Quantitative comparison of the mRNA content of human iPSC‐derived motor neurons and their extracellular vesicles

Extracellular vesicles (EVs) contain various cargo molecules, including RNAs and proteins. EVs, which include exosomes, are predicted to be suitable surrogates of their source cells for liquid biopsy to measure biomarkers. Several studies have performed qualitative comparisons of cargo molecule repertoires between source cells and their EVs. However, quantitative comparisons have not been reported so far. Furthermore, many studies analyzed microRNAs or proteins in EVs, but not mRNAs. In this study, we analyzed mRNAs in motor neurons and their EVs. Normal human‐induced pluripotent stem cells were differentiated into motor neurons, and comprehensive analysis of mRNAs in the cells and their EVs was performed by RNA sequencing. Differential analysis between cellular and EV mRNAs was performed by edgeR after normalization of read count. The results suggest that signatures in the abundance of EV mRNAs were different from those of cellular mRNAs. Comparison of intracellular vesicle and EV mRNA abundance showed negatively and positively biased genes in the EVs. Gene Ontology analysis revealed that the genes showing negatively biased abundance in the EVs were enriched in many functions regarding neuronal development. In contrast, the positively biased genes were enriched in functions regarding cellular metabolism and protein synthesis. These results suggest that mRNAs in motor neurons are loaded into EVs to regulate certain mechanisms, which are yet to be elucidated

Discovery and pharmacological characterization of a new class of prolyl-tRNA synthetase inhibitor for anti-fibrosis therapy

<div><p>Scleroderma has clinical characteristics including skin and other tissue fibrosis, but there is an unmet need for anti-fibrotic therapy. Halofuginone (HF) is a well-known anti-fibrosis agent in preclinical and clinical studies which exerts its effect via inhibition of TGF-β/Smad3 signaling pathway. Recently, prolyl-tRNA synthetase (PRS) was elucidated as a target protein for HF that binds to the proline binding site of the catalytic domain of PRS. Here, we characterized a new class of PRS inhibitor (T-3833261) that is carefully designed in a way that binds to the ATP site of the catalytic domain and does not disrupt binding of proline. The anti-fibrotic activity and the mechanism of action for T-3833261 on TGF-β-induced fibrotic assay were compared with those of HF in primary human skin fibroblast. We evaluated <i>in vivo</i> effect of topical application of T-3833261 and HF on TGF-β-induced fibrotic genes expression in mice. We found that T-3833261 suppressed TGF-β-induced α-smooth muscle actin (α-SMA) and type I collagen α1 (COL1A1) expression through the Smad3 axis in a similar fashion to HF. <i>In vivo</i> topical application of T-3833261 reduced the increase of fibrotic genes expression such as α-Sma, Col1a1 and Col1a2 by TGF-β intradermal injection to the ear of a mouse. We revealed that T-3833261 is more effective than HF under the conditions of high proline concentration, as reported in fibrotic tissues. These results suggest the potential of ATP competitive PRS inhibitors for the treatment of fibrotic diseases such as scleroderma.</p></div

Effect of T-3833261 or Halofuginone on Smad3 protein expression in human skin fibroblasts.

<p>Representative western blots show that Halofuginone or T-3833261 reduces Smad3 and phosphorylated Smad3 (p-Smad3) protein expressions significantly. β-actin shown as simultaneous loading controls. The data of this experiments were repeated three times.</p

Fibrotic genes expression of ear and dorsal skin in a mouse by TGF-β injection.

<p>TGF-β (500 ng/20 μL) or vehicle was intradermally injected 3 or 5 times in the ear (A) or dorsal skin (B) of mice. mRNA expression of α-Sma, Col1a1 and Col1a2 in the ear was measured 3 or 5 days after final TGF-β administration was evaluated. Gene expression levels (normalized to Gapdh) are expressed as the fold change of vehicle-treated control. Values are mean ± SE (n = 2–5). #p<0.05 compared to PBS-injected normal mice, *p<0.05 compared to PBS-injected normal mice.</p

T-3833261 is a potent ATP competitive PRS inhibitor.

<p>(A) Molecular model constructed by available PRS crystal structures bearing adenosine and proline (PDB: 4K87) and Halofuginone (PDB: 4K88), (B) Binding mode of T-3833261 in the ATP binding site, (C) Chemical structure, (D) Biochemical activity of compound in PRS inhibition measured by an ATP/PPi exchange method. Results performed in duplicate are shown as the mean ± SD. The inhibitory activities are expressed as the percentage of vehicle-treated control.</p

Effects of topical T-3833261 or Halofuginone on TGF-β-induced fibrotic genes expression in mouse ear skin.

<p>TGF-β (500 ng/20 μL) or vehicle was intradermally injected 5 times in the ear of mice. T-3833261, Halofuginone or vehicle was locally administered to the mice 1 h before TGF-β injection. Skin biopsies were taken from each sample treated mice. mRNA expression of α-Sma, Col1a1 and Col1a2 in the ear was measured 78 h after final TGF-β administration was evaluated. Gene expression levels (normalized to Gapdh) are expressed as the fold change of vehicle-treated control. Values are mean ± SE (n = 4–8). #p<0.05 compared to PBS-injected normal mice, *p<0.05 compared to TGF-β-injected mice.</p

Effect of T-3833261 or Halofuginone on α-SMA and pro-COL1A1 protein content in TGF-β-treated human skin fibroblast.

<p>Skin fibroblasts were pre-treated with samples (1–300 nM) for 0.5 h, followed by stimulation with TGF-β (1 ng/mL). After incubation for 24 h, α-SMA (A) and pro-COL1A1 (B) protein levels were measured by ELISA. The expression levels are expressed as the percentage of vehicle-treated control. Values are mean ± SD (n = 4). #p<0.05 compared to vehicle-treated control, *p<0.05 compared to TGF-β-treated control. The experiment was repeated by using other fibroblast lots and similar results were obtained.</p

Effects of proline addition on PRS inhibitors (T-3833261 or Halofuginone)-induced reduction of α-SMA mRNA expression in skin fibroblasts.

<p>Skin fibroblasts were treated with or without T-3833261 or Halofuginone (1–300 nM) and/or proline (0.05, 0.2 and 1 mM) for 24 h. After incubation for 24 h, mRNA levels were measured by quantitative real-time RT-PCR. Gene expression levels (normalized to GAPDH) are expressed as the fold change of vehicle-treated control. Values are means ± SD (n = 3).</p

Effects of T-3833261 or Halofuginone on mRNA expression of fibrosis-related genes.

<p>Skin fibroblasts were pre-treated with samples (1–300 nM), followed by stimulation with TGF-β (1 ng/mL). After incubation for 24 h, α-SMA, COL1A1 (A), DDIT3, FGF2 and SMURF2 (B) mRNA levels were measured by real-time quantitative RT-PCR. Gene expression levels (normalized to GAPDH) are expressed as the fold change of vehicle-treated control. Values are mean ± SD (n = 6). #p<0.05 compared to vehicle-treated control, *p<0.05 compared to TGF-β-treated control. The experiment was repeated by using other fibroblast lots and similar results were obtained.</p