ISSLS PRIZE in Basic Science 2024: superiority of nucleus pulposus cell- versus mesenchymal stromal cell-derived extracellular vesicles in attenuating disc degeneration and alleviating pain.

PURPOSE: To investigate the therapeutic potential of extracellular vesicles (EVs) derived from human nucleus pulposus cells (NPCs), with a specific emphasis on Tie2-enhanced NPCs, compared to EVs derived from human bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a coccygeal intervertebral disc degeneration (IDD) rat model. METHODS: EVs were isolated from healthy human NPCs cultured under standard (NPCSTD-EVs) and Tie2-enhancing (NPCTie2+-EVs) conditions. EVs were characterized, and their potential was assessed in vitro on degenerative NPCs in terms of cell proliferation and senescence, with or without 10 ng/mL interleukin (IL)-1ß. Thereafter, 16 Sprague-Dawley rats underwent annular puncture of three contiguous coccygeal discs to develop IDD. Phosphate-buffered saline, NPCSTD-EVs, NPCTie2+-EVs, or BM-MSC-derived EVs were injected into injured discs, and animals were followed for 12 weeks until sacrifice. Behavioral tests, radiographic disc height index (DHI) measurements, evaluation of pain biomarkers, and histological analyses were performed to assess the outcomes of injected EVs. RESULTS: NPC-derived EVs exhibited the typical exosomal morphology and were efficiently internalized by degenerative NPCs, enhancing cell proliferation, and reducing senescence. In vivo, a single injection of NPC-derived EVs preserved DHI, attenuated degenerative changes, and notably reduced mechanical hypersensitivity. MSC-derived EVs showed marginal improvements over sham controls across all measured outcomes. CONCLUSION: Our results underscore the regenerative potential of young NPC-derived EVs, particularly NPCTie2+-EVs, surpassing MSC-derived counterparts. These findings raise questions about the validity of MSCs as both EV sources and cellular therapeutics against IDD. The study emphasizes the critical influence of cell type, source, and culture conditions in EV-based therapeutics.

S-nitrosylation drives cell senescence and aging in mammals by controlling mitochondrial dynamics and mitophagy.

S-nitrosylation, a prototypic redox-based posttranslational modification, is frequently dysregulated in disease. S-nitrosoglutathione reductase (GSNOR) regulates protein S-nitrosylation by functioning as a protein denitrosylase. Deficiency of GSNOR results in tumorigenesis and disrupts cellular homeostasis broadly, including metabolic, cardiovascular, and immune function. Here, we demonstrate that GSNOR expression decreases in primary cells undergoing senescence, as well as in mice and humans during their life span. In stark contrast, exceptionally long-lived individuals maintain GSNOR levels. We also show that GSNOR deficiency promotes mitochondrial nitrosative stress, including excessive S-nitrosylation of Drp1 and Parkin, thereby impairing mitochondrial dynamics and mitophagy. Our findings implicate GSNOR in mammalian longevity, suggest a molecular link between protein S-nitrosylation and mitochondria quality control in aging, and provide a redox-based perspective on aging with direct therapeutic implications.

A novel neuroferritinopathy mouse model (FTL 498InsTC) shows progressive brain iron dysregulation, morphological signs of early neurodegeneration and motor coordination deficits.

Neuroferritinopathy is a rare genetic disease with a dominant autosomal transmission caused by mutations of the ferritin light chain gene (FTL). It belongs to Neurodegeneration with Brain Iron Accumulation, a group of disorders where iron dysregulation is tightly associated with neurodegeneration. We studied the 498-499InsTC mutation which causes the substitution of the last 9 amino acids and an elongation of extra 16 amino acids at the C-terminus of L-ferritin peptide. An analysis with cyclic voltammetry on the purified protein showed that this structural modification severely reduces the ability of the protein to store iron. In order to analyze the impact of the mutation in vivo, we generated mouse models for the some pathogenic human FTL gene in FVB and C57BL/6J strains. Transgenic mice in the FVB background showed high accumulation of the mutated ferritin in brain where it correlated with increased iron deposition with age, as scored by magnetic resonance imaging. Notably, the accumulation of iron-ferritin bodies was accompanied by signs of oxidative damage. In the C57BL/6 background, both the expression of the mutant ferritin and the iron levels were lower than in the FVB strain. Nevertheless, also these mice showed oxidative alterations in the brain. Furthermore, post-natal hippocampal neurons obtained from these mice experienced a marked increased cell death in response to chronic iron overload and/or acute oxidative stress, in comparison to wild-type neurons. Ultrastructural analyses revealed an accumulation of lipofuscin granules associated with iron deposits, particularly enriched in the cerebellum and striatum of our transgenic mice. Finally, experimental subjects were tested throughout development and aging at 2-, 8- and 18-months for behavioral phenotype. Rotarod test revealed a progressive impaired motor coordination building up with age, FTL mutant old mice showing a shorter latency to fall from the apparatus, according to higher accumulation of iron aggregates in the striatum. Our data show that our 498-499InsTC mouse models recapitulate early pathological and clinical traits of the human neuroferritinopathy, thus providing a valuable model for the study of the disease. Finally, we propose a mechanistic model of lipofuscine formation that can account for the etiopathogenesis of human neuroferritinopathy.

Wharton's Jelly mesenchymal stromal cell-derived extracellular vesicles promote nucleus pulposus cell anabolism in an in vitro 3D alginate-bead culture model.

Background: Intradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile microenvironment of the intervertebral disc (IVD) may compromise the survival of implanted cells. Interestingly, studies reported that paracrine factors, such as extracellular vesicles (EVs) released by MSCs, may regenerate the IVD. The aim of this study was to investigate the therapeutic effects of Wharton's Jelly MSC (WJ-MSC)-derived EVs on human nucleus pulposus cells (hNPCs) using an in vitro 3D alginate-bead culture model. Methods: After EV isolation and characterization, hNPCs isolated from surgical specimens were encapsulated in alginate beads and treated with 10, 50, and 100 µg/mL WJ-MSC-EVs. Cell proliferation and viability were assessed by flow cytometry and live/dead staining. Nitrite and glycosaminoglycan (GAG) content was evaluated through Griess and 1,9-dimethylmethylene blue assays. hNPCs in alginate beads were paraffin-embedded and stained for histological analysis (hematoxylin-eosin and Alcian blue) to assess extracellular matrix (ECM) composition. Gene expression levels of catabolic (MMP1, MMP13, ADAMTS5, IL6, NOS2), anabolic (ACAN), and hNPC marker (SOX9, KRT19) genes were analyzed through qPCR. Collagen type I and type II content was assessed with Western blot analysis. Results: Treatment with WJ-MSC-EVs resulted in an increase in cell content and a decrease in cell death in degenerated hNPCs. Nitrite production was drastically reduced by EV treatment compared to the control. Furthermore, proteoglycan content was enhanced and confirmed by Alcian blue histological staining. EV stimulation attenuated ECM degradation and inflammation by suppressing catabolic and inflammatory gene expression levels. Additionally, NPC phenotypic marker genes were also maintained by the EV treatment. Conclusions: WJ-MSC-derived EVs ameliorated hNPC growth and viability, and attenuated ECM degradation and oxidative stress, offering new opportunities for IVD regeneration as an attractive alternative strategy to cell therapy, which may be jeopardized by the harsh microenvironment of the IVD.

Scapula development is governed by genetic interactions of Pbx1 with its family members and with Emx2 via their cooperative control of Alx1.

The genetic pathways underlying shoulder blade development are largely unknown, as gene networks controlling limb morphogenesis have limited influence on scapula formation. Analysis of mouse mutants for Pbx and Emx2 genes has suggested their potential roles in girdle development. In this study, by generating compound mutant mice, we examined the genetic control of scapula development by Pbx genes and their functional relationship with Emx2. Analyses of Pbx and Pbx1;Emx2 compound mutants revealed that Pbx genes share overlapping functions in shoulder development and that Pbx1 genetically interacts with Emx2 in this process. Here, we provide a biochemical basis for Pbx1;Emx2 genetic interaction by showing that Pbx1 and Emx2 can bind specific DNA sequences as heterodimers. Moreover, the expression of genes crucial for scapula development is altered in these mutants, indicating that Pbx genes act upstream of essential pathways for scapula formation. In particular, expression of Alx1, an effector of scapula blade patterning, is absent in all compound mutants. We demonstrate that Pbx1 and Emx2 bind in vivo to a conserved sequence upstream of Alx1 and cooperatively activate its transcription via this potential regulatory element. Our results establish an essential role for Pbx1 in genetic interactions with its family members and with Emx2 and delineate novel regulatory networks in shoulder girdle development.

Micro-fragmented and nanofat adipose tissue derivatives: In vitro qualitative and quantitative analysis.

Introduction: Adipose tissue is widely exploited in regenerative medicine thanks to its trophic properties, mainly based on the presence of adipose-derived stromal cells. Numerous devices have been developed to promote its clinical use, leading to the introduction of one-step surgical procedures to obtain minimally manipulated adipose tissue derivatives. However, only a few studies compared their biological properties. This study aimed to characterize micro-fragmented (MAT) and nanofat adipose tissue (NAT) obtained with two different techniques. Methods: MAT, NAT and unprocessed lipoaspirate were collected from surgical specimens. RNA extraction and collagenase isolation of stromal vascular fraction (SVF) were performed. Tissue sections were analysed by histological and immunohistochemical (collagen type I, CD31, CD34 and PCNA) staining to assess tissue morphology and cell content. qPCR was performed to evaluate the expression of stemness-related (SOX2, NANOG and OCT3/4), extracellular matrix (COL1A1) and inflammatory genes (IL1ß, IL6 and iNOS). Furthermore, multilineage differentiation was assessed following culture in adipogenic and osteogenic media and staining with Oil Red O and Alizarin red. ASC immunophenotype was assessed by flow cytometric analysis of CD90, CD105, CD73 and CD45. Results: Histological and immunohistochemical results showed an increased amount of stroma and a reduction of adipocytes in MAT and NAT, with the latter displaying the highest content of collagen type I, CD31, CD34 and PCNA. From LA to MAT and NAT, an increasing expression of NANOG, SOX2, OCT3/4, COL1A1 and IL6 was noted, while no significant differences in terms of IL1ß and iNOS emerged. No statistically significant differences were noted between NAT and SVF in terms of stemness-related genes, while the latter demonstrated a significantly higher expression of stress-related markers. SVF cells derived from all three samples (LA, MAT, and NAT) showed a similar ASC immunoprofile as well as osteogenic and adipogenic differentiation. Discussion: Our results showed that both MAT and NAT techniques allowed the rapid isolation of ASC-rich grafts with a high anabolic and proliferative potential. However, NAT showed the highest levels of extracellular matrix content, replicating cells, and stemness gene expression. These results may provide precious clues for the use of adipose tissue derivatives in the clinical setting.

Effect of Irisin on Human Nucleus Pulposus Cells: New Insights into the Biological Cross-talk Between Muscle and Intervertebral Disk.

STUDY DESIGN: In vitro study. OBJECTIVE: To investigate the effect of irisin on human nucleus pulposus cells (hNPCs) in vitro. SUMMARY OF BACKGROUND DATA: Physical exercise (PE) favours weight loss and ameliorates function in patients with low back pain. Although there is no biological evidence that the intervertebral disk (IVD) can respond to PE, recent studies have shown that running is associated with increased IVD hydration and hypertrophy. Irisin, a myokine released upon muscle contraction, has demonstrated anabolic effects on different cell types, including chondrocytes. MATERIALS AND METHODS: hNPCs were exposed to 5, 10, and 25 ng/mL irisin. Cell proliferation, glycosaminoglycan (GAG) content, metabolic activity, gene expression of collagen type II (COL2), matrix metalloproteinase (MMP)-13, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-3, aggrecan (ACAN), interleukin (IL)-1ß, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 were assessed. In addition, MTT assay and ADAMTS-5, COL2, TIMP-1, and IL-1ß gene expression were evaluated following incubation with irisin for 24 hours and subsequent culture with 10 ng/mL IL-1ß and vice versa (incubation for 24 hours with IL-1ß and subsequent culture with irisin). RESULTS: Irisin increased hNPC proliferation, metabolic activity, and GAG content, as well as COL2, ACAN, TIMP-1 and TIMP-3 gene expression, while decreasing MMP-13 and IL-1ß mRNA levels. Irisin pretreatment of hNPCs cultured in proinflammatory conditions resulted in a rescue of metabolic activity and a decrease of IL-1ß levels. Similarly, incubation of hNPCs with IL-1ß and subsequent exposure to irisin led to an increment of metabolic activity, COL2 gene expression, and a reduction of IL-1ß and ADAMTS-5 levels. CONCLUSIONS: Irisin increases hNPC proliferation, GAG content, metabolic activity, and promotes anabolic gene expression while reducing catabolic markers. Irisin may be one of the mediators by which PE and muscle tissues modulate IVD metabolism, suggesting the existence of a biological cross-talk between the muscle and IVD.

Mesenchymal stem cell-derived secretome enhances nucleus pulposus cell metabolism and modulates extracellular matrix gene expression in vitro.

Introduction: Intradiscal mesenchymal stromal cell (MSC) therapies for intervertebral disc degeneration (IDD) have been gaining increasing interest due to their capacity to ameliorate intervertebral disc metabolism and relieve low back pain (LBP). Recently, novel investigations have demonstrated that most of MSC anabolic effects are exerted by secreted growth factors, cytokines, and extracellular vesicles, collectively defined as their secretome. In this study, we aimed to evaluate the effect of bone-marrow-MSCs (BM-MSCs) and adipose-derived stromal cells (ADSCs) secretomes on human nucleus pulposus cells (hNPCs) in vitro. Methods: BM-MSCs and ADSCs were characterized according to surface marker expression by flow cytometry and multilineage differentiation by Alizarin red, Red Oil O and Alcian blue staining. After isolation, hNPCs were treated with either BM-MSC secretome, ADSC secretome, interleukin (IL)-1ß followed by BM-MSC secretome or IL-1ß followed by ADSC secretome. Cell metabolic activity (MTT assay), cell viability (LIVE/DEAD assay), cell content, glycosaminoglycan production (1,9-dimethylmethylene blue assay), extracellular matrix and catabolic marker gene expression (qPCR) were assessed. Results: 20% BM-MSC and ADSC secretomes (diluted to normal media) showed to exert the highest effect towards cell metabolism and were then used in further experiments. Both BM-MSC and ADSC secretomes improved hNPC viability, increased cell content and enhanced glycosaminoglycan production in basal conditions as well as after IL-1ß pretreatment. BM-MSC secretome significantly increased ACAN and SOX9 gene expression, while reducing the levels of IL6, MMP13 and ADAMTS5 both in basal conditions and after in vitro inflammation with IL-1ß. Interestingly, under IL-1ß stimulation, ADSC secretome showed a catabolic effect with decreased extracellular matrix markers and increased levels of pro-inflammatory mediators. Discussion: Collectively, our results provide new insights on the biological effect of MSC-derived secretomes on hNPCs, with intriguing implications on the development of cell-free approaches to treat IDD.

Irisin inhibits tenocyte response to inflammation in vitro: New insights into tendon-muscle cross-talk.

Tendinopathy is one of the most common musculoskeletal disorders with significant repercussions on quality of life and sport activities. Physical exercise (PE) is considered the first-line approach to treat tendinopathy due renowned mechanobiological effects on tenocytes. Irisin, a recently identified myokine released during PE, has been recognized for several beneficial effects towards muscle, cartilage, bone, and intervertebral disc tissues. The aim of this study was to evaluate the effects of irisin on human primary tenocytes (hTCs) in vitro. Human tendons were harvested from specimens of patients undergoing anterior cruciate ligament reconstruction (n = 4). After isolation and expansion, hTCs were treated with RPMI medium (negative control), interleukin (IL)-1ß or tumor necrosis factor-α (TNF-α) (positive controls; 10 ng/mL), irisin (5, 10, 25 ng/mL), IL-1ß or TNF-α pretreatment and subsequent co-treatment with irisin, pretreatment with irisin and subsequent co-treatment with IL-1ß or TNF-α. hTC metabolic activity, proliferation, and nitrite production were evaluated. Detection of unphosphorylated and phosphorylated p38 and ERK was performed. Tissue samples were analyzed by histology and immunohistochemistry to evaluate irisin αVß5 receptor expression. Irisin significantly increased hTC proliferation and metabolic activity, while reducing the production of nitrites both before and after the addition of IL-1ß and TNF-α. Interestingly, irisin reduced p-p38 and pERK levels in inflamed hTCs. The αVß5 receptor was uniformly expressed on hTC plasma membranes, supporting the potential binding of irisin. This is the first study reporting the capacity of irisin to target hTCs and modulating their response to inflammatory stresses, possibly orchestrating a biological crosstalk between the muscle and tendon.

Embryonic arrest at midgestation and disruption of Notch signaling produced by the absence of both epsin 1 and epsin 2 in mice.

Epsins are endocytic adaptors with putative functions in general aspects of clathrin-mediated endocytosis as well as in the internalization of specific membrane proteins. We have now tested the role of the ubiquitously expressed epsin genes, Epn1 and Epn2, by a genetic approach in mice. While either gene is dispensable for life, their combined inactivation results in embryonic lethality at E9.5-E10, i.e., at the beginning of organogenesis. Consistent with studies in Drosophila, where epsin endocytic function was linked to Notch activation, developmental defects observed in epsin 1/2 double knockout (DKO) embryos recapitulated those produced by a global impairment of Notch signaling. Accordingly, expression of Notch primary target genes was severely reduced in DKO embryos. However, housekeeping forms of clathrin-mediated endocytosis were not impaired in cells derived from these embryos. These findings support a role of epsin as a specialized endocytic adaptor, with a critical role in the activation of Notch signaling in mammals.

Control of pelvic girdle development by genes of the Pbx family and Emx2.

Genes expressed in the somatopleuric mesoderm, the embryonic domain giving rise to the vertebrate pelvis, appear important for pelvic girdle formation. Among such genes, Pbx family members and Emx2 were found to genetically interact in hindlimb and pectoral girdle formation. Here, we generated compound mutant embryos carrying combinations of mutated alleles for Pbx1, Pbx2, and Pbx3, as well as Pbx1 and Emx2, to examine potential genetic interactions during pelvic development. Indeed, Pbx genes share overlapping functions and Pbx1 and Emx2 genetically interact in pelvic formation. We show that, in compound Pbx1;Pbx2 and Pbx1;Emx2 mutants, pelvic mesenchymal condensation is markedly perturbed, indicative of an upstream control by these homeoproteins. We establish that expression of Tbx15, Prrx1, and Pax1, among other genes involved in the specification and development of select pelvic structures, is altered in our compound mutants. Lastly, we identify potential Pbx1-Emx2-regulated enhancers for Tbx15, Prrx1, and Pax1, using bioinformatics analyses.

Plasmodium falciparum liver stage antigen-1 is cross-linked by tissue transglutaminase.

BACKGROUND: Plasmodium falciparum sporozoites injected by mosquitoes into the blood rapidly enter liver hepatocytes and undergo pre-erythrocytic developmental schizogony forming tens of thousands of merozoites per hepatocyte. Shortly after hepatocyte invasion, the parasite starts to produce Liver Stage Antigen-1 (LSA-1), which accumulates within the parasitophorous vacuole surrounding the mass of developing merozoites. The LSA-1 protein has been described as a flocculent mass, but its role in parasite development has not been determined. METHODS: Recombinant N-terminal, C-terminal or a construct containing both the N- and C- terminal regions flanking two 17 amino acid residue central repeat sequences (LSA-NRC) were subjected to in vitro modification by tissue transglutaminase-2 (TG2) to determine if cross-linking occurred. In addition, tissue sections of P. falciparum-infected human hepatocytes were probed with monoclonal antibodies to the isopeptide ε-(γ-glutamyl)lysine cross-bridge formed by TG2 enzymatic activity to determine if these antibodies co-localized with antibodies to LSA-1 in the growing liver schizonts. RESULTS: This study identified a substrate motif for (TG2) and a putative casein kinase 2 phosphorylation site within the central repeat region of LSA-1. The function of TG2 is the post-translational modification of proteins by the formation of a unique isopeptide ε-(γ-glutamyl)lysine cross-bridge between glutamine and lysine residues. When recombinant LSA-1 protein was crosslinked in vitro by purified TG2 in a calcium dependent reaction, a flocculent mass of protein was formed that was highly resistant to degradation. The cross-linking was not detectably affected by phosphorylation with plasmodial CK2 in vitro. Monoclonal antibodies specific to the very unique TG2 catalyzed ε- lysine cross-bridge co-localized with antibodies to LSA-1 in infected human hepatocytes providing visual evidence that LSA-1 was cross-linked in vivo. CONCLUSIONS: While the role of LSA-1 is still unknown these results suggest that it becomes highly cross-linked which may aid in the protection of the parasite as it develops.

Does Vancomycin Wrapping in Anterior Cruciate Ligament Reconstruction Affect Tenocyte Activity In Vitro?

Knee septic arthritis is a devastating complication following anterior cruciate ligament (ACL) reconstruction. To prevent this issue, intraoperative soaking of ACL grafts with vancomycin is often performed before implantation. Although vancomycin cytotoxicity has been reported several times, little is known about its biological effect on tenocytes. The aim of this study was to evaluate the in vitro effects of vancomycin on human primary tenocytes (hTCs). hTCs were isolated from hamstring grafts of four patients undergoing ACL reconstruction. After expansion, hTCs were treated with different concentrations of vancomycin (0, 2.5, 5, 10, 25, 50 and 100 mg/mL) for 10, 15, 30 and 60 min. In vitro cytotoxicity was evaluated measuring metabolic activity, cell toxicity, and apoptosis. hTC metabolic activity was affected starting from 10 mg/mL vancomycin and decreased markedly at 100 mg/mL. Cell viability remained unaffected only at a concentration of 2.5 mg/mL vancomycin. Vancomycin cytotoxicity was detected from 10 mg/mL after 15 min and at all higher concentrations. Cells died when treated with concentrations higher than 5 mg/mL. The use of this antibiotic on tendons to prevent infections could be useful and safe for resident cells if used at a concentration of 2.5 mg/mL for up to 1 h of treatment.

Irisin Recovers Osteoarthritic Chondrocytes In Vitro.

Physical exercise favors weight loss and ameliorates articular pain and function in patients suffering from osteoarthritis. Irisin, a myokine released upon muscle contraction, has demonstrated to yield anabolic effects on different cell types. This study aimed to investigate the effect of irisin on human osteoarthritic chondrocytes (hOAC) in vitro. Our hypothesis was that irisin would improve hOAC metabolism and proliferation. Cells were cultured in growing media and then exposed to either phosphate-buffered saline (control group) or human recombinant irisin (experimental group). Cell proliferation, glycosaminoglycan content, type II/X collagen gene expression and protein quantification as well as p38/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK), protein kinase B (Akt), c-Jun N-terminal kinase (JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) involvement were evaluated. Furthermore, gene expression of interleukin (IL)-1 and -6, matrix metalloproteinase (MMP)-1 and -13, inducible nitric oxide synthase (iNOS), and tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -3 were investigated following irisin exposure. Irisin increased hOAC cell content and both type II collagen gene expression and protein levels, while decreased type X collagen gene expression and protein levels. Moreover, irisin decreased IL-1, IL-6, MMP-1, MMP-13 and iNOS gene expression, while increased TIMP-1 and TIMP-3 levels. These effects seemed to be mediated by inhibition of p38, Akt, JNK and NFκB signaling pathways. The present study suggested that irisin may stimulate hOAC proliferation and anabolism inhibiting catabolism through p38, Akt, JNK, and NFκB inactivation in vitro, demonstrating the existence of a cross-talk between muscle and cartilage.

Pbx1/Pbx2 govern axial skeletal development by controlling Polycomb and Hox in mesoderm and Pax1/Pax9 in sclerotome.

The post-cranial axial skeleton consists of a metameric series of vertebral bodies and intervertebral discs, as well as adjoining ribs and sternum. Patterning of individual vertebrae and distinct regions of the vertebral column is accomplished by Polycomb and Hox proteins in the paraxial mesoderm, while their subsequent morphogenesis depends partially on Pax1/Pax9 in the sclerotome. In this study, we uncover that Pbx1/Pbx2 are co-expressed during successive stages of vertebral and rib development. Next, by exploiting a Pbx1/Pbx2 loss-of-function mouse, we show that decreasing Pbx2 dosage in the absence of Pbx1 affects axial development more severely than single loss of Pbx1. Pbx1/Pbx2 mutants exhibit a homogeneous vertebral column, with loss of vertebral identity, rudimentary ribs, and rostral hindlimb shifts. Of note, these axial defects do not arise from perturbed notochord function, as cellular proliferation, apoptosis, and expression of regulators of notochord signaling are normal in Pbx1/Pbx2 mutants. While the observed defects are consistent with loss of Pbx activity as a Hox-cofactor in the mesoderm, we additionally establish that axial skeletal patterning and hindlimb positioning are governed by Pbx1/Pbx2 through their genetic control of Polycomb and Hox expression and spatial distribution in the mesoderm, as well as of Pax1/Pax9 in the sclerotome.

Epsins Regulate Mouse Embryonic Stem Cell Exit from Pluripotency and Neural Commitment by Controlling Notch Activation.

Epsins are part of the internalization machinery pivotal to control clathrin-mediated endocytosis. Here, we report that epsin family members are expressed in mouse embryonic stem cells (mESCs) and that epsin1/2 knockdown alters both mESC exits from pluripotency and their differentiation. Furthermore, we show that epsin1/2 knockdown compromises the correct polarization and division of mESC-derived neural progenitors and their conversion into expandable radial glia-like neural stem cells. Finally, we provide evidence that Notch signaling is impaired following epsin1/2 knockdown and that experimental restoration of Notch signaling rescues the epsin-mediated phenotypes. We conclude that epsins contribute to control mESC exit from pluripotency and allow their neural differentiation by appropriate modulation of Notch signaling.

Spatio-temporal expression of Pbx3 during mouse organogenesis.

Pbx3 is a member of the Pbx family of TALE (three amino acid loop extension) class homeodomain transcription factors. These transcription factors are implicated in developmental and transcriptional gene regulation in numerous cell types through their abilities to form hetero-oligomeric DNA-binding complexes. Pbx3 was found to be expressed at high levels in the developing central nervous system (CNS), including a region of the medulla oblongata which is implicated in the control of respiration. Furthermore, as reported, Pbx3-deficient mice develop to term but die within a few hours of birth from central respiratory failure. In this study, we have characterized Pbx3 expression patterns during organogenesis in numerous tissues and organ systems other than the CNS, as a first step toward understanding the potentially overlapping functions of Pbx3 with other Pbx family members during vertebrate development. We have performed in situ hybridization on whole mount and sectioned mouse embryos from gestational day (E) 9 to E16.5. During early organogenesis, until E12.5, Pbx3 expression is found mostly in the embryonic head, forelimbs, and septum transversum, unlike Pbx1 and Pbx2 expression which is more widespread. Conversely, later in organogenesis, Pbx3 expression becomes more widely detectable throughout the developing embryo. Epithelial and mesenchymal tissues, as well as the CNS, represent major sites of Pbx3 expression. The enteric nervous system also expresses high levels of Pbx3, distinctively in the cells of the ganglia of Auerbach's myenteric nerve plexus, that also express Dlx2 and Notch1. Cartilage is also a site of Pbx3 expression. Interestingly, like Pbx1, Pbx3 is highly expressed in proliferating chondrocytes but is lost as chondrocytes become hypertrophic during endochondral ossification. Finally, Pbx3 is expressed only in the forelimb buds during early limb development, while the hindlimb bud is devoid of Pbx3. This finding leads us to add Pbx3 to the sparse list of early forelimb-specific molecular markers.

S-nitrosylation of the Mitochondrial Chaperone TRAP1 Sensitizes Hepatocellular Carcinoma Cells to Inhibitors of Succinate Dehydrogenase.

S-nitrosoglutathione reductase (GSNOR) represents the best-documented denitrosylase implicated in regulating the levels of proteins posttranslationally modified by nitric oxide on cysteine residues by S-nitrosylation. GSNOR controls a diverse array of physiologic functions, including cellular growth and differentiation, inflammation, and metabolism. Chromosomal deletion of GSNOR results in pathologic protein S-nitrosylation that is implicated in human hepatocellular carcinoma (HCC). Here we identify a metabolic hallmark of aberrant S-nitrosylation in HCC and exploit it for therapeutic gain. We find that hepatocyte GSNOR deficiency is characterized by mitochondrial alteration and by marked increases in succinate dehydrogenase (SDH) levels and activity. We find that this depends on the selective S-nitrosylation of Cys(501) in the mitochondrial chaperone TRAP1, which mediates its degradation. As a result, GSNOR-deficient cells and tumors are highly sensitive to SDH inhibition, namely to α-tocopheryl succinate, an SDH-targeting molecule that induced RIP1/PARP1-mediated necroptosis and inhibited tumor growth. Our work provides a specific molecular signature of aberrant S-nitrosylation in HCC, a novel molecular target in SDH, and a first-in-class therapy to treat the disease. Cancer Res; 76(14); 4170-82. ©2016 AACR.

Mature Erythrocytes of Iguana iguana (Squamata, Iguanidae) Possess Functional Mitochondria.

Electron microscopy analyses of Iguana iguana blood preparations revealed the presence of mitochondria within erythrocytes with well-structured cristae. Fluorescence microscopy analyses upon incubation with phalloidin-FITC, Hoechst 33342 and mitochondrial transmembrane potential (Δψm)-sensitive probe MitoTracker Red indicated that mitochondria i) widely occur in erythrocytes, ii) are polarized, and iii) seem to be preferentially confined at a "perinuclear" region, as confirmed by electron microscopy. The analysis of NADH-dependent oxygen consumption showed that red blood cells retain the capability to consume oxygen, thereby providing compelling evidence that mitochondria of Iguana erythrocytes are functional and capable to perform oxidative phosphorylation.

S-nitrosation and ubiquitin-proteasome system interplay in neuromuscular disorders.

Protein S-nitrosation is deemed as a prototype of posttranslational modifications governing cell signaling. It takes place on specific cysteine residues that covalently incorporate a nitric oxide (NO) moiety to form S-nitrosothiol derivatives and depends on the ratio between NO produced by NO synthases and nitrosothiol removal catalyzed by denitrosating enzymes. A large number of cysteine-containing proteins are found to undergo S-nitrosation and, among them, the enzymes catalyzing ubiquitination, mainly the class of ubiquitin E3 ligases and the 20S component of the proteasome, have been reported to be redox modulated in their activity. In this review we will outline the processes regulating S-nitrosation and try to debate whether and how it affects protein ubiquitination and degradation via the proteasome. In particular, since muscle and neuronal health largely depends on the balance between protein synthesis and breakdown, here we will discuss the impact of S-nitrosation in the efficiency of protein quality control system, providing lines of evidence and speculating about its involvement in the onset and maintenance of neuromuscular dysfunctions.