MotomiRs: miRNAs in Motor Neuron Function and Disease

MiRNAs are key regulators of the mammalian transcriptome that have been increasingly linked to degenerative diseases of the motor neurons. Although many of the miRNAs currently incriminated as participants in the pathogenesis of these diseases are also important to the normal development and function of motor neurons, at present there is no knowledge of the complete miRNA profile of motor neurons. In this review, we examine the current understanding with respect to miRNAs that are specifically required for motor neuron development, function and viability, and provide evidence that these should be considered as a functional network of miRNAs which we have collectively termed MotomiRs. We will also summarize those MotomiRs currently known to be associated with both amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), and discuss their potential use as biomarkers

Dysregulation of human NEFM and NEFH mRNA stability by ALS-linked miRNAs

Abstract Neurofilaments (NFs) are the most abundant cytoskeletal component of vertebrate myelinated axons. NFs function by determining axonal caliber, promoting axonal growth and forming a 3-dimensional lattice that supports the organization of cytoplasmic organelles. The stoichiometry of NF protein subunits (NFL, NFM and NFH) has to be tightly controlled to avoid the formation of NF neuronal cytoplasmic inclusions (NCIs), axonal degeneration and neuronal death, all pathological hallmarks of amyotrophic lateral sclerosis (ALS). The post-transcriptional control of NF transcripts is critical for regulating normal levels of NF proteins. Previously, we showed that miRNAs that are dysregulated in ALS spinal cord regulate the levels of NEFL mRNA. In order to complete the understanding of altered NF expression in ALS, in this study we have investigated the regulation of NEFM and NEFH mRNA levels by miRNAs. We observed that a small group of ALS-linked miRNAs that are expressed in human spinal motor neurons directly regulate NEFM and NEFH transcript levels in a manner that is associated with an increase in NFM and NFH protein levels in ALS spinal cord homogenates. In concert with previous observations demonstrating the suppression of NEFL mRNA steady state levels in ALS, these observations provide support for the hypothesis that the dysregulation of miRNAs in spinal motor neurons in ALS fundamentally alters the stoichiometry of NF expression, leading to the formation of pathological NCIs

Novel miR-b2122 regulates several ALS-related RNA-binding proteins

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

Analysis of novel NEFL mRNA targeting microRNAs in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by progressive motor neuron degeneration and neurofilament aggregate formation. Spinal motor neurons in ALS also show a selective suppression in the levels of low molecular weight neurofilament (NEFL) mRNA. We have been interested in investigating the role of microRNAs (miRNAs) in NEFL transcript stability. MiRNAs are small, 20-25 nucleotide, non-coding RNAs that act as post-transcriptional gene regulators by targeting the 3' untranslated region (3'UTR) of mRNA resulting in mRNA decay or translational silencing. In this study, we characterized putative novel miRNAs from a small RNA library derived from control and sporadic ALS (sALS) spinal cords. We detected 80 putative novel miRNAs, 24 of which have miRNA response elements (MREs) within the NEFL mRNA 3'UTR. From this group, we determined by real-time PCR that 10 miRNAs were differentially expressed in sALS compared to controls. Functional analysis by reporter gene assay and relative quantitative RT-PCR showed that two novel miRNAs, miR-b1336 and miR-b2403, were downregulated in ALS spinal cord and that both stabilize NEFL mRNA. We confirmed the direct effect of these latter miRNAs using anit-miR-b1336 and anti-miR-b2403. These results demonstrate that the expression of two miRNAs (miRNAs miR-b1336 and miR-b2403) whose effect is to stabilize NEFL mRNA are down regulated in ALS, the net effect of which is predicted to contribute directly to the loss of NEFL steady state mRNA which is pathognomic of spinal motor neurons in ALS

Additional file 1: Table S1. of Novel miR-b2122 regulates several ALS-related RNA-binding proteins

Site-directed mutagenesis primers for TARDBP, FUS/TLS and RGNEF 3’UTRs. (DOCX 16 kb

Additional file 4: Figure S3. of Novel miR-b2122 regulates several ALS-related RNA-binding proteins

Let-7a has no effect on protein levels of TDP-43, FUS/TLS, or RGNEF within SH-SY5Y cells. Let-7a was transfected into SH-SY5Y cells to determine if it changed the basal protein levels of TDP-43, FUS/TLS or RGNEF, and was compared to a non-transfected control. The data indicated no significant change in the protein levels of either TDP-43 (p=0.71), FUS/TLS (p=0.28), or RGNEF (p=0.87) between the two conditions. Data is expressed as sample mean ± SEM, and significance was determined using a Student’s t-test. (TIFF 215 kb

Additional file 2: Figure S1. of Novel miR-b2122 regulates several ALS-related RNA-binding proteins

3’UTR isoforms of RNA-binding proteins, and miR-194 and miR-b2122 are expressed in SH-SY5Y cells. (A) 3’RACE PCR showing TARDBP, FUS/TLS and RGNEF 3’UTR isoforms expressed in SH-SY5Y cells. FUS/TLS and RGNEF isoforms match those expressed in human spinal cord. TARDBP showed multiple isoforms, but only the 1398b isoform identified in spinal cord could be confirmed by sequencing. (B) Real-time PCR indicating the expression of miR-194 and miR-b2122 in SH-SY5Y cells. (TIFF 171 kb

Patient demographics of samples used in construct the small RNA libraries.

<p>Patient demographics of samples used in construct the small RNA libraries.</p

A group of novel miRNAs dysregulated in sALS regulates the activity of a reporter linked to <i>NEFL</i> 3′UTR.

<p>Luciferase reporter gene assay was performed by transfecting HEK293T cells with pre-miRNAs and a reporter construct expressing Firefly luciferase coupled to <i>NEFL</i> 3′UTR. Data are expressed as relative change compared to empty vector and show positive values as up-regulation and negative values as down-regulation. Experiments were performed in triplicate. Results are shown as mean ± SEM (<i>t</i>-test: p<0.001 = ***; p<0.005 = **).</p