A Stem-Cell Based Bioassay to Critically Assess the Pathology of Dysfunctional Neuromuscular Junctions

Peter H Chipman,Victor F Rafuse,Ying Zhang,Andrew James Grierson

doi:10.1371/journal.pone.0091643

Peter H Chipman, Victor F Rafuse + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0091643

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Journal: PloS one	Publication Date: Mar 13, 2014
Citations: 31	License type: CC BY 4.0

Affiliation: Dalhousie University

Abstract

Pluripotent stem cells can be directed to differentiate into motor neurons and assessed for functionality in vitro. An emerging application of this technique is to model genetically inherited diseases in differentiated motor neurons and to screen for new therapeutic targets. The neuromuscular junction (NMJ) is essential to the functionality of motor neurons and its dysfunction is a primary hallmark of motor neuron disease. However, mature NMJs that possess the functional and morphological characteristics of those formed in vivo have so far not been obtained in vitro. Here we describe the generation and analysis of mature NMJs formed between embryonic stem cell-derived motor neurons (ESCMNs) and primary myotubes. We compared the formation and maturation of NMJs generated by wild-type (NCAM+/+) ESCMNs to those generated by neural cell adhesion molecule null (NCAM-/-) ESCMNs in order to definitively test the sensitivity of this assay to identify synaptic pathology. We find that co-cultures using NCAM-/- ESCMNs replicate key in vivo NCAM-/- phenotypes and reveal that NCAM influences neuromuscular synaptogenesis by controlling the mode of synaptic vesicle endocytosis. Further, we could improve synapse formation and function in NCAM-/- co-cultures by chronic treatment with nifedipine, which blocks an immature synaptic vesicle recycling pathway. Together, our results demonstrate that this ESCMN/myofiber co-culture system is a highly sensitive bioassay for examining molecules postulated to regulate synaptic function and for screening therapeutics that will improve the function of compromised NMJs.

Highlights

Several classes of motor neuron diseases (MNDs) manifest themselves as disorders of the neuromuscular junction (NMJ) prior to overt cell death [1,2,3,4,5,6,7,8]
The present study shows that neural cell adhesion molecule (NCAM)+/+ and NCAM-/- embryonic stem cell-derived motor neurons (ESCMNs) form NMJs with co-cultured myotubes that are remarkably similar to their endogenous counterparts in wild-type and NCAM-/- mice, respectively
Compared to NCAM+/+ ESCMNs, NMJs formed by NCAM-/- ESCMNs had limited growth, reduced neurotransmission and abnormal synaptic vesicle dynamics

Summary

Introduction

Several classes of motor neuron diseases (MNDs) manifest themselves as disorders of the neuromuscular junction (NMJ) prior to overt cell death [1,2,3,4,5,6,7,8]. Even when motor neurons are prevented from dying in a mouse model of amyotrophic lateral sclerosis (ALS), motor axons still degenerate from the motor endplate causing muscle paralysis and death [4]. This degeneration is due, at least in part, to anatomical and/or functional deficits at the NMJ. Large scale screening of therapeutics aimed at improving the function of NMJs in MNDs requires the creation of an in vitro model system that accurately emulates normal development, function and long-term stability of the motor endplate in vivo. The structure and function of the co-cultured system should respond appropriately to pharmacological interventions

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