Publications by authors named "Piergiorgio La Rosa"

25 Publications

  • Page 1 of 1

Nuclear Factor Erythroid 2-Related Factor 2 Activation Might Mitigate Clinical Symptoms in Friedreich's Ataxia: Clues of an "Out-Brain Origin" of the Disease From a Family Study.

Front Neurosci 2021 23;15:638810. Epub 2021 Feb 23.

Unit of Muscular and Neurodegenerative Diseases, Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy.

Friedreich's ataxia (FRDA) is the most frequent autosomal recessive ataxia in western countries, with a mean age of onset at 10-15 years. Patients manifest progressive cerebellar and sensory ataxia, dysarthria, lower limb pyramidal weakness, and other systemic manifestations. Previously, we described a family displaying two expanded GAA alleles not only in the proband affected by late-onset FRDA but also in the two asymptomatic family members: the mother and the younger sister. Both of them showed a significant reduction of frataxin levels, without any disease manifestation. Here, we analyzed if a protective mechanism might contribute to modulate the phenotype in this family. We particularly focused on the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), the first line of antioxidant defense in cells, and on the glutathione (GSH) system, an index of reactive oxygen species (ROS) detoxification ability. Our findings show a great reactivity of the GSH system to the frataxin deficiency, particularly in the asymptomatic mother, where the genes of GSH synthesis [glutamate-cysteine ligase ()] and GSSG detoxification [GSH S-reductase ()] were highly responsive. The was activated even in the asymptomatic sister and in the proband, reflecting the need of buffering the GSSG increase. Furthermore, and contrasting the NRF2 expression documented in FRDA tissues, NRF2 was highly activated in the mother and in the younger sister, while it was constitutively low in the proband. This suggests that, also under frataxin depletion, the endogenous stimulation of NRF2 in asymptomatic FRDA subjects may contribute to protect against the progressive oxidative damage, helping to prevent the onset of neurological symptoms and highlighting an "out-brain origin" of the disease.
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http://dx.doi.org/10.3389/fnins.2021.638810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940825PMC
February 2021

Ferroptosis in Friedreich's Ataxia: A Metal-Induced Neurodegenerative Disease.

Biomolecules 2020 11 13;10(11). Epub 2020 Nov 13.

Unit of Muscular and Neurodegenerative Diseases, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy.

Ferroptosis is an iron-dependent form of regulated cell death, arising from the accumulation of lipid-based reactive oxygen species when glutathione-dependent repair systems are compromised. Lipid peroxidation, mitochondrial impairment and iron dyshomeostasis are the hallmark of ferroptosis, which is emerging as a crucial player in neurodegeneration. This review provides an analysis of the most recent advances in ferroptosis, with a special focus on Friedreich's Ataxia (FA), the most common autosomal recessive neurodegenerative disease, caused by reduced levels of frataxin, a mitochondrial protein involved in iron-sulfur cluster synthesis and antioxidant defenses. The hypothesis is that the iron-induced oxidative damage accumulates over time in FA, lowering the ferroptosis threshold and leading to neuronal cell death and, at last, to cardiac failure. The use of anti-ferroptosis drugs combined with treatments able to activate the antioxidant response will be of paramount importance in FA therapy, such as in many other neurodegenerative diseases triggered by oxidative stress.
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http://dx.doi.org/10.3390/biom10111551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696618PMC
November 2020

The Nrf2 induction prevents ferroptosis in Friedreich's Ataxia.

Redox Biol 2021 Jan 5;38:101791. Epub 2020 Nov 5.

Unit of Neuromuscular and Neurodegenerative Diseases, IRCCS Bambino Gesù Children's Hospital, Rome, Italy. Electronic address:

Ferroptosis is an iron-dependent cell death caused by impaired glutathione metabolism, lipid peroxidation and mitochondrial failure. Emerging evidences report a role for ferroptosis in Friedreich's Ataxia (FRDA), a neurodegenerative disease caused by the decreased expression of the mitochondrial protein frataxin. Nrf2 signalling is implicated in many molecular aspects of ferroptosis, by upstream regulating glutathione homeostasis, mitochondrial function and lipid metabolism. As Nrf2 is down-regulated in FRDA, targeting Nrf2-mediated ferroptosis in FRDA may be an attractive option to counteract neurodegeneration in such disease, thus paving the way to new therapeutic opportunities. In this study, we evaluated ferroptosis hallmarks in frataxin-silenced mouse myoblasts, in hearts of a frataxin Knockin/Knockout (KIKO) mouse model, in skin fibroblasts and blood of patients, particularly focusing on ferroptosis-driven gene expression, mitochondrial impairment and lipid peroxidation. The efficacy of Nrf2 inducers to neutralize ferroptosis has been also evaluated.
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http://dx.doi.org/10.1016/j.redox.2020.101791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7677700PMC
January 2021

Antioxidant Amelioration of Riboflavin Transporter Deficiency in Motoneurons Derived from Patient-Specific Induced Pluripotent Stem Cells.

Int J Mol Sci 2020 Oct 7;21(19). Epub 2020 Oct 7.

Genetics and Rare Diseases Research Division, IRCCS Ospedale Pediatrico Bambino Gesù, 00146 Rome, Italy.

Mitochondrial dysfunction is a key element in the pathogenesis of neurodegenerative disorders, such as riboflavin transporter deficiency (RTD). This is a rare, childhood-onset disease characterized by motoneuron degeneration and caused by mutations in and , encoding riboflavin (RF) transporters (RFVT2 and RFVT3, respectively), resulting in muscle weakness, ponto-bulbar paralysis and sensorineural deafness. Based on previous findings, which document the contribution of oxidative stress in RTD pathogenesis, we tested possible beneficial effects of several antioxidants (Vitamin C, Idebenone, Coenzyme Q and EPI-743, either alone or in combination with RF) on the morphology and function of neurons derived from induced pluripotent stem cells (iPSCs) from two RTD patients. To identify possible improvement of the neuronal morphotype, neurite length was measured by confocal microscopy after β-III tubulin immunofluorescent staining. Neuronal function was evaluated by determining superoxide anion generation by MitoSOX assay and intracellular calcium (Ca) levels, using the Fluo-4 probe. Among the antioxidants tested, EPI-743 restored the redox status, improved neurite length and ameliorated intracellular calcium influx into RTD motoneurons. In conclusion, we suggest that antioxidant supplementation may have a role in RTD treatment.
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http://dx.doi.org/10.3390/ijms21197402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582490PMC
October 2020

Androgen Receptor signaling promotes the neural progenitor cell pool in the developing cortex.

J Neurochem 2020 Sep 21. Epub 2020 Sep 21.

Laboratory of Neuroembryology, IRCCS Fondazione Santa Lucia, Rome, Italy.

Neural Progenitor Cells (NPCs) are multipotent cells that are able to self-renew and differentiate into neurons. The size of the initial pool of NPCs during the brain development strongly affects the number of neurons that compose cortical multi-layer during development. Gonadal hormones can influence the balance between self-renewal and differentiation processes. Herein, we investigated the role of dihydrotestosterone (DHT), the active metabolite of testosterone, in the regulation of NPC stemness and differentiation. First, we evaluated the expression of the androgen receptor (AR), the transcription factor activated by DHT that mediates the physiological effects of androgens, in NPCs. Western blot analysis showed that DHT-mediated activation of AR induces mitogenic signaling pathways (PI3K/AKT and MAPK/ERK) in NPCs, whereas luciferase activity assays demonstrated the induction of AR transcriptional activity. AR activation mediated by DHT treatment strongly increased the proliferation of NPCs and reduced their propensity to differentiate into neurons. Furthermore, the effects of AR activation were mediated, at least in part, by increased expression of Aldehyde Dehydrogenase 1 Family Member A3 enzyme (ALDH1A3). Pharmacological inhibition of ALDH activity with N,N-diethylaminobenzaldehyde (DEAB) reduced the effect of DHT on NPC proliferation in vitro. Furthermore, inhibition of AR activity by Enzalutamide reduced the NPC pool in the developing cortex of male C57/BL6 mouse embryos. These findings indicate that androgens engage an AR-dependent signaling pathway that impact on neurogenesis by increasing the NPC pool in the developing mouse cortex.
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http://dx.doi.org/10.1111/jnc.15192DOI Listing
September 2020

Sam68 splicing regulation contributes to motor unit establishment in the postnatal skeletal muscle.

Life Sci Alliance 2020 10 4;3(10). Epub 2020 Aug 4.

Department of Movement, Human and Health Sciences, University of Rome "Foro Italico," Rome, Italy

RNA-binding proteins orchestrate the composite life of RNA molecules and impact most physiological processes, thus underlying complex phenotypes. The RNA-binding protein Sam68 regulates differentiation processes by modulating splicing, polyadenylation, and stability of select transcripts. Herein, we found that mice display altered regulation of alternative splicing in the spinal cord of key target genes involved in synaptic functions. Analysis of the motor units revealed that Sam68 ablation impairs the establishment of neuromuscular junctions and causes progressive loss of motor neurons in the spinal cord. Importantly, alterations of neuromuscular junction morphology and properties in mice correlate with defects in muscle and motor unit integrity. muscles display defects in postnatal development, with manifest signs of atrophy. Furthermore, fast-twitch muscles in mice show structural features typical of slow-twitch muscles, suggesting alterations in the metabolic and functional properties of myofibers. Collectively, our data identify a key role for Sam68 in muscle development and suggest that proper establishment of motor units requires timely expression of synaptic splice variants.
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http://dx.doi.org/10.26508/lsa.201900637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409371PMC
October 2020

Compound heterozygosity for an expanded (GAA) and a (GAAGGA) repeat at FXN locus: from a diagnostic pitfall to potential clues to the pathogenesis of Friedreich ataxia.

Neurogenetics 2020 10 7;21(4):279-287. Epub 2020 Jul 7.

Dept of Neuroscience, Faculty of Medicine and Surgery, Università Cattolica del Scaro Cuore, L.go F. Vito 1, 000168, Rome, Italy.

Friedreich's ataxia (FRDA) is usually due to a homozygous GAA expansion in intron 1 of the frataxin (FXN) gene. Rarely, uncommon molecular rearrangements at the FXN locus can cause pitfalls in the molecular diagnosis of FRDA. Here we describe a family whose proband was affected by late-onset Friedreich's ataxia (LOFA); long-range PCR (LR-PCR) documented two small expanded GAA alleles both in the proband and in her unaffected younger sister, who therefore received a diagnosis of pre-symptomatic LOFA. Later studies, however, revealed that the proband's unaffected sister, as well as their healthy mother, were both carriers of an expanded GAA allele and an uncommon (GAAGGA) repeat mimicking a GAA expansion at the LR-PCR that was the cause of the wrong initial diagnosis of pre-symptomatic LOFA. Extensive studies in tissues from all the family members, including LR-PCR, assessment of methylation status of FXN locus, MboII restriction analysis and direct sequencing of LR-PCR products, analysis of FXN mRNA, and frataxin protein expression, support the virtual lack of pathogenicity of the rare (GAAGGA) repeat, also providing significant data about the modulation of epigenetic modifications at the FXN locus. Overall, this report highlights a rare but possible pitfall in FRDA molecular diagnosis, emphasizing the need of further analysis in case of discrepancy between clinical and molecular data.
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http://dx.doi.org/10.1007/s10048-020-00620-7DOI Listing
October 2020

Oxidative Stress in DNA Repeat Expansion Disorders: A Focus on NRF2 Signaling Involvement.

Biomolecules 2020 05 1;10(5). Epub 2020 May 1.

Unit of Muscular and Neurodegenerative Diseases, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy.

DNA repeat expansion disorders are a group of neuromuscular and neurodegenerative diseases that arise from the inheritance of long tracts of nucleotide repetitions, located in the regulatory region, introns, or inside the coding sequence of a gene. Although loss of protein expression and/or the gain of function of its transcribed mRNA or translated product represent the major pathogenic effect of these pathologies, mitochondrial dysfunction and imbalance in redox homeostasis are reported as common features in these disorders, deeply affecting their severity and progression. In this review, we examine the role that the redox imbalance plays in the pathological mechanisms of DNA expansion disorders and the recent advances on antioxidant treatments, particularly focusing on the expression and the activity of the transcription factor NRF2, the main cellular regulator of the antioxidant response.
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http://dx.doi.org/10.3390/biom10050702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7277112PMC
May 2020

Correction: Frataxin deficiency induces lipid accumulation and affects thermogenesis in brown adipose tissue.

Cell Death Dis 2020 Mar 3;11(3):165. Epub 2020 Mar 3.

Department Biology, University of Rome Tor Vergata, via della Ricerca Scientifica, Rome, Italy.

Since online publication of this article, the authors noticed that there was a basic citation error in PubMed citation data. Specifically, the name of the author "Piergiorgio La Rosa" is cited as "Rosa P" in the PubMed citation, when it should be "La Rosa P", "La Rosa" being the surname and "Piergiorgio" the name of the author.
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http://dx.doi.org/10.1038/s41419-020-2347-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054354PMC
March 2020

The NRF2 Signaling Network Defines Clinical Biomarkers and Therapeutic Opportunity in Friedreich's Ataxia.

Int J Mol Sci 2020 01 30;21(3). Epub 2020 Jan 30.

Unit of Muscular and Neurodegenerative Diseases, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy.

Friedreich's ataxia (FA) is a trinucleotide repeats expansion neurodegenerative disorder, for which no cure or approved therapies are present. In most cases, GAA trinucleotide repetitions in the first intron of the gene are the genetic trigger of FA, determining a strong reduction of frataxin, a mitochondrial protein involved in iron homeostasis. Frataxin depletion impairs iron-sulfur cluster biosynthesis and determines iron accumulation in the mitochondria. Mounting evidence suggests that these defects increase oxidative stress susceptibility and reactive oxygen species production in FA, where the pathologic picture is worsened by a defective regulation of the expression and signaling pathway modulation of the transcription factor NF-E2 p45-related factor 2 (NRF2), one of the fundamental mediators of the cellular antioxidant response. NRF2 protein downregulation and impairment of its nuclear translocation can compromise the adequate cellular response to the frataxin depletion-dependent redox imbalance. As NRF2 stability, expression, and activation can be modulated by diverse natural and synthetic compounds, efforts have been made in recent years to understand if regulating NRF2 signaling might ameliorate the pathologic defects in FA. Here we provide an analysis of the pharmaceutical interventions aimed at restoring the NRF2 signaling network in FA, elucidating specific biomarkers useful for monitoring therapeutic effectiveness, and developing new therapeutic tools.
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http://dx.doi.org/10.3390/ijms21030916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037688PMC
January 2020

Frataxin deficiency induces lipid accumulation and affects thermogenesis in brown adipose tissue.

Cell Death Dis 2020 01 23;11(1):51. Epub 2020 Jan 23.

Department Biology, University of Rome Tor Vergata, via della Ricerca Scientifica, Rome, Italy.

Decreased expression of mitochondrial frataxin (FXN) causes Friedreich's ataxia (FRDA), a neurodegenerative disease with type 2 diabetes (T2D) as severe comorbidity. Brown adipose tissue (BAT) is a mitochondria-enriched and anti-diabetic tissue that turns excess energy into heat to maintain metabolic homeostasis. Here we report that the FXN knock-in/knock-out (KIKO) mouse shows hyperlipidemia, reduced energy expenditure and insulin sensitivity, and elevated plasma leptin, recapitulating T2D-like signatures. FXN deficiency leads to disrupted mitochondrial ultrastructure and oxygen consumption as well as lipid accumulation in BAT. Transcriptomic data highlights cold intolerance in association with iron-mediated cell death (ferroptosis). Impaired PKA-mediated lipolysis and expression of genes controlling mitochondrial metabolism, lipid catabolism and adipogenesis were observed in BAT of KIKO mice as well as in FXN-deficient T37i brown and primary adipocytes. Significant susceptibility to ferroptosis was observed in adipocyte precursors that showed increased lipid peroxidation and decreased glutathione peroxidase 4. Collectively our data point to BAT dysfunction in FRDA and suggest BAT as promising therapeutic target to overcome T2D in FRDA.
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http://dx.doi.org/10.1038/s41419-020-2253-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978516PMC
January 2020

Targeting NRF2 for the Treatment of Friedreich's Ataxia: A Comparison among Drugs.

Int J Mol Sci 2019 Oct 21;20(20). Epub 2019 Oct 21.

Unit of Muscular and Neurodegenerative Diseases, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy.

NRF2 (Nuclear factor Erythroid 2-related Factor 2) signaling is impaired in Friedreich's Ataxia (FRDA), an autosomal recessive disease characterized by progressive nervous system damage and degeneration of nerve fibers in the spinal cord and peripheral nerves. The loss of frataxin in patients results in iron sulfur cluster deficiency and iron accumulation in the mitochondria, making FRDA a fatal and debilitating condition. There are no currently approved therapies for the treatment of FRDA and molecules able to activate NRF2 have the potential to induce clinical benefits in patients. In this study, we compared the efficacy of six redox-active drugs, some already adopted in clinical trials, targeting NRF2 activation and frataxin expression in fibroblasts obtained from skin biopsies of FRDA patients. All of these drugs consistently increased NRF2 expression, but differential profiles of NRF2 downstream genes were activated. The Sulforaphane and -acetylcysteine were particularly effective on genes involved in preventing inflammation and maintaining glutathione homeostasis, the dimethyl fumarate, omaxevolone, and EPI-743 in counteracting toxic products accumulation, the idebenone in mitochondrial protection. This study may contribute to develop synergic therapies, based on a combination of treatment molecules.
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http://dx.doi.org/10.3390/ijms20205211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829337PMC
October 2019

Nrf2 Induction Re-establishes a Proper Neuronal Differentiation Program in Friedreich's Ataxia Neural Stem Cells.

Front Cell Neurosci 2019 31;13:356. Epub 2019 Jul 31.

Unit of Neuromuscular and Neurodegenerative Diseases, IRCCS Bambino Gesù Children's Hospital, Rome, Italy.

Frataxin deficiency is the pathogenic cause of Friedreich's Ataxia, an autosomal recessive disease characterized by the increase of oxidative stress and production of free radicals in the cell. Although the onset of the pathology occurs in the second decade of life, cognitive differences and defects in brain structure and functional activation are observed in patients, suggesting developmental defects to take place during fetal neurogenesis. Here, we describe impairments in proliferation, stemness potential and differentiation in neural stem cells (NSCs) isolated from the embryonic cortex of the Frataxin Knockin/Knockout mouse, a disease animal model whose slow-evolving phenotype makes it suitable to study pre-symptomatic defects that may manifest before the clinical onset. We demonstrate that enhancing the expression and activity of the antioxidant response master regulator Nrf2 ameliorates the phenotypic defects observed in NSCs, re-establishing a proper differentiation program.
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http://dx.doi.org/10.3389/fncel.2019.00356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685360PMC
July 2019

Post-transcriptional regulation of FUS and EWS protein expression by miR-141 during neural differentiation.

Hum Mol Genet 2017 07;26(14):2732-2746

Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", 00135 Rome, Italy.

Brain development involves proliferation, migration and specification of neural progenitor cells, culminating in neuronal circuit formation. Mounting evidence indicates that improper regulation of RNA binding proteins (RBPs), including members of the FET (FUS, EWS, TAF15) family, results in defective cortical development and/or neurodegenerative disorders. However, in spite of their physiological relevance, the precise pattern of FET protein expression in developing neurons is largely unknown. Herein, we found that FUS, EWS and TAF15 expression is differentially regulated during brain development, both in time and in space. In particular, our study identifies a fine-tuned regulation of FUS and EWS during neuronal differentiation, whereas TAF15 appears to be more constitutively expressed. Mechanistically FUS and EWS protein expression is regulated at the post-transcriptional level during neuron differentiation and brain development. Moreover, we identified miR-141 as a key regulator of these FET proteins that modulate their expression levels in differentiating neuronal cells. Thus, our studies uncover a novel link between post-transcriptional regulation of FET proteins expression and neurogenesis.
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http://dx.doi.org/10.1093/hmg/ddx160DOI Listing
July 2017

Faulty RNA splicing: consequences and therapeutic opportunities in brain and muscle disorders.

Hum Genet 2017 09 22;136(9):1215-1235. Epub 2017 Apr 22.

Department of Biomedicine and Prevention, University of Rome Tor Vergata, Via Montpellier, 1, 00133, Rome, Italy.

Alternative splicing is a powerful mechanism that largely expands the coding potential of eukaryotic genomes. Indeed, its complex and flexible regulation is exploited by cells to adapt to various environmental conditions, through production of protein variants displaying different functions. Such flexibility, however, is accompanied by high risk of errors, and dysregulation of splicing is now recognized as an important factor in human diseases. Notably, the RNA-based nature of splicing, which involves high specificity through base pair recognition, offers a remarkable therapeutic opportunity by allowing design of tools with elevated target selectivity. Herein, we illustrate examples of how defective splicing, obtained by mutations affecting multiple layers of regulation, can result in pathology. In particular, we focus on splicing-related defects occurring in brain and muscle diseases and describe therapeutic approaches currently available for these pathologies.
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http://dx.doi.org/10.1007/s00439-017-1802-yDOI Listing
September 2017

Sam68 promotes self-renewal and glycolytic metabolism in mouse neural progenitor cells by modulating pre-mRNA 3'-end processing.

Elife 2016 11 15;5. Epub 2016 Nov 15.

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

The balance between self-renewal and differentiation of neural progenitor cells (NPCs) dictates neurogenesis and proper brain development. We found that the RNA- binding protein Sam68 (Khdrbs1) is strongly expressed in neurogenic areas of the neocortex and supports the self-renewing potential of mouse NPCs. Knockout of constricted the pool of proliferating NPCs by accelerating their cell cycle exit and differentiation into post-mitotic neurons. Sam68 function was linked to regulation of pre-mRNA 3'-end processing. Binding of Sam68 to an intronic polyadenylation site prevents its recognition and premature transcript termination, favoring expression of a functional enzyme. The lower ALDH1A3 expression and activity in NPCs results in reduced glycolysis and clonogenicity, thus depleting the embryonic NPC pool and limiting cortical expansion. Our study identifies Sam68 as a key regulator of NPC self-renewal and establishes a novel link between modulation of ALDH1A3 expression and maintenance of high glycolytic metabolism in the developing cortex.
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http://dx.doi.org/10.7554/eLife.20750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5122457PMC
November 2016

Xenoestrogens alter estrogen receptor (ER) α intracellular levels.

PLoS One 2014 20;9(2):e88961. Epub 2014 Feb 20.

Department of Science, University Roma Tre, Rome, Italy.

17β-estradiol (E2)-dependent estrogen receptor (ER) α intracellular concentration is a well recognized critical step in the pleiotropic effects elicited by E2 in several target tissues. Beside E2, a class of synthetic and plant-derived chemicals collectively named endocrine disruptors (EDs) or xenoestrogens bind to and modify both nuclear and extra-nuclear ERα activities. However, at the present no information is available on the ability of EDs to hamper ERα intracellular concentration. Here, the effects of bisphenol A (BPA) and naringenin (Nar), prototypes of synthetic and plant-derived ERα ligands, have been evaluated on ERα levels in MCF-7 cells. Both EDs mimic E2 in triggering ERα Ser118 phosphorylation and gene transcription. However, only E2 or BPA induce an increase of cell proliferation; whereas 24 hrs after Nar stimulation a dose-dependent decrease in cell number is reported. E2 or BPA treatment reduces ERα protein and mRNA levels after 24 hrs. Contrarily, Nar stimulation does not alter ERα content but reduces ERα mRNA levels like other ligands. Co-stimulation experiments indicate that 48 hrs of Nar treatment prevents the E2-induced ERα degradation and hijacks the physiological ability of E2:ERα complex to regulate gene transcription. Mechanistically, Nar induces ERα protein accumulation by preventing proteasomal receptor degradation via persistent activation of p38/MAPK pathway. As a whole these data demonstrate that ERα intracellular concentration is an important target through which EDs hamper the hormonal milieu of E2 target cells driving cells to different outcomes or mimicking E2 even in the absence of the hormone.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0088961PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3930606PMC
January 2015

Identification of an estrogen receptor α non covalent ubiquitin-binding surface: role in 17β-estradiol-induced transcriptional activity.

J Cell Sci 2013 Jun 16;126(Pt 12):2577-82. Epub 2013 Apr 16.

Department of Science, Section Biomedical Science and Technologies, University Roma Tre, Viale Guglielmo Marconi, 446, 00146 Rome, Italy.

Ubiquitin (Ub)-binding domains (UBDs) located in Ub receptors decode the ubiquitination signal by non-covalently engaging the Ub modification on their binding partners and transduce the Ub signalling through Ub-based molecular interactions. In this way, inducible protein ubiquitination regulates diverse biological processes. The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that mediates the pleiotropic effects of the sex hormone 17β-estradiol (E2). Fine regulation of E2 pleiotropic actions depends on E2-dependent ERα association with a plethora of binding partners and/or on the E2 modulation of receptor ubiquitination. Indeed, E2-induced ERα polyubiquitination triggers receptor degradation and transcriptional activity, and E2-dependent reduction in ERα monoubiquitination is crucial for E2 signalling. Monoubiquitinated proteins often contain UBDs, but whether non-covalent Ub-ERα binding could occur and play a role in E2-ERα signalling is unknown. Here, we report an Ub-binding surface within the ERα ligand binding domain that directs in vitro the receptor interaction with both ubiquitinated proteins and recombinant Ub chains. Mutational analysis reveals that ERα residues leucine 429 and alanine 430 are involved in Ub binding. Moreover, impairment of ERα association to ubiquitinated species strongly affects E2-induced ERα transcriptional activity. Considering the importance of UBDs in the Ub-based signalling network and the central role of different ERα binding partners in the modulation of E2-dependent effects, our discoveries provide novel insights into ERα activity that could also be relevant for ERα-dependent diseases.
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http://dx.doi.org/10.1242/jcs.123307DOI Listing
June 2013

Palmitoylation regulates 17β-estradiol-induced estrogen receptor-α degradation and transcriptional activity.

Mol Endocrinol 2012 May 22;26(5):762-74. Epub 2012 Mar 22.

Department of Biology, University Roma Tre, I-00146, Rome, Italy.

The estrogen receptor-α (ERα) is a transcription factor that regulates gene expression through the binding to its cognate hormone 17β-estradiol (E2). ERα transcriptional activity is regulated by E2-evoked 26S proteasome-mediated ERα degradation and ERα serine (S) residue 118 phosphorylation. Furthermore, ERα mediates fast cell responses to E2 through the activation of signaling cascades such as the MAPK/ERK and phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog 1 pathways. These E2 rapid effects require a population of the ERα located at the cell plasma membrane through palmitoylation, a dynamic enzymatic modification mediated by palmitoyl-acyl-transferases. However, whether membrane-initiated and transcriptional ERα activities integrate in a unique picture or represent parallel pathways still remains to be firmly clarified. Hence, we evaluated here the impact of ERα palmitoylation on E2-induced ERα degradation and S118 phosphorylation. The lack of palmitoylation renders ERα more susceptible to E2-dependent degradation, blocks ERα S118 phosphorylation and prevents E2-induced ERα estrogen-responsive element-containing promoter occupancy. Consequently, ERα transcriptional activity is prevented and the receptor addressed to the nuclear matrix subnuclear compartment. These data uncover a circuitry in which receptor palmitoylation links E2-dependent ERα degradation, S118 phosphorylation, and transcriptional activity in a unique molecular mechanism. We propose that rapid E2-dependent signaling could be considered as a prerequisite for ERα transcriptional activity and suggest an integrated model of ERα intracellular signaling where E2-dependent early extranuclear effects control late receptor-dependent nuclear actions.
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http://dx.doi.org/10.1210/me.2011-1208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5417099PMC
May 2012

Susceptibility of estrogen receptor rapid responses to xenoestrogens: Physiological outcomes.

Steroids 2012 Aug 5;77(10):910-7. Epub 2012 Mar 5.

Department of Biology, University Roma TRE, viale G. Marconi, 446, I-00146 Rome, Italy.

17β-Estradiol (E2) binding induces rapid modification in the conformation of its cognate receptors (i.e., ERα and ERβ). These allosteric changes allow the association of ERs with cell specific transcriptional cofactors, thus determining cellular contexts specific variations in gene expression. In addition, E2-ER complexes could also interact with membrane and cytosolic signal molecules triggering extra-nuclear signalling pathways. The synergy between these mechanisms is necessary for E2-induced pleiotropic actions in target tissues. Besides E2, the ER ligand binding domains can accommodate many other natural and synthetic ligands. Several of these compounds act as agonist or antagonist of ER transcriptional activity due to their ability to modify the interactions between ERs and transcriptional co-regulators. However, the ability of natural or manmade ER ligands to affect the extra-nuclear interactions of the ERs has been rarely evaluated. Here, the ability of two diet-derived flavonoids (i.e., naringenin and quercetin) and of the synthetic food-contaminant bisphenol A to modulate specifically ER extra-nuclear signalling pathways will be reported. All the tested compounds bind to both ER subtypes even if lesser than E2 activating divergent signal transduction pathways. In fact, in the presence of ERα, both naringenin and quercetin decouple ERα activities by specifically interfering with ERα membrane initiating signals. On the other hand, bisphenol A, but not flavonoids, maintains ERβ at the membrane thus impairing the activation of the downstream kinases. As a whole, extra-nuclear ER signals are highly susceptible to different ligands that, by unbalancing E2-induced cell functions drive cells to different functional endpoints.
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http://dx.doi.org/10.1016/j.steroids.2012.02.019DOI Listing
August 2012

3-hydroxy 3-methylglutaryl coenzyme A reductase inhibition impairs muscle regeneration.

J Cell Biochem 2012 Jun;113(6):2057-63

Department of Biology, University Roma Tre, Viale Marconi 446, 00146 Rome, Italy.

Skeletal muscle has the ability to regenerate new muscle fibers after injury. The process of new muscle formation requires that quiescent mononuclear muscle precursor cells (myoblasts) become activated, proliferate, differentiate, and fuse into multinucleated myotubes which, in turn, undergo further differentiation and mature to form functional muscle fibers. Previous data demonstrated the crucial role played by 3-hydroxy 3-methylglutaryl coenzyme A reductase (HMGR), the rate-limiting enzyme of cholesterol biosynthetic pathway, in fetal rat myoblast (L6) differentiation. This finding, along with epidemiological studies assessing the myotoxic effect of statins, HMGR inhibitors, allowed us to speculate that HMGR could be strongly involved in skeletal muscle repair. Thus, our research was aimed at evaluating such involvement: in vitro and in vivo experiments were performed on both mouse adult satellite cell derived myoblasts (SCDM) and mouse muscles injured with cardiotoxin. Results demonstrate that HMGR inhibition by the statin Simvastatin reduces SCDM fusion index, fast MHC protein levels by 60% and slow MHC by 40%. Most importantly, HMGR inhibition delays skeletal muscle regeneration in vivo. Thus, besides complaining of myopathies, patients given Simvastatin could also undergo an impairment in muscle repair.
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http://dx.doi.org/10.1002/jcb.24077DOI Listing
June 2012

Signaling functions of ubiquitin in the 17β-estradiol (E2):estrogen receptor (ER) α network.

J Steroid Biochem Mol Biol 2011 Nov 30;127(3-5):223-30. Epub 2011 Jul 30.

Department of Biology, University Roma Tre, Viale Guglielmo Marconi, 446, I-00146 Rome, Italy.

Protein posttranslational modifications (PTMs) are signaling alterations that allow coordinating the cellular responses with the changes in the extracellular environment. In this way, the posttranslationally-modified protein becomes a switch node in the transduction network activated by the specific extracellular stimuli. It is now clear that this is the case also for protein ubiquitination: this extremely versatile PTM controls cell physiology through the modulation of protein stability as well as through the modulation of the dynamics of the intracellular signaling cascades. Recent evidence clearly indicates that such a complex scheme appears to be valid also for the 17β-estradiol (E2):estrogen receptor (ER) α signal transduction pathways. Indeed, beside the long standing notion that ERα ubiquitination is required for the regulation of receptor stability, several laboratories, including our own, have clearly indicated that ERα ubiquitination also serves non-degradative functions. This review will reconsider the role of ubiquitination in E2:ERα signaling by particularly highlighting how the functions of the non-degradative ubiquitination impact on ERα activities and contribute to the modulation of E2-dependent physiological processes.
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http://dx.doi.org/10.1016/j.jsbmb.2011.07.008DOI Listing
November 2011

Effects of salmeterol on skeletal muscle cells: metabolic and proapoptotic features.

Med Sci Sports Exerc 2011 Dec;43(12):2259-73

Department of Health Sciences, University of Rome Foro Italico, Rome, Italy.

Purpose: Salmeterol is a β2-adrenergic receptor agonist widely used for the treatment of asthma and chronic obstructive pulmonary disease. It has been shown that salmeterol is also used at supratherapeutic doses as performance-enhancing substance in sport practice. Although the abuse of β-agonists might determine some adverse effects, the molecular effects of salmeterol on skeletal muscle cells remain unclear.

Methods: We evaluated the effects of salmeterol (0.1-10 μM) on both proliferative and differentiated rat L6C5 and mouse C2C12 skeletal muscle cell lines. The metabolic effects were evaluated by glyceraldehyde phosphate dehydrogenase, lactate dehydrogenase, citrate synthase, 3-OH acyl-CoA dehydrogenase, and alanine transglutaminase activities. Cytotoxic and apoptotic effects were analyzed by 3-(4,5-dimethylthiazol-1)-5-(3-carboxymeth-oxyphenyl)-2H-tetrazolium, trypan blue exclusion assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, Western blot analysis, and immunofluorescence staining.

Results: We showed that salmeterol reduced the growth rate of proliferating cells in a dose- and time-dependent manner (6-48 h). An increase in oxidative metabolism was found after 6 h in C2C12 and L6C5 myoblasts and in C2C12 myotubes with respect to control cells, while in L6C5 myotubes, anaerobic metabolism prevailed. Exposure of myoblasts and myotubes for 48 and 72 h at high salmeterol concentrations induced apoptosis by the activation of the intrinsic apoptotic pathway, as confirmed by the modulation of the apoptotic proteins Bcl-xL, caspase-9, and poly (ADP-ribose) polymerase and by the cytoplasmic release of Smac/DIABLO.

Conclusions: Altogether, our results demonstrate that short-term supratherapeutic salmeterol exposure increased oxidative metabolic pathways on skeletal muscle cells, whereas prolonged treatment inhibits cell growth and exerts either a cytostatic or a proapoptotic effect in a time- and dose-dependent way.
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http://dx.doi.org/10.1249/MSS.0b013e3182223094DOI Listing
December 2011

17β-Estradiol-induced cell proliferation requires estrogen receptor (ER) α monoubiquitination.

Cell Signal 2011 Jul 26;23(7):1128-35. Epub 2011 Feb 26.

Department of Biology, University Roma Tre, Viale Guglielmo Marconi 446, Rome, Italy.

Protein monoubiquitination (monoUbq) (i.e., the attachment of one single ubiquitin to the substrate) is a non-proteolytic reversible modification that controls protein functions. Among other proteins, the estrogen receptor α (ERα), which mediates the pleiotropic effects of the cognate hormone 17β-estradiol (E2), is a monoubiquitinated protein. Although it has been demonstrated that E2 rapidly reduces ERα monoUbq in breast cancer cells, the impact of monoUbq in the regulation of the ERα activities is poorly appreciated. Here, we show that mutation of the ERα monoUbq sites prevents the E2-induced ERα phosphorylation in the serine residue 118 (S118), reduces ERα transcriptional activity, and precludes the ERα-mediated extranuclear activation of signaling pathways (i.e., AKT activation) thus impeding the E2-induced cyclin D1 promoter activation and consequently cell proliferation. In addition, the interference with ERα monoUbq deregulates E2-induced association of ERα to the insulin like growth factor receptor (IGF-1-R). Altogether these data demonstrate an inherent role for monoUbq in ERα signaling and point to the physiological function of ERα monoUbq in the regulation of E2-induced cell proliferation.
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http://dx.doi.org/10.1016/j.cellsig.2011.02.006DOI Listing
July 2011

17β-estradiol regulates estrogen receptor α monoubiquitination.

IUBMB Life 2011 Jan;63(1):49-53

Department of Biology, University Roma Tre, Viale Guglielmo Marconi, 446, Rome, Italy.

Monoubiquitination is a nonproteolytic signal involved in a network of several different physiological processes. Recently, monoubiquitination has been discovered as a new post-transductional modification of the estrogen receptor α (ERα). However, at present no information is available about the role of the cognate ligand 17β-estradiol (E2) in modulating this receptor post-transductional modification. Thus, we studied the E2-dependent modulation of ERα monoubiquitination in different cell lines. Here, we report that ERα monoubiquitination isnegatively modulated by E2. These results demonstrate thatERα monoubiquitination represents a new signalling modification that may modulate the E2:ERα-regulated cellular processes.
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http://dx.doi.org/10.1002/iub.414DOI Listing
January 2011