scholarly journals Nanoscopic investigation of C9orf72 poly-GA oligomers on nuclear membrane disruption by photoinducible platform

2021 ◽  
Author(s):  
Hung-Ming Chien ◽  
Ruei-Yu He ◽  
Chi-Chang Lee ◽  
Yung-An Huang ◽  
I-Ju Hung ◽  
...  
Keyword(s):  
Nuclear Membrane ◽  
Cortical Neurons ◽  
Ex Vivo ◽  
Membrane Integrity ◽  
Super Resolution ◽  
Dependent Manner ◽  
Nucleocytoplasmic Shuttling ◽  
Als Patients ◽  
Lateral Sclerosis

Abstract Dipeptide repeats (DPRs) translated from the mutated C9orf72 gene have recently been correlated with amyotrophic lateral sclerosis (ALS). Within these DPRs, the most abundant glycine-alanine (GA) DPRs form insoluble inclusions in C9orf72-ALS patients. While GA DPRs aggregates have been considered as amyloid, the biophysical features and cytotoxicity of GA DPRs oligomers generated during the amyloidogenesis has not yet been explored due to its unstable and fast equilibrium nature. In this study, we develop a photoinducible platform based on methoxynitrobenzene chemistry to enrich GA DPRs that allows to monitor the oligomerization process of GA DPRs in cells in nanoscale. By combining lifetime-based and super-resolution fluorescence microscopies with biophysical tools, we thoroughly examined the GA DPRs oligomerization process nanoscopically in a time-dependent manner. We provide direct ex vivo and in vitro evidences to demonstrate GA DPRs oligomers rather than nanofibrils disrupt nuclear membrane integrity. In addition, we found GA DPRs sabotage Ran protein gradient, hamper nucleocytoplasmic shuttling in neurons, and cause the mislocalization of TAR DNA-binding protein 43 in primary cortical neurons. Our results highlight the nanoscopic properties and toxicity of GA DPRs oligomers in living cells, which is a key step toward elucidating the pathological roles of C9orf72 DPRs in disease.

scholarly journals Nanoscopic investigation of C9orf72 poly-GA oligomers on nuclear membrane disruption by a photoinducible platform

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Hung-Ming Chien ◽  
Ruei-Yu He ◽  
Chi-Chang Lee ◽  
Yung-An Huang ◽  
I-Ju Hung ◽  
...  
Keyword(s):  
Nuclear Membrane ◽  
Cortical Neurons ◽  
Dna Binding Protein ◽  
Nucleocytoplasmic Transport ◽  
Time Dependent ◽  
Membrane Disruption ◽  
Dependent Manner ◽  
Alanine Dipeptide ◽  
Lateral Sclerosis ◽  
In Cells

AbstractGlycine-alanine dipeptide repeats (GA DPRs) translated from the mutated C9orf72 gene have recently been correlated with amyotrophic lateral sclerosis (ALS). While GA DPRs aggregates have been suggested as amyloid, the biophysical features and cytotoxicity of GA DPRs oligomers has not been explored due to its unstable nature. In this study, we develop a photoinducible platform based on methoxynitrobenzene chemistry to enrich GA DPRs that allows monitoring the oligomerization process of GA DPRs in cells. By applying advanced microscopies, we examined the GA DPRs oligomerization process nanoscopically in a time-dependent manner. We provided direct evidences to demonstrate GA DPRs oligomers rather than nanofibrils disrupt nuclear membrane. Moreover, we found GA DPRs hamper nucleocytoplasmic transport in cells and cause cytosolic retention of TAR DNA-binding protein 43 in cortical neurons. Our results highlight the toxicity of GA DPRs oligomers, which is a key step toward elucidating the pathological roles of C9orf72 DPRs.

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 Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

scholarly journals The Carbamate, Physostigmine does not Impair Axonal Transport in Rat Cortical Neurons

2021 ◽  
Vol 16 ◽  
pp. 263310552110202
Author(s):  
Sean X Naughton ◽  
Wayne D Beck ◽  
Zhe Wei ◽  
Guangyu Wu ◽  
Peter W Baas ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Cortical Neurons ◽  
Acetylcholinesterase Inhibitors ◽  
Neurological Deficits ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Rat Cortical Neurons ◽  
Toxicological Mechanism

Among the various chemicals that are commonly used as pesticides, organophosphates (OPs), and to a lesser extent, carbamates, are most frequently associated with adverse long-term neurological consequences. OPs and the carbamate, pyridostigmine, used as a prophylactic drug against potential nerve agent attacks, have also been implicated in Gulf War Illness (GWI), which is often characterized by chronic neurological symptoms. While most OP- and carbamate-based pesticides, and pyridostigmine are relatively potent acetylcholinesterase inhibitors (AChEIs), this toxicological mechanism is inadequate to explain their long-term health effects, especially when no signs of acute cholinergic toxicity are exhibited. Our previous work suggests that a potential mechanism of the long-term neurological deficits associated with OPs is impairment of axonal transport (AXT); however, we had not previously evaluated carbamates for this effect. Here we thus evaluated the carbamate, physostigmine (PHY), a highly potent AChEI, on AXT using an in vitro neuronal live imaging assay that we have previously found to be very sensitive to OP-related deficits in AXT. We first evaluated the OP, diisopropylfluorophosphate (DFP) (concentration range 0.001-10.0 µM) as a reference compound that we found previously to impair AXT and subsequently evaluated PHY (concentration range 0.01-100 nM). As expected, DFP impaired AXT in a concentration-dependent manner, replicating our previously published results. In contrast, none of the concentrations of PHY (including concentrations well above the threshold for impairing AChE) impaired AXT. These data suggest that the long-term neurological deficits associated with some carbamates are not likely due to acute impairments of AXT.

scholarly journals Newly developed dual topoisomerase inhibitor P8-D6 is highly active in ovarian cancer

2021 ◽  
Vol 13 ◽  
pp. 175883592110598
Author(s):  
Inken Flörkemeier ◽  
Tamara N. Steinhauer ◽  
Nina Hedemann ◽  
Magnus Ölander ◽  
Per Artursson ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Topoisomerase I ◽  
Ex Vivo ◽  
Chemotherapy Resistance ◽  
Membrane Integrity ◽  
Three Dimensional ◽  
New Drugs ◽  
Highly Active ◽  
Gynaecologic Neoplasms

Background: Ovarian cancer (OvCa) constitutes a rare and highly aggressive malignancy and is one of the most lethal of all gynaecologic neoplasms. Due to chemotherapy resistance and treatment limitations because of side effects, OvCa is still not sufficiently treatable. Hence, new drugs for OvCa therapy such as P8-D6 with promising antitumour properties have a high clinical need. The benzo[ c]phenanthridine P8-D6 is an effective inductor of apoptosis by acting as a dual topoisomerase I/II inhibitor. Methods: In the present study, the effectiveness of P8-D6 on OvCa was investigated in vitro. In various OvCa cell lines and ex vivo primary cells, the apoptosis induction compared with standard therapeutic agents was determined in two-dimensional monolayers. Expanded by three-dimensional and co-culture, the P8-D6 treated cells were examined for changes in cytotoxicity, apoptosis rate and membrane integrity via scanning electron microscopy (SEM). Likewise, the effects of P8-D6 on non-cancer human ovarian surface epithelial cells and primary human hepatocytes were determined. Results: This study shows a significant P8-D6-induced increase in apoptosis and cytotoxicity in OvCa cells which surpasses the efficacy of well-established drugs like cisplatin or the topoisomerase inhibitors etoposide and topotecan. Non-cancer cells were affected only slightly by P8-D6. Moreover, no hepatotoxic effect in in vitro studies was detected. Conclusion: P8-D6 is a strong and rapid inductor of apoptosis and might be a novel treatment option for OvCa therapy.

scholarly journals Kisspeptin-10 Inhibits Angiogenesis in Human Placental Vessels ex Vivo and Endothelial Cells in Vitro

Endocrinology ◽  
2010 ◽  
Vol 151 (12) ◽  
pp. 5927-5934 ◽  
Author(s):  
Thayalini Ramaesh ◽  
James J. Logie ◽  
Antonia K. Roseweir ◽  
Robert P. Millar ◽  
Brian R. Walker ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Vessels ◽  
Ex Vivo ◽  
Umbilical Vein ◽  
Biologically Active ◽  
Trophoblast Invasion ◽  
In Vitro Assays ◽  
Dependent Manner ◽  
Inhibition Of Proliferation

Recent studies suggest that kisspeptin (a neuropeptide central to the regulation of gonadotrophin secretion) has diverse roles in human physiology, including a putative role in implantation and placental function. Kisspeptin and its receptor are present in human blood vessels, where they mediate vasoconstriction, and kisspeptin is known to inhibit tumor metastasis and trophoblast invasion, both processes involving angiogenesis. We hypothesized that kisspeptin contributes to the regulation of angiogenesis in the reproductive system. The presence of the kisspeptin receptor was confirmed in human placental blood vessels and human umbilical vein endothelial cells (HUVEC) using immunochemistry. The ability of kisspeptin-10 (KP-10) (a shorter biologically active processed peptide) to inhibit angiogenesis was tested in explanted human placental arteries and HUVEC using complementary ex vivo and in vitro assays. KP-10 inhibited new vessel sprouting from placental arteries embedded in Matrigel and tube-like structure formation by HUVEC, in a concentration-dependent manner. KP-10 had no effect on HUVEC viability or apoptosis but induced concentration-dependent inhibition of proliferation and migration. In conclusion, KP-10 has antiangiogenic effects and, given its high expression in the placenta, may contribute to the regulation of angiogenesis in this tissue.

scholarly journals Selective tumor antigen vaccine delivery to human CD169+antigen-presenting cells using ganglioside-liposomes

2020 ◽  
Vol 117 (44) ◽  
pp. 27528-27539
Author(s):  
Alsya J. Affandi ◽  
Joanna Grabowska ◽  
Katarzyna Olesek ◽  
Miguel Lopez Venegas ◽  
Arnaud Barbaria ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Antigen ◽  
Tumor Antigens ◽  
Ex Vivo ◽  
Antigen Presenting Cells ◽  
Dependent Manner ◽  
Cross Presentation ◽  
Vaccine Carrier ◽  
Antigen Presenting

Priming of CD8+T cells by dendritic cells (DCs) is crucial for the generation of effective antitumor immune responses. Here, we describe a liposomal vaccine carrier that delivers tumor antigens to human CD169/Siglec-1+antigen-presenting cells using gangliosides as targeting ligands. Ganglioside-liposomes specifically bound to CD169 and were internalized by in vitro-generated monocyte-derived DCs (moDCs) and macrophages and by ex vivo-isolated splenic macrophages in a CD169-dependent manner. In blood, high-dimensional reduction analysis revealed that ganglioside-liposomes specifically targeted CD14+CD169+monocytes and Axl+CD169+DCs. Liposomal codelivery of tumor antigen and Toll-like receptor ligand to CD169+moDCs and Axl+CD169+DCs led to cytokine production and robust cross-presentation and activation of tumor antigen-specific CD8+T cells. Finally, Axl+CD169+DCs were present in cancer patients and efficiently captured ganglioside-liposomes. Our findings demonstrate a nanovaccine platform targeting CD169+DCs to drive antitumor T cell responses.

scholarly journals Dose-Independent Therapeutic Benefit of Bone Marrow Stem Cell Transplantation after MI in Mice

Biomedicines ◽  
2020 ◽  
Vol 8 (6) ◽  
pp. 157
Author(s):  
Nicole Zarniko ◽  
Anna Skorska ◽  
Gustav Steinhoff ◽  
Robert David ◽  
Ralf Gaebel
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Ex Vivo ◽  
Therapeutic Benefit ◽  
Dependent Manner ◽  
Mesenchymal Progenitors

Several cell populations derived from bone marrow (BM) have been shown to possess cardiac regenerative potential. Among these are freshly isolated CD133+ hematopoietic as well as culture-expanded mesenchymal stem cells. Alternatively, by purifying CD271+ cells from BM, mesenchymal progenitors can be enriched without an ex vivo cultivation. With regard to the limited available number of freshly isolated BM-derived stem cells, the effect of the dosage on the therapeutic efficiency is of particular interest. Therefore, in the present pre-clinical study, we investigated human BM-derived CD133+ and CD271+ stem cells for their cardiac regenerative potential three weeks post-myocardial infarction (MI) in a dose-dependent manner. The improvement of the hemodynamic function as well as cardiac remodeling showed no therapeutic difference after the transplantation of both 100,000 and 500,000 stem cells. Therefore, beneficial stem cell transplantation post-MI is widely independent of the cell dose and detrimental stem cell amplification in vitro can likely be avoided.

scholarly journals Binding of Dipyridamole of Human Platelets and to α-Acid Glycoprotein and its Significance for the Inhibition of Adenosine Uptake

1977 ◽  
Author(s):  
K. Subbarao ◽  
B. Rucinski ◽  
A. Summers ◽  
S. Niewiarowski
Keyword(s):  
Binding Sites ◽  
Ex Vivo ◽  
Human Platelets ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Acid Glycoprotein ◽  
High Affinity ◽  
Adenosine Uptake ◽  
Adenosine Transport

The interactions of dipyridamole with α1-acid glycoprotein of plasma and with human platelets are related to inhibition of adenosine uptake by platelets. One mole of dipyridamole binds to one mole of α1-acid glycoprotein with a dissociation constant (Kd) of 1.3 μM. It was found that platelets contain both high and low affinity binding sites for the drug. The binding of dipyridamole to the high affinity sites follows a Michaelis Menten binding pattern with a Kd of 0.04 μM. Approximately 2x104 dipyridamole molecules are bound at the high affinity sites of each platelet. The lower affinity sites bind the drug with a Kd of 4 μM. In the presence of α1acid glycoprotein the binding of dipyridamole to platelets is inhibited. Correspondingly, the dipyridamole inhibition of adenosine uptake by platelets is reduced 1000-fold by α1acid glycoprotein. Binding of dipyridamole to human platelets is essential for its inhibition of adenosine uptake by platelets. Dipyridamole reduced the [14C]-ATP to [14C]-ADP ratio in the platelets. Purified α1acid glycoprotein reversed these effects of dipyridamole on adenosine metabolism of platelets in a concentration dependent manner. A correlationwas observed between the level of circulating dipyridamole in plasma and the inhibition of [14C]-adenosine uptake by platelets of PRP samples of 12 human volunteers given different amounts of dipyridamole. The in vitro and ex vivo effects of dipyridamole on the [14C]-adenosine uptake by platelets were found to be identical. Our data suggest the presence of dipyridamole binding sites in platelets that regulate adenosine transport across the cell surface.

scholarly journals The Histone Variant MacroH2A1 Regulates Key Genes for Myogenic Cell Fusion in a Splice-Isoform Dependent Manner

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1109
Author(s):  
Sarah Hurtado-Bagès ◽  
Melanija Posavec Marjanovic ◽  
Vanesa Valero ◽  
Roberto Malinverni ◽  
David Corujo ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Ex Vivo ◽  
Histone Variants ◽  
Histone Variant ◽  
C2c12 Cells ◽  
Dependent Manner ◽  
Chronic Injury ◽  
Splice Isoforms ◽  
Splice Isoform

MacroH2A histone variants have functions in differentiation, somatic cell reprogramming and cancer. However, at present, it is not clear how macroH2As affect gene regulation to exert these functions. We have parted from the initial observation that loss of total macroH2A1 led to a change in the morphology of murine myotubes differentiated ex vivo. The fusion of myoblasts to myotubes is a key process in embryonic myogenesis and highly relevant for muscle regeneration after acute or chronic injury. We have focused on this physiological process, to investigate the functions of the two splice isoforms of macroH2A1. Individual perturbation of the two isoforms in myotubes forming in vitro from myogenic C2C12 cells showed an opposing phenotype, with macroH2A1.1 enhancing, and macroH2A1.2 reducing, fusion. Differential regulation of a subset of fusion-related genes encoding components of the extracellular matrix and cell surface receptors for adhesion correlated with these phenotypes. We describe, for the first time, splice isoform-specific phenotypes for the histone variant macroH2A1 in a physiologic process and provide evidence for a novel underlying molecular mechanism of gene regulation.

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