Amyotrophic Lateral Sclerosis (ALS) is a progressive fatal neuromuscular disease characterized by selective motorneurons loss. Since mutations in TARDBP and FUS genes were discovered to cause familial form of ALS and TDP-43 and FUS proteins play important roles in RNA metabolism, transcriptional alterations emerged as potential pathogenic mechanism. RNA metabolism include several aspects of RNA regulation such as RNA transcription, maturations and regulation. In this study we have investigated two different fields of RNA metabolism: the first one concerns to microRNAs (miRNA) which regulate translation of several mRNAs, and the second one is related to a specific muscular and neuronal transcription factor potential involved in ALS. First, we have assessed any selected miRNAs with neuronal functions in human neuroblastoma cell lines expressing the pathological SOD1(G93A) mutation and we found a small group of altered miRNAs. Subsequently, we explored these miRNAs in the spinal cord of transgenic SOD1(G93A) mice identified a panel of targets commonly altered in SOD1 ALS models. Furthermore, we assessed the expression levels of a panel of selected miRNAs in circulating cells obtain from patients affected by sporadic ALS form (sALS). This approach let us to identify two microRNAs (miR129-5p and miR200c) that were up-regulated in both SOD1 ALS models and in blood cells of patients with sporadic form of disease, evidencing two possible parameters potentially involved in the pathogenesis of both the sporadic and the familial form of ALS. Moreover, we also identified HuD protein as a potential molecular target of miR129-5p; this protein has been previously reported to play a role in neuronal plasticity and in recovery from axonal injury. Indeed, in a cell line stably overespressing mir129-5p we found a reduction in neurite outgrowth and decreased expression levels of differentiation markers with respect to control cells. Taken together these data strongly suggest that microRNAs play a role in ALS pathogenesis and in particular that mir129-5p can affect neuronal plasticity by modulating HuD levels. In the second part of the study we investigated the possible involvement of two members of myocyte enhancer factor 2 (MEF2) family in the pathogenesis of ALS. MEF2D and MEF2C are transcriptional factors playing crucial roles both in muscle and in neuron development and maintenance. We have performed gene expression analysis in peripheral blood mononuclear cells (PBMCs), we showed a strong increased in MEF2D and MEF2C levels both in sporadic and in familial ALS (SOD1+) patients and a direct correlation between MEF2D and MEF2C mRNA levels was observed in patients and controls. Although protein levels were unchanged, a different pattern of distribution for MEF2D-MEF2C proteins in patient cells was found, suggesting a possible lack of their function. To evaluate the transcriptional activity of MEF2 proteins mRNA levels of their downstream targets BDNF, KLF6, RUFY3 and NPEPPS were assessed. Our results showed a significant down-regulation of BDNF, KLF6 and RUFY3 levels confirming that transcriptional activity of both MEF2D and MEF2C isoforms was altered in sporadic and familial ALS patients. In conclusion, our results evidenced a systemic alteration of MEF2D and MEF2C pathways in ALS patients independently from the presence of SOD1 gene mutations, highlighting a possible common feature between the sporadic and the familiar form of disease which are characterized by a different clinical phenotype and pathological hallmarks.

(2015). Study of transcriptional alterations in Amyotrophic Lateral Sclerosis. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).

Study of transcriptional alterations in Amyotrophic Lateral Sclerosis

AROSIO, ALESSANDRO
2015

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a progressive fatal neuromuscular disease characterized by selective motorneurons loss. Since mutations in TARDBP and FUS genes were discovered to cause familial form of ALS and TDP-43 and FUS proteins play important roles in RNA metabolism, transcriptional alterations emerged as potential pathogenic mechanism. RNA metabolism include several aspects of RNA regulation such as RNA transcription, maturations and regulation. In this study we have investigated two different fields of RNA metabolism: the first one concerns to microRNAs (miRNA) which regulate translation of several mRNAs, and the second one is related to a specific muscular and neuronal transcription factor potential involved in ALS. First, we have assessed any selected miRNAs with neuronal functions in human neuroblastoma cell lines expressing the pathological SOD1(G93A) mutation and we found a small group of altered miRNAs. Subsequently, we explored these miRNAs in the spinal cord of transgenic SOD1(G93A) mice identified a panel of targets commonly altered in SOD1 ALS models. Furthermore, we assessed the expression levels of a panel of selected miRNAs in circulating cells obtain from patients affected by sporadic ALS form (sALS). This approach let us to identify two microRNAs (miR129-5p and miR200c) that were up-regulated in both SOD1 ALS models and in blood cells of patients with sporadic form of disease, evidencing two possible parameters potentially involved in the pathogenesis of both the sporadic and the familial form of ALS. Moreover, we also identified HuD protein as a potential molecular target of miR129-5p; this protein has been previously reported to play a role in neuronal plasticity and in recovery from axonal injury. Indeed, in a cell line stably overespressing mir129-5p we found a reduction in neurite outgrowth and decreased expression levels of differentiation markers with respect to control cells. Taken together these data strongly suggest that microRNAs play a role in ALS pathogenesis and in particular that mir129-5p can affect neuronal plasticity by modulating HuD levels. In the second part of the study we investigated the possible involvement of two members of myocyte enhancer factor 2 (MEF2) family in the pathogenesis of ALS. MEF2D and MEF2C are transcriptional factors playing crucial roles both in muscle and in neuron development and maintenance. We have performed gene expression analysis in peripheral blood mononuclear cells (PBMCs), we showed a strong increased in MEF2D and MEF2C levels both in sporadic and in familial ALS (SOD1+) patients and a direct correlation between MEF2D and MEF2C mRNA levels was observed in patients and controls. Although protein levels were unchanged, a different pattern of distribution for MEF2D-MEF2C proteins in patient cells was found, suggesting a possible lack of their function. To evaluate the transcriptional activity of MEF2 proteins mRNA levels of their downstream targets BDNF, KLF6, RUFY3 and NPEPPS were assessed. Our results showed a significant down-regulation of BDNF, KLF6 and RUFY3 levels confirming that transcriptional activity of both MEF2D and MEF2C isoforms was altered in sporadic and familial ALS patients. In conclusion, our results evidenced a systemic alteration of MEF2D and MEF2C pathways in ALS patients independently from the presence of SOD1 gene mutations, highlighting a possible common feature between the sporadic and the familiar form of disease which are characterized by a different clinical phenotype and pathological hallmarks.
FERRARESE, CARLO
ALS; microRNA; miR129-5p; MEF2D; MEF2C
MED/26 - NEUROLOGIA
Italian
27-nov-2015
Scuola di Dottorato in Scienze Mediche Sperimentali e Cliniche
NEUROSCIENZE - 17R
28
2014/2015
(2015). Study of transcriptional alterations in Amyotrophic Lateral Sclerosis. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2015).
File in questo prodotto:
File Dimensione Formato  
phd_unimib_701648.pdf

Accesso Aperto

Descrizione: Tesi dottorato
Tipologia di allegato: Doctoral thesis
Dimensione 5.35 MB
Formato Adobe PDF
5.35 MB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/94396
Citazioni
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
Social impact