and Cdc42 in vitro (Hildebrand et al 1996 Mol. Cell Biol. 16: 3169–3178). In this study we sought to determine whether Graf acted at the level of Cdc42, Rho, or both in vivo and whether Graf was a signal terminator or transducer for these proteins. Microinjection of Graf cDNA into subconfluent Swiss 3T3 cells (in the presence of serum) has marked effects on cell shape and actin localization. Graf expression causes clearing of stress fibers followed by formation of long actin based filopodial-like extensions. Similar phenotypes were observed following injection of the Rho-inhibitor, C3 into these cells. The Graf response was dependent on GAP activity, since injection of Graf cDNA containing point mutations in the GAP domain (R236Q or N351V) which block enzymatic activity, does not confer this phenotype. Injection of Graf into Swiss 3T3 cells in which Rho has been down-regulated by serum starvation has no effect on cell morphology. Using this system, we demonstrate that Graf blocks sphingosine-1-phosphate (SPP) stimulated (Rho-mediated) stress fiber formation. Conversely, Graf expression does not inhibit bradykinin stimulated (Cdc42-mediated) filopodial extensions. These data indicate that Graf is a GAP for Rho in vivo. To further substantiate these results we examined the effect of Graf over-expression on Rho-mediated neurite retraction in nerve growth factor (NGF)-differentiated PC12 cells. In PC12 cells, which express relatively high levels of endogenous Graf, overexpression of Graf (but not Graf containing the R236Q mutation) enhances SPP-induced neurite retraction. These data indicate the possibility that Graf may be an effector for Rho in certain cell types.

scholarly journals 64COMPARISON OF DEVELOPMENTAL POTENTIAL OF IN VIVO AND IN VITRO RECIPIENT OOCYTES AFTER NUCLEAR TRANSFER IN GOAT

2004 ◽  
Vol 16 (2) ◽  
pp. 154
Author(s):  
H.S. Park ◽  
M.Y. Lee ◽  
S.P. Hong ◽  
J.I. Jin ◽  
J.K. Park ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Electric Pulse ◽  
Serum Starvation ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

Recent techniques in somatic cell nuclear transfer (SCNT) have been widely used for animal research. In addition, SCNT techniques may allow for the rescue of endangered species. Despite efforts for wildlife preservation, however, some threatened or endangered wild animal species will likely become extinct. As a preliminary experiment of a series in wildlife research, we tried to identify an improved method for the production of more transferable NT embryos in goats. Mature donor animals of Korean native goats (20–25kg) were synchronized with a CIDR (type G; InterAg, New Zealand) vaginal implant for 10 days followed by a total of 8 twice daily injections of 70mg of FSH (Folltropine, London, Ontario, Canada) and 400IU of hCG (Chorulon, Intervet, Moxmeer, The Netherlands). Oocytes were then collected surgically by retograde oviduct flush or direct aspiration from ovarian follicles in vivo at 29–34h after hCG. Oocytes collected from follicles were matured in TCM-199 containing 10% FBS and hormones. Prepared ear skin cells from the goat were cultured in TCM-199 containing 10% FBS at 39°C, 5% CO2 in air, and confluent monolayers were obtained. Oocytes were enucleated and donor cells from serum starvation (0.5%) culture were fused through a single electric pulse (DC 2.36kvcm−1, 17μs), and then activated by a single electric pulse (AC 5vmm−1, 5s+DC 1.56kvcm−1, 30μs) or chemical treatment (5μgmL−1 ionomycin 5min−1, 1.9mM 6-DMAP/4h). Reconstructed oocytes were cultured in M16 medium with 10% goat serum (GS) for 6–7 days. Data were analyzed by chi-square test. In in vitro development, significantly (P<0.05) more oocytes were cleaved (24/30, 80.0%) and developed (7/24, 29.2%) to morula or blastocyst stage, respectively, in NT oocytes activated by Iono + DMAP compared to electric stimulated oocytes (2/21, 40.0%; 0/2, 0%). There was a significant difference in in vitro development of NT embryos by the method of oocyte collection. Cleavage rate was higher (P<0.05) in NT embryos from in vivo oocytes (23/28, 82.1%) than in in vitro matured oocytes (19/35, 54.3%), and further development to morula or blastocyst was also significantly (P<0.05%) higher in NT embryos from in vivo oocytes (7/23, 30.4%) than in NT embryos from in vitro matured oocytes (0/19, 0%). When we compared NT embryos to parthenotes, developmental rate was not significantly different between NT embryos and parthenotes. These results strongly suggest that the in vivo oocytes will have superior developmental potential to oocytes matured in vitro. Table 1 Effect of different oocyte source on in vitro development following caprine SCNT

scholarly journals Salirasib inhibits the growth of hepatocarcinoma cell lines in vitro and tumor growth in vivo through ras and mTOR inhibition

2010 ◽  
Vol 9 (1) ◽  
pp. 256 ◽  
Author(s):  
Nicolas Charette ◽  
Christine De Saeger ◽  
Valérie Lannoy ◽  
Yves Horsmans ◽  
Isabelle Leclercq ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Cell Lines ◽  
Mtor Inhibition ◽  
Hepatocarcinoma Cell

scholarly journals Dual PI3 K/mTOR inhibition reduces prostate cancer bone engraftment altering tumor-induced bone remodeling

Tumor Biology ◽  
2018 ◽  
Vol 40 (4) ◽  
pp. 101042831877177 ◽  
Author(s):  
Andrea Mancini ◽  
Alessandro Colapietro ◽  
Simona Pompili ◽  
Andrea Del Fattore ◽  
Simona Delle Monache ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Patients ◽  
Therapeutic Potential ◽  
Disease Free Survival ◽  
Bone Lesions ◽  
Metastatic Progression ◽  
Mtor Inhibition ◽  
Osteoblast Activity

Morbidity in advanced prostate cancer patients is largely associated with bone metastatic events. The development of novel therapeutic strategies is imperative in order to effectively treat this incurable stage of the malignancy. In this context, Akt signaling pathway represents a promising therapeutic target able to counteract biochemical recurrence and metastatic progression in prostate cancer. We explored the therapeutic potential of a novel dual PI3 K/mTOR inhibitor, X480, to inhibit tumor growth and bone colonization using different in vivo prostate cancer models including the subcutaneous injection of aggressive and bone metastatic (PC3) and non-bone metastatic (22rv1) cell lines and preclinical models known to generate bone lesions. We observed that X480 both inhibited the primary growth of subcutaneous tumors generated by PC3 and 22rv1 cells and reduced bone spreading of PCb2, a high osteotropic PC3 cell derivative. In metastatic bone, X480 inhibited significantly the growth and osteolytic activity of PC3 cells as observed by intratibial injection model. X480 also increased the bone disease-free survival compared to untreated animals. In vitro experiments demonstrated that X480 was effective in counteracting osteoclastogenesis whereas it stimulated osteoblast activity. Our report provides novel information on the potential activity of PI3 K/Akt inhibitors on the formation and progression of prostate cancer bone metastases and supports a biological rationale for the use of these inhibitors in castrate-resistant prostate cancer patients at high risk of developing clinically evident bone lesions.

scholarly journals Extracellular vesicle budding is inhibited by redundant regulators of TAT-5 flippase localization and phospholipid asymmetry

2018 ◽  
Vol 115 (6) ◽  
pp. E1127-E1136 ◽  
Author(s):  
Katharina B. Beer ◽  
Jennifer Rivas-Castillo ◽  
Kenneth Kuhn ◽  
Gholamreza Fazeli ◽  
Birgit Karmann ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Extracellular Vesicle ◽  
Endosomal Trafficking ◽  
Vesicle Budding ◽  
Sorting Nexins ◽  
Dnaj Protein ◽  
Membrane Budding ◽  
Pleiotropic Functions

Cells release extracellular vesicles (EVs) that mediate intercellular communication and repair damaged membranes. Despite the pleiotropic functions of EVs in vitro, their in vivo function is debated, largely because it is unclear how to induce or inhibit their formation. In particular, the mechanisms of EV release by plasma membrane budding or ectocytosis are poorly understood. We previously showed that TAT-5 phospholipid flippase activity maintains the asymmetric localization of the lipid phosphatidylethanolamine (PE) in the plasma membrane and inhibits EV budding by ectocytosis in Caenorhabditis elegans. However, no proteins that inhibit ectocytosis upstream of TAT-5 were known. Here, we identify TAT-5 regulators associated with retrograde endosomal recycling: PI3Kinase VPS-34, Beclin1 homolog BEC-1, DnaJ protein RME-8, and the uncharacterized Dopey homolog PAD-1. PI3Kinase, RME-8, and semiredundant sorting nexins are required for the plasma membrane localization of TAT-5, which is important to maintain PE asymmetry and inhibit EV release. PAD-1 does not directly regulate TAT-5 localization, but is required for the lipid flipping activity of TAT-5. PAD-1 also has roles in endosomal trafficking with the GEF-like protein MON-2, which regulates PE asymmetry and EV release redundantly with sorting nexins independent of the core retromer. Thus, in addition to uncovering redundant intracellular trafficking pathways, our study identifies additional proteins that regulate EV release. This work pinpoints TAT-5 and PE as key regulators of plasma membrane budding, further supporting the model that PE externalization drives ectocytosis.

Ultrastructural analysis of Drosophila ovarian follicles differing in yolk polypeptide (yps) composition

Development ◽  
1993 ◽  
Vol 117 (1) ◽  
pp. 319-328
Author(s):  
F. Giorgi ◽  
P. Lucchesi ◽  
A. Morelli ◽  
M. Bownes
Keyword(s):  
Golgi Apparatus ◽  
Juvenile Hormone ◽  
Developmental Stages ◽  
Ovarian Follicles ◽  
Endocytic Pathway ◽  
Intermediate Cell ◽  
Wild Type ◽  
Vesicular Traffic

Drosophila ovarian follicles were examined ultrastructurally to study the vesicular traffic in the cortical ooplasm. The endocytic pathway leading to the production of yolk spheres was visualized following in vivo or in vitro exposure to peroxidase. The Golgi apparatus and the yolk spheres of wild-type ovarian follicles were preferentially labelled by fixation with osmium zinc iodide (OZI). Labelling of wild-type ovarian follicles was compared to that of several mutant follicles--L186/Basc, fs(2)A17 and ap4--which are defective in vitellogenesis. In these mutants, the Golgi apparatus and the vesicles nearby were either scantly labelled or not labelled at all. In oocytes from flies homozygous for the gene fs(1)1163, the Golgi apparatus was labelled as in the controls, but no yolk spheres appeared to be labelled with OZI at any of the developmental stages. In several Drosophila strains, the pattern of OZI label in the cortical ooplasm was seen to vary in relation to the number of yp structural genes. In starved Drosophila females, OZI labelling of the cortical ooplasm appeared restricted to the Golgi apparatus and to an extended tubular network. A similar labelling pattern was also detected in in vitro cultured vitellogenic follicles. Refeeding, topical application of juvenile hormone analogue to starved females or hormone addition to the culture medium, all caused the yolk spheres to become labelled with OZI and to incorporate peroxidase. These observations prove that impairing endocytic uptake by either mutation or lack of juvenile hormone prevents fusion of coated vesicles and tubules with the yolk spheres and leads them instead to form an intermediate cell compartment with Golgi-derived vesicles.

scholarly journals Primary Cilia Blockage Promotes the Malignant Behaviors of Hepatocellular Carcinoma via Induction of Autophagy

2019 ◽  
Vol 2019 ◽  
pp. 1-14
Author(s):  
Lian Liu ◽  
Jia-Qi Sheng ◽  
Mu-Ru Wang ◽  
Yun Gan ◽  
Xiao-Li Wu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Primary Cilia ◽  
The Cancer Genome Atlas ◽  
Migration And Invasion ◽  
Serum Starvation ◽  
Autophagic Flux ◽  
And Function ◽  
Hcc Cells

Primary cilia are organelles protruding from cell surface into environment that function in regulating cell cycle and modulating cilia-related signal. Primary ciliogenesis and autophagy play important roles in tumorigenesis. However, the functions and interactions between primary cilia and autophagy in hepatocellular carcinoma (HCC) have not been reported yet. Here, we aimed to investigate the relationship and function of primary cilia and autophagy in HCC. In vitro, we showed that serum starvation stimuli could trigger primary ciliogenesis in HCC cells. Blockage of primary ciliogenesis by IFT88 silencing enhanced the proliferation, migration, and invasion ability of HCC cells. In addition, inhibition of primary cilia could positively regulate autophagy. However, the proliferation, migration, and invasion ability which were promoted by IFT88 silencing could be partly reversed by inhibition of autophagy. In vivo, interference of primary cilia led to acceleration of tumor growth and increase of autophagic flux in xenograft HCC mouse models. Moreover, IFT88 high expression or ATG7 low expression in HCC tissues was correlated with longer survival time indicated by the Cancer Genome Atlas (TCGA) analysis. In conclusion, our study demonstrated that blockage of primary ciliogenesis by IFT88 silencing had protumor effects through induction of autophagy in HCC. These findings define a newly recognized role of primary cilia and autophagy in HCC.

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