Dynactin1 depletion leads to neuromuscular synapse instability and functional abnormalities

Valérie Bercier,Céline Revenu,Filippo Del Bene,Thomas O Auer,Kevin Fidelin,Karine Duroure,Claire Wyart,Jeffrey M Hubbard

doi:10.1186/s13024-019-0327-3

Abstract

BackgroundDynactin subunit 1 is the largest subunit of the dynactin complex, an activator of the molecular motor protein complex dynein. Reduced levels of DCTN1 mRNA and protein have been found in sporadic amyotrophic lateral sclerosis (ALS) patients, and mutations have been associated with disease, but the role of this protein in disease pathogenesis is still unknown.MethodsWe characterized a Dynactin1a depletion model in the zebrafish embryo and combined in vivo molecular analysis of primary motor neuron development with live in vivo axonal transport assays in single cells to investigate ALS-related defects. To probe neuromuscular junction (NMJ) function and organization we performed paired motor neuron-muscle electrophysiological recordings and GCaMP calcium imaging in live, intact larvae, and the synapse structure was investigated by electron microscopy.ResultsHere we show that Dynactin1a depletion is sufficient to induce defects in the development of spinal cord motor neurons and in the function of the NMJ. We observe synapse instability, impaired growth of primary motor neurons, and higher failure rates of action potentials at the NMJ. In addition, the embryos display locomotion defects consistent with NMJ dysfunction. Rescue of the observed phenotype by overexpression of wild-type human DCTN1-GFP indicates a cell-autonomous mechanism. Synaptic accumulation of DCTN1-GFP, as well as ultrastructural analysis of NMJ synapses exhibiting wider synaptic clefts, support a local role for Dynactin1a in synaptic function. Furthermore, live in vivo analysis of axonal transport and cytoskeleton dynamics in primary motor neurons show that the phenotype reported here is independent of modulation of these processes.ConclusionsOur study reveals a novel role for Dynactin1 in ALS pathogenesis, where it acts cell-autonomously to promote motor neuron synapse stability independently of dynein-mediated axonal transport.

Highlights

Dynactin subunit 1 is the largest subunit of the dynactin complex, an activator of the molecular motor protein complex dynein
The ratio of microtubule capture, defined by comets terminating their run at putative synapse sites, for mok m632−/− embryos was unchanged when compared with their wild-type siblings (Fig. 2f ). This suggests that synaptic microtubule capture, while dependent on interaction between dynein and the dynactin complex [67], is not affected by Dynactin1a depletion. These results indicate that the growth defects observed in 6dpf caudal primary (CaP) motor neurons upon Dynactin1a depletion do not result from impaired modulation of the actin or microtubule cytoskeleton, and that the cell has both the potential for sensing and the support for trophic signaling in mok m632−/− embryos
As N-Cadherin is known to be involved in synapse stabilization by mediating cell-cell interactions, our results suggest that synapse instability is not due to impaired localization of this adhesion molecule at the synapse, defects could still arise from compromised interactions upon depletion of Dynactin1a

Summary

Introduction

Dynactin subunit 1 is the largest subunit of the dynactin complex, an activator of the molecular motor protein complex dynein. ALS is an adult-onset, neurodegenerative disease affecting upper and lower motor neurons. It leads to denervation at the neuromuscular junction, muscle wasting and progressive paralysis, ending in the demise of the patient two to 5 years after diagnosis. Axonal transport is mediated by ATP-driven molecular motors, which carry vesicles and organelles by moving along the microtubule network. This component of the cell cytoskeleton is composed of protofilaments polymerized from tubulin subunits which are oriented in a highly polarized manner in the axon, resulting in a fast-growing end (+) at the synapse and a slow-growing end (−) oriented toward the nucleus at the soma. The dynein motor complex transports cargo in the retrograde direction (− end directed) [17] and relies on multi-subunit complexes like dynactin for functional versatility [18, 19]

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