The lncRNA ZEB1-AS1 is implicated in sporadic ALS: role in neuronal differentiation and characterization of a novel disease pathway

RNAs dysregulation is becoming more and more relevant in the pathogenesis of sporadic ALS (sALS), 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. We aimed to characterize the role of the lncRNA ZEB1-AS1 in ALS pathogenesis identifying a possible disease-modifying target. Targets expression levels were determined by Real Time PCR, Western Blot and Immunofluorescence. ZEB1-AS1 localization was also identified. Live&Dead Assay and MTT assay were performed to assess cell viability. In PBMCs, undifferentiated SH-SY5Y cells and differentiated SH-SY5Y cells silenced for ZEB1-AS1 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. Our results show an implication for ZEB1-AS1’s pathway in sALS and specifically in neuronal differentiation.