Human neuromuscular junction on micro-structured microfluidic devices implemented with a custom micro electrode array (MEA).

In the neuromuscular system, signal transmission from motor neurons (MNs) to innervated muscle fibers is crucial for their synaptic function, viability, and maintenance. In order to better understand human neuromuscular junction (hNMJ) functionality, it is important to develop on-a-chip devices with human cells. To investigate this cell network, microfluidic platforms are useful to grow different cell types in isolated compartments. Such devices have already been developed to study in vitro neuronal circuitry. Here, we combined microfluidics with two techniques: soft lithography and custom microelectrodes array (MEA). Our goal was to create hNMJs on a specific pattern of electrodes to stimulate pre-synaptic axons and record post-synaptic muscle activity. Micromachining was used to create structurations to guide muscle growth above electrodes, without impairing axon propagation, therefore optimizing the effectiveness of activity recording. Electrodes were also arranged to be aligned with the microfluidic chambers in order to specifically stimulate axons that were growing between the two compartments. Isolation of the two cell types allows for the selective treatment of neurons or muscle fibers to assess NMJ functionality hallmarks. Altogether, this microfluidic/microstructured/MEA platform allowed mature and functional in vitro hNMJ modelling. We demonstrate that electrical activation of MNs can trigger recordable extracellular muscle action potentials. This study provides evidence for a physiologically relevant model to mimic a hNMJ that will in the future be a powerful tool, more sensitive than calcium imaging, to better understand and characterize NMJs and their disruption in neurodegenerative diseases.

Splicing Defects of the Profilin Gene Alter Actin Dynamics in an S. pombe SMN Mutant.

Spinal muscular atrophy (SMA) is a devastating motor neuron disorder caused by mutations in the survival motor neuron (SMN) gene. It remains unclear how SMN deficiency leads to the loss of motor neurons. By screening Schizosaccharomyces pombe, we found that the growth defect of an SMN mutant can be alleviated by deletion of the actin-capping protein subunit gene acp1+. We show that SMN mutated cells have splicing defects in the profilin gene, which thus directly hinder actin cytoskeleton homeostasis including endocytosis and cytokinesis. We conclude that deletion of acp1+ in an SMN mutant background compensates for actin cytoskeleton alterations by restoring redistribution of actin monomers between different types of cellular actin networks. Our data reveal a direct correlation between an impaired function of SMN in snRNP assembly and defects in actin dynamics. They also point to important common features in the pathogenic mechanism of SMA and ALS.

TRIM17 and TRIM28 antagonistically regulate the ubiquitination and anti-apoptotic activity of BCL2A1.

BCL2A1 is an anti-apoptotic member of the BCL-2 family that contributes to chemoresistance in a subset of tumors. BCL2A1 has a short half-life due to its constitutive processing by the ubiquitin-proteasome system. This constitutes a major tumor-suppressor mechanism regulating BCL2A1 function. However, the enzymes involved in the regulation of BCL2A1 protein stability are currently unknown. Here, we provide the first insight into the regulation of BCL2A1 ubiquitination. We present evidence that TRIM28 is an E3 ubiquitin-ligase for BCL2A1. Indeed, endogenous TRIM28 and BCL2A1 bind to each other at the mitochondria and TRIM28 knock-down decreases BCL2A1 ubiquitination. We also show that TRIM17 stabilizes BCL2A1 by blocking TRIM28 from binding and ubiquitinating BCL2A1, and that GSK3 is involved in the phosphorylation-mediated inhibition of BCL2A1 degradation. BCL2A1 and its close relative MCL1 are thus regulated by common factors but with opposite outcome. Finally, overexpression of TRIM28 or knock-out of TRIM17 reduced BCLA1 protein levels and restored sensitivity of melanoma cells to BRAF-targeted therapy. Therefore, our data describe a molecular rheostat in which two proteins of the TRIM family antagonistically regulate BCL2A1 stability and modulate cell death.