Autophagy modulation effect on homotypic transfer of intracellular components via tunneling nanotubes in mesenchymal stem cells

Abstract

Background Recent studies have proved the role of autophagy in mesenchymal stem cell (MSCs) function and regenerative properties. How and by which mechanism autophagy modulation can affect the juxtacrine interaction of MSCs should be addressed. Here, the role of autophagy was investigated in the formation of tunneling nanotubes (TNTs) and homotypic mitochondrial donation. Methods MSCs were incubated with 15 µM Metformin (Met) and/or 3 µM 3-methyladenine (3-MA) for 48 h. The formation of TNTs was assessed using bright-field and SEM images. The mitochondria density and ΔΨ values were monitored using flow cytometry analysis. Using RT-PCR and protein array, the close interaction and shared mediators between autophagy, apoptosis, and Wnt signaling pathways were also monitored. The total fatty acid profile was assessed using gas chromatography. Result Data indicated the increase of TNT length and number, along with other cell projections after the induction of autophagy while these features were blunted in 3-MA-treated MSCs (p < 0.05). Western blotting revealed the significant reduction of Rab8 and p-FAK in 3-MA-treated MSCs (p < 0.05), indicating the inhibition of TNT assembly and vesicle transport. Likewise, the stimulation of autophagy increased autophagic flux and mitochondrial membrane integrity compared to 3-MA-treated MSCs. 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

1. Introduction

During the last decades, the discovery and application of various progenitors and stem cell types have revolutionized human medicine [1, 2]. Mesenchymal stem cells (MSCs) are the most commonly used stem cells in several experiments and human clinical trials [3–7]. These cells can use a multitude of mechanisms to exert their regenerative properties in the injured sites. Secretion of chemokines, cytokines, growth factors, metabolic products, and extracellular matrix, commonly referred to as the paracrine effect, is the main underlying mechanism for reparative outcomes [8]. In stem cell secretome, various extracellular vesicle (EVs) types, including exosomes and microvesicles, can transfer signaling molecules and maintain reciprocal communication between cells [9, 10].

Tunneling nanotubes (TNTs) are cytoskeletal protrusions and maintain juxtacrine cell-to-cell communication [11]. TNTs enable the transmission of intracellular organelles in bi- and uni-directional manners, exhibiting distinct structural and functional characteristics in various cell types [12]. These nano-sized structures are supported by the actin polymer backbone and can generated via the elongation of cellular protrusion and dislodgement [13]. Of note, TNTs can facilitate the transfer of diverse cellular cargo such as mitochondria and Golgi vesicles, as well as vesicles, genetic molecules like microRNA and siRNA, proteins, and ions between the cells [14]. It has been found that TNTs can also distribute various microbial pathogens and factors associated with several pathological conditions such as neurological disorders and cancers [15]. MSCs can generate TNTs as intercellular bridges to transport cargo, i.e. mitochondria, for regenerative purposes [16, 17]. Along with cytoskeletal remodeling, several enzymes and mediators such as Miro1 and 2 (mitochondrial Rho-GTPases), Rab8, TRAK1, TRAK2, and Myo19 are required for organelle donation. It has been found that knockdown of Miro1 inhibits TNT production and blunt regenerative potential of MSCs [17, 18].

Autophagy serves as a crucial intracellular degradation system and is regulated by various autophagy-related (ATG) proteins. By the activation of autophagy, the host cells can recycle abnormal cytoplasmic components.