key: cord-0257132-4i85bqww
authors: Sagar, Shakti; Faizan, Md. Imam; Chaudhary, Nisha; Gheware, Atish; Sharma, Khushboo; Azmi, Iqbal; Singh, Vijay Pal; Kharya, Gaurav; Mabalirajan, Ulaganathan; Agrawal, Anurag; Ahmad, Tanveer; Roy, Soumya Sinha
title: Obesity impairs therapeutic efficacy of mesenchymal stem cells by inhibiting cardiolipin-dependent mitophagy and intercellular mitochondrial transfer in mouse models of allergic airway inflammation
date: 2021-11-28
journal: bioRxiv
DOI: 10.1101/2021.11.27.470183
sha: ff705432619943dc19c3a8723fa85a086480caf5
doc_id: 257132
cord_uid: 4i85bqww

Mesenchymal stem cell (MSC) transplantation alleviates metabolic defects in diseased recipient cells by intercellular mitochondrial transport (IMT). However, the effect of host metabolic conditions on MSCs in general, and IMT in particular, has largely remained unexplored. This study has identified a molecular pathway that primarily governs the metabolic function and IMT of MSCs. We found underlying mitochondrial dysfunction, impaired mitophagy, and reduced IMT in MSCs derived from high-fat diet (HFD)-induced obese mice (MSC-Ob). Mechanistically, MSC-Ob failed to sequester their damaged mitochondria into LC3-dependent autophagosomes due to decrease in mitochondrial cardiolipin content, which we propose as a putative mitophagy receptor for LC3 in MSCs. Functionally, MSC-Ob exhibited diminished potential to rescue metabolic deficits and cell death in stress-induced epithelial cells. In a small molecule screen, we found pyrroloquinoline quinone (PQQ) as a regulator of mitophagy and IMT. Long-term culture of MSC-Ob with PQQ (MSC-ObPQQ) restored cardiolipin content and sequestration of mitochondria to autophagosomes with concomitant activation of mitophagy. Upon co-culture, MSC-ObPQQ rescued cell death in stress-induced epithelial cells by enhancing IMT. The beneficial effect of PQQ was also evident in MSCs derived from human subjects in an in vitro model. In two independent mice models, the transplantation of MSC-ObPQQ restored IMT to airway epithelial cells, improved their mitochondrial metabolism and attenuated features of allergic airway inflammation (AAI). However, unmodulated MSC-Ob failed to do so. In summary, we uncover the molecular mechanism leading to the therapeutic decline of obese-derived MSCs and highlight the importance of pharmacological modulation of these cells for therapeutic intervention.

MSCs are widely being explored as promising cell-based therapies for several human diseases (1, 2) . Clinical trials are underway to explore MSCs for the treatment of complex lung disorders (2, 3) . A recent addition is the successful early phase clinical trial of MSCs to lower mortality due to COVID-19 (4) . The beneficial effect of MSCs is attributed to their intrinsic immunomodulatory activity, generally mediated by their paracrine secretions (5) (6) (7) . Over the last decade, a new paradigm of MSC therapeutics has emerged which is based on their unique ability to donate functional mitochondria to metabolically compromised recipient cells (8) (9) (10) (11) (12) .

The mitochondrial donation by MSCs to injured alveolar and bronchial epithelial cells alleviates acute lung injury and inflammatory airway diseases (8, 13, 14) , independent of their immunomodulatory cytokine secretion and regenerative potential (8) . On the flip side, mitochondrial donation by MSCs enhances bioenergetics in cancer cells (15) (16) (17) and increases their drug-resistance. This mitochondrial donation potential is also retained by MSCs derived from induced pluripotent cells (iPSCs), which alleviate asthmatic features and cigarette smokeinduced COPD (18, 19) . Further, MSCs donate mitochondria directly to immune cells to modulate host cell immune response and alleviate tissue inflammation (20) . Nevertheless, most of the studies exploring the therapeutic efficacy of MSCs have relied on the cells obtained from disease-free conditions. Induction of cell stress in MSC drastically reduces their intercellular mitochondrial donation capacity and compromises their therapeutic efficacy (10) .

Accumulating evidence now show that MSCs derived from disease-specific animal models and human patients exhibit impaired bioenergetics and altered mitochondrial quality control (MQC), which may compromise their long-term therapeutic efficacy (21) (22) (23) (24) (25) .

Human adipose derived stem cells (ASCs) from aged individuals display oxidative stress, reduced multilineage proliferation rate, and decline in differential potential with a concomitant increase in senescence phenotype (26, 27) . Similarly, ASCs derived from human orbital fat show senescent phenotype, reduced differential potential, and impaired stemness properties (28). These age-related changes markedly reduce the number of functional MSCs, which further limits their clinical application (29). Most notably, these changes are associated with a decline in mitochondrial function and elevated oxidative stress (30). Besides donor age, mitochondrial dysfunction is also observed in MSCs obtained from animal models of metabolic syndrome (MetS) and human patients with obesity, diabetes, MetS, and aging (31, 32) . Obese condition also has a pronounced effect on MSC immunomodulatory activity, stem cell 4 property, and therapeutic efficacy (23, 33) . This has important clinical implications for autologous MSC treatment in such patients, especially for indications related to complications of metabolic disorders. Here we examine whether intercellular mitochondrial donation capacity and therapeutic efficacy of MSC from obese subjects is compromised. Further, we examine whether mitochondria-targeted therapy can reverse such defects, enabling autologous treatment.

Generally, dysfunctional mitochondria are selectively removed from the cells by mitophagy that reduces the accumulation of damaged mitochondria to prevent cell death (34, 35) . Mitophagy is normally followed by mitochondrial biogenesis to ensure an adequate number of functional mitochondria through the mitochondrial quality control (MQC) pathway (36) . Under certain disease conditions, mitochondrial dysfunction may not always be accompanied by mitophagy, which results in metabolic decline and eventually cell death (37) .

Classical mitophagy involves stabilization of mitochondrial serine/threonine-protein kinase PINK1 on the outer membrane of depolarized mitochondria and recruitment of cytosolic E3ubiquitin-protein ligase Parkin to these depolarized mitochondria. Though, alternate Parkin independent mitophagy mediated by BNIP3L/NIX and FUNDC1 exists in certain cells, (38) (39) (40) recent studies suggest that MSCs mostly utilize the classical PINK1/Parkin pathway to clear the damaged mitochondria (41, 42) . Conditions such as senescence, obesity, and type II diabetes mitigate autophagy/mitophagy in MSCs, resulting in the accumulation of dysfunctional mitochondria and compromised beneficial effect (43) (44) (45) (46) . However, the mechanism of mitochondrial dysfunction and the role of mitophagy in determining the therapeutic potential of MSCs has largely remained unexplored.

Here, we have systematically evaluated the role of mitophagy in MSCs derived from the (HFD)-induced obese mice and human MSCs and evaluated their therapeutic potential in vitro and in two independent mouse models of allergic asthma. Our findings uncover previously unknown mechanism that determine the mitophagy, IMT and thereupon the therapeutic efficacy of MSCs. Importantly, we have shown that the impaired therapeutic potential of MSC-Ob is reversible with a small molecule PQQ, which restores mitophagy and IMT by enhancing sequestration of dysfunctional mitochondria to autophagosomes.

We developed an 18-week-old (HFD)-induced obese mice model as described by us previously (Fig. 1A) (47) . An increase in body weight (44. 2±0.68 in obese vs. 24.9±00.48 in lean) and changes in biochemical profile confirmed the obesity features in these mice (Fig. S1) . To determine whether MSCs derived from HFD mice maintain stem cell property, we harvested cells from lean (MSC-L) and HFD mice (MSC-Ob) and analyzed them for expression of key stem cell markers (positive: Sca1, CD44; negative: CD11b). Both the groups had similar expression of the stem cell markers (Fig. S2A) . However, MSC-Ob showed a trend towards increase in cell death and cellular senescence but decrease in the rate of cell proliferation ( It has largely remained elusive as to what extent MSCs derived from diseased, particularly from obese patients retain therapeutic efficacy. To probe the same, we used a coculture system to model mitochondrial donation, using mito-GFP expressing MSCs as donors and stressed mouse lung epithelial cells (MLE12) as mitochondria recipients. MLE12 cells were first treated with either vehicle (Veh) or rotenone (Rot) (to induce mitochondrial dysfunction) for 12 hours (hrs) and then stained with cell tracker deep-red before co-culture.

After 24 hrs of co-culture, we performed flow cytometry analysis to measure the percentage of MLE12 cells expressing mito-GFP -an indicator of mitochondrial uptake. A substantial decrease in intercellular mitochondrial transport from MSC-Ob to the MLE12 Rot cells was observed, compared to that from MSC-L (Fig. 1B) . We found a similar trend using imaging studies followed by quantitative analysis (Fig. 1C-D) . This decline in the mitochondrial donation by MSC-Ob was associated with their diminished potential to rescue cell death in MLE12 Rot cells (Fig. 1E) .

We have previously reported that Miro1 regulates the intercellular transfer of mitochondria by MSCs (8) . To examine whether the decrease in mitochondrial donation was due to the differential expression of Miro1, we measured its expression at mRNA and protein levels. However, MSC-L and MSC-Ob did not significantly differ in the Miro1 expression ( Fig. 1F and S 3A) , which is consistent with our previous study that endogenous Miro1 expression does change during epithelial cell stress (8) . Similarly, we did not find any 6 significant changes in tunneling nanotube (TNT) formation, which are the primary mediators of IMT (Fig. S3B, C) (48) .

The decrease in IMT can also attribute to enhanced mitochondrial turn-over or reduced mitochondrial biogenesis. On the contrary, we found a distinctly increased mitochondrial mass, mtDNA content, and mitochondrial number per cell in MSC-Ob, as measured by flow cytometry, imaging analysis, and RT-qPCR ( Fig. 1G-J) . The increase in mitochondrial mass was not due to mitochondrial biogenesis as reflected by unaltered PGC-1α expression (Fig.   1K) . Notably, MSC-Ob displayed smaller and punctate mitochondrial structure with disrupted cristae than the regular tubular network seen in MSC-L ( Fig. 1L-N) . In line with changes in mitochondrial shape, we found trend towards increase in Drp1 (mito-fission protein) with a concomitant decrease in Mfn2 (mito-fusion protein) expression (Fig. O) . These mitochondrial shape and form changes corroborate increased mitochondrial ROS (mtROS) levels, reduced mitochondrial membrane potential (ΔΨm), and decreased bioenergetic flux ( Fig. 1P-R) .

Prominently, ATP levels, basal respiration, maximal respiration, and spare respiratory capacity were significantly reduced (Fig. S4) . Altogether, these results indicate that MSCs derived from obese mice exhibit reduced intercellular mitochondrial donation, apparently due to the accumulation of dysfunctional mitochondria.

Accumulation of dysfunctional mitochondria is generally attributed to their reduced clearance from the cells by mitophagy (49) . Therefore, we looked at the conventional pathway of mitophagy in MSCs, which involves PINK1 and Parkin. MSCs were treated with FCCP (5 and 10 µM) to induce mitochondrial depolarization, and mitochondrial fractions were prepared and subjected to immunoblotting for endogenous PINK1 and Parkin. The quality of mitochondrial fraction was confirmed by checking for the expression of mitochondrial marker and the absence of cytosolic marker (Fig. S5A) . As shown in Fig. 2A , FCCP treatment induced accumulation/stabilization of PINK1 in the mitochondrial fractions derived from MSC-L.

Surprisingly, MSC-Ob showed inherent stabilization of PINK1, with no further increase upon depolarization ( Fig. 2A) . Parkin showed a similar pattern of expression (Fig. 2B) and these results were confirmed by immunofluorescence followed by colocalization analysis (as calculated by Mander's coefficient) (Fig. 2C, D) . These results are thus consistent with the notion that depolarized mitochondria stabilize PINK1 and recruit cytosolic Parkin (34, 50) .

This pattern of expression seen in MSC-Ob indicates their inherently depolarized mitochondria, as shown in Fig. 1Q .

We next assessed the role of essential proteins implicated in autophagosome formation.

Depolarized mitochondria are generally sequestered to the autophagosomes and thereupon to lysosomes for clearance (34, 50) . The autophagosome formation begins with the maturation of diffused LC3 to the puncta forming LC3-II, which is also considered as a marker for autophagosomes. First, we performed immunoblotting to probe any changes in the maturation of LC3-II. As shown in Fig. 2E , FCCP treatment induced LC3-II formation in MSC-L, whereas MSC-Ob had significantly lower levels. Next, we evaluated LC3-dependent autophagosome formation and LC3 colocalization with mitochondria under un-induced and chemicallyinduced depolarization conditions. We used a mild depolarizing agent antimycin A (AMA), which induces mitochondrial dysfunction by enhancing mtROS production (51) . As expected, we found LC3 puncta formation in MSC-L and their colocalization with mitochondria upon depolarization. On the contrary, MSC-Ob showed significantly reduced colocalization with a concomitant decrease in the autophagosome number (Fig. 2F-H and S5B, C) . We also assessed the effect of LC3 overexpression on autophagosome formation and mitochondrial sequestration. However, we did not find any significant changes in LC3 maturation and colocalization with mitochondria in MSC-Ob after LC3 overexpression (Fig. S5D) . These results thus indicate that reduced sequestration of dysfunctional mitochondria into the autophagosomes contributes to their accumulation in MSC-Ob.

To examine the involvement of the general autophagy pathway, we assessed the expression of crucial autophagy regulatory proteins. However, we did not find any significant changes in the expression of Atg5, Atg7, total and phosphorylated Beclin1 (Fig. S6A-C) . Similarly, using an autophagy pathway specific RT-qPCR array, we did not find differential expression in their transcript levels (Fig. S6D) . These results suggest that the expression of general autophagy proteins remains unaltered in MSC-Ob, despite significant alterations in the mitophagy pathway.

The autophagosomes eventually fuse with the lysosomes to form the autophagolysosome. Previous studies have reported impaired mitophagy during lysosomal abnormalities (52) . We thus assessed the status of lysosomes by evaluating the expression of late endosome/lysosome marker LAMP1 and lysosomal content. Surprisingly, we observed a 8 significant increase in basal LAMP1 expression in MSC-Ob. On the contrary, MSC-L showed increased expression only upon depolarization (Fig. 3A, B) . Additionally, we performed livecell imaging of cells stained with lysotracker deep red (LTDR) followed by image analysis. As shown in Fig. 3C , an increase in the LTDR staining in MSC-Ob was observed. These results thus suggest that MSC-Ob has inherently higher lysosomal content.

Given the fact that MSC-Ob have increased lysosomal content and an alternative autophagy pathway exists wherein depolarized mitochondria are directly taken up by the endosomal pathway for clearance (53, 54) , we investigated whether increase in lysosomal content primes these cells to use this alternative for mitochondrial clearance. To explore this possibility, we transduced the cells with lentiviral mito-GFP and subsequently stained them with LAMP1 and LTDR, respectively. MSC-Ob did not show any significant changes in the association of mitochondria with LAMP1 ( Fig. 3D) 

To check whether MSC-Ob has a general defect in the autophagic flux, we used a control autophagy assay based on p62/SQSTM1 turnover (55) . Autophagy was induced in the cells by starvation. Consistent with the results above (Fig. 2) , autophagosome formation was induced in MSC-Ob only upon starvation, albeit lower than MSC-L. Notably, MSC-Ob showed relatively lower p62 colocalization with LC3 and LAMP1 than MSC-L (Fig. S7) . Together, these results illustrate that an increase in lysosomal content observed in MSC-Ob does not correlate with clearance of dysfunctional mitochondria.

For activation of downstream mitophagy, LC3-containing autophagosome binds to the depolarized mitochondria via mitophagy receptors. Altered expression of these receptors impairs sequestration of damaged mitochondria into autophagosomes (36, 56) . We thus evaluated the expression pattern of these receptors. While FUNDC1 and p62 expression did not significantly differ between MSC-L and MSC-Ob, a significant decrease in cardiolipin (stained with NAO) content was observed ( Fig. 3H-K) . We also observed that MSC-Ob cardiolipin-stained mitochondria showed reduced colocalization with LC3 upon depolarization ( Fig. 3L and S8 ). Our findings (including Fig. 1M ) are consistent with previous reports that 9 decrease in cardiolipin causes cristae disruption and impairs mitochondrial clearance due to reduced interaction with LC3-containing autophagosomes (57, 58) . Thus, these results demonstrate that obese-derived MSCs with underlying mitochondrial dysfunction and reduced cardiolipin content fail to sequester their depolarized mitochondria into the autophagosomes.

To correlate our findings in human MSCs, we also evaluated the impact of free fatty acids (FFA) on mitochondrial health, cardiolipin content and mitophagy, in an in vitro model of a human (h) MSC FFA . Cells were harvested from normal healthy subjects with no known history of MetS or obesity and characterized for stem cell markers (Fig. S9A) . The in vitro model depicting features of MetS or obesity was developed by treating the cells with FFA for 24 hrs as described earlier (59) . Upon culturing in FFA, hMSCs showed increased mtROS, pronounced punctate mitochondrial morphology, and increased mitochondrial mass (Fig. S9B-D) . Additionally, hMSC FFA showed a significant reduction in cardiolipin content (Fig. S9E) .

Upon depolarization, LC3 colocalised with cardiolipins to a much higher extent in Veh than hMSC FFA (Fig. S9F) . Collectively, these findings reveal that like MSC-Ob, hMSC FFA also exhibit accumulation of dysfunctional mitochondria and reduced cardiolipin content which may impede the clearance of defective mitochondria.

From the results above, it is evident that MSC-Ob has dysfunctional mitochondria with associated impaired mitophagy. We thus hypothesized that restoring mitochondrial health or autophagy will increase clearance of depolarized mitochondria. We choose drugs that directly or indirectly regulate mitochondrial health, mitophagy and autophagy. As an assay outcome, we used endogenous Tom20 and LC3 as markers to find the effect of these small molecules in inducing sequestration of mitochondria into autophagosomes. Images were taken and subjected to image analysis to determine the extent of colocalization between LC3 and mitochondria.

Interestingly, we found relatively higher colocalization by pyrroloquinoline quinone (PQQ) than other molecules tested (Fig. 4A) . Earlier studies have reported that PQQ attenuates mtROS and improves mitochondrial health, and increases autophagy (60-62), so based on these rational we choose PQQ in subsequent experiments to explore its role in alleviating mitochondrial health of MSC-Ob. Initially, we tested various time points of PQQ to find the optimal dose by looking at its effect in lowering the mtROS. We found that chronic treatment (6 doses at an interval of 48 hrs) of 30 µM PQQ (hereafter referred to as PQQ) significantly reduced mtROS in MSC-Ob PQQ as compared to any other dose and time point tested ( Fig. 4B and S10B). PQQ treatment also restored mitochondrial, ΔΨm, and enhanced mitochondrial bioenergetics ( Fig. 4C-E) . Moreover, MSC-Ob PQQ showed more elongated and tubular-shaped mitochondria than punctate form, with a concomitant decrease in mitochondrial mass and restoration of damaged cristae, suggesting clearance of the damaged mitochondria ( Fig. 4F-H and S10C, D).

We next assessed the effect of PQQ in the clearance of damaged mitochondria. A significant increase in the colocalization of mitochondria with LC3-autophagosomes was observed in MSC-Ob treated with PQQ ( Fig. 4I-K) . This effect of PQQ was corroborated with increase in the cardiolipin content and colocalisation with LC3 ( Fig. S11A-C) with a similar trend observed in hMSC FFA treated with PQQ ( Fig. S11D-E) . Similarly, we found that PQQ enhanced the recruitment of mitochondria to the lysosomes upon AMA treatment (Fig. 4L,   M) . Functionally, the effect of PQQ was evident from the clearance of chemically-induced depolarized mitochondria from MSC-Ob PQQ but not from untreated MSC-Ob, which accumulate the damaged mitochondria (Fig. N) . These results indicate that PQQ induces cardiolipin levels which enhances sequestration of dysfunctional mitochondria into LC3containing autophagosomes.

We and others have previously reported that donation of healthy functional mitochondria is integral to the therapeutic potential of MSCs, while a donation of dysfunctional mitochondria deteriorates the health of recipient cells (8, 63) . Here, we asked whether MSC-Ob PQQ can rescue epithelial cell apoptosis by donating functional mitochondria. As shown in Fig. 5A , in comparison to the untreated cells, PQQ treatment significantly increased IMT potential of MSC-Ob to damaged epithelial cells. This mitochondrial donation was associated with attenuation of mtROS and restoration of ΔΨm in recipient MLE12 cells (Fig. 5B, C) . Further, the mitochondrial donation by MSC-Ob PQQ restored mitochondrial shape, mitochondrial mass, and attenuated cell death in MLE12 cells (Fig. 5D-F) . The effect of PQQ to restore IMT was found independent of its effect on Miro1 expression and TNT formation (Fig. 5G, H) . These results thus suggest that mitochondrial health regulates IMT in MSCs. While partially depolarized mitochondria may still undergo IMT (42) , severe depolarization significantly compromises this property. These findings are consistent with our previous findings that Rot-induced MSCs have impaired mitochondrial donation potential (8) . Notably, the results presented here demonstrate that obesity-associated changes in MSC-Ob are reversible. Their therapeutic efficacy can be restored by enhancing sequestration of dysfunctional mitochondria into autophagosomes. whereas MSC-Ob failed to do so (Fig. S12D) . Further, epithelial cell damage was significantly reduced in the animals treated with MSC-Ob PQQ than MSC-Ob (Fig. S12E) . Notably, MSCs harvested from obese mice which were fed PQQ, did not show significant alleviation in AHR or airway inflammation suggesting that direct in vitro modulation of MSC-Ob is a better approach. 

To find the effect of MSCs on airway remodeling and AHR in the HDM model, we evaluated the effect after 48 hrs of MSC transplantation. PQQ treated MSCs significantly attenuated AHR and reduced airway inflammatory cells, while MSC-Ob group showed a trend towards AHR and increased inflammatory response (Fig. 7A, B) . Further, a decrease in mucus hypersecretion was observed in animals treated with MSC-Ob PQQ but no significant effect was seen with MSC-Ob (Fig. 7C) . To look at the effect of MSC-Ob PQQ on inflammatory cells and pro-inflammatory cytokines, we performed total leucocyte count (TLC), and eosinophil cell count in BALF, and measured the inflammatory Th2 cytokine levels in the lung homogenates. A significant reduction in inflammatory cell number and Th2 cytokine release was found in mice treated with MSC-Ob PQQ (Fig. 7D-H) . However, MSC-Ob failed to attenuate these inflammatory responses.

Together with the results from the above-mentioned Ova-induced model, these results strongly suggest that MSCs derived from obese animals have compromised therapeutic efficacy, which is restored upon long-term culture of these cells in PQQ. Thus, it is imperative to consider metabolic modulation of MSCs derived from patients with metabolic syndrome before their application for therapeutic intervention.

In this study, we have uncovered a critical molecular pathway responsible for diminished therapeutic efficacy of MSCs derived from obese source. We found that MSC-Ob: (1) display mitochondrial dysfunction, with reduced protective intercellular mitochondrial transport; (2) show inadequate activation of mitophagic pathway; and (3) exhibit reduced cardiolipin content, which diminishes the sequestration of depolarized mitochondria into autophagosomes. These metabolic changes are together responsible for the therapeutic decline in these cells. Notably, we demonstrate that treatment with a small antioxidant molecule PQQ reverses this therapeutic deficit both in in vitro and in pre-clinical models, summarized in Fig. 8 .

in mitochondrial form and function (64, 65) . MSCs derived from human adipose tissue were shown to possess mitochondria with lowered oxygen consumption rate and compromised intrinsic mitochondrial respiration parameters (66) . We provide the first direct evidence that a high-fat diet modulates the metabolic state and sensitizes MSCs to apoptosis under culture.

Using an in vitro model of IMT as a rescue assay, we demonstrate that while MSC-L restored mtROS levels, membrane potential, bioenergetics, and apoptosis of metabolically stressed MLE12 cells in coculture, MSC-Ob significantly lacked the mitochondrial donation potential and protective effect. Notably, the decline in mitochondrial donation was independent of Miro1 and changes in TNT formation. This diminished IMT was due to underlying mitochondrial dysfunction which is consistent with our previous study that chemically induced mitochondrial dysfunction diminishes IMT by MSCs (8) .

Normally, dysfunctional mitochondria are eliminated from cells by selective autophagy (mitophagy) to escape cell death (34) . However, MSC-Ob displayed impaired mitophagy with concomitant accumulation of dysfunctional mitochondria. Generally, the mitophagy is initiated by stabilization of PINK1 on the depolarized mitochondria which then recruits the cytosolic Parkin to ubiquitinate the OMM proteins (50) . The ubiquitin-decorated mitochondria are then loaded to LC3-phagophores to form the autophagosome before being eventually cleared by lysosomes (50) . Interestingly, our data shows that despite accumulation of PINK1 and Parkin on the inherently depolarized mitochondria of MSC-Ob, these cells are unable to clear the damaged mitochondria. Evaluating the downstream pathway, we found defect in the sequestration of mitochondria to LC3-containing autophagosomes as the underlying cause.

LC3 binds to mitochondria via mitophagy receptors such as FUNDC, p62, AMBRA1, BNIP3, NIX or via interaction with the ubiquitin chains associated with mitochondrial proteins (67) (68) (69) . While we have not specifically investigated the role of all these proteins, we found that at least FUNDC and p62 may not be directly involved. Previous studies in animal models of (HFD)-induced obese mice have reported that FUNDC1 knockout aggravated obesity and insulin resistance (70) . However, MSC-Ob did not exhibit altered expression of FUNDC1, which led us to explore other non-canonical pathways. Interestingly, we found substantial decrease in the cardiolipin content of MSC-Ob, which is reported to interact with LC3 during mitophagy (58) . We also found significant decrease in the LC3 and cardiolipin colocalization in MSC-Ob and in human MSCs treated with FFA (signal 1). Based on these findings, we propose cardiolipins as putative mitophagy receptors for LC3 in MSCs and an underlying mechanism that prevents sequestration of dysfunctional mitochondrial to LC3autophagosomes. In addition, we found a significant decrease in the induction of LC3autophagosomes in MSC-Ob at basal and chemically depolarized conditions (signal 2).

Contrary to the report that over-fed state reduces autophagy-related gene expression (56), we could not find any significant changes in the expression of key autophagy proteins such as Beclin1, Atg5, and Atg7 or gene expression by performing the autophagy pathway specific expression analysis; we presume that other regulatory mechanisms may exist (46) . Recent studies have reported that cells deficient in LC3-dependent autophagosome formation use alternative pathways to clear damaged mitochondria, such as Rab GTPase mediated endosomal-lysosomal pathway or mitochondrial-derived vesicles (MDV)-lysosomal pathways (42, 53, 54) . We evaluated whether MSC-Ob utilize the former pathway. While we found an increase in late endosomal/lysosomal content in MSC-Ob, surprisingly the lysosomal content did not correlate with mitochondrial clearance, thus negating the possibility of this alternate mitophagy pathways in our system. To eliminate the role of defective lysosomal function, we used a control autophagy assay based on p62 sequestration to lysosomes. We found recruitment of autophagy protein p62 to lysosomes suggesting existence of a functional lysosomal pathway.

Thus, we propose a model wherein a cumulative effect (signal 1 and signal 2) of decrease in sequestration of dysfunctional mitochondria to autophagosomes and reduced induction of these structures leads to impaired mitophagy and decrease in IMT in MSC-Ob. In some instances, as a survival mechanism, MSCs outsource mitophagy to remove their partly depolarized 15 mitochondria (42), or by shedding off damaged regions of mitochondria as MDVs (71) .

Although we did not evaluate these alternate mitophagy pathways, our results encourage future studies to explore these mechanisms.

As it became evident that MSC-Ob has impaired mitophagy, we sought ways to restore the mitochondrial function and induce mitophagy which may subsequently restore IMT. A small-molecule screen led to PQQ identification, which alleviated mitochondrial dysfunction and restored IMT. Notably, PQQ enhances cardiolipin content LC3-dependent autophagosome formation with concomitant induction of mitophagy to eliminate depolarized mitochondria.

These results point toward a new role of PQQ in inducing mitophagy in MSCs, besides regulating mitochondrial biogenesis and mtROS, as shown by others (60, 72). Our findings here are consistent with a recent report showing that PQQ induces autophagy in human microglia by regulating LC3 maturation and Atg5 expression, independent of its effect on mitochondrial biogenesis (61) . Further, in neuroblastoma cells and mice models, PQQ induces AMPK1 expression, which is a crucial regulator of autophagosome formation (62) . Thus, we propose that PQQ may have a multifactorial role (including in mitochondrial biogenesis), with a more pronounced effect on restoring mitochondrial health by regulating mitophagy.

To determine the therapeutic efficacy of MSC-Ob and check whether modulation of these cells with PQQ will be clinically relevant, we tested these cells in two different allergic airway inflammation models. In line with our in vitro findings, MSC-Ob PQQ showed enhanced IMT to lung epithelial cells, restoring their bioenergetics, mitochondrial membrane potential, mtROS, and epithelial cell damage. Moreover, MSC-Ob PQQ alleviated Th2 cytokine levels and inflammatory cell infiltration into the lungs, while MSC-Ob failed to do so. These results are consistent with our previous study that MSCs with chemically induced mitochondrial dysfunction fail to restore epithelial cell damage and airway remodeling (8) . Other studies have also reported the poor therapeutic outcome of MSCs with chemically induced mitochondrial dysfunction (11) . Thus, the metabolic activity of MSCs is emerging as integral to their therapeutic efficacy (73) (74) (75) . In line with our work, a recent study has shown that reduced heparan sulfates in white adipose tissue macrophages reduces their ability to receive the mitochondria from adipocytes in an HFD model (76) . Taken these reports along with our own study into consideration, we propose that obesity has a long-lasting impact on MSCs, which besides reducing their mitochondrial donation capacity, may also hamper their immunomodulatory activity (77) . Reportedly, oxidative stress and mitochondrial dysfunction impair the immunomodulatory activity of MSCs (78, 79).

Our findings have far-reaching clinical applications, particularly when autologous MSC transplantation is required. Autologous MSCs are preferable over allogenic sources for their intrinsic property to alleviate graft vs. host disease (GvHD). While alternate sources such as induced pluripotent cell (iPSC)-derived MSCs (80) are being clinically evaluated as universal cells, their clinical efficacy and long-term safety remains unknown. At present, more than 1000 clinical trials are underway to explore the beneficial effect of MSCs (autologous and allogeneic) for the treatment of a wide range of human diseases, including complex lung disorders and obesity (2, 81) . Thus, it is imperative to evaluate the outcome of these studies by considering the source of MSCs. Based on our findings, we strongly recommend evaluating the impact of tissue source of MSCs and suggest that the cells derived from diseased patients should undergo rational modulation for clinical application. with mitotracker green to find the mitochondrial turn-over, represented as integrated density.

Mean±SEM. ****P < 0.001; ***P < 0.005; **P < 0.01; *P < 0.05; ns (non-significant). Scale bars: 10 µm. 

High-fat diet-induced obesity model was developed using our previously described method (47) . Briefly, four to six-week-old C57BL/6 male mice were divided in two groups and were labelled according to the diet provided as control (Chow diet) and high-fat diet (HFD) (Research Diets, Inc.). All the mice were housed in IVC cage with enrichment facilities. Every week, weight estimation was done to record the weight gain. Animals were sacrificed using the combination of xylazine and thiopentone sodium as per body weight.

Acute mice model of asthma using Ovalbumin (OVA) (Sigma) and House dust mite (HDM) (Greer Laboratories) were developed as previously described (8) 

For initial in vitro screening to find the candidate drug molecule which enhances mitophagy by restoring depolarized mitochondria colocalisation with the LC3-autophagosomes, we used the following drugs: Pyrroloquinoline quinone (PQQ; 30µM); nicotinamide mononucleotide (NAM; 1mM); Urolithin A (UA; 50µM); Mito-Tempo (Mito-T; 100 µM); resveratrol (RSV: 1 µM); 3-methyladenine (3-MA; 5mM); N-acetyl cysteine (NAC; 1mM). After 48 hrs of incubation, the cells were fixed and stained for LC3 and Tom20. Images were taken and subjected to analysis using Image J to calculate Integrated density and Mander's coefficient. Human MSCs were treated with a mixture of FFA such as Palmitic acid, Stearic acid, and Oleic acid, ratio of 1:1:1). Cells were treated with two different concentrations of FFA (750 mM and 1 mM) for 24 hrs (59) . The PQQ treatment (30µM) was done 2 hrs prior to FFA treatment.

Blood glucose was measured using Accu-chek active test strips as earlier described (47) .

Biochemical parameters such as triglycerides and cholesterol level were measured in the blood serum by quantitation kits (BIOVISION) as previously described (47) .

MSCs were seeded in 24 well plates and staining for cellular senescence was performed using manufacturer's protocol (Sigma). Briefly, cells were washed with 1X PBS and fixed with fixative buffer for 7 mins at room temperature. Cells were stained with SA-β-gal staining solution at 37 °C for 4 hrs. Senescent cells were stained blue and counted using phase contrast microscope at 10X magnification. The percentage was calculated from five different view field of each sample in four independent experiments.

The cells were dissociated with 0.125% trypsin-EDTA (Sigma) and then stained with using Dead cell apoptosis kit (Invitrogen) with Annexin V FITC and propidium iodide using manufacturer's protocol. 10,000 cells were acquired using flow cytometry (BD Melody).

The live and dead assay was performed using the LIVE/DEAD™ Viability/Cytotoxicity Kit (Invitrogen) according to the manufacturer's instructions. Briefly, the medium was changed to phenol red-free DMEM with green fluorescent calcein-AM to indicate intracellular esterase activity (live cells) and red-fluorescent ethidium homodimer-1 which indicates loss of plasma membrane integrity (dead cells). Cells were incubated at 37 °C for 30 mins and were analyzed under FLoid™ Cell Imaging Station.

Lentiviral particle packaging was performed using previously described method (84) . Briefly, Plasmid encoding for mitochondria specific protein/ autophagosome specific protein is transfected in HEK293T cells along with packaging vector (pDR8.2; Addgene #8455) and envelope encoding protein (VSVG; Addgene # 8454). Lentiviral particles were collected after 48 hrs of transfection and were used for transducing target cell lines.

MSCs were transduced with lentiviral particles having mitochondria targeting protein and GFP in downstream (Addgene) and lysosome were stained with lysotracker Deep-Red (Invitrogen, USA) followed by 3 times wash with 1X PBS. Imaging was done before and after treating with FCCP (10 uM) for 2 hrs using Nikon confocal Ti2E at 60x magnification. Image analysis was done using Nikon Elements software and Image J.

Quantitation of mitochondrial size in mouse and human MSCs were done by staining with Mito-tracker Red (Invitrogen/Thermo Fisher Scientific) using manufacturer's protocol.

In brief, cells were stained with mito-tracker red for 15 mins at 37 °C, followed by 3 wash with 1X PBS. Imaging was done using Nikon confocal Ti2E at 60x magnification and analysis was done using Image J.

Cells were allowed to adhere to glass coverslip before being fixed with 4% paraformaldehyde (ThermoFisher Scientific) in 1X PBS for 15minutes at room temperature. Permeabilization and blocking was done in a buffer containing 0.1% Triton X-100, 5% goat serum in 1X PBS for 1 hr at RT. Primary antibodies, shown in Table 1 , incubation was done in the buffer containing 0.01% Triton X-100 and 2% goat serum in 1X PBS overnight at 4 °C. Secondary antibody incubation was done in the same buffer using fluorophore tagged secondary antibodies for 1 hr at RT. Cells were washed between steps using 1X PBS for 5 minutes each at RT. The coverslip was then mounted on frosted slides (Corning) using DAPI mountant and allowed to dry before being sealed using colorless nail polish. TNT were visualized by staining F actin with phalloidin 594 (Invitrogen).

Human and mouse MSCs were stained with NAO (Invitrogen) followed by three times wash with 1X PBS and fixed with 4% PFA. After fixation, cells were washed 3 times with 1X PBS, blocked in blocking buffer and primary antibody incubation for overnight at 4 °C followed by secondary antibody incubation for 1 hr. Mounted using DAPI and images were obtained on a Nikon confocal Ti2E and were analyzed using Nikon Elements software and Image J.

Immunofluorescence of Mito-GFP presence in MSC transplanted mice lung tissue was done as previously described (8) . Briefly, tissue sections were deparaffinized following the antigen retrieval. Permeabilization was done in a buffer containing 0.1% Triton X-100 for 30mins followed by blocking with blocking buffer (5% sera in 0.01% Triton X-100) at RT for 1 hr. Primary antibody incubation was done in same buffer for overnight at 4 °C followed by 3 times wash with 1 X PBS. Secondary antibody incubation was done in the same buffer for 1 hr at RT followed by 3 times wash with 1X PBS. Nuclear staining was done using DAPI and mounted with DPX mounting solution using coverslip. The images were obtained on a Zeiss microscope integrated with Apotome 2 (ZEISS Axio Observer 7) and analyzed using Image J.

The microscopy images were first processed that includes splitting of the three-channel image, selecting the green channel (labelled as mitochondria) image, followed by sharpen, despeckle, background subtraction and enhancing the local contrast of the final image. As mitochondria are filamentous tube-like structures, so a filter named as 'tubeness' (sigma = 0.0210) is also applied to enhance the filamentous property of the segmented mitochondria. At last, the image 27 is converted into binary preceded by gaussian blur (sigma radius = 1.000). This image is then used to calculated the mitochondria length.

For integrated density calculation, the image was first split into three different channels.

The green channel image is selected and rest are closed. During the image processing step, the background was subtracted from the image followed by binary conversion. Finally, the image was redirected to measure the integrated density parameter.

For colocalisation analysis, the 'EzColocalization' plugin (85) was used to measure the following parameters. At first step, the three-channel image was again split into different channels, out of which red and green channel image was taken to perform colocalization analysis. Mander's overlap coefficient (or Mander's' coefficient) was calculated between PINK1 and Tom20; Parkin and Tom20; LC3 and Tom20 or mito-GFP; LAMP1 and Tom20; p62 and LC3; p62 and LAMP1; Tom20 and MTDR. Mander's coefficient measures the percentage colocalisation between two channels with values ranging from 0 to 1 as represented in the Figure. 

Mitochondria fraction was prepared using mitochondria isolation kit (Sigma) following the manufacturer' s protocol. Briefly, 2-3 x 10 7 cells were washed with ice cold PBS and centrifuged at 600 x g @ 4 °C. The pellet was resuspended in 1.5ml of 1x extraction buffer A following with the incubation for 10 mins on ice. Cells were homogenized using Dounce homogenizer 10-30 strokes following with a centrifuge at 600x g for 10 mins @ 4 °C. Transfer the supernatant to another tube and centrifuge at 11000x g for 10 mins @ 4 °C. The supernatant contains the cytosolic fraction and the pellet obtained was mitochondria fraction. Washed the pellet with extraction buffer to remove cytosolic contamination from mitochondrial fraction.

Cells were lysed using RIPA buffer (Sigma) supplemented with protease inhibitor cocktail (Sigma). The debris were removed from the lysates by centrifugation and protein estimation was performed by Bicinchoninic Acid (BCA) assay (Sigma). Proteins were resolved by loading 10ug of protein on a 10% and 12% SDS-PAGE gel. Transfer of proteins to the polyvinylidene difluoride (PVDF) membrane was done using a wet transfer technique. Blots were probed with primary antibodies, indicated in Table 1 , for overnight at 4 °C, followed by HRP-conjugated secondary antibodies for 1 hr at room temperature, indicated in Table 1 . The protein bands were visualized with enhanced chemiluminescence (Invitrogen/Thermo scientific).

Mitochondrial respiration in live MSCs were measured using seahorse XF24 Extracellular Flux Analyzer with Mito stress test kit according to manufacturer's protocol (Agilent). In brief, cells were plated at 60,000 cells/ well onto XF24-well microplates the day before analysis. On the day of analysis, the cells were equilibrated in XF buffer and were kept in non-CO2 incubator for 60 minutes. OCR was measured after repeated cycles which includes mixing (3 minutes), the incubation (2 minutes), and measurement (3 minutes) periods. Following basal line measurements, cells were treated with 1 µM oligomycin (ATP synthase inhibitor), 2 µM FCCP (Mitochondrial OXPHOS uncoupler) and mixture of 1 µM rotenone (Complex I inhibitor) and antimycin A (Complex III), and the changes in OCR were recorded. OCR data was normalized by total cell protein and the data was expressed in pmol/min/ug.

Mouse MSCs RT 2 Prolifer PCR array (PAMM-084ZG, Qiagen) was used to evaluate the expression of 84 specific genes related to autophagy using manufacturer's instructions. Briefly, total RNA was isolated from 10 5 to 10 6 cells using the RNeasy Mini kit (Qiagen) using manufacturer's protocol. cDNA synthesis from 500ng of the total RNA using the RT 2 first strand kit (Qiagen). The RT 2 Prolifer PCR array test includes various measures allowing to identify the contamination with genomic DNA, contamination with DNA during the procedure, to control the presence of reverse transcription and PCR inhibitors. After all control test, the samples were analyzed using the RT 2 Prolifer PCR array, altogether 84 different genes simultaneously amplified in the sample. PCR array were performed in 384-well plates on a LightCycler 480 instrument (Roche Applied Science). The reaction mix of 102 ul of sample cDNA was prepared using 2x SA Biosciences RT 2 qPCR Master Mix and 10 ul of this mixture was added into each well of the PCR array. The data was analyzed using Qiagen's online Web analysis tool (https://dataanalysis2.qiagen.com/pcr). A more than twofold change in gene expression compared to control group was considered as the up-or downregulation of a specific gene expression.

The RT-qPCR for Miro1 and PCR for mitochondrial DNA was performed on Rotor gene Q (Qiagen). Relative gene expression of Miro1 and mitochondrial DNA copy number were evaluated using their specific forward and reverse primers as indicated in Table 2 . The analysis and fold change expression were done as previously described (87) .

Examination of MSCs mitochondria using a TEM was performed as described earlier (8) . In brief, cells were fixed overnight at 4ºC in fixative containing 2.5% glutaraldehyde and 4% paraformaldehyde. After washing with 0.1M sodium cacodylate buffer to remove excess fixative, cells were embedded in 2% agar blocks. Samples were post-fixed in 2% osmium tetra oxide for 1 hr, dehydrated in graded series of ethanol (30%, 50%, 70%, and 100%) and 

The quantification of dead cells in lung tissue was performed using an in-situ apoptosis detection DeadEnd Colorimetric TUNEL System (Promega) as previously described (88) .

Briefly, the tissues sections were cut into 5 µm slices and stained as per the protocol.

Hematoxylin was used as a counterstain to visualize the nuclei. The images were taken with LMI microscope using 100X objective (DM-X, LMI microscopes, UK).

ATP levels was measured using ATP assay kit (Colorimetric) (Abcam) following the manufacturer's protocol in lung tissue lysate as previously described (8) .

Histopathology of lung and Airway hyperresponsiveness was measured using the protocol as previously described (8) . The AHR was measured with the flexiVent systems (SCIREQ). The scoring of the H&E slides was done manually as described previously (8) .

The tissues sections were subjected to Periodic acid-Schiff (PAS) staining as described by us previously (8) . Briefly, the tissues sections were deparaffinized and hydrated using decreasing concentrations of ethanol following that the slides were incubated in 0.3% periodic acid. The slides were subsequently incubated in Schiff's reagent (Sigma) and counterstained with hematoxylin and mounted with DPX mountant (Sigma) and images were taken with LMI microscope using 20X objective (DM-X, LMI microscopes, UK). For better visualisation of the signal, the images were processed with Image J software and the images were split into three channels (red, green and blue). The red channel represents mucus secretion and the blue channels which represents nuclei (hematoxylin) were merged. The intensity of the images was calculated as described in the image analysis section and represented as integrated density.

ELISA assay of different cytokines IL-4 (BioLegend), IL-5 (BioLegend) and IL-13 (R&D Systems) was done in Lung lysate using manufacturer's protocol. Briefly, 10 µg of the total cell lysate was used for the measurement. The readings were obtained by the spectrophotometer (Multiskan SkyHigh Microplate Spectrophotometer, Thermofischer scientific). The calculations were done as described by us previously (8) .

For statistical analysis, non-parametric t test was used to compare various groups. The analysis was done using Prism version 8.0 (GraphPad Software). The bar graphs were plotted in the Prism with respective P values. The data is expressed as mean±SEM and the values indicate ****P<0.001; ***P<0.005; **P<0.01; *P<0.05, which were considered as significant. The analysis was done in a minimum of three biological replicates or else as mentioned in the Data is presented as Mean±SEM. ****P < 0.001; **P < 0.01. Mander's coefficient was calculated to determine the degree of colocalization between LC3-YFP and Tom20 (right panel). Data is shown as Mean±SEM. ****P < 0.001; ***P < 0.005; *P < 0.05. Scale bars: 10 µm.

The expression of core autophagy markers is not altered in the MSC-Ob: (LTDR). The degree of colocalization was calculated between P62 with LC3 or LTDR, respectively (below panels). (C) The autophagosome formation was calculated in cells stained for LC3 under normal and SRV conditions. Data is shown as Mean±SEM. ***P < 0.005; **P < 0.01; *P < 0.05; ns (non-significant). Scale bars: 10 µm.

Reduced cardiolipin colocalization with LC3:

The representative images correspond to the insets shown in Figure 3L . 

Mesenchymal stem cell perspective: cell biology to clinical progress

Shattering barriers toward clinically meaningful MSC therapies

Stem cells and regenerative medicine in lung biology and diseases

Further validation of the efficacy of mesenchymal stem cell infusions for reducing mortality in COVID-19 patients with ARDS

Bone marrow stromal cells use TGF-beta to suppress allergic responses in a mouse model of ragweed-induced asthma

Mesenchymal Stem Cell-derived Extracellular Vesicles: Toward Cell-free Therapeutic Applications

Immunomodulation by Mesenchymal Stem Cells (MSCs): Mechanisms of Action of Living, Apoptotic, and Dead MSCs

Miro1 regulates intercellular mitochondrial transport & enhances mesenchymal stem cell rescue efficacy

Mitochondria Donation by Mesenchymal Stem Cells: Current Understanding and Mitochondria Transplantation Strategies

Intercellular mitochondrial transfer as a means of tissue revitalization

Mitochondrial transfer of mesenchymal stem cells effectively protects corneal epithelial cells from mitochondrial damage

Endoplasmic reticulum mediates mitochondrial transfer within the osteocyte dendritic network

Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury

Iron oxide nanoparticles augment the intercellular mitochondrial transfer-mediated therapy

Exogenous mitochondrial transfer and endogenous mitochondrial fission facilitate AML resistance to OxPhos inhibition

Protective mitochondrial transfer from bone marrow stromal cells to acute myeloid leukemic cells during chemotherapy

Activated stromal cells transfer mitochondria to rescue acute lymphoblastic leukemia cells from oxidative stress

Connexin 43-Mediated Mitochondrial Transfer of iPSC-MSCs Alleviates Asthma Inflammation

Mitochondrial transfer of induced pluripotent stem cell-derived mesenchymal stem cells to airway epithelial cells attenuates cigarette smoke-induced damage

Mitochondrial transfer from MSCs to T cells induces Treg differentiation and restricts inflammatory response

Obesity-induced mitochondrial dysfunction in porcine adipose tissuederived mesenchymal stem cells

Stromal Stem Cells: New Insight into EqASCs Isolated from EMS Horses in the Context of Their Aging

Metabolic syndrome increases senescence-associated micro-RNAs in extracellular vesicles derived from swine and human mesenchymal stem/stromal cells

Obesity and Type 2 Diabetes Alters the Immune Properties of Human Adipose Derived Stem Cells

Mechanisms of mitophagy

Mitochondria-Striking a balance between host and endosymbiont

Mitophagy and Quality Control Mechanisms in Mitochondrial Maintenance

Mitochondria and mitophagy: the yin and yang of cell death control

BNIP3L/NIX and FUNDC1-mediated mitophagy is required for mitochondrial network remodeling during cardiac progenitor cell differentiation

Mitochondrial quality control beyond PINK1/Parkin

Regulation of PRKN-independent mitophagy

P53 and Parkin co-regulate mitophagy in bone marrow mesenchymal stem cells to promote the repair of early steroid-induced osteonecrosis of the femoral head

Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs

Melatonin suppresses senescence-derived mitochondrial dysfunction in mesenchymal stem cells via the HSPA1L-mitophagy pathway

Obese-derived ASCs show impaired migration and angiogenesis properties

Diet-induced obesity alters the differentiation potential of stem cells isolated from bone marrow, adipose tissue and infrapatellar fat pad: the effects of free fatty acids

Autophagy: a potential key contributor to the therapeutic action of mesenchymal stem cells

Metabolic Syndrome Is Associated with Increased Oxo-Nitrative Stress and Asthma-Like Changes in Lungs

MICAL2PV suppresses the formation of tunneling nanotubes and modulates mitochondrial trafficking

Impaired mitophagy links mitochondrial disease to epithelial stress in methylmalonyl-CoA mutase deficiency

Mitochondrial quality control mediated by PINK1 and Parkin: links to parkinsonism

Computational classification of mitochondrial shapes reflects stress and redox state

Impaired autophagy bridges lysosomal storage disease and epithelial dysfunction in the kidney

A Rab5 endosomal pathway mediates Parkin-dependent mitochondrial clearance

Discovery of Atg5/Atg7-independent alternative macroautophagy

Selective turnover of p62/A170/SQSTM1 by autophagy

BNIP3L/Nix-induced mitochondrial fission, mitophagy, and impaired myocyte glucose uptake are abrogated by PRKA/PKA phosphorylation

LC3 binds externalized cardiolipin on injured mitochondria to signal mitophagy in neurons: implications for Parkinson disease

Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells

Free fatty acids induce JNKdependent hepatocyte lipoapoptosis

Pyrroloquinoline quinone stimulates mitochondrial biogenesis through cAMP response element-binding protein phosphorylation and increased PGC-1alpha expression

Pyrroloquinoline Quinone Inhibits Rotenone-Induced Microglia Inflammation by Enhancing Autophagy

Pyrroloquinoline quinone promotes mitochondrial biogenesis in rotenone-induced Parkinson's disease model via AMPK activation

Mesenchymal stem cells alleviate oxidative stress-induced mitochondrial dysfunction in the airways

Autophagy receptor OPTN (optineurin) regulates mesenchymal stem cell fate and bone-fat balance during aging by clearing FABP3

The multifaceted contributions of mitochondria to cellular metabolism

Human tissue-specific MSCs demonstrate differential mitochondria transfer abilities that may determine their regenerative abilities

Structural basis for the phosphorylation of FUNDC1 LIR as a molecular switch of mitophagy

Dimerization of mitophagy receptor BNIP3L/NIX is essential for recruitment of autophagic machinery

Molecular mechanisms and physiological functions of mitophagy

Deficiency of mitophagy receptor FUNDC1 impairs mitochondrial quality and aggravates dietary-induced obesity and metabolic syndrome

Mitochondrial-derived vesicles compensate for loss of LC3-mediated mitophagy

Pyrroloquinoline quinone preserves mitochondrial function and prevents oxidative injury in adult rat cardiac myocytes

Metabolism in Human Mesenchymal Stromal Cells: A Missing Link Between hMSC Biomanufacturing and Therapy? Front Immunol

Mitochondria transfer from mesenchymal stem cells structurally and functionally repairs renal proximal tubular epithelial cells in diabetic nephropathy in vivo

Platelets Facilitate the Wound-Healing Capability of Mesenchymal Stem Cells by Mitochondrial Transfer and Metabolic Reprogramming

Intercellular Mitochondria Transfer to Macrophages Regulates White Adipose Tissue Homeostasis and Is Impaired in Obesity

Mechanisms underlying the protective effects of mesenchymal stem cell-based therapy

Effects of Oxidative Stress on Mesenchymal Stem Cell Biology

Oxidative stress-mediated mitochondrial dysfunction facilitates mesenchymal stem cell senescence in ankylosing spondylitis

Production, safety and efficacy of iPSC-derived mesenchymal stromal cells in acute steroid-resistant graft versus host disease: a phase I, multicenter, open-label, dose-escalation study

Mitophagy in degenerative joint diseases

A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone

Fibroblast Growth Factor-2 alone as an efficient inducer for differentiation of human bone marrow mesenchymal stem cells into dopaminergic neurons

STIM1 activation of adenylyl cyclase 6 connects Ca(2+) and cAMP signaling during melanogenesis

EzColocalization: An ImageJ plugin for visualizing and measuring colocalization in cells and organisms

Impaired mitophagy leads to cigarette smoke stress-induced cellular senescence: implications for chronic obstructive pulmonary disease

PCR based determination of mitochondrial DNA copy number in multiple species

Simvastatin improves epithelial dysfunction and airway hyperresponsiveness: from asymmetric dimethyl-arginine to asthma

Urolithin A induces mitophagy and prolongs lifespan in C. elegans and increases muscle function in rodents

Enhancement of Mesenchymal Stem Cell-Driven Bone Regeneration by Resveratrol-Mediated SOX2 Regulation

Adult mesenchymal stem cell ageing interplays with depressed mitochondrial Ndufs6

Pyrroloquinoline quinone stimulates mitochondrial biogenesis through cAMP response element-binding protein phosphorylation and increased PGC-1alpha expression

NAD(+) augmentation restores mitophagy and limits accelerated aging in Werner syndrome

Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase

Synergistic protection of N-acetylcysteine and ascorbic acid 2-phosphate on human mesenchymal stem cells against mitoptosis, necroptosis and apoptosis

We thank the lab members of Dr. Anurag Agrawal, Dr. Soumya Sinha Roy and Dr. Tanveer 

Anti-PGC1 alpha Abcam ab54481 4Anti-MIRO1 Abcam ab83779 5Anti-LC3B Abcam ab48394 6Anti-TOMM20 Abcam ab56783 7Anti-LAMP1 Abcam ab25245 8Recombinant Anti-Beclin 1 Abcam ab207612 9Anti-VDAC1/Porin Abcam ab15895 10Anti-PGC1 alpha Abcam ab106814 11Anti-PINK1 Abcam ab75487 12Anti-COX IV Abcam ab14744 13Anti-FUNDC1 Abcam ab74834 14Anti-DRP1 Abcam ab140494 15Anti-SQSTM1 / p62 Abcam ab56416 16 Anti-Uteroglobin Abcam ab40873 17Recombinant Anti-EpCAM Abcam ab32392 18Anti-alpha Tubulin Abcam ab7291 19Anti-Mitofusin 1 Abcam ab57602 20Anti-LAMP1 Abcam ab24170 21Anti