Abstract
Common pathological features of amyotrophic lateral sclerosis (ALS) include cytoplasmic aggregation of several RNA-binding proteins. Out of these RNA-binding proteins, TDP-43, FUS/TLS and RGNEF have been shown to co-aggregate with one another within motor neurons of sporadic ALS (sALS) patients, suggesting that there may be a common regulatory network disrupted. MiRNAs have been a recent focus in ALS research as they have been identified to be globally down-regulated in the spinal cord of ALS patients. The objective of this study was to identify if there are miRNA(s) dysregulated in sALS that are responsible for regulating the TDP-43, FUS/TLS and RGNEF network. In this study, we identify miR-194 and miR-b2122 to be significantly down-regulated in sALS patients, and were predicted to regulate TARDBP, FUS/TLS and RGNEF expression. Reporter gene assays and RT-qPCR revealed that miR-b2122 down-regulates the reporter gene through direct interactions with either the TARDBP, FUS/TLS, or RGNEF 3’UTR, while miR-194 down-regulates firefly expression when it contained either the TARDBP or FUS/TLS 3’UTR. Further, we showed that miR-b2122 regulates endogenous expression of all three of these genes in a neuronal-derived cell line. Also, an ALS-associated mutation in the FUS/TLS 3’UTR ablates the ability of miR-b2122 to regulate reporter gene linked to FUS/TLS 3’UTR, and sALS samples which showed a down-regulation in miR-b2122 also showed an increase in FUS/TLS protein expression. Overall, we have identified a novel miRNA that is down-regulated in sALS that appears to be a central regulator of disease-related RNA-binding proteins, and thus its dysregulation likely contributes to TDP-43, FUS/TLS and RGNEF pathogenesis in sALS.
Highlights
Amyotrophic lateral sclerosis (ALS) is a progressive motor neurodegenerative disease resulting in paralysis and death within 2–5 years after diagnosis [1, 2]. 5–10% of ALS cases are familial, while the remaining are sporadic ~10–12% of these latter cases have a genetic basis [1, 3, 4]
Our results suggest that the down-regulation of miR-b2122 within sporadic ALS (sALS) cases could result in altered levels of all three of these RNA-binding proteins, contributing to the pathological state of TAR DNA-binding protein (TDP-43), FUS and Rho guanine nucleotide exchange factor (RGNEF) observed within motor neurons of sALS patients
It has been previously shown that the pathogenesis of sALS likely does not rely on the dysregulation of a single RNA-binding protein, but a combination of TDP-43, Fused in sarcoma/translocation in liposarcoma (FUS/TLS) and RGNEF, as they co-aggregate with each other in motor neurons of sALS patients [6]
Summary
Amyotrophic lateral sclerosis (ALS) is a progressive motor neurodegenerative disease resulting in paralysis and death within 2–5 years after diagnosis [1, 2]. 5–10% of ALS cases are familial (fALS), while the remaining are sporadic (sALS) ~10–12% of these latter cases have a genetic basis [1, 3, 4]. It has been shown that miRNAs, essential regulators of mRNA expression and protein synthesis, are globally down-regulated within the spinal cord tissue of sALS patients [13, 14] This down-regulation of miRNA expression has been shown to be motor neuron specific [15], contributing to the concept that altered miRNA homeostasis is a major contributor to the pathogenesis of ALS [16, 17]. The finding of this global down-regulation of miRNAs within sALS patients is intriguing, as TDP-43 and FUS/TLS, two proteins often found to be dysregulated in sALS, are known to be essential components of miRNA biogenesis [18, 19]. This suggests that there may be a disruption in the feedback networks between miRNAs and RNA-binding proteins in ALS, including TDP-43 and FUS/TLS
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