Human-Relevant Sensitivity of iPSC-Derived Human Motor Neurons to BoNT/A1 and B1

The application of botulinum neurotoxins (BoNTs) for medical treatments necessitates a potency quantification of these lethal bacterial toxins, resulting in the use of a large number of test animals. Available alternative methods are limited in their relevance, as they are based on rodent cells or neuroblastoma cell lines or applicable for single toxin serotypes only. Here, human motor neurons (MNs), which are the physiological target of BoNTs, were generated from induced pluripotent stem cells (iPSCs) and compared to the neuroblastoma cell line SiMa, which is often used in cell-based assays for BoNT potency determination. In comparison with the mouse bioassay, human MNs exhibit a superior sensitivity to the BoNT serotypes A1 and B1 at levels that are reflective of human sensitivity. SiMa cells were able to detect BoNT/A1, but with much lower sensitivity than human MNs and appear unsuitable to detect any BoNT/B1 activity. The MNs used for these experiments were generated according to three differentiation protocols, which resulted in distinct sensitivity levels. Molecular parameters such as receptor protein concentration and electrical activity of the MNs were analyzed, but are not predictive for BoNT sensitivity. These results show that human MNs from several sources should be considered in BoNT testing and that human MNs are a physiologically relevant model, which could be used to optimize current BoNT potency testing.

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Split luciferase-based assay to detect botulinum neurotoxins using human motor neurons derived from induced pluripotent stem cell.

10.21203/rs.3.rs-730824/v1 ◽

2021 ◽

Author(s):

Laurent Cotter ◽

Feifan Yu ◽

Juliette Duschene De Lamotte ◽

Min Dong ◽

Johannes Krupp ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Motor Neurons ◽

Pluripotent Stem Cells ◽

Detection System ◽

Detection Sensitivity ◽

Concanamycin A ◽

Botulinum Neurotoxins ◽

Induced Pluripotent

Abstract Botulinum neurotoxins (BoNTs) have been widely used clinically as a muscle relaxant. These toxins target motor neurons and cleave proteins essential for neurotransmitter release like Synaptosomal-associated protein of 25 kDa (SNAP-25). Most in vitro assays for BoNT testing use rodent cells or immortalized cell lines, which showed limitations in accuracy and physiological relevance. Here, we report a cell-based assay for detecting SNAP25-cleaving BoNTs by combining human induced Pluripotent Stem Cells (hiPSC)-derived motor neurons and a luminescent detection system based on split nanoluc luciferase. This assay is convenient, rapid, free-of-specialized antibodies, and can discriminate the potency of different BoNTs, with a detection sensitivity of femtomolar concentrations of toxin and can be used to study the different steps of BoNT intoxication. Abreviations: BoNT, Botulinum neurotoxin, SNAP-25, Synaptosomal-associated protein of 25 kDa, hiPSC, human induced Pluripotent Stem Cells, SNARE, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor, SV2, synaptic vesicle proteins, MLB, mouse lethality bioassay, LD50, toxin’s dose lethal for half of the animal injected, CB-assay, cell-based assays, FRET, Förster resonance energy transfer, Concanamycin A, EC50, Half maximal effective concentration, MNs, motor neurons.

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Novel chemical inhibitor against SOD1 misfolding and aggregation protects neuron-loss and ameliorates disease symptoms in ALS mouse model

Communications Biology ◽

10.1038/s42003-021-02862-z ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Tae-Gyun Woo ◽

Min-Ho Yoon ◽

So-mi Kang ◽

Soyoung Park ◽

Jung-Hyun Cho ◽

...

Keyword(s):

Motor Neurons ◽

Neuroblastoma Cell ◽

Neuroblastoma Cell Line ◽

Human Neuroblastoma Cell ◽

Human Neuroblastoma ◽

Mutant Sod1 ◽

Chemical Screening ◽

Model Mouse ◽

Extended Lifespan ◽

Selective Death

AbstractAmyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective death of motor neurons. Mutations in Cu, Zn-superoxide dismutase (SOD1) causing the gain of its toxic property are the major culprit of familial ALS (fALS). The abnormal SOD1 aggregation in the motor neurons has been suggested as the major pathological hallmark of ALS patients. However, the development of pharmacological interventions against SOD1 still needs further investigation. In this study, using ELISA-based chemical screening with wild and mutant SOD1 proteins, we screened a new small molecule, PRG-A01, which could block the misfolding/aggregation of SOD1 or TDP-43. The drug rescued the cell death induced by mutant SOD1 in human neuroblastoma cell line. Administration of PRG-A01 into the ALS model mouse resulted in significant improvement of muscle strength, motor neuron viability and mobility with extended lifespan. These results suggest that SOD1 misfolding/aggregation is a potent therapeutic target for SOD1 related ALS.

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Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition

10.1101/2020.10.21.346874 ◽

2020 ◽

Author(s):

Katarina Stoklund Dittlau ◽

Emily N. Krasnow ◽

Laura Fumagalli ◽

Tijs Vandoorne ◽

Pieter Baatsen ◽

...

Keyword(s):

Motor Neuron ◽

Neurite Outgrowth ◽

Motor Neurons ◽

Neuromuscular Junctions ◽

Induced Pluripotent Stem Cell ◽

Microfluidic Devices ◽

Motor Units ◽

Human Motor ◽

Induced Pluripotent

AbstractNeuromuscular junctions (NMJs) ensure proper communication between motor neurons and muscle through the release of neurotransmitters. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS), NMJs degenerate resulting in muscle atrophy, paralysis and respiratory failure. The aim of this study was to establish a versatile and reproducible in vitro model of a human motor unit to study the effect of ALS-causing mutations. Therefore, we generated a co-culture of human induced pluripotent stem cell-derived motor neurons and human primary mesoangioblast-derived myotubes in microfluidic devices. A chemotactic and volumetric gradient facilitated the growth of motor neuron neurites through microgrooves resulting in the interaction with myotubes and the formation of NMJs. We observed that ALS-causing FUS mutations resulted in a reduced neurite outgrowth and in a decreased NMJ number. Interestingly, the selective HDAC6 inhibitor, Tubastatin A, improved the neurite outgrowth and the NMJ morphology of FUS-ALS co-cultures, further prompting HDAC6 inhibition as a potential therapeutic strategy for ALS.

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hiPSC-Derived Neurons Provide a Robust and Physiologically Relevant In Vitro Platform to Test Botulinum Neurotoxins

Frontiers in Pharmacology ◽

10.3389/fphar.2020.617867 ◽

2021 ◽

Vol 11 ◽

Author(s):

Juliette Duchesne De Lamotte ◽

Sylvain Roqueviere ◽

Hélène Gautier ◽

Elsa Raban ◽

Céline Bouré ◽

...

Keyword(s):

Motor Neurons ◽

Neurological Diseases ◽

Physiological Relevance ◽

Botulinum Neurotoxins ◽

Zinc Metalloproteases ◽

Induced Pluripotent ◽

Immortalized Cell ◽

Effective Drugs ◽

Human Ipsc

Botulinum neurotoxins (BoNTs) are zinc metalloproteases that block neurotransmitter release at the neuromuscular junction (NMJ). Their high affinity for motor neurons combined with a high potency have made them extremely effective drugs for the treatment of a variety of neurological diseases as well as for aesthetic applications. Current in vitro assays used for testing and developing BoNT therapeutics include primary rodent cells and immortalized cell lines. Both models have limitations concerning accuracy and physiological relevance. In order to improve the translational value of preclinical data there is a clear need to use more accurate models such as human induced Pluripotent Stem Cells (hiPSC)-derived neuronal models. In this study we have assessed the potential of four different human iPSC-derived neuronal models including Motor Neurons for BoNT testing. We have characterized these models in detail and found that all models express all proteins needed for BoNT intoxication and showed that all four hiPSC-derived neuronal models are sensitive to both serotype A and E BoNT with Motor Neurons being the most sensitive. We showed that hiPSC-derived Motor Neurons expressed authentic markers after only 7 days of culture, are functional and able to form active synapses. When cultivated with myotubes, we demonstrated that they can innervate myotubes and induce contraction, generating an in vitro model of NMJ showing dose-responsive sensitivity BoNT intoxication. Together, these data demonstrate the promise of hiPSC-derived neurons, especially Motor Neurons, for pharmaceutical BoNT testing and development.

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HMGB1 Receptors as Potential Novel Targets for Neuroblastoma

Proceedings of IMPRS ◽

10.18060/23467 ◽

2019 ◽

Vol 2 (1) ◽

Author(s):

Christian Garcia ◽

Jared Smith ◽

Peter Malicky ◽

Fletcher White

Keyword(s):

Cell Line ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

High Mobility ◽

Neuroblastoma Cell Line ◽

Receptor Protein ◽

Cellular Mechanisms ◽

Cxcr4 Receptor ◽

Sds Page ◽

Secondary Antibodies

Background and Hypothesis: The pathogenesis of neuroblastoma remains elusive. In order to further understand the pathogenesis, it is necessary to identify molecular and cellular mechanisms of the cancer. One protein of interest is high-mobility group box 1 protein (HMGB1). HMGB1 has been implicated in cancer including tumor growth, invasion, angiogenesis, metastasis, relapse and therapeutic resistance. HMGB1 isoforms signals via different receptors including the chemokine receptor, CXCR4, receptor for advanced glycation end products (RAGE), and the prominent inflammation-associated receptor, toll-like receptor 4 (TLR4). To gain insight into the possible impact of HMGB1 on the SH-SY5Y neuroblastoma cell line, we determined the degree to which CXCR4, TLR4 and RAGE are present on SH-SY5Y cells. We hypothesize that the presence of these receptors in SH-SY5Y cells may mediate proliferation of neuroblastoma cells and other types of cancer. Experimental Design or Project Methods: The SH-SY5Y cell line (ATCC® CRL-2266™) was derived from a metastatic bone tumor in a 4 year old female. Cells were lysed in lysis buffer, electrophoresed on a 10% SDS-PAGE, and blotted onto PVDF membrane. After blocking, the membranes were incubated with primary antibodies against the receptor protein overnight at 4 °C, and then with HRP-conjugated secondary antibodies for 1h. Protein bands were visualized with a SuperSignal West Pico Chemiluminescent Substrate. Results: To be finalized Conclusion and Potential Impact: Investigating SH-SY5Y cell line for the presence of these inflammation-associated receptors could potentially serve as a model to better understand the role of these receptors in cancer research.

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Post-transcriptional remodelling is temporally deregulated during motor neurogenesis in human ALS models

10.1101/180372 ◽

2017 ◽

Author(s):

Raphaelle Luisier ◽

Giulia E. Tyzack ◽

Claire E. Hall ◽

Jernej Ule ◽

Nicholas M. Luscombe ◽

...

Keyword(s):

Motor Neurons ◽

Rna Processing ◽

Neuronal Development ◽

Intron Retention ◽

Length Variation ◽

List Type ◽

Main Mode ◽

Transcriptional Changes ◽

Human Motor ◽

Induced Pluripotent

SUMMARYMutations causing amyotrophic lateral sclerosis (ALS) strongly implicate regulators of RNA-processing that are ubiquitously expressed throughout development. To understand the molecular impact of ALS-causing mutations on early neuronal development and disease, we performed transcriptomic analysis of differentiated human control and VCP-mutant induced pluripotent stem cells (iPSCs) during motor neurogenesis. We identify intron retention (IR) as the predominant splicing change affecting early stages of wild-type neural differentiation, targeting key genes involved in the splicing machinery. Importantly, IR occurs prematurely in VCP-mutant cultures compared with control counterparts; these events are also observed in independent RNAseq datasets from SOD1- and FUS-mutant motor neurons (MNs). Together with related effects on 3’UTR length variation, these findings implicate alternative RNA-processing in regulating distinct stages of lineage restriction from iPSCs to MNs, and reveal a temporal deregulation of such processing by ALS mutations. Thus, ALS-causing mutations perturb the same post-transcriptional mechanisms that underlie human motor neurogenesis.HIGHLIGHTSIntron retention is the main mode of alternative splicing in early differentiation.The ALS-causing VCP mutation leads to premature intron retention.Increased intron retention is seen with multiple ALS-causing mutations.Transcriptional programs are unperturbed despite post-transcriptional defects.eTOC BLURBLuisier et al. identify post-transcriptional changes underlying human motor neurogenesis: extensive variation in 3’ UTR length and intron retention (IR) are the early predominant modes of splicing. The VCP mutation causes IR to occur prematurely during motor neurogenesis and these events are validated in other ALS-causing mutations, SOD1 and FUS.

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Analysis of Motor Neurons Differentiated from Human Induced Pluripotent Stem Cells for the Use in Cell-Based Botulinum Neurotoxin Activity Assays

Toxins ◽

10.3390/toxins12050276 ◽

2020 ◽

Vol 12 (5) ◽

pp. 276

Author(s):

Maren Schenke ◽

Brit-Maren Schjeide ◽

Gerhard P. Püschel ◽

Bettina Seeger

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Motor Neurons ◽

Pluripotent Stem Cells ◽

Neurotransmitter Release ◽

Neuroblastoma Cell ◽

Synaptic Proteins ◽

Detection Methods ◽

Induced Pluripotent

Botulinum neurotoxins (BoNTs) are potent neurotoxins produced by bacteria, which inhibit neurotransmitter release, specifically in their physiological target known as motor neurons (MNs). For the potency assessment of BoNTs produced for treatment in traditional and aesthetic medicine, the mouse lethality assay is still used by the majority of manufacturers, which is ethically questionable in terms of the 3Rs principle. In this study, MNs were differentiated from human induced pluripotent stem cells based on three published protocols. The resulting cell populations were analyzed for their MN yield and their suitability for the potency assessment of BoNTs. MNs produce specific gangliosides and synaptic proteins, which are bound by BoNTs in order to be taken up by receptor-mediated endocytosis, which is followed by cleavage of specific soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins required for neurotransmitter release. The presence of receptors and substrates for all BoNT serotypes was demonstrated in MNs generated in vitro. In particular, the MN differentiation protocol based on Du et al. yielded high numbers of MNs in a short amount of time with high expression of BoNT receptors and targets. The resulting cells are more sensitive to BoNT/A1 than the commonly used neuroblastoma cell line SiMa. MNs are, therefore, an ideal tool for being combined with already established detection methods.

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Bioink Composition and Printing Parameters for 3D Modeling Neural Tissue

Cells ◽

10.3390/cells8080830 ◽

2019 ◽

Vol 8 (8) ◽

pp. 830 ◽

Cited By ~ 10

Author(s):

Valentina Fantini ◽

Matteo Bordoni ◽

Franca Scocozza ◽

Michele Conti ◽

Eveljn Scarian ◽

...

Keyword(s):

Stem Cells ◽

Broad Class ◽

Neuroblastoma Cell ◽

Neural Tissue ◽

Cell Types ◽

Neuroblastoma Cell Line ◽

3D Bioprinting ◽

Vitro Model ◽

Induced Pluripotent

Neurodegenerative diseases (NDs) are a broad class of pathologies characterized by the progressive loss of neurons in the central nervous system. The main problem in the study of NDs is the lack of an adequate realistic experimental model to study the pathogenic mechanisms. Induced pluripotent stem cells (iPSCs) partially overcome the problem, with their capability to differentiate into almost every cell types; even so, these cells alone are not sufficient to unveil the mechanisms underlying NDs. 3D bioprinting allows to control the distribution of cells such as neurons, leading to the creation of a realistic in vitro model. In this work, we analyzed two biomaterials: sodium alginate and gelatin, and three different cell types: a neuroblastoma cell line (SH-SY5Y), iPSCs, and neural stem cells. All cells were encapsulated inside the bioink, printed and cultivated for at least seven days; they all presented good viability. We also evaluated the maintenance of the printed shape, opening the possibility to obtain a reliable in vitro neural tissue combining 3D bioprinting and iPSCs technology, optimizing the study of the degenerative processes that are still widely unknown.

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Botulinum neurotoxins A, B, C, E, and F preferentially enter cultured human motor neurons compared to other cultured human neuronal populations

FEBS Letters ◽

10.1002/1873-3468.13508 ◽

2019 ◽

Vol 593 (18) ◽

pp. 2675-2685 ◽

Cited By ~ 3

Author(s):

Sabine Pellett ◽

William H. Tepp ◽

Eric A. Johnson

Keyword(s):

Motor Neurons ◽

Neuronal Populations ◽

Botulinum Neurotoxins ◽

Human Motor

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Chchd10 or Chchd2 are not Required for Human Motor Neuron Differentiation In Vitro but Modify Synaptic Transcriptomes

10.1101/828376 ◽

2019 ◽

Author(s):

Sandra Harjuhaahto ◽

Tiina S Rasila ◽

Svetlana M Molchanova ◽

Rosa Woldegebriel ◽

Jouni Kvist ◽

...

Keyword(s):

Motor Neurons ◽

Muscular Atrophy ◽

Axonal Neuropathy ◽

Synaptic Function ◽

Control Line ◽

Intermembrane Space ◽

Mitochondrial Intermembrane Space ◽

Human Motor ◽

Induced Pluripotent

ABSTRACTMitochondrial intermembrane space proteins CHCHD2 and CHCHD10 have roles in diseases affecting motor neurons such as amyotrophic lateral sclerosis, spinal muscular atrophy and axonal neuropathy and in Parkinson’s disease, and form a complex of unknown function. Here we address the importance of these two proteins in human motor neurons. We show that gene edited human induced pluripotent stem cells (iPSC) lacking either CHCHD2 or CHCHD10 are viable and can be differentiated into functional motor neurons that fire spontaneous and evoked action potentials. Knockout iPSC and motor neurons sustain mitochondrial ultrastructure and show reciprocal compensatory increases in CHCHD2 or CHCHD10. Knockout motor neurons have largely overlapping transcriptome profiles compared to isogenic control line, in particular for synaptic gene expression. Our results show that absence of CHCHD2 or CHCHD10 does not disrupt functionality, but induces similar modifications in human motor neurons. Thus pathogenic mechanisms may involve loss of synaptic function.

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