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

FEBS Letters ◽  
2019 ◽  
Vol 593 (18) ◽  
pp. 2675-2685 ◽  
Author(s):  
Sabine Pellett ◽  
William H. Tepp ◽  
Eric A. Johnson
Keyword(s):  
Motor Neurons ◽  
Neuronal Populations ◽  
Botulinum Neurotoxins ◽  
Human Motor

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

Toxins ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 585
Author(s):  
Maren Schenke ◽  
Hélène-Christine Prause ◽  
Wiebke Bergforth ◽  
Adina Przykopanski ◽  
Andreas Rummel ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cell Line ◽  
Alternative Methods ◽  
Receptor Protein ◽  
Mouse Bioassay ◽  
Lower Sensitivity ◽  
Botulinum Neurotoxins ◽  
Human Motor ◽  
Induced Pluripotent

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.

scholarly journals Image-based deep learning reveals the responses of human motor neurons to stress and ALS

2021 ◽  
Author(s):  
Colombine Verzat ◽  
Jasmine Harley ◽  
Rickie Patani ◽  
Raphaëlle Luisier
Keyword(s):  
Deep Learning ◽  
Motor Neurons ◽  
Rna Binding ◽  
Relevant Information ◽  
Morphological Attributes ◽  
Fluorescent Markers ◽  
Human Motor ◽  
Microscopy Data ◽  
Key Aspects ◽  
Lateral Sclerosis

SUMMARYAlthough morphological attributes of cells and their substructures are recognized readouts of physiological or pathophysiological states, these have been relatively understudied in amyotrophic lateral sclerosis (ALS) research. In this study we integrate multichannel fluorescence high-content microscopy data with deep-learning imaging methods to reveal - directly from unsegmented images - novel neurite-associated morphological perturbations associated with (ALS-causing) VCP-mutant human motor neurons (MNs). Surprisingly, we reveal that previously unrecognized disease-relevant information is withheld in broadly used and often considered ‘generic’ biological markers of nuclei (DAPI) and neurons (βIII-tubulin). Additionally, we identify changes within the information content of ALS-related RNA binding protein (RBP) immunofluorescence imaging that is captured in VCP-mutant MN cultures. Furthermore, by analyzing MN cultures exposed to different extrinsic stressors, we show that heat stress recapitulates key aspects of ALS. Our study therefore reveals disease-relevant information contained in a range of both generic and more specific fluorescent markers, and establishes the use of image-based deep learning methods for rapid, automated and unbiased testing of biological hypotheses.

scholarly journals Loss of TBK1 activity leads to TDP-43 proteinopathy through lysosomal dysfunction in human motor neurons

2021 ◽  
Author(s):  
Jin Hao ◽  
Michael F Wells ◽  
Gengle Niu ◽  
Irune Guerra San Juan ◽  
Francesco Limone ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Neurodegenerative Disease ◽  
Motor Neurons ◽  
Inhibition Mechanism ◽  
Loss Of Function ◽  
Neurodegenerative Process ◽  
Lysosomal Dysfunction ◽  
Human Motor ◽  
Lateral Sclerosis ◽  
Lysosomal Acidification

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron loss accompanied by cytoplasmic localization of TDP-43 proteins and their insoluble accumulations. Haploinsufficiency of TBK1 has been found to associate with or cause ALS. However, the cell-autonomous mechanisms by which reduced TBK1 activity contributes to human motor neuron pathology remain elusive. Here, we generated a human cellular model harboring loss-of-function mutations of TBK1 by gene editing and found that TBK1 deficiency was sufficient to cause TDP-43 pathology in human motor neurons. In addition to its functions in autophagy, we found that TBK1 interacted with endosomes and was required for normal endosomal maturation and subsequent lysosomal acidification. Surprisingly, TDP-43 pathology resulted more from the dysfunctional endo-lysosomal pathway than the previously recognized autophagy inhibition mechanism. Restoring TBK1 levels ameliorated lysosomal dysfunction and TDP-43 pathology and maintained normal motor neuron homeostasis. Notably, using patient-derived motor neurons, we found that haploinsufficiency of TBK1 sensitized neurons to lysosomal stress, and chemical regulators of endosomal maturation rescued the neurodegenerative process. Together, our results revealed the mechanism of TBK1 in maintaining TDP-43 and motor neuron homeostasis and suggested that modulating endosomal maturation was able to rescue neurodegenerative disease phenotypes caused by TBK1 deficiency.

scholarly journals Single cell transcriptome profiling of the human developing spinal cord reveals a conserved genetic programme with human specific features

2021 ◽  
Author(s):  
Teresa Rayon ◽  
Rory J. Maizels ◽  
Christopher Barrington ◽  
James Briscoe
Keyword(s):  
Gene Expression ◽  
Spinal Cord ◽  
Single Cell ◽  
Motor Neurons ◽  
Neural Tube ◽  
Transcriptome Profiling ◽  
Cell Types ◽  
Human Embryos ◽  
Neuronal Populations ◽  
Cell Transcriptome

AbstractThe spinal cord receives input from peripheral sensory neurons and controls motor output by regulating muscle innervating motor neurons. These functions are carried out by neural circuits comprising molecularly and physiologically distinct neuronal subtypes that are generated in a characteristic spatial-temporal arrangement from progenitors in the embryonic neural tube. The systematic mapping of gene expression in mouse embryos has provided insight into the diversity and complexity of cells in the neural tube. For human embryos, however, less information has been available. To address this, we used single cell mRNA sequencing to profile cervical and thoracic regions in four human embryos of Carnegie Stages (CS) CS12, CS14, CS17 and CS19 from Gestational Weeks (W) 4-7. In total we recovered the transcriptomes of 71,219 cells. Analysis of progenitor and neuronal populations from the neural tube, as well as cells of the peripheral nervous system, in dorsal root ganglia adjacent to the neural tube, identified dozens of distinct cell types and facilitated the reconstruction of the differentiation pathways of specific neuronal subtypes. Comparison with existing mouse datasets revealed the overall similarity of mouse and human neural tube development while highlighting specific features that differed between species. These data provide a catalogue of gene expression and cell type identity in the developing neural tube that will support future studies of sensory and motor control systems and can be explored at https://shiny.crick.ac.uk/scviewer/neuraltube/.

scholarly journals Botulinum and Tetanus Neurotoxins

2019 ◽  
Vol 88 (1) ◽  
pp. 811-837 ◽  
Author(s):  
Min Dong ◽  
Geoffrey Masuyer ◽  
Pål Stenmark
Keyword(s):  
Motor Neurons ◽  
Host Proteins ◽  
Tetanus Neurotoxin ◽  
Synaptic Vesicle Exocytosis ◽  
Receptor Recognition ◽  
Toxin Complex ◽  
Vesicle Exocytosis ◽  
Botulinum Neurotoxins ◽  
Distinct Features ◽  
Progenitor Toxin

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the most potent toxins known and cause botulism and tetanus, respectively. BoNTs are also widely utilized as therapeutic toxins. They contain three functional domains responsible for receptor-binding, membrane translocation, and proteolytic cleavage of host proteins required for synaptic vesicle exocytosis. These toxins also have distinct features: BoNTs exist within a progenitor toxin complex (PTC), which protects the toxin and facilitates its absorption in the gastrointestinal tract, whereas TeNT is uniquely transported retrogradely within motor neurons. Our increasing knowledge of these toxins has allowed the development of engineered toxins for medical uses. The discovery of new BoNTs and BoNT-like proteins provides additional tools to understand the evolution of the toxins and to engineer toxin-based therapeutics. This review summarizes the progress on our understanding of BoNTs and TeNT, focusing on the PTC, receptor recognition, new BoNT-like toxins, and therapeutic toxin engineering.

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