Abstract
Motor neurons project axons from the hindbrain and spinal cord to muscle, where they induce myofibre contractions through neurotransmitter release at neuromuscular junctions. Studies of neuromuscular junction formation and homeostasis have been largely confined to in vivo models. In this study we have merged three powerful tools - pluripotent stem cells, optogenetics and microfabrication - and designed an open microdevice in which motor axons grow from a neural compartment containing embryonic stem cell-derived motor neurons and astrocytes through microchannels to form functional neuromuscular junctions with contractile myofibers in a separate compartment. Optogenetic entrainment of motor neurons in this reductionist neuromuscular circuit enhanced neuromuscular junction formation more than two-fold, mirroring the activity-dependence of synapse development in vivo. We incorporated an established motor neuron disease model into our system and found that coculture of motor neurons with SOD1G93A astrocytes resulted in denervation of the central compartment and diminished myofiber contractions, a phenotype which was rescued by the Receptor Interacting Serine/Threonine Kinase 1 (RIPK1) inhibitor Necrostatin. This coculture system replicates key aspects of nerve-muscle connectivity in vivo and represents a rapid and scalable alternative to animal models of neuromuscular function and disease.
Highlights
Motor neurons project axons from the hindbrain and spinal cord to muscle, where they induce myofibre contractions through neurotransmitter release at neuromuscular junctions
Gliaderived neurotrophic factor”[27] (Gdnf)-ACs were enriched by magnetic-activated cell sorting (MACS) (Figure 1A,B) using the CD14 cell-surface reporter and plated as a monolayer along with ChR2-motor neurons (MNs), which were MACS-enriched with the MN-specific Hb9::CD14-IRES-GFP reporter.[25]
Gdnf expression in sorted Gdnf-ACs was confirmed by immunocytochemistry (Figure 1C) and quantitative polymerase chain reaction analysis (Table S1, Supporting Information)
Summary
Motor neurons project axons from the hindbrain and spinal cord to muscle, where they induce myofibre contractions through neurotransmitter release at neuromuscular junctions. Vertebrates, MNs emerge from ventral progenitor domains in the caudal neural tube[1] and project axons into the surrounding mesenchyme.[2] Motor axon growth cones navigate toward threonine kinase 1 inhibitor necrostatin This coculture system replicates their specific target muscle in a series of key aspects of nerve–muscle connectivity in vivo and represents a rapid and scalable alternative to animal models of neuromuscular function and disease. A key advance in modeling both normal MN biology and MN disease was the establishment of in vitro derivation protocols capable of generating large numbers (>106) of MNs from pluripotent stem cells (PSCs).[13] These protocols use chemical cues that govern normal neurogenesis in the spinal cord and emulate the developmental program of MN specification, allowing the study of transcriptional programs of MN differentiation,[14] cellular mechanisms of MN degeneration,[15] and local spinal circuit formation.[16] MNs in these models lack their normal functional context, because they are not connected to myofibres through NMJs. In vitro cocultures of primary MNs and myofibres have long been used to model neuromuscular circuits. This deterioration of motor innervation resembles the early peripheral pathology of ALS seen in human patients and rodent models
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