Publications by authors named "Marcello Ceci"

30 Publications

  • Page 1 of 1

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

Biochim Biophys Acta Mol Basis Dis 2021 Apr 28;1867(4):166046. Epub 2020 Dec 28.

Department of Ecological and Biological Sciences, University of Tuscia, Viterbo, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbadis.2020.166046DOI Listing
April 2021

Defects of full-length dystrophin trigger retinal neuron damage and synapse alterations by disrupting functional autophagy.

Cell Mol Life Sci 2021 Feb 4;78(4):1615-1636. Epub 2020 Aug 4.

Department for Innovation in Biological, Agro-Food and Forest Systems (DIBAF), Università degli Studi della Tuscia, largo dell'Università snc, 01100, Viterbo, Italy.

Dystrophin (dys) mutations predispose Duchenne muscular disease (DMD) patients to brain and retinal complications. Although different dys variants, including long dys products, are expressed in the retina, their function is largely unknown. We investigated the putative role of full-length dystrophin in the homeostasis of neuro-retina and its impact on synapsis stabilization and cell fate. Retinas of mdx mice, the most used DMD model which does not express the 427-KDa dys protein (Dp427), showed overlapped cell death and impaired autophagy. Apoptotic neurons in the outer plexiform/inner nuclear layer and the ganglion cell layer had an impaired autophagy with accumulated autophagosomes. The autophagy dysfunction localized at photoreceptor axonal terminals and bipolar, amacrine, and ganglion cells. The absence of Dp427 does not cause a severe phenotype but alters the neuronal architecture, compromising mainly the pre-synaptic photoreceptor terminals and their post-synaptic sites. The analysis of two dystrophic mutants of the fruit fly Drosophila melanogaster, the homozygous Dys and Dys, lacking functional large-isoforms of dystrophin-like protein, revealed rhabdomere degeneration. Structural damages were evident in the internal network of retina/lamina where photoreceptors make the first synapse. Both accumulated autophagosomes and apoptotic features were detected and the visual system was functionally impaired. The reactivation of the autophagosome turnover by rapamycin prevented neuronal cell death and structural changes of mutant flies and, of interest, sustained autophagy ameliorated their response to light. Overall, these findings indicate that functional full-length dystrophin is required for synapsis stabilization and neuronal survival of the retina, allowing also proper autophagy as a prerequisite for physiological cell fate and visual properties.
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http://dx.doi.org/10.1007/s00018-020-03598-5DOI Listing
February 2021

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

Biochim Biophys Acta Mol Basis Dis 2020 11 15;1866(11):165896. Epub 2020 Jul 15.

Dept of Ecology & Biology Sciences, University of Tuscia, Viterbo, Italy.

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.
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http://dx.doi.org/10.1016/j.bbadis.2020.165896DOI Listing
November 2020

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

Cell Signal 2020 06 29;70:109591. Epub 2020 Feb 29.

Department of Ecological and Biological Science (DEB), University of Tuscia, Largo dell'Università, 01100 Viterbo, Italy. Electronic address:

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 HO 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.
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http://dx.doi.org/10.1016/j.cellsig.2020.109591DOI Listing
June 2020

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

Cell Signal 2019 01 1;53:102-110. Epub 2018 Oct 1.

Department of Ecological and Biological Sciences (DEB), University of Tuscia, Viterbo, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cellsig.2018.09.020DOI Listing
January 2019

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

Aquat Toxicol 2018 Nov 22;204:144-159. Epub 2018 Sep 22.

Department Ecological and Biological Sciences, University of Tuscia, Tuscia University, Viterbo, 01100, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.aquatox.2018.09.010DOI Listing
November 2018

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

Rev Neurosci 2018 12;30(1):45-66

Department of Ecological and Biological Sciences, University of Tuscia, largo dell'Università, I-01100 Viterbo, Italy.

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.
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http://dx.doi.org/10.1515/revneuro-2018-0020DOI Listing
December 2018

The face of epicardial and endocardial derived cells in zebrafish.

Exp Cell Res 2018 08 25;369(1):166-175. Epub 2018 May 25.

Department of Ecological and Biological Sciences, University of Tuscia, Viterbo, Italy.

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.
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http://dx.doi.org/10.1016/j.yexcr.2018.05.022DOI Listing
August 2018

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

Cell Death Discov 2018 12;4:41. Epub 2018 Mar 12.

1Department of Ecological and Biological Sciences, University of Tuscia, Viterbo, Italy.

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.
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http://dx.doi.org/10.1038/s41420-018-0041-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849881PMC
March 2018

Upregulation of eIF6 inhibits cardiac hypertrophy induced by phenylephrine.

Biochem Biophys Res Commun 2018 01 8;495(1):601-606. Epub 2017 Nov 8.

Department of Ecology and Biology (DEB), Tuscia University, 01100 Viterbo, Italy. Electronic address:

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/p27 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 eIF2β.
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http://dx.doi.org/10.1016/j.bbrc.2017.11.046DOI Listing
January 2018

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

Hum Mol Genet 2017 04;26(8):1407-1418

Department of Ecology and Biology, Tuscia University, Viterbo 01100, Italy.

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.
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http://dx.doi.org/10.1093/hmg/ddx035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6075552PMC
April 2017

Neutralization of nerve growth factor impairs proliferation and differentiation of adult neural progenitors in the subventricular zone.

Stem Cells 2014 Sep;32(9):2516-28

Institute of Translational Pharmacology, National Research Council, Rome, Italy; European Brain Research Institute (EBRI), Rome, Italy.

Adult neurogenesis is a multistep process regulated by several extrinsic factors, including neurotrophins. Among them, little is known about the role of nerve growth factor (NGF) in the neurogenic niches of the mouse. Here we analyzed the biology of adult neural stem cells (NSCs) from the subventricular zone (SVZ) of AD11 anti-NGF transgenic mice, in which the expression of the recombinant antibody aD11 leads to a chronic postnatal neutralization of endogenous NGF. We showed that AD11-NSCs proliferate 10-fold less, with respect to their control counterparts, and display a significant impairment in their ability to differentiate into β-tubulin positive neurons. We found a considerable reduction in the number of SVZ progenitors and neuroblasts also in vivo, which correlates with a lower number of newborn neurons in the olfactory bulbs of AD11 mice and a severe deficit in the ability of these mice to discriminate between different odors. We also demonstrated that, in AD11 mice, the morphology of both SVZ-resident and neurosphere-derived astrocytes is significantly altered. We were able to reproduce the AD11 phenotype in vitro, by acutely treating wild type NSCs with the anti-NGF antibody, further demonstrating that both the proliferation and the differentiation defects are due to the NGF deprivation. Consistently, the proliferative impairment of AD11 progenitors, as well as the atrophic morphology of AD11 astrocytes, can be partly rescued in vitro and in vivo by exogenous NGF addition. Altogether, our results demonstrate a causal link between NGF signaling and proper proliferation and differentiation of neural stem cells from the SVZ.
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http://dx.doi.org/10.1002/stem.1744DOI Listing
September 2014

Intranasal "painless" human Nerve Growth Factor [corrected] slows amyloid neurodegeneration and prevents memory deficits in App X PS1 mice.

PLoS One 2012 30;7(5):e37555. Epub 2012 May 30.

European Brain Research Institute, Rome, Italy.

Nerve Growth Factor (NGF) is being considered as a therapeutic candidate for Alzheimer's disease (AD) treatment but the clinical application is hindered by its potent pro-nociceptive activity. Thus, to reduce systemic exposure that would induce pain, in recent clinical studies NGF was administered through an invasive intracerebral gene-therapy approach. Our group demonstrated the feasibility of a non-invasive intranasal delivery of NGF in a mouse model of neurodegeneration. NGF therapeutic window could be further increased if its nociceptive effects could be avoided altogether. In this study we exploit forms of NGF, mutated at residue R100, inspired by the human genetic disease HSAN V (Hereditary Sensory Autonomic Neuropathy Type V), which would allow increasing the dose of NGF without triggering pain. We show that "painless" hNGF displays full neurotrophic and anti-amyloidogenic activities in neuronal cultures, and a reduced nociceptive activity in vivo. When administered intranasally to APPxPS1 mice ( n = 8), hNGFP61S/R100E prevents the progress of neurodegeneration and of behavioral deficits. These results demonstrate the in vivo neuroprotective and anti-amyloidogenic properties of hNGFR100 mutants and provide a rational basis for the development of "painless" hNGF variants as a new generation of therapeutics for neurodegenerative diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037555PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3364340PMC
October 2012

RACK1 is a ribosome scaffold protein for β-actin mRNA/ZBP1 complex.

PLoS One 2012 16;7(4):e35034. Epub 2012 Apr 16.

European Brain Research Institute (EBRI), Rome, Italy.

In neurons, specific mRNAs are transported in a translationally repressed manner along dendrites or axons by transport ribonucleic-protein complexes called RNA granules. ZBP1 is one RNA binding protein present in transport RNPs, where it transports and represses the translation of cotransported mRNAs, including β-actin mRNA. The release of β-actin mRNA from ZBP1 and its subsequent translation depends on the phosphorylation of ZBP1 by Src kinase, but little is known about how this process is regulated. Here we demonstrate that the ribosomal-associated protein RACK1, another substrate of Src, binds the β-actin mRNA/ZBP1 complex on ribosomes and contributes to the release of β-actin mRNA from ZBP1 and to its translation. We identify the Src binding and phosphorylation site Y246 on RACK1 as the critical site for the binding to the β-actin mRNA/ZBP1 complex. Based on these results we propose RACK1 as a ribosomal scaffold protein for specific mRNA-RBP complexes to tightly regulate the translation of specific mRNAs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035034PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3327689PMC
August 2012

Taking pain out of NGF: a "painless" NGF mutant, linked to hereditary sensory autonomic neuropathy type V, with full neurotrophic activity.

PLoS One 2011 Feb 28;6(2):e17321. Epub 2011 Feb 28.

European Brain Research Institute, Rome, Italy.

During adulthood, the neurotrophin Nerve Growth Factor (NGF) sensitizes nociceptors, thereby increasing the response to noxious stimuli. The relationship between NGF and pain is supported by genetic evidence: mutations in the NGF TrkA receptor in patients affected by an hereditary rare disease (Hereditary Sensory and Autonomic Neuropathy type IV, HSAN IV) determine a congenital form of severe pain insensitivity, with mental retardation, while a mutation in NGFB gene, leading to the aminoacid substitution R100W in mature NGF, determines a similar loss of pain perception, without overt cognitive neurological defects (HSAN V). The R100W mutation provokes a reduced processing of proNGF to mature NGF in cultured cells and a higher percentage of neurotrophin secreted is in the proNGF form. Moreover, using Surface Plasmon Resonance we showed that the R100W mutation does not affect NGF binding to TrkA, while it abolishes NGF binding to p75NTR receptors. However, it remains to be clarified whether the major impact of the mutation is on the biological function of proNGF or of mature NGF and to what extent the effects of the R100W mutation on the HSAN V clinical phenotype are developmental, or whether they reflect an impaired effectiveness of NGF to regulate and mediate nociceptive transmission in adult sensory neurons. Here we show that the R100 mutation selectively alters some of the signaling pathways activated downstream of TrkA NGF receptors. NGFR100 mutants maintain identical neurotrophic and neuroprotective properties in a variety of cell assays, while displaying a significantly reduced pain-inducing activity in vivo (n = 8-10 mice/group). We also show that proNGF has a significantly reduced nociceptive activity, with respect to NGF. Both sets of results jointly contribute to elucidating the mechanisms underlying the clinical HSAN V manifestations, and to clarifying which receptors and intracellular signaling cascades participate in the pain sensitizing action of NGF.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017321PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3046150PMC
February 2011

MTORC1 regulates cardiac function and myocyte survival through 4E-BP1 inhibition in mice.

J Clin Invest 2010 Aug 19;120(8):2805-16. Epub 2010 Jul 19.

Department of Medicine, University of California San Diego, La Jolla, California 92093-0613, USA.

Mechanistic target of rapamycin (MTOR) plays a critical role in the regulation of cell growth and in the response to energy state changes. Drugs inhibiting MTOR are increasingly used in antineoplastic therapies. Myocardial MTOR activity changes during hypertrophy and heart failure (HF). However, whether MTOR exerts a positive or a negative effect on myocardial function remains to be fully elucidated. Here, we show that ablation of Mtor in the adult mouse myocardium results in a fatal, dilated cardiomyopathy that is characterized by apoptosis, autophagy, altered mitochondrial structure, and accumulation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). 4E-BP1 is an MTOR-containing multiprotein complex-1 (MTORC1) substrate that inhibits translation initiation. When subjected to pressure overload, Mtor-ablated mice demonstrated an impaired hypertrophic response and accelerated HF progression. When the gene encoding 4E-BP1 was ablated together with Mtor, marked improvements were observed in apoptosis, heart function, and survival. Our results demonstrate a role for the MTORC1 signaling network in the myocardial response to stress. In particular, they highlight the role of 4E-BP1 in regulating cardiomyocyte viability and in HF. Because the effects of reduced MTOR activity were mediated through increased 4E-BP1 inhibitory activity, blunting this mechanism may represent a novel therapeutic strategy for improving cardiac function in clinical HF.
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http://dx.doi.org/10.1172/JCI43008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2912201PMC
August 2010

In vitro receptor binding properties of a "painless" NGF mutein, linked to hereditary sensory autonomic neuropathy type V.

Biochem Biophys Res Commun 2010 Jan 27;391(1):824-9. Epub 2009 Nov 27.

Lay Line Genomics S.p.A., Via Prospero Colonna 32, 00149 Rome, Italy.

Nerve Growth Factor (NGF) signalling is mediated by the TrkA and p75NTR receptors. Besides its neurotrophic and survival activities, NGF displays a potent pro-nociceptive activity. Recently, a missense point mutation was found in the NGFB gene (C661T, leading to the aminoacid substitution R100W) of individuals affected by a form of hereditary loss of pain perception (hereditary sensory and autonomic neuropathy type V, HSAN V). In order to gain insights into the functional consequences of the HSAN V NGF mutation, two sets of hNGFR100 mutants were expressed in Escherichia coli and purified, as mature NGF or proNGF, for in vitro receptor binding studies. Here, we show by Surface Plasmon Resonance analysis that the R100 mutation selectively disrupts binding of hNGF to p75NTR receptor, to an extent which depends on the substituting residue at position 100, while the affinity of hNGFR100 mutants for TrkA receptor is not affected. As for unprocessed hproNGF, the binding of the R100 variants to p75NTR receptor shows only a limited impairment, showing that the impact of the R100 mutation on p75NTR receptor binding is greater in the context of mature, processed hNGF. These results provide a basis for elucidating the mechanisms underlying the clinical manifestations of HSAN V patients, and provide a basis for the development of "painless" hNGF molecules with therapeutic potential.
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http://dx.doi.org/10.1016/j.bbrc.2009.11.146DOI Listing
January 2010

Akt increases sarcoplasmic reticulum Ca2+ cycling by direct phosphorylation of phospholamban at Thr17.

J Biol Chem 2009 Oct 19;284(41):28180-7. Epub 2009 Aug 19.

Istituto di Ricovero e Cura a Carattere Scientifico Multimedica, Milan 20138, Italy.

Cardiomyocytes adapt to physical stress by increasing their size while maintaining cell function. The serine/threonine kinase Akt plays a critical role in this process of adaptation. We previously reported that transgenic overexpression of an active form of Akt (Akt-E40K) in mice results in increased cardiac contractility and cell size, as well as improved sarcoplasmic reticulum (SR) Ca(2+) handling. Because it is not fully elucidated, we decided to study the molecular mechanism by which Akt-E40K overexpression improves SR Ca(2+) handling. To this end, SR Ca(2+) uptake and the phosphorylation status of phospholamban (PLN) were evaluated in heart extracts from wild-type and Akt-E40K mice and mice harboring inducible and cardiac specific knock-out of phosphatidylinositol-dependent kinase-1, the upstream activator of Akt. Moreover, the effect of Akt was assessed in vitro by overexpressing a mutant Akt targeted preferentially to the SR, and by biochemical assays to evaluate potential interaction with PLN. We found that when activated, Akt interacts with and phosphorylates PLN at Thr(17), the Ca(2+)-calmodulin-dependent kinase IIdelta site, whereas silencing Akt signaling, through the knock-out of phosphatidylinositol-dependent kinase-1, resulted in reduced phosphorylation of PLN at Thr(17). Furthermore, overexpression of SR-targeted Akt in cardiomyocytes improved Ca(2+) handling without affecting cell size. Thus, we describe here a new mechanism whereby the preferential translocation of Akt to the SR is responsible for enhancement of contractility without stimulation of hypertrophy.
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http://dx.doi.org/10.1074/jbc.M109.036566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2788869PMC
October 2009

Interval training normalizes cardiomyocyte function, diastolic Ca2+ control, and SR Ca2+ release synchronicity in a mouse model of diabetic cardiomyopathy.

Circ Res 2009 Sep 13;105(6):527-36. Epub 2009 Aug 13.

Norwegian University of Science and Technology, Department of Circulation and Medical Imaging, Olav Kyrres gt. 9, 7489 Trondheim, Norway.

Rationale: In the present study we explored the mechanisms behind excitation-contraction (EC) coupling defects in cardiomyocytes from mice with type-2 diabetes (db/db).

Objective: We determined whether 13 weeks of aerobic interval training could restore cardiomyocyte Ca(2+) cycling and EC coupling.

Methods And Results: Reduced contractility in cardiomyocytes isolated from sedentary db/db was associated with increased diastolic sarcoplasmic reticulum (SR)-Ca(2+) leak, reduced synchrony of Ca(2+) release, reduced transverse (T)-tubule density, and lower peak systolic and diastolic Ca(2+) and caffeine-induced Ca(2+) release. Additionally, the rate of SR Ca(2+) ATPase-mediated Ca(2+) uptake during diastole was reduced, whereas a faster recovery from caffeine-induced Ca(2+) release indicated increased Na(+)/Ca(2+)-exchanger activity. The increased SR-Ca(2+) leak was attributed to increased Ca(2+)-calmodulin-dependent protein kinase (CaMKIIdelta) phosphorylation, supported by the normalization of SR-Ca(2+) leak on inhibition of CaMKIIdelta (AIP). Exercise training restored contractile function associated with restored SR Ca(2+) release synchronicity, T-tubule density, twitch Ca(2+) amplitude, SR Ca(2+) ATPase and Na(+)/Ca(2+)-exchanger activities, and SR-Ca(2+) leak. The latter was associated with reduced phosphorylation of cytosolic CaMKIIdelta. Despite normal contractile function and Ca(2+) handling after the training period, phospholamban was hyperphosphorylated at Serine-16. Protein kinase A inhibition (H-89) in cardiomyocytes from the exercised db/db group abolished the differences in SR-Ca(2+) load when compared with the sedentary db/db mice. EC coupling changes were observed without changes in serum insulin or glucose levels, suggesting that the exercise training-induced effects are not via normalization of the diabetic condition.

Conclusions: These data demonstrate that aerobic interval training almost completely restored the contractile function of the diabetic cardiomyocyte to levels close to sedentary wild type.
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http://dx.doi.org/10.1161/CIRCRESAHA.109.199810DOI Listing
September 2009

Carbon monoxide levels experienced by heavy smokers impair aerobic capacity and cardiac contractility and induce pathological hypertrophy.

Inhal Toxicol 2008 May;20(7):635-46

Department of Circulation and Medical Imaging, Faculty of Medicine, The Norwegian University of Science and Technology (NTNU), Trondheim, Norway.

Cigarette smoke contains hundreds of potentially toxic compounds and is an important risk factor for cardiovascular disease. However, the key components responsible for endothelial and myocardial dysfunction have not been fully identified. The objective of the present study was to determine the cardiovascular effects of long-term inhalation of carbon monoxide (CO) administrated to give concentrations in the blood similar to those observed in heavy smokers. Female rats were exposed to either CO or air (control group) (n = 12). The CO group was exposed to 200 ppm CO (100 h/wk) for 18 mo. Rats exposed to CO had 24% lower maximal oxygen uptake, longer (145 vs. 123 microm) and wider (47 vs. 25 microm) cardiomyocytes, reduced cardiomyocyte fractional shortening (12 vs. 7%), and 26% longer time to 50% re-lengthening than controls. In addition, cardiomyocytes from CO-exposed rats had 48% lower intracellular calcium (Ca2 +) amplitude, 22% longer time to Ca2 + decay, 34% lower capacity of sarcoplasmic reticulum Ca2 +-ATPase (SERCA2a), and 37% less t-tubule area compared to controls. Phosphorylation levels of phospholamban at Ser16 and Thr17 were significantly reduced in the CO group, whereas total concentration of phospholamban and SERCA2a were unchanged. Cardiac atrial natriuretic peptide, vascular endothelial growth factor, cyclic guanosine monophosphate, calcineurin, calmodulin, pERK, and pS6 increased, whereas pAkt and pCaMKII delta remained unchanged by CO. Endothelial function and systemic blood pressure were not affected by CO exposure. Long-term CO exposure reduces aerobe capacity and contractile function and leads to pathological hypertrophy. Impaired Ca2 + handling and increased growth factor signaling seem to be responsible for these pathological changes.
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http://dx.doi.org/10.1080/08958370701883821DOI Listing
May 2008

Myocardial sarcoplasmic reticulum Ca2+ ATPase function is increased by aerobic interval training.

Eur J Cardiovasc Prev Rehabil 2008 Apr;15(2):145-8

Institute of Biomedical and Life Sciences, University of Glasgow, UK.

Objective: Reduced activity of the sarcoplasmic reticulum Ca ATPase-2a (SERCA-2a) contributes to myocardial dysfunction. Exercise training improves myocardial Ca-handling, but SERCA-2a function is uncertain. We assessed SERCA-2a activity after exercise training.

Methods: SERCA-2a function was assessed by sarcoplasmic reticulum Ca uptake in cardiomyocytes with other Ca uptake mechanisms blocked, in mice after aerobic interval training versus sedentary controls.

Results: We established protocols to assess SERCA-2a function, and show that aerobic interval training increases the maximal rate of Ca uptake by 30%. This is at least partly explained by reduced phospholamban-to-SERCA-2a ratio.

Conclusion: Aerobic interval training improves myocardial SERCA-2a performance, explaining at least partly why myocardial Ca-handling improves after exercise training.
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http://dx.doi.org/10.1097/HJR.0b013e3282efd4e0DOI Listing
April 2008

Activation or inactivation of cardiac Akt/mTOR signaling diverges physiological from pathological hypertrophy.

J Cell Physiol 2008 Feb;214(2):316-21

Institute of Biomedical and Life Sciences, University of Glasgow, UK.

Cardiomyocyte hypertrophy differs according to the stress exerted on the myocardium. While pressure overload-induced cardiomyocyte hypertrophy is associated with depressed contractile function, physiological hypertrophy after exercise training associates with preserved or increased inotropy. We determined the activation state of myocardial Akt signaling with downstream substrates and fetal gene reactivation in exercise-induced physiological and pressure overload-induced pathological hypertrophies. C57BL/6J mice were either treadmill trained for 6 weeks, 5 days/week, at 85-90% of maximal oxygen uptake (VO(2max)), or underwent transverse aortic constriction (TAC) for 1 or 8 weeks. Total and phosphorylated protein levels were determined with SDS-PAGE, and fetal genes by real-time RT-PCR. In the physiologically hypertrophied heart after exercise training, total Akt protein level was unchanged, but Akt was chronically hyperphosphorylated at serine 473. This was accompanied by activation of the mammalian target of rapamycin (mTOR), measured as phosphorylation of its two substrates: the ribosomal protein S6 kinase-1 (S6K1) and the eukaryotic translation initiation factor-4E binding protein-1 (4E-BP1). Exercise training did not reactivate the fetal gene program (beta-myosin heavy chain, atrial natriuretic factor, skeletal muscle actin). In contrast, pressure overload after TAC reactivated fetal genes already after 1 week, and partially inactivated the Akt/mTOR pathway and downstream substrates after 8 weeks. In conclusion, changes in opposite directions of the myocardial Akt/mTOR signal pathway appears to distinguish between physiological and pathological hypertrophies; exercise training associating with activation and pressure overload associating with inactivation of the Akt/mTOR pathway.
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http://dx.doi.org/10.1002/jcp.21197DOI Listing
February 2008

Aerobic interval training enhances cardiomyocyte contractility and Ca2+ cycling by phosphorylation of CaMKII and Thr-17 of phospholamban.

J Mol Cell Cardiol 2007 Sep 10;43(3):354-61. Epub 2007 Jul 10.

Department of Circulation and Medical Imaging, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway.

Cardiac adaptation to aerobic exercise training includes improved cardiomyocyte contractility and calcium handling. Our objective was to determine whether cytosolic calcium/calmodulin-dependent kinase II and its downstream targets are modulated by exercise training. A six-week aerobic interval training program by treadmill running increased maximal oxygen uptake by 35% in adult mice, whereupon left ventricular cardiomyocyte function was studied and myocardial tissue samples were used for biochemical analysis. Cardiomyocytes from trained mice had enhanced contractility and faster relaxation rates, which coincided with larger amplitude and faster decay of the calcium transient, but not increased peak systolic calcium levels. These changes were associated with reduced phospholamban expression relative to sarcoplasmic reticulum calcium ATPase and constitutively increased phosphorylation of phospholamban at the threonine 17, but not at the serine 16 site. Calcium/calmodulin-dependent kinase IIdelta phosphorylation was increased at threonine 287, indicating activation. To investigate the physiological role of calcium/calmodulin-dependent kinase IIdelta phosphorylation, this kinase was blocked specifically by autocamtide-2 related inhibitory peptide II. This maneuver completely abolished training-induced improvements of cardiomyocyte contractility and calcium handling and blunted, but did not completely abolish the training-induced increase in Ca(2+) sensitivity. Also, inhibition of calcium/calmodulin-dependent kinase II reduced the greater frequency-dependent acceleration of relaxation that was observed after aerobic interval training. These observations indicate that calcium/calmodulin-dependent kinase IIdelta contributes significantly to the functional adaptation of the cardiomyocyte to regular exercise training.
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http://dx.doi.org/10.1016/j.yjmcc.2007.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2995493PMC
September 2007

Increased phospholamban phosphorylation limits the force-frequency response in the MLP-/- mouse with heart failure.

J Mol Cell Cardiol 2006 Mar 20;40(3):350-60. Epub 2006 Jan 20.

Laboratory of Experimental Cardiology, University of Leuven, Campus Gasthuisberg O/N 704, Herestraat 49, B-3000 Leuven, Belgium.

Reduced Ca(2+) release from the sarcoplasmic reticulum (SR) and a negative force-frequency relation characterize end-stage human heart failure. The MLP(-/-) mouse with dilated cardiomyopathy is used as a model to explore novel therapeutic interventions but the alterations in Ca(2+) handling in MLP(-/-) remain incompletely understood. We studied [Ca(2+)](i) in left ventricular myocytes from MLP(-/-) and WT mice (3-4 months old; whole-cell voltage clamp, 30 degrees C). At 1 Hz stimulation, the amplitude of [Ca(2+)](i) transients was similar. However, in contrast to WT, at higher frequencies the [Ca(2+)](i) transient amplitude declined in MLP(-/-) and there was no increase in SR Ca(2+) content. Unexpectedly, the decline of [Ca(2+)](i) was faster in MLP(-/-) than in WT (at 1 Hz, tau of 80 +/- 9 vs. 174 +/- 29 ms, P < 0.001) and the frequency-dependent acceleration of the decline was abolished suggesting an enhanced basal SERCA activity. Indeed, the Ca(2+) affinity of SR Ca(2+) uptake in homogenates was higher in MLP(-/-), with the maximal uptake rate similar to WT. Phosphorylation of phospholamban in MLP(-/-) was increased (2.3-fold at Ser(16) and 2.9-fold at the Thr(17) site, P < 0.001) with similar SERCA and total phospholamban protein levels. On increasing stimulation frequency to 4 Hz, WT, but not MLP(-/-), myocytes had a net gain of Ca(2+), suggesting inadequate Ca(2+) sequestration in MLP(-/-). In conclusion, increased baseline phosphorylation of phospholamban in MLP(-/-) leads to a reduced reserve for frequency-dependent increase of Ca(2+) release. This represents a novel paradigm for altered Ca(2+) handling in heart failure, underscoring the importance of phosphorylation pathways.
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http://dx.doi.org/10.1016/j.yjmcc.2005.12.002DOI Listing
March 2006

Sen34p depletion blocks tRNA splicing in vivo and delays rRNA processing.

Biochem Biophys Res Commun 2005 Nov;337(1):89-94

Molecular Histology and Cell Growth, DIBIT-HSR, 20132 Milan, Italy.

Tif6p (eIF6) is necessary for 60S biogenesis, rRNA maturation and must be released from 60S to permit 80S assembly and translation. We characterized Tif6p interactors. Tif6p is mostly on 66S-60S pre-ribosomes, partly free. Tif6p complex(es) contain nucleo-ribosomal factors and Asc1p. Surprisingly, Tif6p particle contains the low-abundance endonuclease Sen34p. We analyzed Sen34p role on rRNA/tRNA synthesis, in vivo. Sen34p depletion impairs tRNA splicing and causes unexpected 80S accumulation. Accordingly, Sen34p overexpression causes 80S decrease and increased polysomes which suggest increased translational efficiency. With delayed kinetics, Sen34p depletion impairs rRNA processing. We conclude that Sen34p is absolutely required for tRNA splicing and that it is a rate-limiting element for efficient translation. Finally, we confirm that Tif6p accompanies 27S pre-rRNA maturation to 25S rRNA and we suggest that Sen34p endonuclease in Tif6p complex may affect also rRNA maturation.
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http://dx.doi.org/10.1016/j.bbrc.2005.09.012DOI Listing
November 2005

Molecular determinants of the physiological adaptation to stress in the cardiomyocyte: a focus on AKT.

J Mol Cell Cardiol 2004 Nov;37(5):905-12

San Raffaele Biomedical Science Park, Rome 00128, Italy.

Cardiomyocytes (CMCs) adapt to physiological or pathological stimuli by undergoing molecular changes which differentiate according to the specificity of the stimulus and eventually generate a phenotype with peculiar molecular characteristics. Here, we review the literature on the molecular mechanisms activated in the CMC during physiologic adaptation to stress, as opposed to maladaptation. The critical role of the IGF-1 receptor/PI3K/Akt signaling pathway during this process is described, including effector targets regulating inotropism and cell size.
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http://dx.doi.org/10.1016/j.yjmcc.2004.06.020DOI Listing
November 2004

Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly.

Nature 2003 Dec;426(6966):579-84

Molecular Histology Unit, DIBIT-HSR, 20132 Milano, Italy.

The assembly of 80S ribosomes requires joining of the 40S and 60S subunits, which is triggered by the formation of an initiation complex on the 40S subunit. This event is rate-limiting for translation, and depends on external stimuli and the status of the cell. Here we show that 60S subunits are activated by release of eIF6 (also termed p27BBP). In the cytoplasm, eIF6 is bound to free 60S but not to 80S. Furthermore, eIF6 interacts in the cytoplasm with RACK1, a receptor for activated protein kinase C (PKC). RACK1 is a major component of translating ribosomes, which harbour significant amounts of PKC. Loading 60S subunits with eIF6 caused a dose-dependent translational block and impairment of 80S formation, which were reversed by expression of RACK1 and stimulation of PKC in vivo and in vitro. PKC stimulation led to eIF6 phosphorylation, and mutation of a serine residue in the carboxy terminus of eIF6 impaired RACK1/PKC-mediated translational rescue. We propose that eIF6 release regulates subunit joining, and that RACK1 provides a physical and functional link between PKC signalling and ribosome activation.
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http://dx.doi.org/10.1038/nature02160DOI Listing
December 2003

Formation of nuclear matrix filaments by p27(BBP)/eIF6.

Biochem Biophys Res Commun 2002 Jul;295(2):295-9

University Vita-Salute San Raffaele School of Medicine, Milan, Italy.

p27(BBP)/eIF6 is an evolutionarily conserved protein necessary for ribosome biogenesis which was cloned in mammals for its ability to bind the cytodomain of beta 4 integrin. In cultured cells, a conspicuous fraction of p27(BBP)/eIF6 is associated with the intermediate filaments/nuclear matrix (IF/NM) cytoskeleton. The mechanism of this association is not known. Here we show that in epidermis p27(BBP)/eIF6 is naturally associated with IF/NM. To analyze the intrinsic capability of p27(BBP)/eIF6 to generate cytoskeletal networks, the properties of the pure, recombinant, untagged protein were studied. Recombinant p27(BBP)/eIF6 binds beta 4 integrin. Upon dialysis against IF buffer, p27(BBP)/eIF6 forms polymers which, strikingly, have a morphology identical to NM filaments. Cross-linking experiments suggested that polymerization is favored by the formation of disulphide bridges. These data suggest that p27(BBP)/eIF6 is associated with the cytoskeleton, and contributes to formation of NM filaments. These findings help to settle the controversy on nuclear matrix.
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http://dx.doi.org/10.1016/s0006-291x(02)00671-xDOI Listing
July 2002