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Adenosine diphosphate released from stressed cells triggers mitochondrial transfer to achieve tissue homeostasis [1]

['Hao Li', 'Department Of Orthopaedics', 'Shanghai Sixth People S Hospital Affiliated To Shanghai Jiao Tong University School Of Medicine', 'Shanghai', 'Institute Of Microsurgery On Extremities', 'Department Of Orthopedic Surgery', 'Hongping Yu', 'The First Affiliated Hospital Of Xiamen University', 'School Of Medicine', 'Xiamen University']

Date: 2024-08

Cell-to-cell mitochondrial transfer has recently been shown to play a role in maintaining physiological functions of cell. We previously illustrated that mitochondrial transfer within osteocyte dendritic network regulates bone tissue homeostasis. However, the mechanism of triggering this process has not been explored. Here, we showed that stressed osteocytes in mice release adenosine diphosphate (ADP), resulting in triggering mitochondrial transfer from healthy osteocytes to restore the oxygen consumption rate (OCR) and to alleviate reactive oxygen species accumulation. Furthermore, we identified that P2Y2 and P2Y6 transduced the ADP signal to regulate osteocyte mitochondrial transfer. We showed that mitochondrial metabolism is impaired in aged osteocytes, and there were more extracellular nucleotides release into the matrix in aged cortical bone due to compromised membrane integrity. Conditioned medium from aged osteocytes triggered mitochondrial transfer between osteocytes to enhance the energy metabolism. Together, using osteocyte as an example, this study showed new insights into how extracellular ADP triggers healthy cells to rescue energy metabolism crisis in stressed cells via mitochondrial transfer in tissue homeostasis.

During aging, mitochondrial membrane undergoes profound architectural changes, including inner membrane vesiculation and ATP synthase dimer dissociation, which in turn impairs adenosine diphosphate (ADP) turnover in cells. Subsequent reactive oxygen species (ROS) accumulation initiates various types of cellular damage [ 14 , 15 ]. Mitochondrial dysfunction plays a vital role in various physiological and pathological processes, including age-related degeneration. Abnormalities in mitochondrial structures, components, and functions precipitate diseases of nervous [ 16 ], cardiovascular [ 17 ], digestive [ 18 ], and immune system [ 19 ]. In this study, we demonstrated a mechanism of triggering mitochondrial transfer between cells. It is also shown that mitochondrial transfer play roles in maintaining bone homeostasis. Extracellular ADP acts as a chemical signal to trigger intercellular mitochondrial transfer between osteocytes, interacting with the nucleotide receptors, P2Y2 and P2Y6, on neighboring cells in a paracrine manner. Mitochondrial transfer is initiated to rescue energy metabolism crisis in stressed cells. Our results revealed a novel mechanism of cell–cell interaction in maintaining tissue homeostasis through mitochondrial transfer.

Increasing evidence indicated recently that mitochondria undergo dynamic intercellular transfer between cells [ 1 ]. The horizontal mitochondrial transfer has been observed in vivo and proved to play roles in physiological [ 2 , 3 ] and pathophysiological conditions [ 4 ]. Their impact in physiological and pathological conditions has been implied in various tissues and organs [ 5 ]. Therapeutic strategies for restoration of mitochondrial functions have been developed. One of the emerging trends is mitochondrial transfer and transplantation, which have become a promising therapeutic option for treatment of diseases such as ischemic diseases [ 6 , 7 ], reperfusion injuries [ 8 ], and neural disorders [ 9 ]. Our previous study demonstrated that osteocytes sustain their viability by transferring healthy mitochondria to neighboring stressed osteocytes [ 10 ]. While several studies have shown the role of intercellular mitochondria movement toward cells with dysfunctional mitochondria in tissue homeostasis [ 11 – 13 ], it is not clear what triggers the transfer of mitochondria in cells.

Results

Extracellular ADP mediates healthy cells rescuing stressed cells through restoration of the respiratory chain capacity Next, we explored the role of released nucleotides in maintaining the homeostasis of the osteocyte network. We adopted an MLO-Y4 cells co-culture system for the mitochondrial stress test using different ratios of healthy and stressed cells. The mitochondrial stress test showed that oligomycin treatment decreased mitochondrial activity, as the oxygen consumption rate (OCR) dropped drastically in these stressed cells (Fig 1K–1N). When the number of healthy MLO-Y4 cells decreased, the OCR dropped dramatically, indicating that there were fewer healthy cells to maintain homeostasis (Fig 1O–1T). Moreover, ADP supplementation enhances mitochondrial respiratory activity. However, when there were less than 50% healthy cells within the system, the effect of ADP treatment was less obvious (Fig 1O–1T). Next, we examine whether ADP administration could alleviate ROS levels in the same co-culture system by conducting cytometry quantification. We showed that after adding ADP, the ROS level (MitoSOX) decreased compared to that of the same system without ADP treatment. Similarly, when there were less than 50% healthy cells, this effect was not obvious (Fig 1U). Moreover, the effect of ADP in alleviating ROS could not be observed in stressed cells (S3 Fig). These results suggested that the effect of ADP restoring the respiratory chain capacity requires the presence of a sufficient number of healthy cells.

Extracellular ADP enhances endoplasmic reticulum-mitochondria contacts To explore the details of mitochondrial transfer after ADP binds to P2Y2/P2Y6 receptors, an RNA-seq assay on ADP-treated MLO-Y4 cells was performed. In low concentration (0.2 μm) ADP-treated cells, GO analysis indicated that endoplasmic reticulum (ER)-associated genes were most significantly regulated (Fig 5A), and in high concentration (2 μm) ADP-treated cells, dendrite-related genes were most obviously regulated (Fig 5B). Furthermore, RNA-seq analysis of P2Y2/P2Y6 knockdown cells was conducted to validate the effect of these 2 receptors. We combined the results of RNA-seq analysis on both wild-type and knockdown cells. Two subsets of genes were obtained by overlapping gene sets according to the results of RNA-seq analysis. Subset A and subset B contained up-regulated and down-regulated genes in WT cells after stimulation with ADP but were not up-regulated or down-regulated in knockdown cells (Fig 5C). The expression of genes in these 2 sets is not regulated by the binding of ADP to P2Y2/P2Y6 receptors. GO analysis of genes in set A and set B showed that ER- and mitochondria-associated terms were among the top enriched GO terms (Fig 5D and 5E). The results indicated that ER and osteocyte dendritic processes may be correlated with ADP-triggered mitochondrial transfer between osteocytes. Next, we investigated the status of the ER in dendrites. As the ER can directly interact with mitochondria, we stained both the ER and mitochondria by using ER Tracker Green and MitoTracker Red (MTR), respectively. Confocal live cell imaging was used to visualize the distributions of both the ER and mitochondria in MLO-Y4 cells dendrites (Fig 5F), and the levels of ER-mitochondria contact were calculated according to the length of the contact area and the perimeter of mitochondria. Although the average ER areas in dendrites were not different (Fig 5G), the length of ER-mitochondria contacts as well as the contact levels per mitochondria in dendrites were significantly increased in the high-concentration ADP-treated group (Fig 5H and 5I). Next, contact levels were further validated by the stabilized ER-mitochondria tether proteins Mfn2 and Miro1 (Fig 5J and 5K). Mfn2 mediates ER-mitochondria contacts, which have been shown to regulate mitochondrial transfer between osteocytes [18]. These results suggested that extracellular ADP triggers mitochondrial transfer by regulating ER-mitochondria contacts. PPT PowerPoint slide

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TIFF original image Download: Fig 5. The activation of P2Y2/P2Y6 receptors promotes mitochondrial motility through Mfn2. (A and B) RNA-Seq GO analysis of significantly regulated genes showed the top regulated genes in MLO-Y4 cells treated with 0.2 μm and 2 μm ADP. (C) Venn diagram of the selection of gene subsets that might participate in the promotion of mitochondrial transfer through the membrane nucleotide receptors P2Y2 and P2Y6. Subset A: The upper left circle represents genes that are up-regulated in wild-type MLO-Y4 cells after treatment with additional ADP; the upper right and lower circles represent genes that are not up-regulated in P2Y2/P2Y6 knockdown MLO-Y4 cells after treatment with additional ADP. Subset A is the overlap of the 3 sets of genes. Subset B: The upper left circle represents genes that are down-regulated in wild-type MLO-Y4 cells after treatment with additional ADP; the upper right and lower circles represent genes that are not down-regulated in P2Y2/P2Y6 knockdown MLO-Y4 cells after treatment with additional ADP. Subset B is the overlap of the 3 sets of genes. (D and E) GO enrichment analysis of enriched cellular compartment terms of genes in subset A and subset B. (F) Representative images of dendrites with mitochondria (MTR) and endoplasm (ERTG) labeling in MLO-Y4 cells treated with vehicle and ADP at 0.2 μm or 2 μm. (G–I) Morphological parameter analysis based on confocal images, including the average area of ER per dendrite, length of ER-mitochondria contacts, and contact level per mitochondria. (J) Immunoblotting for Mfn2, Miro1, and Actin in MLO-Y4 cells treated with vehicle, 0.2 μm ADP and 2 μm ADP. (K) Immunoblotting for Miro1 in the osteocytes of Mfn2fl/fl and Dmp1CreMfn2fl/fl mice with or without ADP treatment. The data underlying the graphs shown in the figure can be found in S5 Data. ADP, adenosine diphosphate; ER, endoplasmic reticulum; GO, gene ontology. https://doi.org/10.1371/journal.pbio.3002753.g005

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002753

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