Real Time Videomicroscopy and Semiautomated Analysis of Brain Cell Culture Models of Trinucleotide Repeat Expansion Diseases

2019 ◽  
pp. 217-240
Author(s):  
Sandra O. Braz ◽  
Diana M. Dinca ◽  
Geneviève Gourdon ◽  
Mário Gomes-Pereira
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Trinucleotide Repeat ◽  
Brain Cell ◽  
Repeat Expansion ◽  
Trinucleotide Repeat Expansion ◽  
Culture Models ◽  
Brain Cell Culture ◽  
Cell Culture Models

scholarly journals Chronic Exposure to Cadmium and Antioxidants Does Not Affect the Dynamics of Expanded CAG•CTG Trinucleotide Repeats in a Mouse Cell Culture System of Unstable DNA

2021 ◽  
Vol 14 ◽  
Author(s):  
Mário Gomes-Pereira ◽  
Darren G. Monckton
Keyword(s):  
Cell Culture ◽  
Disease Severity ◽  
Chronic Exposure ◽  
Trinucleotide Repeat ◽  
Excision Repair ◽  
Clinical Manifestations ◽  
Mouse Cell ◽  
Repeat Expansion ◽  
Dna Repeats ◽  
Trinucleotide Repeat Expansion

More than 30 human disorders are caused by the expansion of simple sequence DNA repeats, among which triplet repeats remain the most frequent. Most trinucleotide repeat expansion disorders affect primarily the nervous system, through mechanisms of neurodysfunction and/or neurodegeneration. While trinucleotide repeat tracts are short and stably transmitted in unaffected individuals, disease-associated expansions are highly dynamic in the germline and in somatic cells, with a tendency toward further expansion. Since longer repeats are associated with increasing disease severity and earlier onset of symptoms, intergenerational repeat size gains account for the phenomenon of anticipation. In turn, higher levels of age-dependent somatic expansion have been linked with increased disease severity and earlier age of onset, implicating somatic instability in the onset and progression of disease symptoms. Hence, tackling the root cause of symptoms through the control of repeat dynamics may provide therapeutic modulation of clinical manifestations. DNA repair pathways have been firmly implicated in the molecular mechanism of repeat length mutation. The demonstration that repeat expansion depends on functional DNA mismatch repair (MMR) proteins, points to MMR as a potential therapeutic target. Similarly, a role of DNA base excision repair (BER) in repeat expansion has also been suggested, particularly during the removal of oxidative lesions. Using a well-characterized mouse cell model system of an unstable CAG•CTG trinucleotide repeat, we tested if expanded repeat tracts can be stabilized by small molecules with reported roles in both pathways: cadmium (an inhibitor of MMR activity) and a variety of antioxidants (capable of neutralizing oxidative species). We found that chronic exposure to sublethal doses of cadmium and antioxidants did not result in significant reduction of the rate of trinucleotide repeat expansion. Surprisingly, manganese yielded a significant stabilization of the triplet repeat tract. We conclude that treatment with cadmium and antioxidants, at doses that do not interfere with cell survival and cell culture dynamics, is not sufficient to modify trinucleotide repeat dynamics in cell culture.

scholarly journals Factors to consider when interrogating 3D culture models with plate readers or automated microscopes

2021 ◽  
Author(s):  
Terry Riss ◽  
O. Joseph Trask
Keyword(s):  
Cell Culture ◽  
Three Dimensional ◽  
3D Culture ◽  
Fluorescent Imaging ◽  
Culture Models ◽  
Assay Methods ◽  
Diffusion Rates ◽  
Cell Culture Models ◽  
Dimensional Cell ◽  
Cells Cultured

AbstractAlong with the increased use of more physiologically relevant three-dimensional cell culture models comes the responsibility of researchers to validate new assay methods that measure events in structures that are physically larger and more complex compared to monolayers of cells. It should not be assumed that assays designed using monolayers of cells will work for cells cultured as larger three-dimensional masses. The size and barriers for penetration of molecules through the layers of cells result in a different microenvironment for the cells in the outer layer compared to the center of three-dimensional structures. Diffusion rates for nutrients and oxygen may limit metabolic activity which is often measured as a marker for cell viability. For assays that lyse cells, the penetration of reagents to achieve uniform cell lysis must be considered. For live cell fluorescent imaging assays, the diffusion of fluorescent probes and penetration of photons of light for probe excitation and fluorescent emission must be considered. This review will provide an overview of factors to consider when implementing assays to interrogate three dimensional cell culture models.

scholarly journals Trinucleotide repeat expansion length as a predictor of the clinical progression of Fuchs’ Endothelial Corneal Dystrophy

PLoS ONE ◽  
2019 ◽  
Vol 14 (1) ◽  
pp. e0210996 ◽  
Author(s):  
Yu Qiang Soh ◽  
Gary Peh Swee Lim ◽  
Hla Myint Htoon ◽  
Xin Gong ◽  
V. Vinod Mootha ◽  
...  
Keyword(s):  
Trinucleotide Repeat ◽  
Corneal Dystrophy ◽  
Repeat Expansion ◽  
Clinical Progression ◽  
Trinucleotide Repeat Expansion

scholarly journals Comparative Proteomic Characterization of 4 Human Liver-Derived Single Cell Culture Models Reveals Significant Variation in the Capacity for Drug Disposition, Bioactivation, and Detoxication

2015 ◽  
Vol 147 (2) ◽  
pp. 412-424 ◽  
Author(s):  
Rowena L. C. Sison-Young ◽  
Dimitra Mitsa ◽  
Rosalind E. Jenkins ◽  
David Mottram ◽  
Eliane Alexandre ◽  
...  
Keyword(s):  
Cell Culture ◽  
Single Cell ◽  
Significant Variation ◽  
Human Liver ◽  
Drug Disposition ◽  
Culture Models ◽  
Single Cell Culture ◽  
Cell Culture Models

scholarly journals Turnover of the Active Fraction of IRS1 Involves Raptor-mTOR- and S6K1-Dependent Serine Phosphorylation in Cell Culture Models of Tuberous Sclerosis

2006 ◽  
Vol 26 (17) ◽  
pp. 6425-6434 ◽  
Author(s):  
O. Jameel Shah ◽  
Tony Hunter
Keyword(s):  
Cell Culture ◽  
Tuberous Sclerosis ◽  
High Speed ◽  
Serine Phosphorylation ◽  
Feedback Signal ◽  
Insulin Receptor Substrates ◽  
Igf I ◽  
Culture Models ◽  
Cell Culture Models

ABSTRACT The TSC1-TSC2/Rheb/Raptor-mTOR/S6K1 cell growth cassette has recently been shown to regulate cell autonomous insulin and insulin-like growth factor I (IGF-I) sensitivity by transducing a negative feedback signal that targets insulin receptor substrates 1 and 2 (IRS1 and -2). Using two cell culture models of the familial hamartoma syndrome, tuberous sclerosis, we show here that Raptor-mTOR and S6K1 are required for phosphorylation of IRS1 at a subset of serine residues frequently associated with insulin resistance, including S307, S312, S527, S616, and S636 (of human IRS1). Using loss- and gain-of-function S6K1 constructs, we demonstrate a requirement for the catalytic activity of S6K1 in both direct and indirect regulation of IRS1 serine phosphorylation. S6K1 phosphorylates IRS1 in vitro on multiple residues showing strong preference for RXRXXS/T over S/T,P sites. IRS1 is preferentially depleted from the high-speed pellet fraction in TSC1/2-deficient mouse embryo fibroblasts or in HEK293/293T cells overexpressing Rheb. These studies suggest that, through serine phosphorylation, Raptor-mTOR and S6K1 cell autonomously promote the depletion of IRS1 from specific intracellular pools in pathological states of insulin and IGF-I resistance and thus potentially in lesions associated with tuberous sclerosis.

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