scholarly journals High-Throughput Phenotypic Assay for Compounds That Influence Mitochondrial Health Using iPSC-Derived Human Neurons

2021 ◽  
pp. 247255522110006
Author(s):  
Courtney MacMullen ◽  
Ronald L. Davis
Keyword(s):  
Neurodegenerative Diseases ◽  
High Throughput ◽  
High Throughput Screening ◽  
Mitochondrial Dynamics ◽  
Live Cell ◽  
Phenotypic Assay ◽  
Microtiter Plates ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
And Function

There is a critical need to develop high-throughput assays to identify compounds that offer therapy for individuals suffering from neurodegenerative diseases. Most brain disorders, including neurodegenerative diseases, share the common neuropathology of mitochondria dysfunction, which can lead to apoptosis of neurons, overproduction of reactive oxygen species (ROS), and other cellular neuropathologies characteristic of these diseases. Human induced pluripotent stem cells (iPSCs) with a stable genomic insertion of the neurogenin-2 transcription factor under the control of the TetOn promoter can be differentiated into excitatory human neurons (i3Neurons) within 3 days of exposure to doxycycline. These neurons have been used to develop and validate a live-cell assay for parameters of mitochondrial dynamics and function using two compounds known to promote mitochondrial elongation in mouse neurons, 4-hydroxychalcone and 2,4-dihyrdroxychalcone. The assay involves plating the neurons in 384-well microtiter plates, treating them with known or unknown substances, and then capturing morphological information for the neuronal mitochondria using a lentivirus vector to express a mitochondrial-targeted fluorescence reporter. The i3Neuron cultures exposed to these two compounds for 24 h exhibit significantly decreased circularity and significantly increased length compared to controls, two morphological parameters correlated with increased mitochondrial health. The assay is rapid, with results obtained after a one-week-long i3Neuron culture or one month if neurons are co-cultured with astrocytes. This live-cell, mitochondrial phenotypic assay can be used for high-throughput screening or as an orthogonal assay for compounds obtained via other high-throughput screening campaigns.

scholarly journals A high-throughput screening assay based on automated microscopy for monitoring antibiotic susceptibility of Mycobacterium tuberculosis phenotypes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sadaf Kalsum ◽  
Blanka Andersson ◽  
Jyotirmoy Das ◽  
Thomas Schön ◽  
Maria Lerm
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Imaging System ◽  
Aggregate Size ◽  
Live Cell ◽  
Screening Assay ◽  
High Throughput Screening Assay

Abstract Background Efficient high-throughput drug screening assays are necessary to enable the discovery of new anti-mycobacterial drugs. The purpose of our work was to develop and validate an assay based on live-cell imaging which can monitor the growth of two distinct phenotypes of Mycobacterium tuberculosis and to test their susceptibility to commonly used TB drugs. Results Both planktonic and cording phenotypes were successfully monitored as fluorescent objects using the live-cell imaging system IncuCyte S3, allowing collection of data describing distinct characteristics of aggregate size and growth. The quantification of changes in total area of aggregates was used to define IC50 and MIC values of selected TB drugs which revealed that the cording phenotype grew more rapidly and displayed a higher susceptibility to rifampicin. In checkerboard approach, testing pair-wise combinations of sub-inhibitory concentrations of drugs, rifampicin, linezolid and pretomanid demonstrated superior growth inhibition of cording phenotype. Conclusions Our results emphasize the efficiency of using automated live-cell imaging and its potential in high-throughput whole-cell screening to evaluate existing and search for novel antimycobacterial drugs.

scholarly journals Non-invasive and high-throughput interrogation of exon-specific isoform expression

2021 ◽  
Author(s):  
Dong-Jiunn Jeffery Truong ◽  
Teeradon Phlairaharn ◽  
Bianca Eßwein ◽  
Christoph Gruber ◽  
Deniz Tümen ◽  
...  
Keyword(s):  
Stem Cells ◽  
High Throughput ◽  
High Throughput Screening ◽  
Embryonic Stem ◽  
High Sensitivity ◽  
Therapeutic Interventions ◽  
Dominant Factor ◽  
Reporter System ◽  
Isoform Expression ◽  
Induced Pluripotent

AbstractExpression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.

scholarly journals Fragile X syndrome patient-derived neurons developing in the mouse brain show FMR1 -dependent phenotypes

2021 ◽  
Author(s):  
Marine A Krzisch ◽  
Hao A Wu ◽  
Bingbing Yuan ◽  
Troy W. Whitfield ◽  
X. Shawn Liu ◽  
...  
Keyword(s):  
Fragile X Syndrome ◽  
Neuronal Development ◽  
Fragile X ◽  
In Vitro Models ◽  
Synaptic Activity ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
And Function ◽  
The Brain

Abnormal neuronal development in Fragile X syndrome (FXS) is poorly understood. Data on FXS patients remain scarce and FXS animal models have failed to yield successful therapies. In vitro models do not fully recapitulate the morphology and function of human neurons. Here, we co-injected neural precursor cells (NPCs) from FXS patient-derived and corrected isogenic control induced pluripotent stem cells into the brain of neonatal immune-deprived mice. The transplanted cells populated the brain and a proportion differentiated into neurons and glial cells. Single-cell RNA sequencing of transplanted cells revealed upregulated excitatory synaptic transmission and neuronal differentiation pathways in FXS neurons. Immunofluorescence analyses showed accelerated maturation of FXS neurons after an initial delay. Additionally, increased percentages of Arc- and Egr1-positive FXS neurons and wider dendritic protrusions of mature FXS striatal medium spiny neurons pointed to an increase in synaptic activity and synaptic strength as compared to control. This transplantation approach provides new insights into the alterations of neuronal development in FXS by facilitating physiological development of cells in a 3D context, and could be used to test new therapeutic compounds correcting neuronal development defects in FXS.

High-throughput screening of human induced pluripotent stem cell-derived brain organoids

2020 ◽  
Vol 335 ◽  
pp. 108627 ◽  
Author(s):  
Madel Durens ◽  
Jonathan Nestor ◽  
Madeline Williams ◽  
Kevin Herold ◽  
Robert F. Niescier ◽  
...  
Keyword(s):  
Stem Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Induced Pluripotent

scholarly journals A 96-multiplex capillary electrophoresis screening platform for product based evolution of P450 BM3

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Anna Gärtner ◽  
Anna Joëlle Ruff ◽  
Ulrich Schwaneberg
Keyword(s):  
Capillary Electrophoresis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Product Formation ◽  
Resonance Spectroscopy ◽  
P450 Bm3 ◽  
Microtiter Plates ◽  
Main Challenge ◽  
Total Turnover ◽  
Screening Systems

Abstract The main challenge that prevents a broader application of directed enzyme evolution is the lack of high-throughput screening systems with universal product analytics. Most directed evolution campaigns employ screening systems based on colorimetric or fluorogenic surrogate substrates or universal quantification methods such as nuclear magnetic resonance spectroscopy or mass spectrometry, which have not been advanced to achieve a high-throughput. Capillary electrophoresis with a universal UV-based product detection is a promising analytical tool to quantify product formation. Usage of a multiplex system allows the simultaneous measurement with 96 capillaries. A 96-multiplexed capillary electrophoresis (MP-CE) enables a throughput that is comparable to traditional direct evolution campaigns employing 96-well microtiter plates. Here, we report for the first time the usage of a MP-CE system for directed P450 BM3 evolution towards increased product formation (oxidation of alpha-isophorone to 4-hydroxy-isophorone; highest reached total turnover number after evolution campaign: 7120 mol4-OH molP450−1). The MP-CE platform was 3.5-fold more efficient in identification of beneficial variants than the standard cofactor (NADPH) screening system.

scholarly journals Actin-binding compounds, previously discovered by FRET-based high-throughput screening, differentially affect skeletal and cardiac muscle

2020 ◽  
Vol 295 (41) ◽  
pp. 14100-14110 ◽  
Author(s):  
Piyali Guhathakurta ◽  
Lien A. Phung ◽  
Ewa Prochniewicz ◽  
Sarah Lichtenberger ◽  
Anna Wilson ◽  
...  
Keyword(s):  
Cardiac Muscle ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cell Types ◽  
Actin Binding ◽  
Time Resolved ◽  
Biochemical Assays ◽  
Numerous Cell ◽  
Cardiac Muscles ◽  
And Function

Actin's interactions with myosin and other actin-binding proteins are essential for cellular viability in numerous cell types, including muscle. In a previous high-throughput time-resolved FRET (TR-FRET) screen, we identified a class of compounds that bind to actin and affect actomyosin structure and function. For clinical utility, it is highly desirable to identify compounds that affect skeletal and cardiac muscle differently. Because actin is more highly conserved than myosin and most other muscle proteins, most such efforts have not targeted actin. Nevertheless, in the current study, we tested the specificity of the previously discovered actin-binding compounds for effects on skeletal and cardiac α-actins as well as on skeletal and cardiac myofibrils. We found that a majority of these compounds affected the transition of monomeric G-actin to filamentous F-actin, and that several of these effects were different for skeletal and cardiac actin isoforms. We also found that several of these compounds affected ATPase activity differently in skeletal and cardiac myofibrils. We conclude that these structural and biochemical assays can be used to identify actin-binding compounds that differentially affect skeletal and cardiac muscles. The results of this study set the stage for screening of large chemical libraries for discovery of novel compounds that act therapeutically and specifically on cardiac or skeletal muscle.

scholarly journals Discovery of SERCA2a/PLB Activators and Inhibitors by Structure-Based High-throughput Screening using Live Cell FRET Biosensors

2018 ◽  
Vol 114 (3) ◽  
pp. 147a
Author(s):  
Daniel R. Stroik ◽  
Samantha L. Yuen ◽  
Kevyn A. Janicek ◽  
Tory M. Schaaf ◽  
Razvan L. Cornea ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Live Cell ◽  
Fret Biosensors

scholarly journals Development of a Scalable, High-Throughput-Compatible Assay to Detect Tau Aggregates Using iPSC-Derived Cortical Neurons Maintained in a Three-Dimensional Culture Format

2016 ◽  
Vol 21 (8) ◽  
pp. 804-815 ◽  
Author(s):  
X. Medda ◽  
L. Mertens ◽  
S. Versweyveld ◽  
A. Diels ◽  
L. Barnham ◽  
...  
Keyword(s):  
High Throughput ◽  
Cortical Neurons ◽  
High Throughput Screening ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Novel Treatments ◽  
Entry Points ◽  
Induced Pluripotent ◽  
Tau Aggregation

Tau aggregation is the pathological hallmark that best correlates with the progression of Alzheimer’s disease (AD). The presence of neurofibrillary tangles (NFTs), formed of hyperphosphorylated tau, leads to neuronal dysfunction and loss, and is directly associated with the cognitive decline observed in AD patients. The limited success in targeting β-amyloid pathologies has reinforced the hypothesis of blocking tau phosphorylation, aggregation, and/or spreading as alternative therapeutic entry points to treat AD. Identification of novel therapies requires disease-relevant and scalable assays capable of reproducing key features of the pathology in an in vitro setting. Here we use induced pluripotent stem cells (iPSCs) as a virtually unlimited source of human cortical neurons to develop a robust and scalable tau aggregation model compatible with high-throughput screening (HTS). We downscaled cell culture conditions to 384-well plate format and used Matrigel to introduce an extra physical protection against cell detachment that reduces shearing stress and better recapitulates pathological conditions. We complemented the assay with AlphaLISA technology for the detection of tau aggregates in a high-throughput-compatible format. The assay is reproducible across users and works with different commercially available iPSC lines, representing a highly translational tool for the identification of novel treatments against tauopathies, including AD.

scholarly journals Upscaling of hiPS Cell–Derived Neurons for High-Throughput Screening

2016 ◽  
Vol 22 (3) ◽  
pp. 274-286
Author(s):  
Stefanie Traub ◽  
Heiko Stahl ◽  
Holger Rosenbrock ◽  
Eric Simon ◽  
Ralf Heilker
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Detection Sensitivity ◽  
Neuronal Markers ◽  
Downstream Target ◽  
Electrophysiological Recordings ◽  
Downstream Target Gene ◽  
The Central Nervous System ◽  
Induced Pluripotent

The advent of human-induced pluripotent stem (hiPS) cell–derived neurons promised to provide better model cells for drug discovery in the context of the central nervous system. This work demonstrates both the upscaling of cellular expansion and the acceleration of neuronal differentiation to accommodate the immense material needs of a high-throughput screening (HTS) approach. Using GRowth factor–driven expansion and INhibition of NotCH (GRINCH) during maturation, the derived cells are here referred to as GRINCH neurons. GRINCH cells displayed neuronal markers, and their functional activity could be demonstrated by electrophysiological recordings. In an application of GRINCH neurons, the brain-derived neurotrophic factor (BDNF)–mediated activation of tropomyosin receptor kinase (TrkB) was investigated as a promising drug target to treat synaptic dysfunctions. To assess the phosphorylation of endogenous TrkB in the GRINCH cells, the highly sensitive amplified luminescent proximity homogeneous assay LISA (AlphaLISA) format was established as a primary screen. A high-throughput reverse transcription (RT)–PCR format was employed as a secondary assay to analyze TrkB-mediated downstream target gene expression. In summary, an optimized differentiation protocol, highly efficient cell upscaling, and advanced assay miniaturization, combined with increased detection sensitivity, pave the way for a new generation of predictive cell-based drug discovery.

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