Protective effects of mesenchymal stem cells-derived extracellular vesicles against ischemia-reperfusion injury of hearts donated after circulatory death: Preliminary study in a pig model.

INTRODUCTION: Insufficient supply of cardiac grafts represents a severe obstacle in heart transplantation. Donation after Circulatory Death (DCD), in addition to conventional donation after brain death, is one promising option to overcome the organ shortage. However, DCD organs undergo an inevitable more extended period of warm unprotected ischemia between circulatory arrest and graft procurement. Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have shown remarkable protective effects against ischemia-reperfusion injury. Thus, we aimed to enhance grafts preservation from DCD donors, through treatment with MSC-EVs. METHODS: Female pigs were euthanized by barbiturate overdose and after 20 min of a flat EKG, the chest was opened, the heart harvested and subsequently connected to an extracorporeal perfusion machine. MSC-EVs, isolated by ion exchange chromatography, were added to the perfusion solution (1×1011 particles) and the heart was perfused for 2 h. Then, heart tissue biopsies were taken to assess histological changes, mitochondrial morphology, antioxidant enzyme activity and inflammation mediators' expression. Biochemical parameters of myocardial viability were assessed in the perfusate. RESULTS: The treatment with MSC-EVs significantly prevented mitochondria swelling, mitochondrial cristae loss and oxidative stress in cardiac tissue. The protective effect of MSC-EVs was confirmed by the delayed increase of the cardiac-specific enzymes CK and TnC in the perfusate and the reduction of caspase-3+ cells in tissue sections. CONCLUSION: MSC-EVs improve graft quality by preserving the mitochondrial ultrastructure protecting the myocardium against oxidative stress, reducing apoptosis of cardiac cells and preventing the increase of pro-inflammatory cytokines.

Profile of matrix-entrapped extracellular vesicles of microenvironmental and infiltrating cell origin in decellularized colorectal cancer and adjacent mucosa.

Cellular elements that infiltrate and surround tumours and pre-metastatic tissues have a prominent role in tumour invasion and growth. The extracellular vesicles specifically entrapped and stored within the extracellular matrix (ECM-EVs) may reflect the different populations of the tumour microenvironment and their change during tumour progression. However, their profile is at present unknown. To elucidate this aspect, we isolated and characterized EVs from decellularized surgical specimens of colorectal cancer and adjacent colon mucosa and analyzed their surface marker profile. ECM-EVs in tumours and surrounding mucosa mainly expressed markers of lymphocytes, natural killer cells, antigen-presenting cells, and platelets, as well as epithelial cells, representing a multicellular microenvironment. No difference in surface marker expression was observed between tumour and mucosa ECM-EVs in stage II-III tumours. At variance, in the colon mucosa adjacent to stage IV carcinomas, ECM-EV profile showed a significantly increased level of immune, epithelial and platelet markers in comparison to the matrix of the corresponding tumour. The increase of EVs from immune cells and platelets was not observed in the mucosa adjacent to low-stage tumours. In addition, CD25, a T-lymphocyte marker, resulted specifically overexpressed by ECM-EVs from stage IV carcinomas, possibly correlated with the pro-tolerogenic environment found in the corresponding tumour tissue. These results outline the tissue microenvironmental profile of EVs in colorectal carcinoma-derived ECM and unveil a profound change in the healthy mucosa adjacent to high-stage tumours.

Extracellular Vesicles From Mesenchymal Umbilical Cord Cells Exert Protection Against Oxidative Stress and Fibrosis in a Rat Model of Bronchopulmonary Dysplasia.

Oxidative stress and fibrosis are important stress responses that characterize bronchopulmonary dysplasia (BPD), a disease for which only a therapy but not a cure has been developed. In this work, we investigated the effects of mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) on lung and brain compartment in an animal model of hyperoxia-induced BPD. Rat pups were intratracheally injected with MSC-EVs produced by human umbilical cord-derived MSC, following the Good Manufacturing Practice-grade (GMP-grade). After evaluating biodistribution of labelled MSC-EVs in rat pups left in normoxia and hyperoxia, oxidative stress and fibrosis investigation were performed. Oxidative stress protection by MSC-EVs treatment was proved both in lung and in brain. The lung epithelial compartment ameliorated glycosaminoglycan and surfactant protein expression in MSC-EVs-injected rat pups compared to untreated animals. Pups under hyperoxia exhibited a fibrotic phenotype in lungs shown by increased collagen deposition and also expression of profibrotic genes. Both parameters were reduced by treatment with MSC-EVs. We established an in vitro model of fibrosis and another of oxidative stress, and we proved that MSC-EVs suppressed the induction of αSMA, influencing collagen deposition and protecting from the oxidative stress. In conclusion, intratracheal administration of clinical-grade MSC-EVs protect from oxidative stress, improves pulmonary epithelial function, and counteracts the development of fibrosis. In the future, MSC-EVs could represent a new cure to prevent the development of BPD.

Expression of targets of the RNA-binding protein AUF-1 in human airway epithelium indicates its role in cellular senescence and inflammation.

Introduction: The RNA-binding protein AU-rich-element factor-1 (AUF-1) participates to posttranscriptional regulation of genes involved in inflammation and cellular senescence, two pathogenic mechanisms of chronic obstructive pulmonary disease (COPD). Decreased AUF-1 expression was described in bronchiolar epithelium of COPD patients versus controls and in vitro cytokine- and cigarette smoke-challenged human airway epithelial cells, prompting the identification of epithelial AUF-1-targeted transcripts and function, and investigation on the mechanism of its loss. Results: RNA immunoprecipitation-sequencing (RIP-Seq) identified, in the human airway epithelial cell line BEAS-2B, 494 AUF-1-bound mRNAs enriched in their 3'-untranslated regions for a Guanine-Cytosine (GC)-rich binding motif. AUF-1 association with selected transcripts and with a synthetic GC-rich motif were validated by biotin pulldown. AUF-1-targets' steady-state levels were equally affected by partial or near-total AUF-1 loss induced by cytomix (TNFα/IL1ß/IFNγ/10 nM each) and siRNA, respectively, with differential transcript decay rates. Cytomix-mediated decrease in AUF-1 levels in BEAS-2B and primary human small-airways epithelium (HSAEC) was replicated by treatment with the senescence- inducer compound etoposide and associated with readouts of cell-cycle arrest, increase in lysosomal damage and senescence-associated secretory phenotype (SASP) factors, and with AUF-1 transfer in extracellular vesicles, detected by transmission electron microscopy and immunoblotting. Extensive in-silico and genome ontology analysis found, consistent with AUF-1 functions, enriched RIP-Seq-derived AUF-1-targets in COPD-related pathways involved in inflammation, senescence, gene regulation and also in the public SASP proteome atlas; AUF-1 target signature was also significantly represented in multiple transcriptomic COPD databases generated from primary HSAEC, from lung tissue and from single-cell RNA-sequencing, displaying a predominant downregulation of expression. Discussion: Loss of intracellular AUF-1 may alter posttranscriptional regulation of targets particularly relevant for protection of genomic integrity and gene regulation, thus concurring to airway epithelial inflammatory responses related to oxidative stress and accelerated aging. Exosomal-associated AUF-1 may in turn preserve bound RNA targets and sustain their function, participating to spreading of inflammation and senescence to neighbouring cells.

Fumarate induces vesicular release of mtDNA to drive innate immunity.

Mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell carcinoma1. Loss of FH in the kidney elicits several oncogenic signalling cascades through the accumulation of the oncometabolite fumarate2. However, although the long-term consequences of FH loss have been described, the acute response has not so far been investigated. Here we generated an inducible mouse model to study the chronology of FH loss in the kidney. We show that loss of FH leads to early alterations of mitochondrial morphology and the release of mitochondrial DNA (mtDNA) into the cytosol, where it triggers the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-TANK-binding kinase 1 (TBK1) pathway and stimulates an inflammatory response that is also partially dependent on retinoic-acid-inducible gene I (RIG-I). Mechanistically, we show that this phenotype is mediated by fumarate and occurs selectively through mitochondrial-derived vesicles in a manner that depends on sorting nexin 9 (SNX9). These results reveal that increased levels of intracellular fumarate induce a remodelling of the mitochondrial network and the generation of mitochondrial-derived vesicles, which allows the release of mtDNAin the cytosol and subsequent activation of the innate immune response.

Long-term proliferation of immature hypoxia-dependent JMML cells supported by a 3D in vitro system.

Juvenile myelomonocytic leukemia (JMML) is a rare clonal stem cell disorder that occurs in early childhood and is characterized by the hyperactivation of the RAS pathway in 95% of the patients. JMML is characterized by a hyperproliferation of granulocytes and monocytes, and little is known about the heterogeneous nature of leukemia-initiating cells, as well as of the cellular hierarchy of the JMML bone marrow. In this study, we report the generation and characterization of a novel patient-derived three-dimensional (3D) in vitro JMML model, called patient-derived JMML Atypical Organoid (pd-JAO), sustaining the long-term proliferation of JMML cells with stem cell features and patient-specific hallmarks. JMML cells brewed in a 3D model under different microenvironmental conditions acquired proliferative and survival advantages when placed under low oxygen tension. Transcriptomic and microscopic analyses revealed the activation of specific metabolic energy pathways and the inactivation of processes leading to cell death. Furthermore, we demonstrated the pd-JAO-derived cells' migratory, propagation, and self-renewal capacities. Our study contributes to the development of a robust JMML 3D in vitro model for studying and defining the impact of microenvironmental stimuli on JMML disease and the molecular mechanisms that regulate JMML initiating and propagating cells. Pd-JAO may become a promising model for compound tests focusing on new therapeutic interventions aimed at eradicating JMML progenitors and controlling JMML disease.

Small intestinal submucosa-derived extracellular matrix as a heterotopic scaffold for cardiovascular applications.

Structural cardiac lesions are often surgically repaired using prosthetic patches, which can be biological or synthetic. In the current clinical scenario, biological patches derived from the decellularization of a xenogeneic scaffold are gaining more interest as they maintain the natural architecture of the extracellular matrix (ECM) after the removal of the native cells and remnants. Once implanted in the host, these patches can induce tissue regeneration and repair, encouraging angiogenesis, migration, proliferation, and host cell differentiation. Lastly, decellularized xenogeneic patches undergo cell repopulation, thus reducing host immuno-mediated response against the graft and preventing device failure. Porcine small intestinal submucosa (pSIS) showed such properties in alternative clinical scenarios. Specifically, the US FDA approved its use in humans for urogenital procedures such as hernia repair, cystoplasties, ureteral reconstructions, stress incontinence, Peyronie's disease, penile chordee, and even urethral reconstruction for hypospadias and strictures. In addition, it has also been successfully used for skeletal muscle tissue reconstruction in young patients. However, for cardiovascular applications, the results are controversial. In this study, we aimed to validate our decellularization protocol for SIS, which is based on the use of Tergitol 15 S 9, by comparing it to our previous and efficient method (Triton X 100), which is not more available in the market. For both treatments, we evaluated the preservation of the ECM ultrastructure, biomechanical features, biocompatibility, and final bioinductive capabilities. The overall analysis shows that the SIS tissue is macroscopically distinguishable into two regions, one smooth and one wrinkle, equivalent to the ultrastructure and biochemical and proteomic profile. Furthermore, Tergitol 15 S 9 treatment does not modify tissue biomechanics, resulting in comparable to the native one and confirming the superior preservation of the collagen fibers. In summary, the present study showed that the SIS decellularized with Tergitol 15 S 9 guarantees higher performances, compared to the Triton X 100 method, in all the explored fields and for both SIS regions: smooth and wrinkle.

The Flavone Cirsiliol from Salvia x jamensis Binds the F1 Moiety of ATP Synthase, Modulating Free Radical Production.

Several studies have shown that mammalian retinal rod outer segments (OS) are peculiar structures devoid of mitochondria, characterized by ectopic expression of the molecular machinery for oxidative phosphorylation. Such ectopic aerobic metabolism would provide the chemical energy for the phototransduction taking place in the OS. Natural polyphenols include a large variety of molecules having pleiotropic effects, ranging from anti-inflammatory to antioxidant and others. Our goal in the present study was to investigate the potential of the flavonoid cirsiliol, a trihydroxy-6,7-dimethoxyflavone extracted from Salvia x jamensis, in modulating reactive oxygen species production by the ectopic oxidative phosphorylation taking place in the OS. Our molecular docking analysis identified cirsiliol binding sites inside the F1 moiety of the nanomotor F1Fo-ATP synthase. The experimental approach was based on luminometry, spectrophotometry and cytofluorimetry to evaluate ATP synthesis, respiratory chain complex activity and H2O2 production, respectively. The results showed significant dose-dependent inhibition of ATP production by cirsiliol. Moreover, cirsiliol was effective in reducing the free radical production by the OS exposed to ambient light. We report a considerable protective effect of cirsiliol on the structural stability of rod OS, suggesting it may be considered a promising compound against oxidative stress.

Cell cycle alterations due to perfluoroalkyl substances PFOS, PFOA, PFBS, PFBA and the new PFAS C6O4 on bottlenose dolphin (Tursiops truncatus) skin cell.

Per- and polyfluoroalkyl substances (PFAS) have become ubiquitous environmental contaminants in aquatic ecosystems worldwide. Marine mammals, as top predators, are constantly exposed to several PFAS compounds that accumulate in different tissues. As a proxy to assess cytotoxicity of PFAS in the bottlenose dolphin (Tursiops truncatus), we generated a new immortalized cell line derived from skin samples of bottlenose dolphin. Using high content imaging, we assessed the effects of increasing concentrations of PFOS, PFOA, PFBS, PFBA and C6O4 on cell viability and cell cycle phases. In particular, we classified all cells based on multiple morphometric differences of the nucleus in three populations, named respectively "Normal" (nuclei in G0, S and M phase); "Large" (nuclei showing characteristics of senescence) and "Small" (nuclei with fragmentation and condensed chromatin). Combining this approach with cell cycle analysis we determined which phases of the cell cycle were influenced by PFAS. The results revealed that the presence of PFOS, PFBS and PFBA could increase the number of cells in G0+G1 phase and decrease the number of those in the S phase. Moreover, PFOS and PFBS lowered the fraction of cells in the M phase. Interestingly PFOS, PFBS and PFBA reduced the prevalence of the senescence phenotype ("large" nuclei), suggesting a potential tumorigenic effect. Besides, the presence of PFOS and PFBS correlated also with a significant decrease in the number of "small" nuclei. The C6O4 exposure did not highlighted morphometric alteration or cell cycle modification bottlenose dolphin skin cell nuclei. While the effects of PFAS on cell cycle was clear, no significant change was detected either in term of cell proliferation or of viability. This study fosters the overall knowledge on the cellular effects of perfluoroalkyl substances in marine mammals.

Temperature-Related Effects of Myocardial Protection Strategies in Swine Hearts after Prolonged Warm Ischemia.

Insufficient supply of cardiac grafts represents a severe obstacle in heart transplantation. Donation after circulatory death (DCD), in addition to conventional donation after brain death, is one promising option to overcome the organ shortage. However, DCD organs undergo an inevitably longer period of unprotected warm ischemia between circulatory arrest and graft procurement. In this scenario, we aim to improve heart preservation after a warm ischemic period of 20 min by testing different settings of myocardial protective strategies. Pig hearts were collected from a slaughterhouse and assigned to one of the five experimental groups: baseline (BL), cold cardioplegia (CC), cold cardioplegia + adenosine (CC-ADN), normothermic cardioplegia (NtC + CC) or normothermic cardioplegia + cold cardioplegia + adenosine (NtC-ADN + CC). After treatment, tissue biopsies were taken to assess mitochondrial morphology, antioxidant enzyme activity, lipid peroxidation and cytokine and chemokine expressions. NtC + CC treatment significantly prevented mitochondria swelling and mitochondrial cristae loss. Moreover, the antioxidant enzyme activity was lower in this group, as was lipid peroxidation, and the pro-inflammatory chemokine GM-CSF was diminished. Finally, we demonstrated that normothermic cardioplegia preserved mitochondria morphology, thus preventing oxidative stress and the subsequent inflammatory response. Therefore, normothermic cardioplegia is a better approach to preserve the heart after a warm ischemia period, with respect to cold cardioplegia, before transplantation.

Extracellular Vesicles Secreted by Mesenchymal Stromal Cells Exert Opposite Effects to Their Cells of Origin in Murine Sodium Dextran Sulfate-Induced Colitis.

Several reports have described a beneficial effect of Mesenchymal Stromal Cells (MSCs) and of their secreted extracellular vesicles (EVs) in mice with experimental colitis. However, the effects of the two treatments have not been thoroughly compared in this model. Here, we compared the effects of MSCs and of MSC-EV administration in mice with colitis induced by dextran sulfate sodium (DSS). Since cytokine conditioning was reported to enhance the immune modulatory activity of MSCs, the cells were kept either under standard culture conditions (naïve, nMSCs) or primed with a cocktail of pro-inflammatory cytokines, including IL1ß, IL6 and TNFα (induced, iMSCs). In our experimental conditions, nMSCs and iMSCs administration resulted in both clinical and histological worsening and was associated with pro-inflammatory polarization of intestinal macrophages. However, mice treated with iEVs showed clinico-pathological improvement, decreased intestinal fibrosis and angiogenesis and a striking increase in intestinal expression of Mucin 5ac, suggesting improved epithelial function. Moreover, treatment with iEVs resulted in the polarization of intestinal macrophages towards and anti-inflammatory phenotype and in an increased Treg/Teff ratio at the level of the intestinal lymph node. Collectively, these data confirm that MSCs can behave either as anti- or as pro-inflammatory agents depending on the host environment. In contrast, EVs showed a beneficial effect, suggesting a more predictable behavior, a safer therapeutic profile and a higher therapeutic efficacy with respect to their cells of origin.

Muscle functional recovery is driven by extracellular vesicles combined with muscle extracellular matrix in a volumetric muscle loss murine model.

Biological scaffolds derived from decellularized tissues are being investigated as a promising approach to repair volumetric muscle losses (VML). Indeed, extracellular matrix (ECM) from decellularized tissues is highly biocompatible and mimics the original tissue. However, the development of fibrosis and the muscle stiffness still represents a major problem. Intercellular signals mediating tissue repair are conveyed via extracellular vesicles (EVs), biologically active nanoparticles secreted by the cells. This work aimed at using muscle ECM and human EVs derived from Wharton Jelly mesenchymal stromal cells (MSC EVs) to boost tissue regeneration in a VML murine model. Mice transplanted with muscle ECM and treated with PBS or MSC EVs were analyzed after 7 and 30 days. Flow cytometry, tissue analysis, qRT-PCR and physiology test were performed. We demonstrated that angiogenesis and myogenesis were enhanced while fibrosis was reduced after EV treatment. Moreover, the inflammation was directed toward tissue repair. M2-like, pro-regenerative macrophages were significantly increased in the MSC EVs treated group compared to control. Strikingly, the histological improvements were associated with enhanced functional recovery. These results suggest that human MSC EVs can be a naturally-derived boost able to ameliorate the efficacy of tissue-specific ECM in muscle regeneration up to the restored tissue function.

The diterpene Manool extracted from Salvia tingitana lowers free radical production in retinal rod outer segments by inhibiting the extramitochondrial F1 Fo ATP synthase.

Uncontrolled oxidative stress production, especially in the outer retina is one of the causes of retinal degenerations. Mitochondria are considered the principal source of oxidative stress. However, a Reactive Oxygen Intermediates (ROI) production in the retinal photoreceptor layer seems to depend also on the expression of an extramitochondrial oxidative phosphorylation (OxPhos) machinery in the rod outer segments (OS). In fact, OS conduct aerobic metabolism, producing ATP through oxygen consumption, although it is devoid of mitochondria. As diterpenes display an antioxidant effect, we have evaluated the effect Manool, extracted from Salvia tingitana, on the extramitochondrial OxPhos and the ROI production in the retinal rod OS. Results confirm that the OxPhos machinery is ectopically expressed in the OS and that F1 Fo -ATP synthase is a target of Manool, which inhibited the OS ATP synthesis, binding the F1 moiety with high affinity, as analysed by molecular docking. Moreover, the overall slowdown of OxPhos metabolism reduced the ROI production elicited in the OS by light exposure, in vitro. In conclusion, data are consistent with the antioxidant properties of Salvia spp., suggesting its ability to lower oxidative stress production, a primary risk factor for degenerative retinal diseases. SIGNIFICANCE OF THE STUDY: Here we show that Manool, a diterpene extracted from Salvia tingitana has the potential to lower the free radical production by light-exposed rod outer segments in vitro, by specifically targeting the rod OS F1 Fo -ATP synthase belonging to the extramitochondrial OxPhos expressed on the disk membrane. The chosen experimental model allowed to show that the rod OS is a primary producer of oxidative stress linked to the pathogenesis of degenerative retinal diseases. Data are also consistent with the antioxidant and anti-inflammatory action of Salvia spp., suggesting a beneficial effect also in vivo.

Autophagic flux inhibition enhances cytotoxicity of the receptor tyrosine kinase inhibitor ponatinib.

BACKGROUND: Despite reported advances, acquired resistance to tyrosine kinase inhibitors still represents a serious problem in successful cancer treatment. Among this class of drugs, ponatinib (PON) has been shown to have notable long-term efficacy, although its cytotoxicity might be hampered by autophagy. In this study, we examined the likelihood of PON resistance evolution in neuroblastoma and assessed the extent to which autophagy might provide survival advantages to tumor cells. METHODS: The effects of PON in inducing autophagy were determined both in vitro, using SK-N-BE(2), SH-SY5Y, and IMR-32 human neuroblastoma cell lines, and in vivo, using zebrafish and mouse models. Single and combined treatments with chloroquine (CQ)-a blocking agent of lysosomal metabolism and autophagic flux-and PON were conducted, and the effects on cell viability were determined using metabolic and immunohistochemical assays. The activation of the autophagic flux was analyzed through immunoblot and protein arrays, immunofluorescence, and transmission electron microscopy. Combination therapy with PON and CQ was tested in a clinically relevant neuroblastoma mouse model. RESULTS: Our results confirm that, in neuroblastoma cells and wild-type zebrafish embryos, PON induces the accumulation of autophagy vesicles-a sign of autophagy activation. Inhibition of autophagic flux by CQ restores the cytotoxic potential of PON, thus attributing to autophagy a cytoprotective nature. In mice, the use of CQ as adjuvant therapy significantly improves the anti-tumor effects obtained by PON, leading to ulterior reduction of tumor masses. CONCLUSIONS: Together, these findings support the importance of autophagy monitoring in the treatment protocols that foresee PON administration, as this may predict drug resistance acquisition. The findings also establish the potential for combined use of CQ and PON, paving the way for their consideration in upcoming treatment protocols against neuroblastoma.

Sclareol modulates free radical production in the retinal rod outer segment by inhibiting the ectopic f1fo-atp synthase.

We have previously shown that the retinal rod outer segments (OS) produce reactive oxygen species in the function of illumination in vitro, establishing a relationship among the extra-mitochondrial oxidative phosphorylation and phototransduction. This source of oxidative stress in the OS can be modulated by polyphenols, acting as inhibitors of F1Fo-ATP synthase. The present study aimed at exploring whether sclareol, a diterpene, interacts with F1Fo-ATP synthase mitigating the light-induced free radical production in the rod OS. Characterization of bovine retinal sections was conducted by immunogold analysis. Reactive oxygen intermediates production, oxygen consumption, the activity of the four respiratory complexes and ATP synthesis were evaluated in purified bovine rod OS. Molecular docking analyses were also conducted. Sclareol reduced free radical production by light-exposed rod OS. Such antioxidant effect was associated with an inhibition of the respiratory complexes and oxygen consumption (OCR), in coupled conditions. Sclareol also inhibited the rod OS ATP synthetic ability. Since the inhibitor effect on respiratory complexes and OCR is not observed in uncoupled conditions, it is supposed that the modulating effect of sclareol on the ectopic oxidative phosphorylation in the rod OS targets specifically the F1Fo-ATP synthase. This hypothesis is confirmed by the in silico molecular docking analyses, which shows that sclareol binds the F1 moiety of ATP synthase with high affinity. In conclusion, a beneficial effect of sclareol can be envisaged as a modulator of oxidative stress in the photoreceptor, a risk factor for the degenerative retinal diseases, suggestive of its potential beneficial action also in vivo.

Interaction Between Mitochondrial DNA Variants and Mitochondria/Endoplasmic Reticulum Contact Sites: A Perspective Review.

Mitochondria contain their own genome, mitochondrial DNA (mtDNA), essential to support their fundamental intracellular role in ATP production and other key metabolic and homeostatic pathways. Mitochondria are highly dynamic organelles that communicate with all the other cellular compartments, through sites of high physical proximity. Among all, their crosstalk with the endoplasmic reticulum (ER) appears particularly important as its derangement is tightly implicated with several human disorders. Population-specific mtDNA variants clustered in defining the haplogroups have been shown to exacerbate or mitigate these pathological conditions. The exact mechanisms of the mtDNA background-modifying effect are not completely clear and a possible explanation is the outcome of mitochondrial efficiency on retrograde signaling to the nucleus. However, the possibility that different haplogroups shape the proximity and crosstalk between mitochondria and the ER has never been proposed neither investigated. In this study, we pose and discuss this question and provide preliminary data to answer it. Besides, we also address the possibility that single, disease-causing mtDNA point mutations may act also by reshaping organelle communication. Overall, this perspective review provides a theoretical platform for future studies on the interaction between mtDNA variants and organelle contact sites.

Differential expression of the five redox complexes in the retinal mitochondria or rod outer segment disks is consistent with their different functionality.

PURPOSE: The retinal rod outer segment (OS) disk membranes, devoid of mitochondria, conducts oxidative phosphorylation (OxPhos). This study aimed at identifying which proteins expressed in the retinal rod OS disks determined the considerable adenosine-5'-triphosphate production and oxygen consumption observed in comparison with retinal mitochondria. PROCEDURES: Characterization was conducted by immunogold transmission electron microscopy on retinal sections. OxPhos was studied by oximetry and luminometry. The proteomes of OS disks and mitochondria purified from bovine retinas were studied by mass spectrometry. Statistical and bioinformatic analyses were conducted by univariate, multivariate, and machine learning methods. RESULTS: Weighted gene coexpression network analysis identified two protein expression profile modules functionally associated with either retinal mitochondria or disk samples, in function of a strikingly different ability of each sample to utilized diverse substrate for F1Fo-ATP synthase. The OS disk proteins correlated better than mitochondria with the tricarboxylic acids cycle and OxPhos proteins. CONCLUSIONS: The differential enrichment of the expression profile of the OxPhos proteins in the disks versus mitochondria suggests that these proteins may represent a true proteome component of the former, with different functionality. These findings may shed new light on the pathogenesis of rod-driven retinal degenerative diseases.

JNK1 and ERK1/2 modulate lymphocyte homeostasis via BIM and DRP1 upon AICD induction.

The Activation-Induced Cell Death (AICD) is a stimulation-dependent form of apoptosis used by the organism to shutdown T-cell response once the source of inflammation has been eliminated, while allowing the generation of immune memory. AICD is thought to progress through the activation of the extrinsic Fas/FasL pathway of cell death, leading to cytochrome-C release through caspase-8 and Bid activation. We recently described that, early upon AICD induction, mitochondria undergo structural alterations, which are required to promote cytochrome-C release and execute cell death. Here, we found that such alterations do not depend on the Fas/FasL pathway, which is instead only lately activated to amplify the cell death cascade. Instead, such alterations are primarily dependent on the MAPK proteins JNK1 and ERK1/2, which, in turn, regulate the activity of the pro-fission protein Drp1 and the pro-apoptotic factor Bim. The latter regulates cristae disassembly and cooperate with Drp1 to mediate the Mitochondrial Outer Membrane Permeabilization (MOMP), leading to cytochrome-C release. Interestingly, we found that Bim is also downregulated in T-cell Acute Lymphoblastic Leukemia (T-ALL) cells, this alteration favouring their escape from AICD-mediated control.

USP14 inhibition corrects an in vivo model of impaired mitophagy.

Mitochondrial autophagy or mitophagy is a key process that allows selective sequestration and degradation of dysfunctional mitochondria to prevent excessive reactive oxygen species, and activation of cell death. Recent studies revealed that ubiquitin-proteasome complex activity and mitochondrial membrane rupture are key steps preceding mitophagy, in combination with the ubiquitination of specific outer mitochondrial membrane (OMM) proteins. The deubiquitinating enzyme ubiquitin-specific peptidase 14 (USP14) has been shown to modulate both proteasome activity and autophagy. Here, we report that genetic and pharmacological inhibition of USP14 promotes mitophagy, which occurs in the absence of the well-characterised mediators of mitophagy, PINK1 and Parkin. Critical to USP14-induced mitophagy is the exposure of the LC3 receptor Prohibitin 2 by mitochondrial fragmentation and mitochondrial membrane rupture. Genetic or pharmacological inhibition of USP14 in vivo corrected mitochondrial dysfunction and locomotion behaviour of PINK1/Parkin mutant Drosophila model of Parkinson's disease, an age-related progressive neurodegenerative disorder that is correlated with diminished mitochondrial quality control. Our study identifies a novel therapeutic target that ameliorates mitochondrial dysfunction and in vivo PD-related symptoms.