scholarly journals Longevity-Extending MetAP2 Inhibitors Induce Caloric Restriction Through P53-Dependent Induction of GDF-15

2020 ◽  
Vol 4 (Supplement_1) ◽  
pp. 125-126
Author(s):  
Michael MacArthur ◽  
Sarah Mitchell ◽  
Jon Jung ◽  
Margaret Torrence ◽  
Alice Kane ◽  
...  
Keyword(s):  
Food Intake ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Brain Regions ◽  
Target Tissue ◽  
Dependent Manner ◽  
P53 Signaling ◽  
P53 Activation ◽  
Visceral Adipose

Abstract MetAP2 is a 67kDa protein which sits at the translation initiation complex and cleaves N-terminal methionine off of nascent peptides. Inhibitors of MetAP2 cause profound weight loss secondary to decreased food intake. These inhibitors also significantly extend longevity in mice in late-life intervention. However, the exact mechanism of action causing decreased food intake is not known. Here we investigated the molecular mechanism and target tissue of a MetAP2 inhibitor’s (Zgn1062) anorectic effects. First we identified the target tissue by testing targeted Zgn1062 delivery to specific brain regions. Delivery to the medio-basal hypothalamus did not have a significant effect but delivery to the lateral ventricle resulted in significantly decrease food intake and body weight after 2 and 14 hours. When we delivered a neuron-targeted AAV encoding MetAP2 shRNA we saw decreased efficacy of MetAP2 confirming the required for neuronal MetAP2 for anorectic effects. To determine the molecular mechanisms we performed RNAseq of wildtype and MetAP2 KO HT1080 cells across a timecourse of Zgn1062 treatment. The main pathway activated across timepoints in MetAP2-dependent manner was P53 signaling. A main P53 target that was upregulated was the known anorectic peptide GDF15. We confirmed GDF15 increases in vivo at both mRNA (liver and intestines) and protein level (serum) in response to Zgn1062. We also found that Zgn1062 treatment reduces senescent cell burden in visceral adipose tissue in vivo and reduces SASP gene expression in fat explants ex vivo. We hypothesize that Zgn1062’s potent P53 activation may play a role in clearance of senescent cells.

scholarly journals Understanding the Effects of General Anesthetics on Cortical Network Activity Using Ex Vivo Preparations

2019 ◽  
Vol 130 (6) ◽  
pp. 1049-1063 ◽  
Author(s):  
Logan J. Voss ◽  
Paul S. García ◽  
Harald Hentschke ◽  
Matthew I. Banks
Keyword(s):  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Brain Slices ◽  
Brain Regions ◽  
Cortical Network ◽  
Network Activity ◽  
Common Mechanism ◽  
General Anesthetics ◽  
Neural Basis

Abstract General anesthetics have been used to ablate consciousness during surgery for more than 150 yr. Despite significant advances in our understanding of their molecular-level pharmacologic effects, comparatively little is known about how anesthetics alter brain dynamics to cause unconsciousness. Consequently, while anesthesia practice is now routine and safe, there are many vagaries that remain unexplained. In this paper, the authors review the evidence that cortical network activity is particularly sensitive to general anesthetics, and suggest that disruption to communication in, and/or among, cortical brain regions is a common mechanism of anesthesia that ultimately produces loss of consciousness. The authors review data from acute brain slices and organotypic cultures showing that anesthetics with differing molecular mechanisms of action share in common the ability to impair neurophysiologic communication. While many questions remain, together, ex vivo and in vivo investigations suggest that a unified understanding of both clinical anesthesia and the neural basis of consciousness is attainable.

Nicotinamide Enhances the Polyploidization of Primary Megakaryocytes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1366-1366
Author(s):  
Lisa M. Giammona ◽  
Eleftherios Papoutsakis ◽  
William M. Miller
Keyword(s):  
Dna Content ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Annexin V ◽  
Dependent Manner ◽  
Vitamin B3 ◽  
Ploidy Levels ◽  
High Ploidy

Abstract Megakaryocyte (Mk) maturation includes the development of polyploid cells via endomitosis. In vitro models of Mk differentiation can be used to gain a better understanding of the molecular mechanisms controlling this process. However, it is challenging to achieve ploidy levels in cultured human cells that are as high as those observed in vivo. Others have recently reported the use of chemical inhibitors to increase Mk ploidy (Lannutti et al., Blood 105:3875, 2005). Here, we show that nicotinamide (NIC), a form of vitamin B3, enhances the normal process of Mk polyploidization and leads to both a greater fraction of high ploidy cells and a greater degree of polyploidization. Human mobilized peripheral blood CD34+ cells were cultured in serum-free medium supplemented with thrombopoietin (TPO) to induce Mk differentiation. Beginning on day 5 of culture, cells were treated with nicotinamide (3 and 6.25 mM) and monitored for DNA content, growth, apoptosis, and surface marker expression. NIC treatment resulted in a greater fraction of Mks with high ploidy (DNA content greater than or equal to 8N). The ploidy of NIC treated cells continued to increase over the duration of the 13-day culture, whereas the ploidy of untreated cells peaked at day 9. On day 13 (8 days of NIC exposure), the percentages of high ploidy Mks for the untreated, 3 mM NIC, and 6.25 mM NIC conditions were 23%, 48%, and 63%, respectively. Furthermore, cells treated with NIC reached ploidy levels of 64N and 32N for 6.25 and 3 mM NIC, respectively, compared to 16N for untreated cells. NIC-treated cells also displayed dramatic differences in morphology - characterized by an increase in cell size, the presence of a more highly lobated nucleus, and an increased frequency of proplatelet-forming cells. Nicotinamide is known to inhibit poly(ADP-ribose) polymerase (PARP) and Sir2, which are both NAD+ dependent enzymes. Preliminary experiments show that PARP activity is low in cultured Mks and is not affected by addition of 6.25 mM NIC. Continued exposure (beginning at day 5) to the PARP inhibitors (and nicotinamide analogs) 3-aminobenzamide (3-AB) and benzamide at concentrations of 1, 3, and 6.25 mM was toxic to cells in a dose dependent manner. Interestingly, high doses of NIC (25 and 50 mM) were also toxic to cells. Remarkably, while Mk polyploidization and apoptosis are typically correlated, the increase in DNA content observed for NIC-treated cells occurred without significantly affecting the percentage of apoptotic Mks (assessed by Annexin V staining). These data suggest that it may be possible to partially decouple Mk apoptosis and polyploidization. Furthermore, while 6.25 mM NIC inhibited cell proliferation by ~35%, total expansion of cells cultured with 3 mM NIC was similar to that of untreated cells. This, combined with similar Mk commitment, as defined by a similar percentage of CD41+ cells, resulted in a greater overall number of high ploidy Mks in cultures treated with NIC. Since there is a direct correlation between Mk DNA content and platelet production (Mattia et al., Blood 99:888, 2002), these results suggest a possible therapeutic benefit of NIC for the management of thrombocytopenia. Similarly, NIC could also be used as an additive to ex vivo Mk cultures destined for transplantation. Figure Figure

Ethanol Extract of Achillea fragrantissima Enhances Angiogenesis through Stimulation of VEGF Production

2020 ◽  
Vol 21 ◽  
Author(s):  
Hana M. Hammad ◽  
Amer Imraish ◽  
Maysa Al-Hussaini ◽  
Malek Zihlif ◽  
Amani A. Harb ◽  
...  
Keyword(s):  
Wound Healing ◽  
Rural Communities ◽  
Ex Vivo ◽  
Ethanol Extract ◽  
Aortic Ring ◽  
Treated Group ◽  
Control Group ◽  
Dependent Manner ◽  
Achillea Fragrantissima

Objective: Achillea fragrantissima L. (Asteraceae) is a traditionally used medicinal herb in the rural communities of Jordan. Methods: The present study evaluated the efficacy of the ethanol extract of this species on angiogenesis in both, ex vivo using rat aortic ring assay and in vivo using rat excision wound model. Results: In concentrations of 50 and 100 µg/ml, the ethanol extract showed angiogenic stimulatory effect and significantly increased length of capillary protrusions around aorta rings of about 60% in comparison to those of untreated aorta rings. In MCF-7 cells, the ethanol extract of A. fragrantissima stimulates the production of VEGF in a dose-dependent manner. 1% and 5% of ethanol extract of A. fragrantissima containing vaseline based ointment was applied on rat excision wounds for six days and was found to be effective in wound healing and maturation of the scar. Both preparations resulted in better wound healing when compared to the untreated control group and vaseline-treated group. This effect was comparable to that induced by MEBO, the positive control. Conclusion: The results indicate that A. fragrantissima has a pro-angiogenic effect, which may act through the VEGF signaling pathway.

scholarly journals Growth Factor Therapy for Parkinson’s Disease: Alternative Delivery Systems

2021 ◽  
pp. 1-7
Author(s):  
Sarah Jarrin ◽  
Abrar Hakami ◽  
Ben Newland ◽  
Eilís Dowd
Keyword(s):  
Parkinson’S Disease ◽  
Gene Therapy ◽  
Parkinson's Disease ◽  
Growth Factors ◽  
Growth Factor ◽  
Ex Vivo ◽  
Brain Regions ◽  
Delivery Systems ◽  
Alternative Delivery

Despite decades of research and billions in global investment, there remains no preventative or curative treatment for any neurodegenerative condition, including Parkinson’s disease (PD). Arguably, the most promising approach for neuroprotection and neurorestoration in PD is using growth factors which can promote the growth and survival of degenerating neurons. However, although neurotrophin therapy may seem like the ideal approach for neurodegenerative disease, the use of growth factors as drugs presents major challenges because of their protein structure which creates serious hurdles related to accessing the brain and specific targeting of affected brain regions. To address these challenges, several different delivery systems have been developed, and two major approaches—direct infusion of the growth factor protein into the target brain region and in vivo gene therapy—have progressed to clinical trials in patients with PD. In addition to these clinically evaluated approaches, a range of other delivery methods are in various degrees of development, each with their own unique potential. This review will give a short overview of some of these alternative delivery systems, with a focus on ex vivo gene therapy and biomaterial-aided protein and gene delivery, and will provide some perspectives on their potential for clinical development and translation.

scholarly journals Decoding the temporal nature of brain GR activity in the NFκB signal transition leading to depressive-like behavior

2021 ◽  
Author(s):  
Young-Min Han ◽  
Min Sun Kim ◽  
Juyeong Jo ◽  
Daiha Shin ◽  
Seung-Hae Kwon ◽  
...  
Keyword(s):  
Inhibitory Action ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Late Phase ◽  
Fine Tuning ◽  
Dependent Manner ◽  
Middle Phase ◽  
Temporal Shift

AbstractThe fine-tuning of neuroinflammation is crucial for brain homeostasis as well as its immune response. The transcription factor, nuclear factor-κ-B (NFκB) is a key inflammatory player that is antagonized via anti-inflammatory actions exerted by the glucocorticoid receptor (GR). However, technical limitations have restricted our understanding of how GR is involved in the dynamics of NFκB in vivo. In this study, we used an improved lentiviral-based reporter to elucidate the time course of NFκB and GR activities during behavioral changes from sickness to depression induced by a systemic lipopolysaccharide challenge. The trajectory of NFκB activity established a behavioral basis for the NFκB signal transition involved in three phases, sickness-early-phase, normal-middle-phase, and depressive-like-late-phase. The temporal shift in brain GR activity was differentially involved in the transition of NFκB signals during the normal and depressive-like phases. The middle-phase GR effectively inhibited NFκB in a glucocorticoid-dependent manner, but the late-phase GR had no inhibitory action. Furthermore, we revealed the cryptic role of basal GR activity in the early NFκB signal transition, as evidenced by the fact that blocking GR activity with RU486 led to early depressive-like episodes through the emergence of the brain NFκB activity. These results highlight the inhibitory action of GR on NFκB by the basal and activated hypothalamic-pituitary-adrenal (HPA)-axis during body-to-brain inflammatory spread, providing clues about molecular mechanisms underlying systemic inflammation caused by such as COVID-19 infection, leading to depression.

scholarly journals Uptake and Distribution of Administered Bone Marrow Mesenchymal Stem Cell Extracellular Vesicles in Retina

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 730
Author(s):  
Biji Mathew ◽  
Leianne A. Torres ◽  
Lorea Gamboa Gamboa Acha ◽  
Sophie Tran ◽  
Alice Liu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Extracellular Vesicles ◽  
Time Course ◽  
Ganglion Cells ◽  
Ex Vivo ◽  
Dependent Manner ◽  
Paracrine Effects

Cell replacement therapy using mesenchymal (MSC) and other stem cells has been evaluated for diabetic retinopathy and glaucoma. This approach has significant limitations, including few cells integrated, aberrant growth, and surgical complications. Mesenchymal Stem Cell Exosomes/Extracellular Vesicles (MSC EVs), which include exosomes and microvesicles, are an emerging alternative, promoting immunomodulation, repair, and regeneration by mediating MSC’s paracrine effects. For the clinical translation of EV therapy, it is important to determine the cellular destination and time course of EV uptake in the retina following administration. Here, we tested the cellular fate of EVs using in vivo rat retinas, ex vivo retinal explant, and primary retinal cells. Intravitreally administered fluorescent EVs were rapidly cleared from the vitreous. Retinal ganglion cells (RGCs) had maximal EV fluorescence at 14 days post administration, and microglia at 7 days. Both in vivo and in the explant model, most EVs were no deeper than the inner nuclear layer. Retinal astrocytes, microglia, and mixed neurons in vitro endocytosed EVs in a dose-dependent manner. Thus, our results indicate that intravitreal EVs are suited for the treatment of retinal diseases affecting the inner retina. Modification of the EV surface should be considered for maintaining EVs in the vitreous for prolonged delivery.

scholarly journals Endothelial insulin receptors differentially control insulin signaling kinetics in peripheral tissues and brain of mice

2017 ◽  
Vol 114 (40) ◽  
pp. E8478-E8487 ◽  
Author(s):  
Masahiro Konishi ◽  
Masaji Sakaguchi ◽  
Samuel M. Lockhart ◽  
Weikang Cai ◽  
Mengyao Ella Li ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Endothelial Cells ◽  
Food Intake ◽  
Insulin Signaling ◽  
Insulin Action ◽  
Insulin Receptors ◽  
Brain Regions ◽  
Peripheral Tissues ◽  
Systemic Insulin Resistance

Insulin receptors (IRs) on endothelial cells may have a role in the regulation of transport of circulating insulin to its target tissues; however, how this impacts on insulin action in vivo is unclear. Using mice with endothelial-specific inactivation of the IR gene (EndoIRKO), we find that in response to systemic insulin stimulation, loss of endothelial IRs caused delayed onset of insulin signaling in skeletal muscle, brown fat, hypothalamus, hippocampus, and prefrontal cortex but not in liver or olfactory bulb. At the level of the brain, the delay of insulin signaling was associated with decreased levels of hypothalamic proopiomelanocortin, leading to increased food intake and obesity accompanied with hyperinsulinemia and hyperleptinemia. The loss of endothelial IRs also resulted in a delay in the acute hypoglycemic effect of systemic insulin administration and impaired glucose tolerance. In high-fat diet-treated mice, knockout of the endothelial IRs accelerated development of systemic insulin resistance but not food intake and obesity. Thus, IRs on endothelial cells have an important role in transendothelial insulin delivery in vivo which differentially regulates the kinetics of insulin signaling and insulin action in peripheral target tissues and different brain regions. Loss of this function predisposes animals to systemic insulin resistance, overeating, and obesity.

scholarly journals Pathological Relationship between Intracellular Superoxide Metabolism and p53 Signaling in Mice

2021 ◽  
Vol 22 (7) ◽  
pp. 3548
Author(s):  
Kenji Watanabe ◽  
Shuichi Shibuya ◽  
Yusuke Ozawa ◽  
Toshihiko Toda ◽  
Takahiko Shimizu
Keyword(s):  
Apoptotic Cell ◽  
Oxygen Species ◽  
Double Knockout ◽  
In Vivo Experiments ◽  
P53 Signaling ◽  
Tissue Degeneration ◽  
Age Related ◽  
Tissue Changes ◽  
P53 Activation

Intracellular superoxide dismutases (SODs) maintain tissue homeostasis via superoxide metabolism. We previously reported that intracellular reactive oxygen species (ROS), including superoxide accumulation caused by cytoplasmic SOD (SOD1) or mitochondrial SOD (SOD2) insufficiency, induced p53 activation in cells. SOD1 loss also induced several age-related pathological changes associated with increased oxidative molecules in mice. To evaluate the contribution of p53 activation for SOD1 knockout (KO) (Sod1−/−) mice, we generated SOD1 and p53 KO (double-knockout (DKO)) mice. DKO fibroblasts showed increased cell viability with decreased apoptosis compared with Sod1−/− fibroblasts. In vivo experiments revealed that p53 insufficiency was not a great contributor to aging-like tissue changes but accelerated tumorigenesis in Sod1−/− mice. Furthermore, p53 loss failed to improve dilated cardiomyopathy or the survival in heart-specific SOD2 conditional KO mice. These data indicated that p53 regulated ROS-mediated apoptotic cell death and tumorigenesis but not ROS-mediated tissue degeneration in SOD-deficient models.

scholarly journals Fishing for Targets of Alien Metabolites: A Novel Peroxisome Proliferator-Activated Receptor (PPAR) Agonist from a Marine Pest

Marine Drugs ◽  
2018 ◽  
Vol 16 (11) ◽  
pp. 431 ◽  
Author(s):  
Rosa Vitale ◽  
Enrico D'Aniello ◽  
Stefania Gorbi ◽  
Andrea Martella ◽  
Cristoforo Silvestri ◽  
...  
Keyword(s):  
Native Species ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Invasion Biology ◽  
In Vitro Assays ◽  
Bioactive Metabolites ◽  
Peroxisome Proliferator ◽  
Invasive Pests

Although the chemical warfare between invasive and native species has become a central problem in invasion biology, the molecular mechanisms by which bioactive metabolites from invasive pests influence local communities remain poorly characterized. This study demonstrates that the alkaloid caulerpin (CAU)—a bioactive component of the green alga Caulerpa cylindracea that has invaded the entire Mediterranean basin—is an agonist of peroxisome proliferator-activated receptors (PPARs). Our interdisciplinary study started with the in silico prediction of the ligand-protein interaction, which was then validated by in vivo, ex vivo and in vitro assays. On the basis of these results, we candidate CAU as a causal factor of the metabolic and behavioural disorders observed in Diplodus sargus, a native edible fish of high ecological and commercial relevance, feeding on C. cylindracea. Moreover, given the considerable interest in PPAR activators for the treatment of relevant human diseases, our findings are also discussed in terms of a possible nutraceutical/pharmacological valorisation of the invasive algal biomasses, supporting an innovative strategy for conserving biodiversity as an alternative to unrealistic campaigns for the eradication of invasive pests.

scholarly journals ADP-ribosylation factor–like 4A interacts with Robo1 to promote cell migration by regulating Cdc42 activation

2019 ◽  
Vol 30 (1) ◽  
pp. 69-81 ◽  
Author(s):  
Tsai-Shin Chiang ◽  
Ming-Chieh Lin ◽  
Meng-Chen Tsai ◽  
Chieh-Hsin Chen ◽  
Li-Ting Jang ◽  
...  
Keyword(s):  
Cell Migration ◽  
Molecular Mechanisms ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Amino Acid Residues ◽  
Adp Ribosylation ◽  
Promote Cell Migration ◽  
Cytoskeleton Remodeling ◽  
Adp Ribosylation Factor

Cell migration is a highly regulated event that is initiated by cell membrane protrusion and actin reorganization. Robo1, a single-pass transmembrane receptor, is crucial for neuronal guidance and cell migration. ADP-ribosylation factor (Arf)–like 4A (Arl4A), an Arf small GTPase, functions in cell morphology, cell migration, and actin cytoskeleton remodeling; however, the molecular mechanisms of Arl4A in cell migration are unclear. Here, we report that the binding of Arl4A to Robo1 modulates cell migration by promoting Cdc42 activation. We found that Arl4A interacts with Robo1 in a GTP-dependent manner and that the Robo1 amino acid residues 1394–1398 are required for this interaction. The Arl4A-Robo1 interaction is essential for Arl4A-induced cell migration and Cdc42 activation but not for the plasma membrane localization of Robo1. In addition, we show that the binding of Arl4A to Robo1 decreases the association of Robo1 with the Cdc42 GTPase-activating protein srGAP1. Furthermore, Slit2/Robo1 binding down-regulates the Arl4A-Robo1 interaction in vivo, thus attenuating Cdc42-mediated cell migration. Therefore, our study reveals a novel mechanism by which Arl4A participates in Slit2/Robo1 signaling to modulate cell motility by regulating Cdc42 activity.

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