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

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.

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

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.

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 Correction of amyotrophic lateral sclerosis related phenotypes in induced pluripotent stem cell-derived motor neurons carrying a hexanucleotide expansion mutation in C9orf72 by CRISPR/Cas9 genome editing using homology-directed repair

2019 ◽  
Author(s):  
Nidaa Ababneh ◽  
Jakub Scaber ◽  
Rowan Flynn ◽  
Andrew Douglas ◽  
Martin R. Turner ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Stem Cell ◽  
Genome Editing ◽  
Motor Neurons ◽  
Pluripotent Stem Cell ◽  
Intron Retention ◽  
Induced Pluripotent Stem Cell ◽  
Homology Directed Repair ◽  
Induced Pluripotent ◽  
Lateral Sclerosis

AbstractThe G4C2 hexanucleotide repeat expansion (HRE) in C9orf72 is the commonest cause of familial amyotrophic lateral sclerosis (ALS). A number of different methods have been used to generate isogenic control lines using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 and non-homologous end-joining (NHEJ) by deleting the repeat region with the risk of creating indels and genomic instability. In this study we demonstrate complete correction of an induced pluripotent stem cell (iPSC) line derived from a C9orf72-HRE positive ALS/FTD patient using CRISPR/Cas9 genome editing and homology directed repair (HDR), resulting in replacement of the excised region with a donor template carrying the wild-type repeat size to maintain the genetic architecture of the locus. The isogenic correction of the C9orf72 HRE restored normal expression and methylation at the C9orf72 locus, reduced intron retention in the edited lines, and abolished pathological phenotypes associated with the C9orf72 HRE expansion in iPSC derived motor neurons (iPSMNs).RNA sequencing of the mutant line identified 2220 differentially expressed genes compared to its isogenic control. Enrichment analysis demonstrated an over-representation of ALS relevant pathways, including calcium ion dependent exocytosis, synaptic transport and the KEGG ALS pathway, as well as new targets of potential relevance to ALS pathophysiology.Complete correction of the C9orf72 HRE in iPSMNs by CRISPR/Cas9 mediated HDR provides an ideal model to study the earliest effects of the hexanucleotide expansion on cellular homeostasis and the key pathways implicated in ALS pathophysiology.

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

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.

scholarly journals The Link between VAPB Loss of Function and Amyotrophic Lateral Sclerosis

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1865
Author(s):  
Nica Borgese ◽  
Nicola Iacomino ◽  
Sara Francesca Colombo ◽  
Francesca Navone
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Motor Neurons ◽  
Adaptor Proteins ◽  
Loss Of Function ◽  
Membrane Contact Sites ◽  
Physiological Processes ◽  
Main Driver ◽  
Single Allele ◽  
Induced Pluripotent ◽  
Lateral Sclerosis

The VAP proteins are integral adaptor proteins of the endoplasmic reticulum (ER) membrane that recruit a myriad of interacting partners to the ER surface. Through these interactions, the VAPs mediate a large number of processes, notably the generation of membrane contact sites between the ER and essentially all other cellular membranes. In 2004, it was discovered that a mutation (p.P56S) in the VAPB paralogue causes a rare form of dominantly inherited familial amyotrophic lateral sclerosis (ALS8). The mutant protein is aggregation-prone, non-functional and unstable, and its expression from a single allele appears to be insufficient to support toxic gain-of-function effects within motor neurons. Instead, loss-of-function of the single wild-type allele is required for pathological effects, and VAPB haploinsufficiency may be the main driver of the disease. In this article, we review the studies on the effects of VAPB deficit in cellular and animal models. Several basic cell physiological processes are affected by downregulation or complete depletion of VAPB, impinging on phosphoinositide homeostasis, Ca2+ signalling, ion transport, neurite extension, and ER stress. In the future, the distinction between the roles of the two VAP paralogues (A and B), as well as studies on motor neurons generated from induced pluripotent stem cells (iPSC) of ALS8 patients will further elucidate the pathogenic basis of p.P56S familial ALS, as well as of other more common forms of the disease.

scholarly journals Establishment of a Human Induced Pluripotent Stem Cell-Derived Neuromuscular Co-Culture Under Optogenetic Control

2020 ◽  
Author(s):  
Elliot W. Swartz ◽  
Greg Shintani ◽  
Jijun Wan ◽  
Joseph S. Maffei ◽  
Sarah H. Wang ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Calcium Flux ◽  
Electrode Arrays ◽  
Spinal Motor Neurons ◽  
Culture Model ◽  
Skeletal Myotubes ◽  
Induced Pluripotent

SummaryThe failure of the neuromuscular junction (NMJ) is a key component of degenerative neuromuscular disease, yet how NMJs degenerate in disease is unclear. Human induced pluripotent stem cells (hiPSCs) offer the ability to model disease via differentiation toward affected cell types, however, the re-creation of an in vitro neuromuscular system has proven challenging. Here we present a scalable, all-hiPSC-derived co-culture system composed of independently derived spinal motor neurons (MNs) and skeletal myotubes (sKM). In a model of C9orf72-associated disease, co-cultures form functional NMJs that can be manipulated through optical stimulation, eliciting muscle contraction and measurable calcium flux in innervated sKM. Furthermore, co-cultures grown on multi-electrode arrays (MEAs) permit the pharmacological interrogation of neuromuscular physiology. Utilization of this co-culture model as a tunable, patient-derived system may offer significant insights into NMJ formation, maturation, repair, or pathogenic mechanisms that underlie NMJ dysfunction in disease.

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.

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