scholarly journals Optogenetically controlled human functional motor endplate for testing botulinum neurotoxins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Juliette Duchesne de Lamotte ◽  
Jérôme Polentes ◽  
Florine Roussange ◽  
Léa Lesueur ◽  
Pauline Feurgard ◽  
...  
Keyword(s):  
Calcium Imaging ◽  
Muscle Cells ◽  
Motor Endplate ◽  
Cellular Models ◽  
Botulinum Neurotoxins ◽  
Muscle Nerve ◽  
Human Motor ◽  
Nerve System ◽  
The Impact

Abstract Background The lack of physiologically relevant and predictive cell-based assays is one of the major obstacles for testing and developing botulinum neurotoxins (BoNTs) therapeutics. Human-induced pluripotent stem cells (hiPSCs)-derivatives now offer the opportunity to improve the relevance of cellular models and thus the translational value of preclinical data. Methods We investigated the potential of hiPSC-derived motor neurons (hMNs) optical stimulation combined with calcium imaging in cocultured muscle cells activity to investigate BoNT-sensitivity of an in vitro model of human muscle-nerve system. Results Functional muscle-nerve coculture system was developed using hMNs and human immortalized skeletal muscle cells. Our results demonstrated that hMNs can innervate myotubes and induce contractions and calcium transient in muscle cells, generating an in vitro human motor endplate showing dose-dependent sensitivity to BoNTs intoxication. The implementation of optogenetics combined with live calcium imaging allows to monitor the impact of BoNTs intoxication on synaptic transmission in human motor endplate model. Conclusions Altogether, our findings demonstrate the promise of optogenetically hiPSC-derived controlled muscle-nerve system for pharmaceutical BoNTs testing and development.

scholarly journals Sigma 1 Receptor is Overexpressed in Hepatocellular Adenoma: Involvement of ERα and HNF1α

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2213
Author(s):  
Laure Villemain ◽  
Sylvie Prigent ◽  
Aurélie Abou-Lovergne ◽  
Laura Pelletier ◽  
Magali Chiral ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Hepatocellular Adenoma ◽  
Integral Membrane Protein ◽  
Hepatocyte Proliferation ◽  
Sigma 1 Receptor ◽  
Cellular Models ◽  
Huh7 Cells ◽  
The Impact

Sigma receptor 1 (SigR1) is an endoplasmic reticulum resident integral membrane protein whose functions remain unclear. Although the liver shows the highest expression of SigR1, its role in this organ is unknown. SigR1 is overexpressed in many cancers and its expression is correlated to hormonal status in hormone-dependent cancers. To better understand the role of SigR1 in hepatocytes we focused our work on the regulation of its expression in tumoral liver. In this context, hepatocellular adenomas, benign hepatic tumors associated with estrogen intake are of particular interest. The expression of SigR1 mRNA was assessed in hepatocellular adenoma (HCA) patients using qPCR. The impact of estrogen on the expression of SigR1 was studied in vivo (mice) and in vitro (HepG2 and Huh7 cells). The effect of HNF1α on the expression of SigR1 was studied in vivo by comparing wild type mice to HNF1 knockout mice. Estrogen enhanced SigR1 expression through its nuclear receptor ERα. HNF1α mutated HCA (H-HCA) significantly overexpressed SigR1 compared to all other HCA subtypes. HNF1 knockout mice showed an increase in SigR1 expression. Overexpressing SigR1 in cellular models increases proliferation rate and storage of lipid droplets, which phenocopies the H-HCA phenotype. SigR1 is involved in hepatocyte proliferation and steatosis and may play an important role in the control of the H-HCA phenotype.

scholarly journals Differential toxicities of albendazole and its two main metabolites to Balb/c 3T3, HepG2, and FaO lines and rat hepatocytes

2016 ◽  
Vol 60 (4) ◽  
pp. 495-500 ◽  
Author(s):  
Lidia Radko ◽  
Maria Minta ◽  
Sylwia Stypuła-Trębas
Keyword(s):  
Hepatoma Cell ◽  
Rat Hepatocytes ◽  
Neutral Red ◽  
In Vitro Toxicity ◽  
Isolated Rat Hepatocytes ◽  
Hepatoma Cell Lines ◽  
Cellular Models ◽  
The Impact ◽  
Isolated Rat

Abstract Introduction: The cytotoxicity of anthelmintic agent, albendazole (ABZ) and its two major metabolites, sulfoxide (ABZSO) and sulfone (ABZ-SO2), on non-hepatic Balb/c 3T3 line, two hepatoma cell lines (FaO, HepG2), and isolated rat hepatocytes was investigated. Material and Methods: Cell cultures were exposed for 24, 48, and 72 h to eight concentrations of the compounds ranging from 0.05 to 100 μg/mL (ABZ) and from 0.78 to 100 μg/mL (ABZ-SO and ABZ-SO2). Three different assays were applied in which various biochemical endpoints were assessed: lysosomal activity - neutral red uptake (NRU) assay, proliferation - total protein contents (TPC) assay and lactate dehydrogenase (LDH) leakage assay. Results: The most toxic was albendazole whose EC50 values calculated from the concentration effect curves ranged from 0.2 to 0.5 μg/mL (Balb/c 3T3 ) and from 0.4 to 73.3 μg/mL (HepG2). Rat hepatoma line and isolated rat hepatocytes were less sensitive to the impact of ABZ. Toxic action expressed as EC50 was recorded after 72 h exposure only in LDH release assay at 0.8 μg/mL and 9.7 μg/mL respectively. The toxicity of metabolites was much lower. The most sensitive to ABZ-SO were fibroblasts and EC50-72h values were similar in all three assays used, i.e. NRU (14.1 μg/mL), TPC (15.8 μg/mL), and LDH (20.9 μg/mL). In the case of ABZ-SO2 the mean effective concentrations were the highest, and could be reached only in one LDH assay. These values (μg/mL) were as follows: 65.3 (FaO), 65.4 (HepG2), 75.8 (hepatocytes), and 77.4 (Balb/c 3T3). Conclusion: The differences in in vitro toxicity of albendazole depend on metabolic ability of the cellular models. Primary cultured rat hepatocytes represent a valuable tool to study the impact of biotransformation on the cytotoxicity of drugs.

scholarly journals Three Novel Candidate Probiotic Strains with Prophylactic Properties in a Murine Model of Cow's Milk Allergy

2016 ◽  
Vol 82 (6) ◽  
pp. 1722-1733 ◽  
Author(s):  
Elodie Neau ◽  
Johanne Delannoy ◽  
Candice Marion ◽  
Charles-Henry Cottart ◽  
Chantal Labellie ◽  
...  
Keyword(s):  
Murine Model ◽  
Food Allergies ◽  
T Helper ◽  
Murine Splenocytes ◽  
Content Type ◽  
Cellular Models ◽  
Probiotic Strains ◽  
The Impact

ABSTRACTFood allergies can have significant effects on morbidity and on quality of life. Therefore, the development of efficient approaches to reduce the risk of developing food allergies is of considerable interest. The aim of this study was to identify and select probiotic strains with preventive properties against allergies using a combination ofin vitroandin vivoapproaches. To that end, 31 strains of bifidobacteria and lactic acid bacteria were screened for their immunomodulatory properties in two cellular models, namely, human peripheral blood mononuclear cells (PBMCs) and T helper 2 (Th2)-skewed murine splenocytes. Six strains inducing a high interleukin-10 (IL-10)/IL-12p70 ratio and a low secretion of IL-4 on the two cellular models were selected, and their protective impact was testedin vivoin a murine model of food allergy to β-lactoglobulin. Three strains showed a protective impact on sensitization, with a decrease in allergen-specific IgE, and on allergy, with a decrease in mast cell degranulation. Analysis of the impact of these three strains on the T helper balance revealed different mechanisms of action. TheLactobacillus salivariusLA307 strain proved to block Th1 and Th2 responses, while theBifidobacterium longumsubsp.infantisLA308 strain induced a pro-Th1 profile and theLactobacillus rhamnosusLA305 strain induced pro-Th1 and regulatory responses. These results demonstrate that a combination ofin vitroandin vivoscreening is effective in probiotic strain selection and allowed identification of three novel probiotic strains that are active against sensitization in mice.

scholarly journals Monosodium Iodoacetate delays regeneration and inhibits hypertrophy in skeletal muscle cells in vitro

2019 ◽  
Author(s):  
Rowan P. Rimington ◽  
Darren J. Player ◽  
Neil R.W. Martin ◽  
Mark P. Lewis
Keyword(s):  
Skeletal Muscle ◽  
Muscle Cells ◽  
Rodent Model ◽  
Skeletal Muscle Cells ◽  
Skeletal Muscle Regeneration ◽  
Index Number ◽  
Monosodium Iodoacetate ◽  
The Impact

AbstractObjectiveOsteoarthritis (OA) is a musculoskeletal disease which contributes to severe morbidity. The monosodium iodoacetate (MIA) rodent model of OA is now well established, however the effect of MIA on surrounding tissues post injection has not been investigated and as such the impact on phenotypic development is unknown. The aim of this investigation was to examine the impact of MIA incubation on skeletal muscle cells in vitro, to provide an indication as to the potential influence of MIA administration of skeletal muscle in vivo.MethodsC2C12 skeletal muscle myotubes were treated with either 4.8μM MIA or 10μM Dexamethasone (DEX, positive atrophic control) up to 72hrs post differentiation and sampled for morphological and mRNA analyses.ResultsSignificant morphological effects (fusion index, number of myotubes and myotube width, p<0.05) were evident, demonstrating a hypertrophic phenotype in control (CON) compared to a hyperplasic phenotype in MIA and DEX. Increases in MAFbx mRNA were also evident between conditions, with post-hoc analysis demonstrating significance between CON and DEX (p<0.001), but not between CON and MIA (p>0.05).ConclusionsThese data indicate a significant impact of both DEX and MIA on regeneration and hypertrophy in vitro and suggest differential activating mechanisms. Future investigations should determine whether skeletal muscle regeneration and hypertrophy is affected in the in vivo rodent model and the potential impact this has on the OA phenotypic outcome.

scholarly journals Neurons derived from individual early Alzheimer′s disease patients reflect clinical vulnerability

2021 ◽  
Author(s):  
Bryan Ng ◽  
Helen Rowland ◽  
Tina Wei ◽  
Kanisa Arunasalam ◽  
Emma Mee Hayes ◽  
...  
Keyword(s):  
Amyloid Beta ◽  
Brain Activity ◽  
Activity Levels ◽  
Cellular Models ◽  
Novel Approach ◽  
Induced Pluripotent ◽  
Cellular Phenotypes ◽  
The Impact

Modelling sporadic Alzheimer′s disease (sAD) with patient-derived induced pluripotent stem cells (iPSCs) is challenging yet remains an important step in understanding the disease. Here we report a novel approach of sAD modelling with patient iPSC-derived neurons by integrating cellular and clinical phenotypes from individual early symptomatic sAD patients. We establish a correlation between cellular vulnerability to extrinsic amyloid-beta in vitro measured by synapse loss with clinical vulnerability to amyloid-beta burden in vivo measured by cognitive decline and brain activity levels. Our findings indicate that patient iPSC-derived neurons not only preserve some pathological phenotypes of disease measured in the people they were derived from, but also preserve, from people to cells, the impact of those pathological phenotypes on function. Cellular models that reflect an individual′s in-life clinical vulnerability thus represent a tractable method of sAD modelling using clinical data in combination with cellular phenotypes.

Multiple muscle wasting-related transcription factors are acetylated in dexamethasone-treated muscle cells

2012 ◽  
Vol 90 (2) ◽  
pp. 200-208 ◽  
Author(s):  
Wei Chamberlain ◽  
Patricia Gonnella ◽  
Nima Alamdari ◽  
Zaira Aversa ◽  
Per-Olof Hasselgren
Keyword(s):  
Transcription Factors ◽  
Muscle Mass ◽  
Posttranslational Modifications ◽  
Muscle Wasting ◽  
Muscle Cells ◽  
L6 Myotubes ◽  
Vitro Model ◽  
L6 Muscle Cells ◽  
The Impact

Recent studies suggest that the expression and activity of the histone acetyltransferase p300 are upregulated in catabolic muscle allowing for acetylation of cellular proteins. The function of transcription factors is influenced by posttranslational modifications, including acetylation. It is not known if transcription factors involved in the regulation of muscle mass are acetylated in atrophying muscle. We determined cellular levels of acetylated C/EBPβ, C/EBPδ, FOXO1, FOXO3a, and NF-kB/p65 in dexamethasone-treated L6 muscle cells, a commonly used in vitro model of muscle wasting. The role of p300 in dexamethasone-induced transcription factor acetylation and myotube atrophy was examined by transfecting muscle cells with p300 siRNA. Treatment of L6 myotubes with dexamethasone resulted in increased cellular levels of acetylated C/EBPβ and δ, FOXO1 and 3a, and p65. Downregulation of p300 with p300 siRNA reduced acetylation of transcription factors and decreased dexamethasone-induced myotube atrophy and expression of the ubiquitin ligase MuRF1. The results suggest that several muscle wasting-related transcription factors are acetylated supporting the concept that posttranslational modifications of proteins regulating gene transcription may be involved in the loss of muscle mass. The results also suggest that acetylation of the transcription factors is at least in part regulated by p300 and plays a role in glucocorticoid-induced muscle atrophy. Targeting molecules that regulate acetylation of transcription factors may help reduce the impact of muscle wasting.

Increasing donor age adversely impacts beneficial effects of bone marrow but not smooth muscle myocardial cell therapy

2005 ◽  
Vol 289 (5) ◽  
pp. H2089-H2096 ◽  
Author(s):  
Hao Zhang ◽  
Shafie Fazel ◽  
Hai Tian ◽  
Donald A. G. Mickle ◽  
Richard D. Weisel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Smooth Muscle ◽  
Cardiac Function ◽  
Cellular Therapy ◽  
Muscle Cells ◽  
Control Group ◽  
Donor Age ◽  
Fresh Tissue ◽  
The Impact

We evaluated the impact of donor age on the efficacy of myocardial cellular therapy for ischemic cardiomyopathy. Characteristics of smooth muscle cells (SMC), bone marrow stromal cells (MSCs), and skeletal muscle cells (SKMCs) from young, adult, and old rats were compared in vitro. Three weeks after coronary ligation, 3.5 million SMCs ( n = 11) or MSCs ( n = 9) from old syngenic rats or culture medium ( n = 6) were injected into the ischemic region. Five weeks after implantation, cardiac function was assessed by echocardiography and the Langendorff apparatus. In the in vitro study, the numbers and proliferation of MSCs from fresh bone marrow and SKMCs from fresh tissue but not SMCs were markedly diminished in old animals ( P < 0.05 both groups). SKMCs from old animals did not reach confluence. After treatment with 5-azacytidine (azacitidine), the myogenic potential of old MSCs was decreased compared with young MSCs. In the in vivo study, both SMC and MSC transplantation induced significant angiogenesis compared with media injections ( P < 0.05 both groups). Transplantation of SMCs but not MSCs prevented scar thinning ( P = 0.03) and improved ejection fraction and fractional shortening ( P < 0.05). Load-independent indices of cardiac function in a Langendorff preparation confirmed improved function in the aged SMC group ( P = 0.01) but not in the MSC group compared with the control group. In conclusion, donor age adversely impacts the efficacy of cellular therapy for myocardial regeneration and is cell-type dependent. SMCs from old donors retain their ability to improve cardiac function after implantation into ischemic myocardium.

scholarly journals FMR1 loss results in early changes to intrinsic membrane excitability in human cellular models

2020 ◽  
Author(s):  
Sara G. Susco ◽  
Mario A. Arias-Garcia ◽  
Violeta G. Lopez-Huerta ◽  
Amanda Beccard ◽  
Anne M. Bara ◽  
...  
Keyword(s):  
Stem Cell ◽  
Synaptic Transmission ◽  
Cortical Neurons ◽  
Fragile X ◽  
Membrane Excitability ◽  
Cellular Models ◽  
Cortical Hyperexcitability ◽  
The Impact ◽  
Over Time

AbstractFragile X mental retardation 1 (FMR1) encodes the RNA binding protein FMRP. Loss of FMRP drives Fragile X syndrome (FXS), the leading inherited cause of intellectual disability and a leading monogenic cause of autism. Cortical hyperexcitability is a hallmark of FXS, however, the underlying mechanisms reported, including alterations in synaptic transmission and ion channel expression and properties, are heterogeneous and at times contradictory. Here, we generated isogenic FMR1y/+ and FMR1y/- human pluripotent stem cell (hPSC) lines using CRISPR-Cas9, differentiated these stem cell tools into excitatory cortical neurons and systematically assessed the impact of FMRP loss on intrinsic membrane and synaptic properties over the course of in vitro differentiation. Using whole-cell patch clamp analyses at five separate time-points, we observed significant changes in multiple metrics following FMRP loss, including decreased membrane resistance, increased capacitance, decreased action potential half-width and higher maximum frequency, consistent with FMR1y/- neurons overall showing an increased intrinsic membrane excitability compared with age-matched FMR1y/+ controls. Surprisingly, a majority of these changes emerged early during in vitro differentiation and some were not stable over time. Although we detected significant differences in intrinsic properties, no discernable alterations were observed in synaptic transmission. Collectively, this study provides a new isogenic hPSC model to study the mechanisms of FMR1 gene function, identifies electrophysiological impacts of FMRP loss on human excitatory cortical neurons over time in vitro, and underscores that early developmental changes to intrinsic membrane properties may be a critical cellular pathology contributing to cortical hyperexcitability in FXS.

Biophysical groundwork as a hinge to unravel the biology of α-synuclein aggregation and toxicity

2014 ◽  
Vol 47 (1) ◽  
pp. 1-48 ◽  
Author(s):  
Nicoletta Plotegher ◽  
Elisa Greggio ◽  
Marco Bisaglia ◽  
Luigi Bubacco
Keyword(s):  
Point Mutations ◽  
Alpha Synuclein ◽  
Aggregation Process ◽  
Gene Encoding ◽  
Cellular Models ◽  
Transient Complex ◽  
Biophysical Studies ◽  
Heterogeneous Ensemble ◽  
The Impact

AbstractAlpha-synuclein (aS) and its aggregation properties are central in the development and spread of Parkinson's disease. Point mutations and multiplications of the SNCA gene encoding aS cause autosomal dominant forms of the disorder. Moreover, protein inclusions found in the surviving neurons of parkinsonian brains consist mainly of a fibrillar form of aS. Aggregates of aS, which form a transient, complex and heterogeneous ensemble, participate in a wide variety of toxic mechanisms that may be amplified by aS spreading among neighbouring neurons. Recently, significant effort has been directed into the study of the aS aggregation process and the impact of aS aggregates on neuron survival. In this review, we present state-of-the-art biophysical studies on the aS aggregation process in vitro and in cellular models. We comprehensively review the new insights generated by the recent biophysical investigations, which could provide a solid basis from which to design future biomedical studies. The diverse cellular models of aS toxicity and their potential use in the biophysical investigation are also discussed.

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