Pathogenic TDP-43 Disrupts Axon Initial Segment Structure and Neuronal Excitability in a Human iPSC Model of ALS

Harley, Peter; Neves, Guilherme; Riccio, Federica; Machado, Carolina Barcellos; Cheesbrough, Aimee; R’Bibo, Lea; Burrone, Juan; Lieberam, Ivo

doi:10.1101/2022.05.16.492186

Cited by 3 publications

(11 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Subsequently, hiPSC-derived myoblasts (4 × 10 4 cells per well) were seeded onto the elastomer nanofibers and grown for 3 d. Myofibers were derived by forward programming of hiPSCs carrying a dox-inducible PAX7 transgene [ 24 , 25 ]. One neural aggregate per well, comprising of 1 × 10 4 magnetic-activated cell sorting (MACS)-enriched hiPSC-motor neurons suitable for optogenetics (ChR2-YFP + ) (supplementary figure S2), and 5 × 10 3 GDNF-expressing mouse embryonic stem cell (mESC)-derived astrocytes was seeded on top of the hiPSC-myofibers (figure 3 (a), supplementary figure S3).…”

Section: Resultsmentioning

confidence: 99%

A scalable human iPSC-based neuromuscular disease model on suspended biobased elastomer nanofiber scaffolds

Cheesbrough,

Harley,

Riccio

et al. 2023

Biofabrication

Self Cite

View full text Add to dashboard Cite

Many devastating neuromuscular diseases currently lack effective treatments. This is in part due to a lack of drug discovery platforms capable of assessing complex human neuromuscular disease phenotypes in a scalable manner. A major obstacle has been generating scaffolds to stabilise mature contractile myofibers in a multi-well assay format amenable to high content image analysis (HCI). This study describes the development of a scalable human iPSC-neuromuscular disease model, whereby suspended elastomer nanofibers support long-term stability, alignment, maturation, and repeated contractions of iPSC-myofibers, innervated by iPSC-motor neurons in 96-well assay plates. In this platform, optogenetic stimulation of the motor neurons elicits robust myofiber-contractions, providing a functional readout of neuromuscular transmission. Additionally, HCI provides rapid and automated quantification of axonal outgrowth, myofiber morphology, and neuromuscular synapse number and morphology. By incorporating amyotrophic lateral sclerosis (ALS)-related TDP-43G298S mutant motor neurons and CRISPR-corrected controls, key neuromuscular disease phenotypes are recapitulated, including weaker myofiber contractions, reduced axonal outgrowth, and reduced number of neuromuscular synapses. Treatment with a candidate ALS drug, the receptor-interacting protein kinase-1 (RIPK1)-inhibitor necrostatin-1, rescues these phenotypes in a dose-dependent manner, highlighting the potential of this platform to screen novel treatments for neuromuscular diseases.

show abstract

Section: Resultsmentioning

confidence: 99%

A scalable human iPSC-based neuromuscular disease model on suspended biobased elastomer nanofiber scaffolds

Cheesbrough,

Harley,

Riccio

et al. 2023

Biofabrication

Self Cite

View full text Add to dashboard Cite

show abstract

“…Other notable protocols for generating human PSC-motor neurons and astrocytes include (Julia et al, 2017;Canals et al, 2018;Fernandopulle et al, 2018). For the protocol used for these experiments, follow those outlined in (Harley et al, 2022). Consult the original protocol to determine the best point to dissociate and replate the motor neurons/astrocytes.…”

Section: B Generating Neural Spheroidsmentioning

confidence: 99%

“…The next day, dissociate motor neurons and astrocytes into single cells using TrypLE or accutase, respectively. For motor neuron dissociation choose a stage in the protocol that allows live cell sorting (Harley et al, 2022) and replating into different culture formats; if motor neurons are too mature with large axonal projections, then dissociation will result in cell death. Astrocyte dissociation may require longer incubations (5-20 min) and addition of DNase-I to prevent cell clumping.…”

Section: B Generating Neural Spheroidsmentioning

confidence: 99%

“…While mouse models have been widely used for studying neuromuscular physiology, growing evidence shows that there are substantial differences between human and mouse neuromuscular synapses, including: contrasting synaptic morphologies, divergent transcriptomes and proteomes, and altered homeostasis and maintenance (Jones et al, 2017). With the development of differentiation protocols to derive enriched populations of motor neurons and myofibers (MFs) from human pluripotent stem cells (PSCs) (Du et al, 2015;Chal et al, 2016;Fernandopulle et al, 2018;Rao et al, 2018;Cheesbrough et al, 2022;Harley et al, 2022), a number of neuromuscular co-culture platforms have been established. A major challenge for generating human PSC-derived neuromuscular co-cultures has been stabilising contractile myofibers in order to prevent detachment upon contraction and allow long -term culture and maturation (Abd Al Samid et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

“…Likewise, it allowed us to study phenotypes of the same motor neurons innervating an isogenic pair of DMD and CRISPR-corrected myofibers (Paredes-Redondo et al, 2021). Furthermore, incorporation of ALS-related TDP-43 G298S motor neurons and CRISPR-corrected controls enabled us to recapitulate key ALSrelated neuromuscular phenotypes (Harley et al, 2022). In all these experiments, motor neurons were embedded in a scaffold of mouse embryonic stem cell (ESC)-derived astrocytes to promote maturation and provide neurotrophic support.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

3D Compartmentalised Human Pluripotent Stem Cell–derived Neuromuscular Co-cultures

Harley¹,

Paredes‐Redondo²,

Grenci³

et al. 2023

BIO-PROTOCOL

View full text Add to dashboard Cite

Human neuromuscular diseases represent a diverse group of disorders with unmet clinical need, ranging from muscular dystrophies, such as Duchenne muscular dystrophy (DMD), to neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS). In many of these conditions, axonal and neuromuscular synapse dysfunction have been implicated as crucial pathological events, highlighting the need for in vitro disease models that accurately recapitulate these aspects of human neuromuscular physiology. The protocol reported here describes the co -culture of neural spheroids composed of human pluripotent stem cell (PSC)-derived motor neurons and astrocytes, and human PSC-derived myofibers in 3D compartmentalised microdevices to generate functional human neuromuscular circuits in vitro. In this microphysiological model, motor axons project from a central nervous system (CNS)-like compartment along microchannels to innervate skeletal myofibers plated in a separate muscle compartment. This mimics the spatial organization of neuromuscular circuits in vivo. Optogenetics, particle image velocimetry (PIV) analysis, and immunocytochemistry are used to control, record, and quantify functional neuromuscular transmission, axonal outgrowth, and neuromuscular synapse number and morphology. This approach has been applied to study diseasespecific phenotypes for DMD and ALS by incorporating patient-derived and CRISPR-corrected human PSC-derived motor neurons and skeletal myogenic progenitors into the model, as well as testing candidate drugs for rescuing pathological phenotypes. The main advantages of this approach are: i) its simple design; ii) the in vivo-like anatomical separation between CNS and peripheral muscle; and iii) the amenability of the approach to high power imaging. This opens up the possibility for carrying out live axonal transport and synaptic imaging assays in future studies, in addition to the applications reported in this study.

show abstract

Neuronal Circuit Dysfunction in Amyotrophic Lateral Sclerosis

Salzinger,

Ramesh,

Das Sharma

et al. 2024

Cells

View full text Add to dashboard Cite

The primary neural circuit affected in Amyotrophic Lateral Sclerosis (ALS) patients is the corticospinal motor circuit, originating in upper motor neurons (UMNs) in the cerebral motor cortex which descend to synapse with the lower motor neurons (LMNs) in the spinal cord to ultimately innervate the skeletal muscle. Perturbation of these neural circuits and consequent loss of both UMNs and LMNs, leading to muscle wastage and impaired movement, is the key pathophysiology observed. Despite decades of research, we are still lacking in ALS disease-modifying treatments. In this review, we document the current research from patient studies, rodent models, and human stem cell models in understanding the mechanisms of corticomotor circuit dysfunction and its implication in ALS. We summarize the current knowledge about cortical UMN dysfunction and degeneration, altered excitability in LMNs, neuromuscular junction degeneration, and the non-cell autonomous role of glial cells in motor circuit dysfunction in relation to ALS. We further highlight the advances in human stem cell technology to model the complex neural circuitry and how these can aid in future studies to better understand the mechanisms of neural circuit dysfunction underpinning ALS.

show abstract

Pathogenic TDP-43 Disrupts Axon Initial Segment Structure and Neuronal Excitability in a Human iPSC Model of ALS

Cited by 3 publications

References 53 publications

A scalable human iPSC-based neuromuscular disease model on suspended biobased elastomer nanofiber scaffolds

A scalable human iPSC-based neuromuscular disease model on suspended biobased elastomer nanofiber scaffolds

3D Compartmentalised Human Pluripotent Stem Cell–derived Neuromuscular Co-cultures

Neuronal Circuit Dysfunction in Amyotrophic Lateral Sclerosis

Contact Info

Product

Resources

About