RACK1 is evolutionary conserved in satellite stem cell activation and adult skeletal muscle regeneration.

Skeletal muscle growth and regeneration involves the activity of resident adult stem cells, namely satellite cells (SC). Despite numerous mechanisms have been described, different signals are emerging as relevant in SC homeostasis. Here we demonstrated that the Receptor for Activated C-Kinase 1 (RACK1) is important in SC function. RACK1 was expressed transiently in the skeletal muscle of post-natal mice, being abundant in the early phase of muscle growth and almost disappearing in adult mature fibers. The presence of RACK1 in interstitial SC was also detected. After acute injury in muscle of both mouse and the fruit fly Drosophila melanogaster (used as alternative in vivo model) we found that RACK1 accumulated in regenerating fibers while it declined with the progression of repair process. To note, RACK1 also localized in the active SC that populate recovering tissue. The dynamics of RACK1 levels in isolated adult SC of mice, i.e., progressively high during differentiation and low compared to proliferating conditions, and RACK1 silencing indicated that RACK1 promotes both the formation of myotubes and the accretion of nascent myotubes. In Drosophila with depleted RACK1 in all muscle cells or, specifically, in SC lineage we observed a delayed recovery of skeletal muscle after physical damage as well as the low presence of active SC in the wound area. Our results also suggest the coupling of RACK1 to muscle unfolded protein response during SC activation. Collectively, we provided the first evidence that transient levels of the evolutionarily conserved factor RACK1 are critical for adult SC activation and proper skeletal muscle regeneration, favoring the efficient progression of SC from a committed to a fully differentiated state.

Ribosomal RACK1 Regulates the Dendritic Arborization by Repressing FMRP Activity.

FMRP is an RNA-binding protein that represses the translation of specific mRNAs. In neurons, its depletion determines the exaggerated translation of mRNAs leading to dendritic and axonal aberrant development, two peculiar features of Fragile X syndrome patients. However, how FMRP binds to translational machinery to regulate the translation of its mRNA targets is not yet fully understood. Here, we show that FMRP localizes on translational machinery by interacting with the ribosomal binding protein, Receptor for Activated C Kinase 1 (RACK1). The binding of FMRP to RACK1 removes the translational repressive activity of FMRP and promotes the translation of PSD-95 mRNA, one specific target of FMRP. This binding also results in a reduction in the level of FMRP phosphorylation. We also find that the morphological abnormalities induced by Fmr1 siRNA in cortical neurons are rescued by the overexpression of a mutant form of RACK1 that cannot bind ribosomes. Thus, these results provide a new mechanism underlying FMRP activity that contributes to altered development in FXS. Moreover, these data confirm the role of ribosomal RACK1 as a ribosomal scaffold for RNA binding proteins.

Can Blebbistatin block the hypertrophy status in the zebrafish ex vivo cardiac model?

Ex-vivo simple models are powered tools to study cardiac hypertrophy. It is possible to control the activation of critical genes and thus test the effects of drug therapies before the in vivo tests. A zebrafish cardiac hypertrophy developed by 500 µM phenylephrine (PE) treatment in ex vivo culture has been demonstrated to activate the essential expression of the embryonal genes. These genes are the same as those described in several previous pieces of research on hypertrophic pathology in humans. The efficacy of the chemical drug Blebbistatin (BL) on hypertrophy induced ex vivo cultured hearts is studied in this research. BL can inhibit the myosins and the calcium wave in counteracting the hypertrophy status caused by PE. Samples treated with PE, BL and PE simultaneously, or pre/post-treatment with BL, have been analysed for the embryonal gene activation concerning the hypertrophy status. The qRTPCR has shown an inhibitory effect of BL treatments on the microRNAs downregulation with the consequent low expression of essential embryonal genes. In particular, BL seems to be effective in blocking the hyperplasia of the epicardium but less effective in myocardium hypertrophy. The model can make it possible to obtain knowledge on the transduction pathways activated by BL and investigate the potential use of this drug in treating cardiac hypertrophy in humans.

Human Adipose-Derived Stem Cell-Conditioned Medium Promotes Vascularization of Nanostructured Scaffold Transplanted into Nude Mice.

Several studies have been conducted on the interaction between three-dimensional scaffolds and mesenchymal stem cells for the regeneration of damaged tissues. Considering that stem cells do not survive for sufficient time to directly sustain tissue regeneration, it is essential to develop cell-free systems to be applied in regenerative medicine. In this work, by in vivo experiments, we established that a collagen-nanostructured scaffold, loaded with a culture medium conditioned with mesenchymal stem cells derived from adipose tissue (hASC-CM), exerts a synergic positive effect on angiogenesis, fundamental in tissue regeneration. To this aim, we engrafted athymic BALB-C nude mice with four different combinations: scaffold alone; scaffold with hASCs; scaffold with hASC crude protein extract; scaffold with hASC-CM. After their removal, we verified the presence of blood vessels by optical microscopy and confirmed the vascularization evaluating, by real-time PCR, several vascular growth factors: CD31, CD34, CD105, ANGPT1, ANGPT2, and CDH5. Our results showed that blood vessels were absent in the scaffold grafted alone, while all the other systems appeared vascularized, a finding supported by the over-expression of CD31 and CDH5 mRNA. In conclusion, our data sustain the capability of hASC-CM to be used as a therapeutic cell-free approach for damaged tissue regeneration.

The role of RNA-binding and ribosomal proteins as specific RNA translation regulators in cellular differentiation and carcinogenesis.

Tight control of mRNA expression is required for cell differentiation; imbalanced regulation may lead to developmental disorders and cancer. The activity of the translational machinery (including ribosomes and translation factors) regulates the rate (slow or fast) of translation of encoded proteins, and the quality of these proteins highly depends on which mRNAs are available for translation. Specific RNA-binding and ribosomal proteins seem to play a key role in controlling gene expression to determine the differentiation fate of the cell. This demonstrates the important role of RNA-binding proteins, specific ribosome-binding proteins and microRNAs as key molecules in controlling the specific proteins required for the differentiation or dedifferentiation of cells. This delicate balance between specific proteins (in terms of quality and availability) and post-translational modifications occurring in the cytoplasm is crucial for cell differentiation, dedifferentiation and oncogenic potential. In this review, we report how defects in the regulation of mRNA translation can be dependent on specific proteins and can induce an imbalance between differentiation and dedifferentiation in cell fate determination.

Are microRNAs responsible for cardiac hypertrophy in fish and mammals? What we can learn in the activation process in a zebrafish ex vivo model.

Recent studies have correlated dysregulated miRNA expression with diseased hearts. With the aim of developing an easily manipulated experimental model, phenylephrine (PE) was added to cultured zebrafish hearts to study the expression of miR1 and miR133a by qRT-PCR. Both miRs were downregulated, with greater downregulation leading to higher hypertrophy. The involvement of this miRs was confirmed by the in-vivo inoculation of complementary sequences (AmiR1 and AmiR133a). HSP70 (involved in transporting proteins and in anti-apoptosis processes) was increased in both treatments. Hyperplasia was observed in the epicardium based on WT1 expression (embryonic epicardial cell marker) in both the PE treatment and AmiR133a treatment. The treatment with AmiR1 showed only cardiomyocyte hypertrophy. This ex-vivo model revealed that miR1 and miR133a play a key role in activating early processes leading to myocardium hypertrophy and epicardium hyperplasia and confirmed the expected similarities with hypertrophic disease that occurs in humans.

ALS skin fibroblasts reveal oxidative stress and ERK1/2-mediated cytoplasmic localization of TDP-43.

The main hallmark of many forms of familiar and sporadic amyotrophic lateral sclerosis (ALS) is a reduction in nuclear TDP-43 protein and its inclusion in cytoplasmic aggregates in motor neurons. In order to understand which cellular and molecular mechanisms underlie the mislocalization of TDP-43, we examined human skin fibroblasts from two individuals with familial ALS, both with mutations in TDP-43, and two individuals with sporadic ALS, both without TDP-43 mutations or mutations in other ALS related genes. We found that all ALS fibroblasts had a partially cytoplasmic localization of TDP-43 and had reduced cell metabolism as compared to fibroblasts from apparently healthy individuals. ALS fibroblasts showed an increase in global protein synthesis and an increase in 4E-BP1 and rpS6 phosphorylation, which is indicative of mTORC1 activity. We also observed a decrease in glutathione (GSH), which suggests that oxidative stress is elevated in ALS. ERK1/2 activity regulated the extent of oxidative stress and the localization of TDP-43 in the cytoplasm in all ALS fibroblasts. Lastly, ALS fibroblasts showed reduced stress granule formation in response to H2O2 stress. In conclusion, these findings identify specific cellular and molecular defects in ALS fibroblasts, thus providing insight into potential mechanisms that may also occur in degenerating motor neurons.

Ribosomal RACK1 promotes proliferation of neuroblastoma cells independently of global translation upregulation.

Neuroblastoma is the most frequent solid tumor among those diagnosed during infancy and like most tumors, it is characterized by elevated rates of cell proliferation, migration and invasion. RACK1 is among the top 10 genes identified for unfavorable prognosis at 5 years in neuroblastoma cases and its depletion negatively affects proliferation, invasion and migration. Here, we show that the ribosomal localization of RACK1 modulates the proliferation of SH-SY5Y neuroblastoma cells by regulating the expression of cell cycle genes, such as Cyclin D1, D3 and B1 independently of global translation increase. Ribosomal RACK1 is not involved in general protein synthesis, which is instead dependent on total RACK1 and PKC but independent from mTOR. Thus, ribosomal RACK1 may represent a new target to develop more efficient therapies for neuroblastoma treatment.

Environmental pollution and toxic substances: Cellular apoptosis as a key parameter in a sensible model like fish.

The industrial wastes, sewage effluents, agricultural run-off and decomposition of biological waste may cause high environmental concentration of chemicals that can interfere with the cell cycle activating the programmed process of cells death (apoptosis). In order to provide a detailed understanding of environmental pollutants-induced apoptosis, here we reviewed the current knowledge on the interactions of environmental chemicals and programmed cell death. Metals (aluminum, arsenic, cadmium, chromium, cobalt, zinc, copper, mercury and silver) as well as other chemicals including bleached kraft pulp mill effluent (BKME), persistent organic pollutants (POPs), and pesticides (organo-phosphated, organo-chlorinated, carbamates, phyretroids and biopesticides) were evaluated in relation to apoptotic pathways, heat shock proteins and metallothioneins. Although research performed over the past decades has improved our understanding of processes involved in apoptosis in fish, yet there is lack of knowledge on associations between environmental pollutants and apoptosis. Thus, this review could be useful tool to study the cytotoxic/apoptotic effects of different pollutants in fish species.

Zebrafish as a translational regeneration model to study the activation of neural stem cells and role of their environment.

The review is an overview of the current knowledge of neuronal regeneration properties in mammals and fish. The ability to regenerate the damaged parts of the nervous tissue has been demonstrated in all vertebrates. Notably, fish and amphibians have the highest capacity for neurogenesis, whereas reptiles and birds are able to only regenerate specific regions of the brain, while mammals have reduced capacity for neurogenesis. Zebrafish (Danio rerio) is a promising model of study because lesions in the brain or complete cross-section of the spinal cord are followed by an effective neuro-regeneration that successfully restores the motor function. In the brain and the spinal cord of zebrafish, stem cell activity is always able to re-activate the molecular programs required for central nervous system regeneration. In mammals, traumatic brain injuries are followed by reduced neurogenesis and poor axonal regeneration, often insufficient to functionally restore the nervous tissue, while spinal injuries are not repaired at all. The environment that surrounds the stem cell niche constituted by connective tissue and stimulating factors, including pro-inflammation molecules, seems to be a determinant in triggering stem cell proliferation and/or the trans-differentiation of connective elements (mainly fibroblasts). Investigating and comparing the neuronal regeneration in zebrafish and mammals may lead to a better understanding of the mechanisms behind neurogenesis, and the failure of the regenerative response in mammals, first of all, the role of inflammation, considered the main inhibitor of the neuronal regeneration.

The face of epicardial and endocardial derived cells in zebrafish.

Zebrafish hearts can regenerate through activation of growth factors and trans-differentiation of fibroblasts, epicardial, myocardial and endocardial cells, all positive for GATA4 during the process. A possible model of regeneration of the whole heart and the regenerating cells in ex-vivo culture is presented here by a stimulation of cocktail of growth factors. In ex-vivo growth-factors-supplemented culture the heart regeneration was quite complete without signs of fibrosis. Epicardial- and endocardial-derived cells have been analyzed by electron microscopy evidencing two main types: 1) larger/prismatic and 2) small/rounded. Type (1) showed on the surface protein-sculptures, while type(2) was smooth with sparse globular proteins. To confirm their nature we have contemporarily analyzed their proliferative capability and markers-positivity. The cells treated by growth factors have at least two-fold more proliferation with GATA4-positivity. The type (1) cell evidenced WT1+(marker of embryonic epicardium); the type (2) showed NFTA2+(marker of embryonic endocardium); whereas cTNT-cardiotroponin was negative. Under growth factors stimulation, GATA4+/WT1+ and GATA4+/NFTA2+ could be suitable candidates to be the cells with capability to move in/out of the tissue, probably by using their integrins, and it opens the possibility to have long term selected culture to future characterization.

Micro RNAs are involved in activation of epicardium during zebrafish heart regeneration.

Zebrafish could be an interesting translational model to understand and improve the post-infarction trial and possible regeneration in humans. The adult zebrafish is able to regenerate efficiently after resecting nearly 20% of the ventricular apex. This process requires the concert activation of the epicardium and endocardium, as well as trans-differentiation of pre-existing cardiomyocytes that together replace the lost tissue. The molecular mechanisms involved in this activation process are not completely clarified. In this work, in order to investigate if the downregulation of these miRNAs (miRs) are linked with the activation of epicardium, the expressions of miR-133a, b and miR-1 during regeneration were analysed. qPCR analyses in whole-heart, or from distinct dissected epicardial cells comparing to regenerative clot (containing cardiomyocytes, fibroblasts and endocardial cells) by a laser-micro-dissector, have indicated that already at 24 h there is a downregulation of miRs: (1) miR-133a and miR-1 in the epicardium and (2) miR-133b and miR-1 in the regenerative clot. All the miRs remain downregulated until 7 days post-surgery. With the aim to visualize the activations of heart component in combination with miRs, we developed immunohistochemistry using antibodies directed against common markers in mammals as well as zebrafish: Wilms tumour 1 (WT1), a marker of epicardium; heat-shock protein 70 (HSP70), a chaperon activated during regeneration; and the Cardiac Troponin T (cTnT), a marker of differentiated cardiomyocytes. All these markers are directly or indirectly linked to the investigated miRs. WT1 and HSP70 strongly marked the regeneration site just at 2-3 days postventricular resection. In coherence, cTnT intensively marked the regenerative portion from 7 days onwards. miRs-1 and -133 (a,b) have been strongly involved in the activation of epicardium and regenerative clot during the regeneration process in zebrafish. This study can be a useful translational model to understand the early epicardial activation in which miRs-133a and miR-1 seem to play a central role as observed in the human heart.

Upregulation of eIF6 inhibits cardiac hypertrophy induced by phenylephrine.

Cardiac hypertrophy is determined by an increase of cell size in cardiomyocytes (CMCs). Among the cellular processes regulating the growth of cell size, the increase of protein synthesis rate represents a critical event. Most of translational factors promoting protein synthesis stimulate cardiac hypertrophy. In contrast, activity of translational repressor factors, in cardiac hypertrophy, is not fully determined yet. Here we report the effect of a translational modulator, eIF6/p27BBP in the hypertrophy of neonatal rat CMCs. The increase of eIF6 levels surprisingly prevent the growth of cell size induced by phenylephrine, through a block of protein synthesis without affecting skeletal rearrangement and ANF mRNA expression. Thus, this work uncovers a new translational cardiac regulator independent by other well-known factors such as mTOR signalling or eIF2ß.

Computational modeling of immune system of the fish for a more effective vaccination in aquaculture.

MOTIVATION: A computational model equipped with the main immunological features of the sea bass (Dicentrarchus labrax L.) immune system was used to predict more effective vaccination in fish. The performance of the model was evaluated by using the results of two in vivo vaccinations trials against L. anguillarum and P. damselae. RESULTS: Tests were performed to select the appropriate doses of vaccine and infectious bacteria to set up the model. Simulation outputs were compared with the specific antibody production and the expression of BcR and TcR gene transcripts in spleen. The model has shown a good ability to be used in sea bass and could be implemented for different routes of vaccine administration even with more than two pathogens. The model confirms the suitability of in silico methods to optimize vaccine doses and the immune response to them. This model could be applied to other species to optimize the design of new vaccination treatments of fish in aquaculture. AVAILABILITY AND IMPLEMENTATION: The method is available at http://www.iac.cnr.it/∼filippo/c-immsim/. CONTACT: nromano@unitus.it. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Increased cytoplasmic TDP-43 reduces global protein synthesis by interacting with RACK1 on polyribosomes.

TDP-43 is a well known RNA binding protein involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Dementia (FTLD). In physiological conditions, TDP-43 mainly localizes in the nucleus and shuttles, at least in neurons, to the cytoplasm to form TDP-43 RNA granules. In the nucleus, TDP-43 participates to the expression and splicing of RNAs, while in the cytoplasm its functions range from transport to translation of specific mRNAs. However, if loss or gain of these TDP-43 functions are affected in ALS/FTLD pathogenesis is not clear. Here, we report that TDP-43 localizes on ribosomes not only in primary neurons but also in SH-SY5Y human neuroblastoma cells. We find that binding of TDP-43 to the translational machinery is mediated by an interaction with a specific ribosomal protein, RACK1, and that an increase in cytoplasmic TDP-43 represses global protein synthesis, an effect which is rescued by overexpression of RACK1. Ribosomal loss of RACK1, which excludes TDP-43 from the translational machinery, remarkably reduces formation of TDP-43 cytoplasmic inclusions in neuroblastoma cells. Finally, we corroborate the interaction between TDP-43 and RACK1 on polyribosomes of neuroblastoma cells with mis-localization of RACK1 on TDP-43 positive cytoplasmic inclusions in motor neurons of ALS patients. In conclusions, results from this study suggest that TDP-43 represents a translational repressor not only for specific mRNAs but for overall translation and that its binding to polyribosomes through RACK1 may promote, under conditions inducing ALS pathogenesis, the formation of cytoplasmic inclusions.

Extracellular component hyaluronic acid and its receptor Hmmr are required for epicardial EMT during heart regeneration.

AIMS: After injury, the adult zebrafish can regenerate the heart. This requires the activation of the endocardium and epicardium as well as the proliferation of pre-existing cardiomyocytes to replace the lost tissue. However, the molecular mechanisms involved in this process are not completely resolved. In this work, we aim to identify the proteins involved in zebrafish heart regeneration and to explore their function. METHODS AND RESULTS: Using a proteomic approach, we identified Hyaluronan-mediated motility receptor (Hmmr), a hyaluronic acid (HA) receptor, to be expressed following ventricular resection in zebrafish. Moreover, enzymes that produce HA, hyaluronic acid synthases (has), were also expressed following injury, suggesting that this pathway may serve important functions in the regenerating heart. Indeed, suppression of HA production, as well as depletion of Hmmr, blocked cardiac regeneration. Mechanistically, HA and Hmmr are required for epicardial cell epithelial-mesenchymal transition (EMT) and their subsequent migration into the regenerating ventricle. Furthermore, chemical inhibition of Focal Adhesion Kinase (FAK) or inhibition of Src kinases, downstream effectors of Hmmr, also prevented epicardial cell migration, implicating a HA/Hmmr/FAK/Src pathway in this process. In a rat model of myocardial infarction, both HA and HMMR were up-regulated and localized in the infarct area within the first few days following damage, suggesting that this pathway may also play an important role in cardiac repair in mammals. CONCLUSION: HA and Hmmr are required for activated epicardial cell EMT and migration involving the FAK/Src pathway for proper heart regeneration.

Defensive response of European sea bass (Dicentrarchus labrax) against Listonella anguillarum or Photobacterium damselae subsp. piscicida experimental infection.

Sea bass were experimentally infected with Listonella anguillarum or Photobacterium damselae subsp. piscicida (Phdp). At 24 and 72h post-infection, the expression analysis of immune-relevant genes (IL-1ß, IL-6, IL-8, Hepcidin), the transcriptional level and detection of HSP70, and the quantification of serum iron were investigated in association with the histological analysis and the bacterial recognition in tissues by immunohistochemistry. At 15 days post-infection, the specific antibody response was detected in surviving fish, as well as the transcriptional levels of TcR and BcR sequences. Both experimental infections were characterized by a similar acute response, whereas different histological and immunohistochemistry evidences were observed. In particular, the early reaction appeared suitable for the clearance of L. anguillarum, thus limiting the histological lesions, the bacterial dissemination and the further development of acquired immunity in surviving fish. On the contrary, the innate response appeared not enough to resolve the Phdp infection, which was characterized by tissue damage, bacterial widespread and substantial detection of specific humoral immunity in surviving fish, also associated to lymphocytes clonal expansion. Besides the opportunistic conditions involved in fish vibriosis and pasteurellosis, the comparison between these experimental infection models seems to suggest that the rate of development of the acquired immunity is strictly linked to the activation of the host innate response combined to the degree of bacterial virulence.

Apoptosis in thymus of teleost fish.

The presence and distribution of apoptotic cells during thymus development and in adult were studied by in situ end-labelling of fragmented DNA in three temperate species carp (Cyprinus carpio), sea bass (Dicentrarchus labrax) and dusky grouper (Epinephelus marginatus) and in the adult thymus of three Antarctic species belonging to the genus Trematomus spp. During thymus development some few isolated apoptotic cell (AC) firstly appeared in the central-external part of the organ (carp: 5 days ph; sea bass: 35 days ph grouper: 43 days ph). Initially the cells were isolated and then increased in number and aggregated in small groups in the outer-cortical region of the thymus larvae. The high density of apoptotic cells was observed in the junction between cortex and medulla from its appearance (border between cortex and medulla, BCM). ACs decreased in number in juveniles and adult as well as the ACs average diameter. In late juveniles and in adulthood, the apoptosis were restricted to the cortex. In Antarctic species the thymus is highly adapted to low temperature (high vascularisation to effort the circulation of glycoproteins enriched plasma and strongly compact parenchyma). The apoptosis process was more extended (4-7 fold) as compare with the thymus of temperate species, even if the distribution of ACs was similar in all examined species. Data suggested a common process of T lymphocyte negative-selection in BCM of thymus during the ontogeny. The selection process seems to be still active in adult polar fish, but restricted mainly in the cortex zone.