Publications by authors named "Gabriella Dobrowolny"

30 Publications

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A longitudinal study defined circulating microRNAs as reliable biomarkers for disease prognosis and progression in ALS human patients.

Cell Death Discov 2021 Jan 11;7(1). Epub 2021 Jan 11.

DAHFMO-Unit of Histology and Medical Embryology, Sapienza University of Rome, Laboratory Affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Via A. Scarpa 14, 00161, Rome, Italy.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease associated with motor neuron degeneration, muscle atrophy and paralysis. To date, multiple panels of biomarkers have been described in ALS patients and murine models. Nevertheless, none of them has sufficient specificity and thus the molecular signature for ALS prognosis and progression remains to be elucidated. Here we overcome this limitation through a longitudinal study, analyzing serum levels of circulating miRNAs, stable molecules that are recently used as promising biomarkers for many types of human disorders, in ALS patients during the progression of the pathology. We performed next-generation sequencing (NGS) analysis and absolute RT quantification of serum samples of ALS patients and healthy controls. The expression levels of five selected miRNAs were quantitatively analyzed during disease progression in each patient and we demonstrated that high levels of miR-206, miR-133a and miR-151a-5p can predict a slower clinical decline of patient functionality. In particular, we found that miR-206 and miR-151a-5p serum levels were significantly up-regulated at the mild stage of ALS pathology, to decrease in the following moderate and severe stages, whereas the expression levels of miR-133a and miR-199a-5p remained low throughout the course of the disease, showing a diagnostic significance in moderate and severe stages for miR-133a and in mild and terminal ones for miR-199a-5p. Moreover, we found that miR-423-3p and 151a-5p were significantly downregulated respectively in mild and terminal stages of the disease. These data suggest that these miRNAs represent potential prognostic markers for ALS disease.
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http://dx.doi.org/10.1038/s41420-020-00397-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7801652PMC
January 2021

Taurine Attenuates Catabolic Processes Related to the Onset of Sarcopenia.

Int J Mol Sci 2020 Nov 23;21(22). Epub 2020 Nov 23.

Sezione di Istologia ed Embriologia, Dipartimento di Scienze della Vita e Sanità Pubblica, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Roma, Italy.

Sarcopenia that occurs with advancing age is characterized by a gradual loss of muscle protein component due to the activation of catabolic pathways, increased level of inflammation, and mitochondrial dysfunction. Experimental evidence demonstrates that several physio-pathological processes involved in the onset of sarcopenia may be counteracted by the intake of specific amino acids or antioxidant molecules, suggesting that diet may represent an effective strategy for improving the anabolic response of muscle during aging. The non-essential amino acid taurine is highly expressed in several mammalian tissues, including skeletal muscle where it is involved in the ion channel regulation, in the modulation of intracellular calcium concentration, and where it plays an important role as an antioxidant and anti-inflammatory factor. Here, with the purpose to reproduce the chronic low-grade inflammation characteristics of senescent muscle in an in vitro system, we exploited the role of Tumor Necrosis Factor α (TNF) and we analyzed the effect of taurine in the modulation of different signaling pathways known to be dysregulated in sarcopenia. We demonstrated that the administration of high levels of taurine in myogenic L6 cells stimulates the differentiation process by downregulating the expression of molecules involved in inflammatory pathways and modulating processes such as autophagy and apoptosis. Although further studies are currently ongoing in our laboratory to better elucidate the molecular mechanisms responsible for the positive effect of taurine on myogenic differentiation, this study suggests that taurine supplementation may represent a strategy to delay the loss of mass and functionality characteristic of senescent muscles.
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http://dx.doi.org/10.3390/ijms21228865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7700215PMC
November 2020

Neuromuscular Junction as an Entity of Nerve-Muscle Communication.

Cells 2019 08 16;8(8). Epub 2019 Aug 16.

Laboratory affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, DAHFMO-Unit of Histology and Medical Embryology, Sapienza University of Rome, Via A. Scarpa, 14, 00161 Rome, Italy.

One of the crucial systems severely affected in several neuromuscular diseases is the loss of effective connection between muscle and nerve, leading to a pathological non-communication between the two tissues. The neuromuscular junction (NMJ) represents the critical region at the level of which muscle and nerve communicate. Defects in signal transmission between terminal nerve endings and muscle membrane is a common feature of several physio-pathologic conditions including aging and Amyotrophic Lateral Sclerosis (ALS). Nevertheless, controversy exists on whether pathological events beginning at the NMJ precede or follow loss of motor units. In this review, the role of NMJ in the physio-pathologic interplay between muscle and nerve is discussed.
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http://dx.doi.org/10.3390/cells8080906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721719PMC
August 2019

Effects of IGF-1 isoforms on muscle growth and sarcopenia.

Aging Cell 2019 06 5;18(3):e12954. Epub 2019 Apr 5.

DAHFMO-Unit of Histology and Medical Embryology, Laboratory affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy.

The decline in skeletal muscle mass and strength occurring in aging, referred as sarcopenia, is the result of many factors including an imbalance between protein synthesis and degradation, changes in metabolic/hormonal status, and in circulating levels of inflammatory mediators. Thus, factors that increase muscle mass and promote anabolic pathways might be of therapeutic benefit to counteract sarcopenia. Among these, the insulin-like growth factor-1 (IGF-1) has been implicated in many anabolic pathways in skeletal muscle. IGF-1 exists in different isoforms that might exert different role in skeletal muscle. Here we study the effects of two full propeptides IGF-1Ea and IGF-1Eb in skeletal muscle, with the aim to define whether and through which mechanisms their overexpression impacts muscle aging. We report that only IGF-1Ea expression promotes a pronounced hypertrophic phenotype in young mice, which is maintained in aged mice. Nevertheless, examination of aged transgenic mice revealed that the local expression of either IGF-1Ea or IGF-1Eb transgenes was protective against age-related loss of muscle mass and force. At molecular level, both isoforms activate the autophagy/lysosome system, normally altered during aging, and increase PGC1-α expression, modulating mitochondrial function, ROS detoxification, and the basal inflammatory state occurring at old age. Moreover, morphological integrity of neuromuscular junctions was maintained and preserved in both MLC/IGF-1Ea and MLC/IGF-1Eb mice during aging. These data suggest that IGF-1 is a promising therapeutic agent in staving off advancing muscle weakness.
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http://dx.doi.org/10.1111/acel.12954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6516183PMC
June 2019

Elucidating the Contribution of Skeletal Muscle Ion Channels to Amyotrophic Lateral Sclerosis in search of new therapeutic options.

Sci Rep 2019 02 28;9(1):3185. Epub 2019 Feb 28.

Department of Pharmacy-Drug Sciences, University of Bari Aldo Moro, 70125, Bari, Italy.

The discovery of pathogenetic mechanisms is essential to identify new therapeutic approaches in Amyotrophic Lateral Sclerosis (ALS). Here we investigated the role of the most important ion channels in skeletal muscle of an ALS animal model (MLC/SOD1) carrying a mutated SOD1 exclusively in this tissue, avoiding motor-neuron involvement. Ion channels are fundamental proteins for muscle function, and also to sustain neuromuscular junction and nerve integrity. By a multivariate statistical analysis, using machine learning algorithms, we identified the discriminant genes in MLC/SOD1 mice. Surprisingly, the expression of ClC-1 chloride channel, present only in skeletal muscle, was reduced. Also, the expression of Protein Kinase-C, known to control ClC-1 activity, was increased, causing its inhibition. The functional characterization confirmed the reduction of ClC-1 activity, leading to hyperexcitability and impaired relaxation. The increased expression of ion channel coupled AMPA-receptor may contribute to sustained depolarization and functional impairment. Also, the decreased expression of irisin, a muscle-secreted peptide protecting brain function, may disturb muscle-nerve connection. Interestingly, the in-vitro application of chelerythrine or acetazolamide, restored ClC-1 activity and sarcolemma hyperexcitability in these mice. These findings show that ion channel function impairment in skeletal muscle may lead to motor-neuron increased vulnerability, and opens the possibility to investigate on new compounds as promising therapy.
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http://dx.doi.org/10.1038/s41598-019-39676-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395744PMC
February 2019

Neuromuscular magnetic stimulation counteracts muscle decline in ALS patients: results of a randomized, double-blind, controlled study.

Sci Rep 2019 02 26;9(1):2837. Epub 2019 Feb 26.

Rare Neuromuscular Diseases Centre, Department of Human Neuroscience, Sapienza University, Rome, Italy.

The aim of the study was to verify whether neuromuscular magnetic stimulation (NMMS) improves muscle function in spinal-onset amyotrophic lateral sclerosis (ALS) patients. Twenty-two ALS patients were randomized in two groups to receive, daily for two weeks, NMMS in right or left arm (referred to as real-NMMS, rNMMS), and sham NMMS (sNMMS) in the opposite arm. All the patients underwent a median nerve conduction (compound muscle action potential, CMAP) study and a clinical examination that included a handgrip strength test and an evaluation of upper limb muscle strength by means of the Medical Research Council Muscle Scale (MRC). Muscle biopsy was then performed bilaterally on the flexor carpi radialis muscle to monitor morpho-functional parameters and molecular changes. Patients and physicians who performed examinations were blinded to the side of real intervention. The primary outcome was the change in the muscle strength in upper arms. The secondary outcomes were the change from baseline in the CMAP amplitudes, in the nicotinic ACh currents, in the expression levels of a selected panel of genes involved in muscle growth and atrophy, and in histomorphometric parameters of ALS muscle fibers. The Repeated Measures (RM) ANOVA with a Greenhouse-Geisser correction (sphericity not assumed) showed a significant effect [F(3, 63) = 5.907, p < 0.01] of rNMMS on MRC scale at the flexor carpi radialis muscle, thus demonstrating that the rNMMS significantly improves muscle strength in flexor muscles in the forearm. Secondary outcomes showed that the improvement observed in rNMMS-treated muscles was associated to counteracting muscle atrophy, down-modulating the proteolysis, and increasing the efficacy of nicotinic ACh receptors (AChRs). We did not observe any significant difference in pre- and post-stimulation CMAP amplitudes, evoked by median nerve stimulation. This suggests that the improvement in muscle strength observed in the stimulated arm is unlikely related to reinnervation. The real and sham treatments were well tolerated without evident side effects. Although promising, this is a proof of concept study, without an immediate clinical translation, that requires further clinical validation.
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http://dx.doi.org/10.1038/s41598-019-39313-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391419PMC
February 2019

Levetiracetam enhances the temozolomide effect on glioblastoma stem cell proliferation and apoptosis.

Cancer Cell Int 2018 10;18:136. Epub 2018 Sep 10.

1Istituto di Istologia ed Embriologia, Università Cattolica del Sacro Cuore, Fondazione Policlinico Universitario A. Gemelli IRCCS, Roma, Italia.

Background: Glioblastoma multiforme (GBM) is a highly aggressive brain tumor in which cancer cells with stem cell-like features, called cancer stem cells (CSCs), were identified. Two CSC populations have been previously identified in GBM, one derived from the GBM area called enhanced lesion (GCSCs) and the other one from the brain area adjacent to the tumor margin (PCSCs) that greatly differ in their growth properties and tumor-initiating ability. To date the most effective chemotherapy to treat GBM is represented by alkylating agents such as temozolomide (TMZ), whose activity can be regulated by histone deacetylases (HDACs) inhibitors through the modulation of O6-methylguanine-DNA methyltransferase (MGMT) expression. Levetiracetam (LEV), a relatively new antiepileptic drug, modulates HDAC levels ultimately silencing MGMT, thus increasing TMZ effectiveness. However, an improvement in the therapeutic efficacy of TMZ is needed.

Methods: Cell proliferation was investigated by BrdU cell proliferation assay and by Western Blot analysis of PCNA expression. Apoptosis was evaluated by Western Blot and Immunofluorescence analysis of the cleaved Caspase-3 expression. MGMT and HDAC4 expression was analyzed by Western Blotting and Immunofluorescence. Statistical analysis was performed using the Student's test and Mann-Whitney test.

Results: Here we evaluated the effect of TMZ on the proliferation rate of the IDH-wildtype GCSCs and PCSCs derived from six patients, in comparison with the effects of other drugs such as etoposide, irinotecan and carboplatin. Our results demonstrated that TMZ was less effective compared to the other agents; hence, we verified the possibility to increase the effect of TMZ by combining it with LEV. Here we show that LEV enhances the effect of TMZ on GCSCs proliferation (being less effective on PCSCs) by decreasing MGMT expression, promoting HDAC4 nuclear translocation and activating apoptotic pathway.

Conclusions: Although further studies are needed to determine the exact mechanism by which LEV makes GBM stem cells more  sensitive to TMZ, these results suggest that the clinical therapeutic efficacy of TMZ in GBM might be enhanced by the combined treatment with LEV.
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http://dx.doi.org/10.1186/s12935-018-0626-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6131782PMC
September 2018

Molecular Insights into Muscle Homeostasis, Atrophy and Wasting.

Curr Genomics 2018 Aug;19(5):356-369

Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Rome, Italy.

Muscle homeostasis is guaranteed by a delicate balance between synthesis and degradation of cell proteins and its alteration leads to muscle wasting and diseases. In this review, we describe the major anabolic pathways that are involved in muscle growth and homeostasis and the proteolytic systems that are over-activated in muscle pathologies. Modulation of these pathways comprises an attractive target for drug intervention.
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http://dx.doi.org/10.2174/1389202919666180101153911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030854PMC
August 2018

Metabolic Changes Associated With Muscle Expression of SOD1.

Front Physiol 2018 10;9:831. Epub 2018 Jul 10.

Laboratory affiliated to Istituto Pasteur - Fondazione Cenci Bolognetti, DAHFMO - Unit of Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder, classified into sporadic or familial forms and characterized by motor neurons death, muscle atrophy, weakness, and paralysis. Among the familial cases of ALS, approximately 20% are caused by dominant mutations in the gene coding for superoxide dismutase (SOD1) protein. Of note, mutant SOD1 toxicity is not necessarily limited to the central nervous system. ALS is indeed a multi-systemic and multifactorial disease that affects whole body physiology and induces severe metabolic changes in several tissues, including skeletal muscle. Nevertheless, whether alterations in the plasticity, heterogeneity, and metabolism of muscle fibers are the result of motor neuron degeneration or alternatively occur independently of it remain to be elucidated. To address this issue, we made use of a mouse model (MLC/SOD1) that overexpresses the SOD1 mutant gene selectively in skeletal muscle. We found an alteration in the metabolic properties of skeletal muscle characterized by alteration in fiber type composition and metabolism. Indeed, we observed an alteration of muscle glucose metabolism associated with the induction of Phosphofructokinases and Pyruvate dehydrogenase kinase 4 expression. The upregulation of Pyruvate dehydrogenase kinase 4 led to the inhibition of Pyruvate conversion into Acetyl-CoA. Moreover, we demonstrated that the MLC/SOD1 transgene was associated with an increase of lipid catabolism and with the inhibition of fat deposition inside muscle fibers. All together these data demonstrate that muscle expression of the SOD1 gene induces metabolic changes, along with a preferential use of lipid energy fuel by muscle fibers. We provided evidences that muscle metabolic alterations occurred before disease symptoms and independently of motor neuron degeneration, indicating that skeletal muscle is likely an important therapeutic target in ALS.
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http://dx.doi.org/10.3389/fphys.2018.00831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048270PMC
July 2018

Muscle Expression of SOD1 Triggers the Dismantlement of Neuromuscular Junction via PKC-Theta.

Antioxid Redox Signal 2018 04 30;28(12):1105-1119. Epub 2017 Oct 30.

1 Center for Life Nano Science at Sapienza , Istituto Italiano di Tecnologia, Rome, Italy .

Aim: Neuromuscular junction (NMJ) represents the morphofunctional interface between muscle and nerve. Several chronic pathologies such as aging and neurodegenerative diseases, including muscular dystrophy and amyotrophic lateral sclerosis, display altered NMJ and functional denervation. However, the triggers and the molecular mechanisms underlying the dismantlement of NMJ remain unclear.

Results: Here we provide evidence that perturbation in redox signaling cascades, induced by muscle-specific accumulation of mutant SOD1 in transgenic MLC/SOD1 mice, is causally linked to morphological alterations of the neuromuscular presynaptic terminals, high turnover rate of acetylcholine receptor, and NMJ dismantlement. The analysis of potential molecular mechanisms that mediate the toxic activity of SOD1 revealed a causal link between protein kinase Cθ (PKCθ) activation and NMJ disintegration.

Innovation: The study discloses the molecular mechanism that triggers functional denervation associated with the toxic activity of muscle SOD1 expression and suggests the possibility of developing a new strategy to counteract age- and pathology-associated denervation based on pharmacological inhibition of PKCθ activity.

Conclusions: Collectively, these data indicate that muscle-specific accumulation of oxidative damage can affect neuromuscular communication and induce NMJ dismantlement through a PKCθ-dependent mechanism. Antioxid. Redox Signal. 28, 1105-1119.
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http://dx.doi.org/10.1089/ars.2017.7054DOI Listing
April 2018

Noise Enhances Action Potential Generation in Mouse Sensory Neurons via Stochastic Resonance.

PLoS One 2016 15;11(8):e0160950. Epub 2016 Aug 15.

Institute Pasteur-Cenci Bolognetti Foundation, Dept. Physiology and Pharmacology, Sapienza University, Rome, Italy.

Noise can enhance perception of tactile and proprioceptive stimuli by stochastic resonance processes. However, the mechanisms underlying this general phenomenon remain to be characterized. Here we studied how externally applied noise influences action potential firing in mouse primary sensory neurons of dorsal root ganglia, modelling a basic process in sensory perception. Since noisy mechanical stimuli may cause stochastic fluctuations in receptor potential, we examined the effects of sub-threshold depolarizing current steps with superimposed random fluctuations. We performed whole cell patch clamp recordings in cultured neurons of mouse dorsal root ganglia. Noise was added either before and during the step, or during the depolarizing step only, to focus onto the specific effects of external noise on action potential generation. In both cases, step + noise stimuli triggered significantly more action potentials than steps alone. The normalized power norm had a clear peak at intermediate noise levels, demonstrating that the phenomenon is driven by stochastic resonance. Spikes evoked in step + noise trials occur earlier and show faster rise time as compared to the occasional ones elicited by steps alone. These data suggest that external noise enhances, via stochastic resonance, the recruitment of transient voltage-gated Na channels, responsible for action potential firing in response to rapid step-wise depolarizing currents.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160950PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4985147PMC
July 2017

Progressive impairment of CaV1.1 function in the skeletal muscle of mice expressing a mutant type 1 Cu/Zn superoxide dismutase (G93A) linked to amyotrophic lateral sclerosis.

Skelet Muscle 2016 23;6:24. Epub 2016 Jun 23.

Department of Medicine-Cardiology Division, University of Colorado School of Medicine, 12700 East 19th Avenue, B-139, Aurora, CO 80045 USA.

Background: Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder that is typically fatal within 3-5 years of diagnosis. While motoneuron death is the defining characteristic of ALS, the events that underlie its pathology are not restricted to the nervous system. In this regard, ALS muscle atrophies and weakens significantly before presentation of neurological symptoms. Since the skeletal muscle L-type Ca(2+) channel (CaV1.1) is a key regulator of both mass and force, we investigated whether CaV1.1 function is impaired in the muscle of two distinct mouse models carrying an ALS-linked mutation.

Methods: We recorded L-type currents, charge movements, and myoplasmic Ca(2+) transients from dissociated flexor digitorum brevis (FDB) fibers to assess CaV1.1 function in two mouse models expressing a type 1 Cu/Zn superoxide dismutase mutant (SOD1(G93A)).

Results: In FDB fibers obtained from "symptomatic" global SOD1(G93A) mice, we observed a substantial reduction of SR Ca(2+) release in response to depolarization relative to fibers harvested from age-matched control mice. L-type current and charge movement were both reduced by ~40 % in symptomatic SOD1(G93A) fibers when compared to control fibers. Ca(2+) transients were not significantly reduced in similar experiments performed with FDB fibers obtained from "early-symptomatic" SOD1(G93A) mice, but L-type current and charge movement were decreased (~30 and ~20 %, respectively). Reductions in SR Ca(2+) release (~35 %), L-type current (~20 %), and charge movement (~15 %) were also observed in fibers obtained from another model where SOD1(G93A) expression was restricted to skeletal muscle.

Conclusions: We report reductions in EC coupling, L-type current density, and charge movement in FDB fibers obtained from symptomatic global SOD1(G93A) mice. Experiments performed with FDB fibers obtained from early-symptomatic SOD1(G93A) and skeletal muscle autonomous MLC/SOD1(G93A) mice support the idea that events occurring locally in the skeletal muscle contribute to the impairment of CaV1.1 function in ALS muscle independently of innervation status.
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http://dx.doi.org/10.1186/s13395-016-0094-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918102PMC
October 2016

Muscle Expression of SOD1(G93A) Modulates microRNA and mRNA Transcription Pattern Associated with the Myelination Process in the Spinal Cord of Transgenic Mice.

Front Cell Neurosci 2015 1;9:463. Epub 2015 Dec 1.

DAHFMO-Unit of Histology and Medical Embryology, Institute Pasteur-Cenci Bolognetti, IIM, Sapienza University of Rome Rome, Italy ; Center for Life Nano Science at Sapienza, Istituto Italiano di Tecnologia Rome, Italy.

A crucial system severely affected in several neuromuscular diseases is the loss of effective connection between muscle and nerve, leading to a pathological non-communication between the two tissues. One of the best examples of impaired interplay between muscle and nerve is Amyotrophic Lateral Sclerosis, a neurodegenerative disease characterized by degeneration of motor neurons and muscle atrophy. Increasing evidences suggest that damage to motor neurons is enhanced by alterations in the neighboring non-neuronal cells and indicate that altered skeletal muscle might be the source of signals that impinge motor neuron activity and survival. Here we investigated whether muscle selective expression of SOD1(G93A) mutant gene modulates mRNAs and miRNAs expression at the level of spinal cord of MLC/SOD1(G93A) mice. Using a Taqman array, the Affymetrix Mouse Gene 2.0 ST approach and the MiRwalk 2.0 database, which provides information on miRNA and their predicted target genes, we revealed that muscle specific expression of SOD1(G93A) modulates relevant molecules of the genetic and epigenetic circuitry of myelin homeostasis in spinal cord of transgenic mice. Our study provides insights into the pathophysiological interplay between muscle and nerve and supports the hypothesis that muscle is a source of signals that can either positively or negatively affect the nervous system.
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http://dx.doi.org/10.3389/fncel.2015.00463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4664730PMC
December 2015

Postmitotic Expression of SOD1(G93A) Gene Affects the Identity of Myogenic Cells and Inhibits Myoblasts Differentiation.

Mediators Inflamm 2015 28;2015:537853. Epub 2015 Sep 28.

Institute Pasteur-Cenci Bolognetti, DAHFMO-Unit of Histology and Medical Embryology, IIM, Sapienza University of Rome, Via A. Scarpa 16, 00161 Rome, Italy ; Center for Life Nano Science at Sapienza, Istituto Italiano di Tecnologia, Viale Regina Elena 291, 00161 Rome, Italy ; Edith Cowan University, Perth, WA 6027, Australia.

To determine the role of mutant SOD1 gene (SOD1(G93A)) on muscle cell differentiation, we derived C2C12 muscle cell lines carrying a stably transfected SOD1(G93A) gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC/SOD1(G93A) in C2C12 cells resulted in dramatic inhibition of myoblast differentiation. Transfected SOD1(G93A) gene expression in postmitotic skeletal myocytes downregulated the expression of relevant markers of committed and differentiated myoblasts such as MyoD, Myogenin, MRF4, and the muscle specific miRNA expression. The inhibitory effects of SOD1(G93A) gene on myogenic program perturbed Akt/p70 and MAPK signaling pathways which promote differentiation cascade. Of note, the inhibition of the myogenic program, by transfected SOD1(G93A) gene expression, impinged also the identity of myogenic cells. Expression of MLC/SOD1(G93A) in C2C12 myogenic cells promoted a fibro-adipogenic progenitors (FAPs) phenotype, upregulating HDAC4 protein and preventing the myogenic commitment complex BAF60C-SWI/SNF. We thus identified potential molecular mediators of the inhibitory effects of SOD1(G93A) on myogenic program and disclosed potential signaling, activated by SOD1(G93A), that affect the identity of the myogenic cell population.
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http://dx.doi.org/10.1155/2015/537853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4600922PMC
August 2016

R-spondin 1/dickkopf-1/beta-catenin machinery is involved in testicular embryonic angiogenesis.

PLoS One 2015 24;10(4):e0124213. Epub 2015 Apr 24.

Department of Experimental Medicine-Histology and Embryology Laboratory, Second University of Naples, Naples, Italy.

Testicular vasculogenesis is one of the key processes regulating male gonad morphogenesis. The knowledge of the molecular cues underlining this phenomenon is one of today's most challenging issues and could represent a major contribution toward a better understanding of the onset of testicular morphogenetic disorders. R-spondin 1 has been clearly established as a candidate for mammalian ovary determination. Conversely, very little information is available on the expression and role of R-spondin 1 during testicular morphogenesis. This study aims to clarify the distribution pattern of R-spondin 1 and other partners of its machinery during the entire period of testicular morphogenesis and to indicate the role of this system in testicular development. Our whole mount immunofluorescence results clearly demonstrate that R-spondin 1 is always detectable in the testicular coelomic partition, where testicular vasculature is organized, while Dickkopf-1 is never detectable in this area. Moreover, organ culture experiments of embryonic male UGRs demonstrated that Dickkopf-1 acted as an inhibitor of testis vasculature formation. Consistent with this observation, real-time PCR analyses demonstrated that DKK1 is able to slightly but significantly decrease the expression level of the endothelial marker Pecam1. The latter experiments allowed us to observe that DKK1 administration also perturbs the expression level of the Pdgf-b chain, which is consistent with some authors' observations relating this factor with prenatal testicular patterning and angiogenesis. Interestingly, the DKK1 induced inhibition of testicular angiogenesis was rescued by the co-administration of R-spondin 1. In addition, R-spondin 1 alone was sufficient to enhance, in culture, testicular angiogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0124213PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4409372PMC
January 2016

Melatonin down-regulates MDM2 gene expression and enhances p53 acetylation in MCF-7 cells.

J Pineal Res 2014 Aug 10;57(1):120-9. Epub 2014 Jul 10.

Department of Surgery "P. Valdoni", "Sapienza" University of Rome, Rome, Italy.

Compelling evidence demonstrated that melatonin increases p53 activity in cancer cells. p53 undergoes acetylation to be stabilized and activated for driving cells destined for apoptosis/growth inhibition. Over-expression of p300 induces p53 acetylation, leading to cell growth arrest by increasing p21 expression. In turn, p53 activation is mainly regulated in the nucleus by MDM2. MDM2 also acts as E3 ubiquitin ligase, promoting the proteasome-dependent p53 degradation. MDM2 entry into the nucleus is finely tuned by two different modulations: the ribosomal protein L11, acts by sequestering MDM2 in the cytosol, whereas the PI3K-AkT-dependent MDM2 phosphorylation is mandatory for MDM2 translocation across the nuclear membrane. In addition, MDM2-dependent targeting of p53 is regulated in a nonlinear fashion by MDM2/MDMX interplay. Melatonin induces both cell growth inhibition and apoptosis in MCF7 breast cancer cells. We previously reported that this effect is associated with reduced MDM2 levels and increased p53 activity. Herein, we demonstrated that melatonin drastically down-regulates MDM2 gene expression and inhibits MDM2 shuttling into the nucleus, given that melatonin increases L11 and inhibits Akt-PI3K-dependent MDM2 phosphorylation. Melatonin induces a 3-fold increase in both MDMX and p300 levels, decreasing simultaneously Sirt1, a specific inhibitor of p300 activity. Consequently, melatonin-treated cells display significantly higher values of both p53 and acetylated p53. Thus, a 15-fold increase in p21 levels was observed in melatonin-treated cancer cells. Our results provide evidence that melatonin enhances p53 acetylation by modulating the MDM2/MDMX/p300 pathway, disclosing new insights for understanding its anticancer effect.
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http://dx.doi.org/10.1111/jpi.12150DOI Listing
August 2014

Microenvironment promotes tumor cell reprogramming in human breast cancer cell lines.

PLoS One 2013 30;8(12):e83770. Epub 2013 Dec 30.

Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy ; Italian Space Agency (ASI), Rome, Italy.

The microenvironment drives mammary gland development and function, and may influence significantly both malignant behavior and cell growth of mammary cancer cells. By restoring context, and forcing cells to properly interpret native signals from the microenvironment, the cancer cell aberrant behavior can be quelled, and organization re-established. In order to restore functional and morphological differentiation, human mammary MCF-7 and MDA-MB-231 cancer cells were allowed to grow in a culture medium filled with a 10% of the albumen (EW, Egg White) from unfertilized chicken egg. That unique microenvironment behaves akin a 3D culture and induces MCF-7 cells to produce acini and branching duct-like structures, distinctive of mammary gland differentiation. EW-treated MDA-MB-231 cells developed buds of acini and duct-like structures. Both MCF-7 and MDA-MB-231 cells produced β-casein, a key milk component. Furthermore, E-cadherin expression was reactivated in MDA-MB-231 cells, as a consequence of the increased cdh1 expression; meanwhile β-catenin - a key cytoskeleton component - was displaced behind the inner cell membrane. Such modification hinders the epithelial-mesenchymal transition in MDA-MB-231 cells. This differentiating pathway is supported by the contemporary down-regulation of canonical pluripotency markers (Klf4, Nanog). Given that egg-conditioned medium behaves as a 3D-medium, it is likely that cancer phenotype reversion could be ascribed to the changed interactions between cells and their microenvironment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0083770PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3875474PMC
August 2014

Paracrine effects of IGF-1 overexpression on the functional decline due to skeletal muscle disuse: molecular and functional evaluation in hindlimb unloaded MLC/mIgf-1 transgenic mice.

PLoS One 2014 3;8(6):e65167. Epub 2013 Jun 3.

Section of Pharmacology, Department of Pharmacy & Drug Sciences, University of Bari Aldo Moro, Bari, Italy.

Slow-twitch muscles, devoted to postural maintenance, experience atrophy and weakness during muscle disuse due to bed-rest, aging or spaceflight. These conditions impair motion activities and can have survival implications. Human and animal studies demonstrate the anabolic role of IGF-1 on skeletal muscle suggesting its interest as a muscle disuse countermeasure. Thus, we tested the role of IGF-1 overexpression on skeletal muscle alteration due to hindlimb unloading (HU) by using MLC/mIgf-1 transgenic mice expressing IGF-1 under the transcriptional control of MLC promoter, selectively activated in skeletal muscle. HU produced atrophy in soleus muscle, in terms of muscle weight and fiber cross-sectional area (CSA) reduction, and up-regulation of atrophy gene MuRF1. In parallel, the disuse-induced slow-to-fast fiber transition was confirmed by an increase of the fast-type of the Myosin Heavy Chain (MHC), a decrease of PGC-1α expression and an increase of histone deacetylase-5 (HDAC5). Consistently, functional parameters such as the resting chloride conductance (gCl) together with ClC-1 chloride channel expression were increased and the contractile parameters were modified in soleus muscle of HU mice. Surprisingly, IGF-1 overexpression in HU mice was unable to counteract the loss of muscle weight and the decrease of fiber CSA. However, the expression of MuRF1 was recovered, suggesting early effects on muscle atrophy. Although the expression of PGC-1α and MHC were not improved in IGF-1-HU mice, the expression of HDAC5 was recovered. Importantly, the HU-induced increase of gCl was fully contrasted in IGF-1 transgenic mice, as well as the changes in contractile parameters. These results indicate that, even if local expression does not seem to attenuate HU-induced atrophy and slow-to-fast phenotype transition, it exerts early molecular effects on gene expression which can counteract the HU-induced modification of electrical and contractile properties. MuRF1 and HDAC5 can be attractive therapeutic targets for pharmacological countermeasures and then deserve further investigations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065167PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3670938PMC
January 2015

IPLEX administration improves motor neuron survival and ameliorates motor functions in a severe mouse model of spinal muscular atrophy.

Mol Med 2012 Sep 25;18:1076-85. Epub 2012 Sep 25.

Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy.

Spinal muscular atrophy (SMA) is an inherited neurodegenerative disorder and the first genetic cause of death in childhood. SMA is caused by low levels of survival motor neuron (SMN) protein that induce selective loss of α-motor neurons (MNs) in the spinal cord, resulting in progressive muscle atrophy and consequent respiratory failure. To date, no effective treatment is available to counteract the course of the disease. Among the different therapeutic strategies with potential clinical applications, the evaluation of trophic and/or protective agents able to antagonize MNs degeneration represents an attractive opportunity to develop valid therapies. Here we investigated the effects of IPLEX (recombinant human insulinlike growth factor 1 [rhIGF-1] complexed with recombinant human IGF-1 binding protein 3 [rhIGFBP-3]) on a severe mouse model of SMA. Interestingly, molecular and biochemical analyses of IGF-1 carried out in SMA mice before drug administration revealed marked reductions of IGF-1 circulating levels and hepatic mRNA expression. In this study, we found that perinatal administration of IPLEX, even if does not influence survival and body weight of mice, results in reduced degeneration of MNs, increased muscle fiber size and in amelioration of motor functions in SMA mice. Additionally, we show that phenotypic changes observed are not SMN-dependent, since no significant SMN modification was addressed in treated mice. Collectively, our data indicate IPLEX as a good therapeutic candidate to hinder the progression of the neurodegenerative process in SMA.
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http://dx.doi.org/10.2119/molmed.2012.00056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3474434PMC
September 2012

Adaptation of mouse skeletal muscle to long-term microgravity in the MDS mission.

PLoS One 2012 28;7(3):e33232. Epub 2012 Mar 28.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

The effect of microgravity on skeletal muscles has so far been examined in rat and mice only after short-term (5-20 day) spaceflights. The mice drawer system (MDS) program, sponsored by Italian Space Agency, for the first time aimed to investigate the consequences of long-term (91 days) exposure to microgravity in mice within the International Space Station. Muscle atrophy was present indistinctly in all fiber types of the slow-twitch soleus muscle, but was only slightly greater than that observed after 20 days of spaceflight. Myosin heavy chain analysis indicated a concomitant slow-to-fast transition of soleus. In addition, spaceflight induced translocation of sarcolemmal nitric oxide synthase-1 (NOS1) into the cytosol in soleus but not in the fast-twitch extensor digitorum longus (EDL) muscle. Most of the sarcolemmal ion channel subunits were up-regulated, more in soleus than EDL, whereas Ca(2+)-activated K(+) channels were down-regulated, consistent with the phenotype transition. Gene expression of the atrophy-related ubiquitin-ligases was up-regulated in both spaceflown soleus and EDL muscles, whereas autophagy genes were in the control range. Muscle-specific IGF-1 and interleukin-6 were down-regulated in soleus but up-regulated in EDL. Also, various stress-related genes were up-regulated in spaceflown EDL, not in soleus. Altogether, these results suggest that EDL muscle may resist to microgravity-induced atrophy by activating compensatory and protective pathways. Our study shows the extended sensitivity of antigravity soleus muscle after prolonged exposition to microgravity, suggests possible mechanisms accounting for the resistance of EDL, and individuates some molecular targets for the development of countermeasures.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033232PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314659PMC
August 2012

Muscle atrophy induced by SOD1G93A expression does not involve the activation of caspase in the absence of denervation.

Skelet Muscle 2011 Jan 24;1(1). Epub 2011 Jan 24.

Institute Pasteur Cenci-Bolognetti, DAHFMO-Unit of Histology and Medical Embryology, IIM; Sapienza University of Rome, Via A. Scarpa, 14 Rome I-00161, Italy.

Background: The most remarkable feature of skeletal muscle is the capacity to adapt its morphological, biochemical and molecular properties in response to several factors. Nonetheless, under pathological conditions, skeletal muscle loses its adaptability, leading to atrophy or wasting. Several signals might function as physiopathological triggers of muscle atrophy. However, the specific mechanisms underlying the atrophic phenotype under different pathological conditions remain to be fully elucidated. In this paper, we address the involvement of caspases in the induction of muscle atrophy in experimental models of amyotrophic lateral sclerosis (ALS) expressing the mutant SOD1G93A transgene either locally or ubiquitously.

Results: We demonstrate that SOD1G93A-mediated muscle atrophy is independent from caspase activity. In particular, the expression of SOD1G93A promotes a reduction of the phosphatidylinositol 3-kinase/Akt pathway associated with activation of forkhead box O3. In contrast, the activation of caspases occurs later and is causally linked to motor neuron degeneration, which is associated with exacerbation of the atrophic phenotype and a shift in fiber-type composition.

Conclusion: This study suggests that muscle atrophy induced by the toxic effect of SOD1G93A is independent from the activation of apoptotic markers and that caspase-mediated apoptosis is a process activated upon muscle denervation.
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http://dx.doi.org/10.1186/2044-5040-1-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3143901PMC
January 2011

Muscle involvement and IGF-1 signaling in genetic disorders: new therapeutic approaches.

Endocr Dev 2009 27;14:29-37. Epub 2009 Feb 27.

Department of Histology and Medical Embryology, Sapienza University of Rome, Rome, Italy.

In the last decade, dramatic progress has been made in elucidating the molecular defects underlying a number of muscle diseases. With the characterization of mutations responsible for muscle dysfunction in several inherited pathologies, and the identification of novel signaling pathways, subtle alterations in which can lead to significant defects in muscle metabolism, the field is poised to devise successful strategies for treatment of this debilitating and often fatal group of human ailments. Yet progress has been slow in therapeutic applications of our newly gained knowledge. The complexity of muscle types, the intimate relationship between structural integrity and mechanical function, and the sensitivity of skeletal muscle to metabolic perturbations have impeded rapid progress in successful clinical intervention. The relatively poor regenerative properties of striated muscle compound the devastating effects of muscle degeneration. Perhaps the most difficult hurdle is the sheer volume of tissue that must be treated to effect a significant improvement in quality of life. Recent studies on the role of insulin-like growth factor-1 in skeletal muscle growth and homeostasis have excited new interest in this important mediator of anabolic pathways and suggest promising new avenues for intervention in catabolic disease. In this review, we will discuss the potential therapeutic role of local insulin-like growth factor 1 in the treatment of muscle wasting associated with muscle diseases.
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http://dx.doi.org/10.1159/000207474DOI Listing
May 2009

Localized accumulation of oxidative stress causes muscle atrophy through activation of an autophagic pathway.

Autophagy 2009 May 28;5(4):527-9. Epub 2009 May 28.

Institute Pasteur Cenci-Bolognetti, Department of Histology and Medical Embryology, CE-BEMM and IIM, Sapienza University of Rome,Via A. Scarpa 14, Rome, Italy.

A crucial system severely affected in different chronic diseases is the antioxidative defense, leading to accumulation of reactive oxygen species (ROS). The discovery that deletion in the antioxidant genes shortens significantly the mouse life span, and that mutation in the major antioxidant enzyme SOD1 is associated with neurodegenerative diseases, has placed oxidative stress as a central mechanism in the pathogenesis of many pathological conditions. However, how such an oxidative insult plays a role in the disease-related decrease of muscle performance and mass remains largely unknown. We recently demonstrated that autophagy plays a dominant role in the promotion of muscle atrophy associated with local alteration in the activity of the antioxidant enzyme SOD1. In particular, transcription of autophagy-related genes, such as those encoding LC3, Cathepsin-L and Bnip3, is activated in response to localized accumulation of oxidative stress and is mediated by FoxO3. In addition, our study documents how the T-tubule might be the potential donor of membrane that forms sequestering autophagic vesicles. Here we discuss the sequence of events leading to muscle atrophy.
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http://dx.doi.org/10.4161/auto.5.4.7962DOI Listing
May 2009

Skeletal muscle is a primary target of SOD1G93A-mediated toxicity.

Cell Metab 2008 Nov;8(5):425-36

Institute Pasteur Cenci-Bolognetti, Department of Histology and Medical Embryology, CE-BEMM and IIM, Sapienza University of Rome,Via A. Scarpa, 14 Rome 00161, Italy.

The antioxidant enzyme superoxide dismutase 1 (SOD1) is a critical player of the antioxidative defense whose activity is altered in several chronic diseases, including amyotrophic lateral sclerosis. However, how oxidative insult affects muscle homeostasis remains unclear. This study addresses the role of oxidative stress on muscle homeostasis and function by the generation of a transgenic mouse model expressing a mutant SOD1 gene (SOD1(G93A)) selectively in skeletal muscle. Transgenic mice developed progressive muscle atrophy, associated with a significant reduction in muscle strength, alterations in the contractile apparatus, and mitochondrial dysfunction. The analysis of molecular pathways associated with muscle atrophy revealed that accumulation of oxidative stress served as signaling molecules to initiate autophagy, one of the major intracellular degradation mechanisms. These data demonstrate that skeletal muscle is a primary target of SOD1(G93A) -mediated toxicity and disclose the molecular mechanism whereby oxidative stress triggers muscle atrophy.
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http://dx.doi.org/10.1016/j.cmet.2008.09.002DOI Listing
November 2008

Local expression of mIgf-1 modulates ubiquitin, caspase and CDK5 expression in skeletal muscle of an ALS mouse model.

Neurol Res 2008 Mar;30(2):131-6

Department of Histology and Medical Embryology, Centro Eccellenza BEMM and IIM, University of Rome La Sapienza, Via A. Scarpa, 14 Rome 00161, Italy.

Objective: The functional connection between muscle and nerve is often altered in several neuromuscular diseases, including amyotrophic lateral sclerosis (ALS). Knowledge about the molecular and cellular mechanisms involved in the restorative reactions is important to our understanding of the processes involved in neuromuscular maintenance. We previously reported that muscle-restricted expression of a localized Igf-1 isoform maintained muscle integrity, stabilized neuromuscular junctions, reduced inflammation in the spinal cord and enhanced motor neuronal survival in SOD(G93A) mice, delaying the onset and progression of the disease. In this study, we analysed potential molecular pathways that are modulated by mIgf-1 to counteract muscle wasting and to preserve motor neurons activity.

Methods: We performed molecular and morphologic analysis to address the specific proposed questions.

Results And Discussion: Ubiquitin expression and caspase activity resulted markedly increased in SOD(G93A) muscle but maintained at very low levels in the SOD(G93A) x MLC/mIgf-1 (SOD(G93A)/mIgf-1) transgenic muscle. In addition, CDK5 expression, a serine-threonine protein kinase that has been implicated in a number of physiologic processes in nerve and muscle cells, was reduced in SOD(G93A) muscle but increased in SOD(G93A)/mIgf-1 muscle. Notably, while the toxic p25 protein accumulated in SOD(G93A) muscle, no accumulation was evident in the SOD(G93A)/mIgf-1 muscle. The maintenance of muscle phenotype was also associated with maintenance of a normal peripheral nerve, and a greater number of myelinated axons.

Conclusion: These observations offer novel insights into the role of mIgf-1 in the attenuation of muscle wasting in the mouse model of ALS disease.
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http://dx.doi.org/10.1179/174313208X281235DOI Listing
March 2008

The neuroprotective effects of a locally acting IGF-1 isoform.

Exp Gerontol 2007 Jan-Feb;42(1-2):76-80. Epub 2006 Jun 19.

Department of Histology and Medical Embryology, CE-BEMM and Interuniversity Institute of Myology, University of Rome La Sapienza, Italy.

In the last decade, dramatic progress has been made in elucidating the molecular defects underlying a number of neuromuscular diseases. With the characterization of mutations responsible for muscle and nerve dysfunction in several inherited pathologies, and the identification of novel signaling pathways, in which subtle alterations can lead to significant defects in tissue metabolism, the field is poised to devise successful strategies for treatment of this debilitating and often fatal group of human ailments. Yet progress in therapeutic application has been slow despite our newly gained knowledge of basic biology. Hence, where direct therapeutic approaches to address the primary diseases are still sub-optimal, it may be more effective to focus on strategies for improving neuromuscular function. Among potential candidates, insulin-like growth factor (IGF-1) has been involved in several anabolic pathways in both skeletal muscle and the nervous system and it is a promising candidate to attenuate neuromuscular diseases. In this review, we will discuss the role of IGF-1 isoforms in neuromuscular diseases and the contribution of muscle-produced IGF-1 (mIGF-1) to motor neuron survival and activity.
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http://dx.doi.org/10.1016/j.exger.2006.05.004DOI Listing
October 2007

Muscle expression of a local Igf-1 isoform protects motor neurons in an ALS mouse model.

J Cell Biol 2005 Jan;168(2):193-9

Department of Histology and Medical Embryology, CE-BEMM and Interuniversity Institute of Myology, University of Rome "La Sapienza", 14 00161 Rome, Italy.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by a selective degeneration of motor neurons, atrophy, and paralysis of skeletal muscle. Although a significant proportion of familial ALS results from a toxic gain of function associated with dominant SOD1 mutations, the etiology of the disease and its specific cellular origins have remained difficult to define. Here, we show that muscle-restricted expression of a localized insulin-like growth factor (Igf) -1 isoform maintained muscle integrity and enhanced satellite cell activity in SOD1(G93A) transgenic mice, inducing calcineurin-mediated regenerative pathways. Muscle-specific expression of local Igf-1 (mIgf-1) isoform also stabilized neuromuscular junctions, reduced inflammation in the spinal cord, and enhanced motor neuronal survival in SOD1(G93A) mice, delaying the onset and progression of the disease. These studies establish skeletal muscle as a primary target for the dominant action of inherited SOD1 mutation and suggest that muscle fibers provide appropriate factors, such as mIgf-1, for neuron survival.
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http://dx.doi.org/10.1083/jcb.200407021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2171577PMC
January 2005

Stem cell-mediated muscle regeneration is enhanced by local isoform of insulin-like growth factor 1.

Proc Natl Acad Sci U S A 2004 Feb 26;101(5):1206-10. Epub 2004 Jan 26.

Department of Histology and Medical Embryology and Centro Eccellenza Biologia e Medicina Molecolare, University of Rome "La Sapienza," 00161 Rome, Italy.

We investigated the mechanism whereby expression of a transgene encoding a locally acting isoform of insulin-like growth factor 1 (mIGF-1) enhances repair of skeletal muscle damage. Increased recruitment of proliferating bone marrow cells to injured MLC/mIgf-1 transgenic muscles was accompanied by elevated bone marrow stem cell production in response to distal trauma. Regenerating MLC/mIgf-1 transgenic muscles contained increased cell populations expressing stem cell markers, exhibited accelerated myogenic differentiation, expressed markers of regeneration and readily converted cocultured bone marrow to muscle. These data implicate mIGF-1 as a powerful enhancer of the regeneration response, mediating the recruitment of bone marrow cells to sites of tissue damage and augmenting local repair mechanisms.
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http://dx.doi.org/10.1073/pnas.0303792101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC337031PMC
February 2004

Biomonitoring of primary aluminium industry workers: detection of micronuclei and repairable DNA lesions by alkaline SCGE.

Mutat Res 2002 Apr;516(1-2):63-70

Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanita', Viale Regina Elena 299, I-00161 Rome, Italy.

The genetic effects of occupational exposure to low polycyclic aromatic hydrocarbon (PAH) concentrations were investigated in primary aluminium industry workers. The study subjects were employed in a plant that uses pre-baked anode cells, and has relatively low PAH contamination. Forty-two male workers belonging to different job categories (anode fabrication, baking, rodding, electrolysis, maintenance), together with 16 male local residents with no occupational exposure to PAHs were selected for the analysis of micronuclei and DNA lesions in peripheral lymphocytes. The incidence of micronuclei determined in 1000 cytokinesis-blocked cells in each subject was not significantly different between workers and controls (8.5+/-5.4 per thousand versus 9.7+/-4.9 per thousand, respectively), nor between smokers and non-smokers (8.3+/-5.8 per thousand versus 9.2+/-5.1 per thousand), but was significantly (P<0.05) related to the subjects' age. Also the analysis of DNA damage in unstimulated and mitogen-stimulated lymphocytes by single cell gel electrophoresis (SCGE) did not show significant differences between the studied groups (average tail moment values were 0.53+/-0.53 and 0.49+/-0.45 microm in exposed subjects and controls, respectively). However, when lymphocytes were cultured in the presence of cytosine arabinoside (Ara-C, 1 microg/ml for 16h) the SCGE analysis revealed a significant (P=0.018) difference in tail moment values between aluminium workers and controls (1.73+/-1.05 microm versus 0.93+/-0.88 microm, respectively). This difference may highlight an excess of relatively stable DNA lesions, that do not affect strand integrity, and are expressed as intermediates of excision repair in stimulated cells, when gap refilling is inhibited by cytosine arabinoside (Ara-C).
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http://dx.doi.org/10.1016/s1383-5718(02)00028-1DOI Listing
April 2002

Influence of donor age on vinblastine-induced chromosome malsegregation in cultured peripheral lymphocytes.

Mutagenesis 2002 Jan;17(1):83-8

Laboratory of Comparative Toxicology and Ecotoxicology,Istituto Superiore Sanità, Viale Regina Elena 299, I-00161 Rome, Italy.

The incidence of spontaneous aneuploidy in human somatic and germ cells is known to be positively associated with aging. However, the influence of age on the individual susceptibility to chemically induced chromosome malsegregation has not been elucidated. In this study the spindle poison vinblastine (VBL) was used as a model compound to assess the influence of donor age on chemically induced chromosome malsegregation in cultured lymphocytes. Blood cultures from 20 female donors belonging to two different age groups (10 <30 years and 10 >50 years) were treated with VBL (7.5 ng/ml) from 43 h after mitogen stimulation until harvest at 60 h, i.e. during the time interval corresponding to G2/M. In order to block cytokinesis, cytochalasin B (6 microg/ml) was added to cultures at 44 h. For each donor the incidence of micronuclei, polyploidy and malsegregation (non-disjunction and loss) of chromosomes X and 8 was determined using fluorescence in situ hybridization with chromosome-specific centromeric probes. Both background incidence of micronuclei and spontaneous chromosome X non-disjunction were significantly elevated in older donors. Individual responses to VBL treatment showed wide interindividual variability, which was not significantly associated with the age of the donor. In both age classes chromosome X was more susceptible than chromosome 8 to both spontaneous and VBL-induced malsegregation. These results indicate that donor age has a limited influence on the aneugenic effects exerted by VBL in peripheral lymphocytes in vitro. Other factors have to be considered to account for the large interindividual variation in sensitivity to VBL challenge observed in this work.
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http://dx.doi.org/10.1093/mutage/17.1.83DOI Listing
January 2002