Introduction: Alterations in the expression levels of RNAs in the pathogenesis of sporadic ALS (sALS) are becoming increasingly relevant, with RNA-seq data highlighting numerous deregulated long non-coding RNAs (lncRNAs) in tissues derived from sALS patients. The oncogenic lncRNA ZEB1-AS1 emerged as strongly downregulated in peripheral blood mononuclear cells (PBMCs) of sALS patients. In cancer-derived cell lines, ZEB1-AS1 has been shown to act in a feedback negative loop with mir200c, acting as a molecular sponge for this miRNA. Furthermore, ZEB1-AS1’s interaction with mir200c results in the upregulation of the downstream molecule BMI1. Objective: The aim of our work was thus to characterize the role of the lncRNA ZEB1-AS1 in ALS pathogenesis identifying a possible disease-modifying target. Methods: Total RNA was extracted using TRIZOL reagent and the genes’ expression levels were determined by Real Time PCR. Nuclear and cytoplasmic RNA was extracted using the Cytoplasmic & Nuclear RNA Purification Kit and genes’ expression levels were measured by ddPCR. Western Blot analysis and immunofluorescence were also performed to assess protein expression levels. Live&Dead Assay and MTT assay were performed to assess cell viability. Results: In PBMCs, undifferentiated SH-SY5Y cells and differentiated SH-SY5Y cells silenced for ZEB1-AS1 we validated the downregulation of ZEB1-AS1’s expression but we did not see a concordant downregulation of its sense gene ZEB1. We observed an increase of mir200c and a decrease of BMI1, in an opposite pattern to what is observed in cancer, suggesting a specific pathway in sALS. Furthermore, we observed an upregulation of BMI1’s downstream mediator GSK3b, which results inactivated. The silencing of ZEB1-AS1 in SH-SY5Y did not impact on cellular viability. Moreover, we found that ZEB1 and ZEB1-AS1’s levels change during neuronal differentiation, suggesting an implication for the lncRNA in this process. Indeed, ZEB1-AS1’s silencing results in an alteration in neurite length. We demonstrated that ZEB1-AS1 can bind the ALS-implicated RNA binding protein FUS, both in SH-SY5Y cells and in PBMCs, and in this last tissue we found a reduction in the amount of ZEB1-AS1 bound to FUS in sALS patients. Conclusions: Our results show an implication for ZEB1-AS1’s pathway in sALS and specifically in neuronal differentiation. We also report an impaired interaction of ZEB1-AS1 with FUS in sALS patients, suggesting this is the mechanism connecting ZEB1-AS1 to sALS pathology.

Implications for the lncRNA ZEB1-AS1 in sporadic ALS : deregulation in neuronal differentiation and characterization of a novel disease pathway / F. Rey, C. Pandini, B. Barzaghini, E. Maghraby, R. Launi, M. Bordoni, M. Teresa Raimondi, O. Pansarasa, S. Gagliardi, C. Cereda, G.V. Zuccotti, S. Carelli. ((Intervento presentato al convegno ENCALS - European network to cure ALS tenutosi a online nel 2021.

Implications for the lncRNA ZEB1-AS1 in sporadic ALS : deregulation in neuronal differentiation and characterization of a novel disease pathway

F. Rey;C. Pandini;M. Bordoni;G.V. Zuccotti;S. Carelli
2021

Abstract

Introduction: Alterations in the expression levels of RNAs in the pathogenesis of sporadic ALS (sALS) are becoming increasingly relevant, with RNA-seq data highlighting numerous deregulated long non-coding RNAs (lncRNAs) in tissues derived from sALS patients. The oncogenic lncRNA ZEB1-AS1 emerged as strongly downregulated in peripheral blood mononuclear cells (PBMCs) of sALS patients. In cancer-derived cell lines, ZEB1-AS1 has been shown to act in a feedback negative loop with mir200c, acting as a molecular sponge for this miRNA. Furthermore, ZEB1-AS1’s interaction with mir200c results in the upregulation of the downstream molecule BMI1. Objective: The aim of our work was thus to characterize the role of the lncRNA ZEB1-AS1 in ALS pathogenesis identifying a possible disease-modifying target. Methods: Total RNA was extracted using TRIZOL reagent and the genes’ expression levels were determined by Real Time PCR. Nuclear and cytoplasmic RNA was extracted using the Cytoplasmic & Nuclear RNA Purification Kit and genes’ expression levels were measured by ddPCR. Western Blot analysis and immunofluorescence were also performed to assess protein expression levels. Live&Dead Assay and MTT assay were performed to assess cell viability. Results: In PBMCs, undifferentiated SH-SY5Y cells and differentiated SH-SY5Y cells silenced for ZEB1-AS1 we validated the downregulation of ZEB1-AS1’s expression but we did not see a concordant downregulation of its sense gene ZEB1. We observed an increase of mir200c and a decrease of BMI1, in an opposite pattern to what is observed in cancer, suggesting a specific pathway in sALS. Furthermore, we observed an upregulation of BMI1’s downstream mediator GSK3b, which results inactivated. The silencing of ZEB1-AS1 in SH-SY5Y did not impact on cellular viability. Moreover, we found that ZEB1 and ZEB1-AS1’s levels change during neuronal differentiation, suggesting an implication for the lncRNA in this process. Indeed, ZEB1-AS1’s silencing results in an alteration in neurite length. We demonstrated that ZEB1-AS1 can bind the ALS-implicated RNA binding protein FUS, both in SH-SY5Y cells and in PBMCs, and in this last tissue we found a reduction in the amount of ZEB1-AS1 bound to FUS in sALS patients. Conclusions: Our results show an implication for ZEB1-AS1’s pathway in sALS and specifically in neuronal differentiation. We also report an impaired interaction of ZEB1-AS1 with FUS in sALS patients, suggesting this is the mechanism connecting ZEB1-AS1 to sALS pathology.
Settore BIO/11 - Biologia Molecolare
European network to cure ALS-Amyotrophic Lateral Sclerosis
Implications for the lncRNA ZEB1-AS1 in sporadic ALS : deregulation in neuronal differentiation and characterization of a novel disease pathway / F. Rey, C. Pandini, B. Barzaghini, E. Maghraby, R. Launi, M. Bordoni, M. Teresa Raimondi, O. Pansarasa, S. Gagliardi, C. Cereda, G.V. Zuccotti, S. Carelli. ((Intervento presentato al convegno ENCALS - European network to cure ALS tenutosi a online nel 2021.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/844122
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