Investigating the effect of autophagy odulation on mitochondrial donation in human mesenchymal stem cells

Abstract

Recent studies have revealed the role of autophagy in mesenchymal stem cells function and regenerative properties. How and by which mechanism autophagy modulation can affect the juxtacrine interaction of mesenchymal stem cells should be addressed. In this study, the role of autophagy was investigated in the formation of tunneling nanotubes and mitochondrial donation between homotypic cells. Methods: mesenchymal stem cells were treated with 15 μM Metformin (stimulator of autophagy) and/or 3 μM 3-methyladenine (inhibitor of autophagy) for 48 hours. The formation of tunneling nanotubes was assessed using inverted microscope images and scanning electron microscop. The levels of proteins related to the autophagy pathway, tunneling nanotubes and cytoskeleton remodling were studied by western blotting. The mitochondria density and mitochondrial membrane potential (ΔΨ) values were monitored using flow cytometry analysis. Using RT-PCR and protein array, the close interaction and common mediators between autophagy, apoptosis, and Wnt signaling pathways were also monitored. The total fatty acid profile was assessed using gas chromatography. Results: Data indicated the increase of tunneling nanotubes length and number, along with other cell projections after the induction of autophagy while these features were inhibited in 3-methyladenine treated mesenchymal stem cells(p<0.05). Western blotting revealed the significant reduction of Rab8 and p-FAK in 3-methyladenine-treated mesenchymal stem cells (p<0.05), indicating the inhibition of tunneling nanotubes assembly and vesicle transport. Likewise, the stimulation of autophagy increased autophagic flux and mitochondrial membrane integrity compared to mesenchymal stem cells treated with 3-methyladenine. Despite these findings, protein levels of mitochondrial membrane Miro1 and 2 were unchanged after autophagy inhibition/stimulation (p>0.05). We found that the inhibition/stimulation of autophagy can affect the protein, and transcription levels of several mediators related to Wnt and apoptosis signaling pathways involved in different cell biological activities. Data confirmed the increase in the ratio of total unsaturated fatty acids to saturated fatty acids in mesenchymal stem cells exposed to the autophagy stimulator.