MNIM-EOH cells can trigger AChR clustering and form functional connections with cocultured myotubes.

<p>(<b>A</b>) Fluorescence images of MNIM-EOH cells at 1 day after cocultured with C2C12 myotubes. α-BTX staining revealed AChR clustering on C2C12 muscle fibers. Confocal Z-stack imaging, in a line through the region of apparent colocalization, confirmed EGFP+ axons in close proximity to AChRs (arrows). Scale bars: 10 µm. (<b>B</b>) End-plate currents (EPCs) were recorded from myotubes located in close proximity to MNIM-EOH cells. EPCs were blocked from the same cell after application of 15 µM pancuronium. However, after washing out of pancuronium, EPCs could be recorded again. The bars represent mean±SEM. * <i>P</i><0.05. The significance was determined by Student’s <i>t</i> test.</p

MN marker gene expression in hMSCs expressing Olig2 and Hb9 in the presence of MNIM.

<p>(<b>A</b>) Schematic illustration of the protocol used for MN induction of hMSCs. (<b>B</b>) RT-PCR analyses of hMSCs expressing EGFP (E), EGFP-Olig2 (EO), EGFP-Hb9 (EH), and EGFP-Olig2-Hb9 (EOH) with MNIM treatment were preformed. Quantification of NF-M (<b>C</b>), Islet-1 (<b>D</b>), and VAChT (<b>E</b>) PCR products. The mRNA level of a given gene was quantified by densitometry and normalized to the corresponding β-actin level. The bars represent mean±SEM of at least three independent experiments. *<i>P</i><0.05 versus uninduced E. The significance was determined by Mann-Whitney test with Fisher’s LSD <i>post hoc</i> test.</p

Increased expression of mature neuronal/motoneuronal markers and cell-cycle arrest occur in MNIM-treated EOH

<p><b>cells.</b> (<b>A</b>) Immunocytochemistry analysis of neuronal and MN-specific markers in uninduced EOH cells, MNIM-EOH cells, and MNIM-EOH cells further cultured in growth medium for 2 days after complete induction. Each row represents NeuN (red), NF-M (red), ChAT (red), and Islet-1 (red) staining with EGFP (green). Scale bar: 50 µm. (<b>B</b>) Quantitative analysis of NeuN, NF-M, ChAT, and Islet-1 expression. Fifty to 150 cells per group were analyzed in randomly chosen fields. The bars represent mean±SEM of at least three independent experiments. * <i>P</i><0.05, ** <i>P</i><0.001 versus GM. † <i>P</i><0.05, †† <i>P</i><0.001 versus MNIM. The significance was determined by ANOVA followed by Fisher’s LSD <i>post hoc</i> test. (<b>C</b>) Immunocytochemistry of BrdU incorporation (red) and EGFP (green) in EOH cells cultured in GM, MNIM, and MNIM+GM. (<b>D</b>) Quantitative analysis of BrdU incorporation. Fifty to 150 cells per group were analyzed in randomly chosen fields. The bars represent mean±SEM of at least three independent experiments. * <i>P</i><0.05 versus GM. The significance was determined by ANOVA followed by Fisher’s LSD <i>post hoc</i> test.</p

Excitable properties of MNIM-treated EOH cells.

<p>Representative perforated patch clamp recordings from uninduced EOH cells (<b>A</b>) or MNIM-EOH cells (<b>B</b>). The cells were held at −60 mV and currents elicited by stepping from −90 to +30 mV in 10 mV steps. MNIM-EOH cells express robust outward K+ currents but very small inward Na+ currents (filled upright triangle). Current injection (200 pA, 100 ms) induced action potential in MNIM-EOH cells, not in uninduced EOH cells. (<b>C</b>) Resting membrane potential in uninduced EOH cells and MNIM-EOH cells. The bars represent mean ± SEM. * <i>P</i><0.05. The significance was determined by Student’s <i>t</i> test.</p

Quantitative analysis of cell morphology (% of total cells).

<p>The values are expressed as the mean±SEM.</p

Polymerase chain reaction primer pairs.

<p>Polymerase chain reaction primer pairs.</p

Changes in cell morphology of hMSCs expressing Olig2-Hb9 after the induction.

<p>Bright-field images of uninduced EOH cells (<b>A</b>), EOH cells induced for 5 days (<b>B</b>), completely induced EOH cells (<b>C</b>), and induced EOH cells further cultured in growth medium for 2 days after complete induction (<b>E</b>). (<b>D</b>) A higher magnification of the corresponding box shown in (<b>C</b>). (<b>F</b>) GFP (green), Olig2 (blue) and Hb9 (red) were expressed in EOH cells. Scale bar: 100 µm.</p

MNIM-EOH cells express mature neuronal/MN markers after transplanted into the injured spinal cord slice culture.

<p>Confocal microscopy analysis of MNIM-EOH cells (green) immunostained with anti-NeuN (red) (<b>A</b>), anti-NF-M (red) (<b>B</b>), and anti-ChAT (red) (<b>C</b>) antibodies. In the slices, 37 of 472 EGFP+ cells were NeuN+, 20 of 316 EGFP+ cells were NF-M+, and 14 of 469 EGFP+ cells were ChAT+. Colocalization of EGFP and NeuN, NF-M, or ChAT in a single cell was confirmed by z-axis stack analysis. At least 4 slices were analyzed. Scale bars: 50 µm.</p

<i>Ex vivo</i> assessment of EOH cells or MNIM-EOH cells after transplantation into the spinal cord slice culture.

<p>Confocal images of uninduced EOH cells (green) (<b>A-C</b>) or MNIM-EOH cells (green) (<b>D-F</b>) at 2 days after transplantation into the ventral horn area (vh) of a spinal cord slice. (<b>B</b>) and (<b>E</b>) are a higher magnification of (<b>A</b>) and (<b>D</b>), respectively. (<b>C</b>) Confocal image of transplanted EOH cells with a flattened and symmetrical fibroblast-like morphology. (<b>F</b>) Confocal image of transplanted MNIM-EOH cells with a neuron-like morphology. Scale bars: 100 µm.</p

<i>Drosophila</i> Gyf/GRB10 interacting GYF protein is an autophagy regulator that controls neuron and muscle homeostasis

<p>Autophagy is an essential process for eliminating ubiquitinated protein aggregates and dysfunctional organelles. Defective autophagy is associated with various degenerative diseases such as Parkinson disease. Through a genetic screening in <i>Drosophila</i>, we identified <i>CG11148</i>, whose product is orthologous to GIGYF1 (GRB10-interacting GYF protein 1) and GIGYF2 in mammals, as a new autophagy regulator; we hereafter refer to this gene as <i>Gyf</i>. Silencing of <i>Gyf</i> completely suppressed the effect of Atg1-Atg13 activation in stimulating autophagic flux and inducing autophagic eye degeneration. Although <i>Gyf</i> silencing did not affect Atg1-induced Atg13 phosphorylation or Atg6-Pi3K59F (class III PtdIns3K)-dependent Fyve puncta formation, it inhibited formation of Atg13 puncta, suggesting that Gyf controls autophagy through regulating subcellular localization of the Atg1-Atg13 complex. <i>Gyf</i> silencing also inhibited Atg1-Atg13-induced formation of Atg9 puncta, which is accumulated upon active membrane trafficking into autophagosomes. <i>Gyf</i>-null mutants also exhibited substantial defects in developmental or starvation-induced accumulation of autophagosomes and autolysosomes in the larval fat body. Furthermore, heads and thoraxes from <i>Gyf</i>-null adults exhibited strongly reduced expression of autophagosome-associated Atg8a-II compared to wild-type (WT) tissues. The decrease in Atg8a-II was directly correlated with an increased accumulation of ubiquitinated proteins and dysfunctional mitochondria in neuron and muscle, which together led to severe locomotor defects and early mortality. These results suggest that Gyf-mediated autophagy regulation is important for maintaining neuromuscular homeostasis and preventing degenerative pathologies of the tissues. Since human mutations in the <i>GIGYF2</i> locus were reported to be associated with a type of familial Parkinson disease, the homeostatic role of Gyf-family proteins is likely to be evolutionarily conserved.</p