Original Research ArticlemiR-129-5p: A key factor and therapeutic target in amyotrophic lateral sclerosis
Introduction
Amyotrophic lateral sclerosis (ALS) is a relentless neurodegenerative disease with no effective therapeutic options. Phenotypic variability and lack of predictive models are major issues in ALS, and the need for state-specific biomarkers is therefore high. Furthermore, despite the considerable amount of data on the pathogenic mechanisms from different ALS models, a common pathophysiological mechanism that would promote meaningful therapeutic advances remains to be identified.
The genetic and environmental causes of ALS are still under investigation, but 90 % of ALS cases are classified as sporadic, and only approximately 10 % of patients have a familial history (Chia et al., 2018; Renton et al., 2014). The best-studied genetic causes of ALS are mutations in or deletion of the Cu/Zn Super Oxide Dismutase 1 (SOD1) gene (Rosen et al., 1993). Recently, using advanced genomic screening tools, researchers identified several other genes associated with ALS, including TARDBP, encoding TDP-43; Fused in Sarcoma (FUS); and C9ORF72 (DeJesus-Hernandez et al., 2011; Kwiatkowski et al., 2009; Vance et al., 2009; Yokoseki et al., 2008; Kabashi et al., 2008; Sreedharan et al., 2008). While C9ORF72 has been identified as the most prevalent mutated gene among ALS patients, with 40 % of familial ALS (fALS) patients carrying a mutation in this gene (Majounie et al., 2012), the abundance and variety of identified SOD1 mutations, which are found in 20 % of fALS cases, have made this a widespread experimental paradigm (Renton et al., 2014).
Interestingly, TDP-43 and FUS are RNA-binding proteins that function in mRNA and miRNA biogenesis (Kawahara and Mieda-Sato, 2012; Buratti et al., 2010; Morlando et al., 2012). miRNAs are small noncoding RNAs that regulate eukaryotic gene expression at the post-transcriptional level, mainly exerting a repressive function by governing the translation and degradation of target mRNAs (Loffreda et al., 2015). Several observations support the importance of miRNAs in neuronal physiology [reviewed in Sun et al. (2013)]. Importantly, the disruption of miRNA expression in Purkinje cells by postnatal ablation of DICER, a crucial miRNA maturation factor, was shown to lead to neurodegeneration (Schaefer et al., 2007), indicating an essential role of miRNAs in the survival of differentiated post-mitotic neurons. Moreover, evidence is accumulating for a critical role of specific miRNAs in neurodegenerative disorders, as in the case of miR-206/miR-153 in Alzheimer’s disease (Lee et al., 2012; Liang et al., 2012) or miR-9/miR-9* in Huntington’s disease (Packer et al., 2008). miRNA expression has also been repeatedly investigated in motor neuron (MN) diseases (Vance et al., 2009; Haramati et al., 2010; Parisi et al., 2016; Butovsky et al., 2015a; Emde et al., 2015), including ALS, where a global reduction of mature miRNAs and alterations in miRNA processing were found in post-mortem spinal cord samples of patients (Figueroa-Romero et al., 2016). In addition, specific miRNAs were found to be dysregulated in the cerebrospinal fluid, serum and leukocytes of ALS patients (Benigni, 2016; De Felice et al., 2014; Takahashi et al., 2015; Freischmidt et al., 2015; Tasca et al., 2016).
Because they regulate multiple biological processes, miRNAs have gained increasing attention as promising candidates for novel biomarkers (Gaughwin et al., 2011; Miyachi et al., 2010; Galimberti et al., 2014; Keller et al., 2011; Cloutier et al., 2015) and therapeutic targets. Currently, several miRNA-based therapeutic strategies are being investigated for the treatment of human cancers (Rupaimoole and Slack, 2017). Two miRNA-based therapeutic strategies are being explored in vivo: the restoration of miRNA expression using miRNA mimics and inhibition with anti-miRNA molecules to block the function of the miRNA of interest. Similar approaches could be envisaged for neurodegenerative diseases, although delivery to the CNS represents an additional challenge. Nevertheless, the recent FDA approval of an antisense oligonucleotide (ASO)-based therapeutic strategy for spinal muscular atrophy (SMA) provides a successful model of intervention for other MN diseases, including ALS (Parente and Corti, 2018).
Here, we identified an upregulated miRNA, miR-129-5p, whose expression was consistently increased in different models of SOD1-linked ALS and in peripheral blood mononuclear cells (PBMCs) of sporadic ALS (sALS) patients. We demonstrated that miR-129-5p targets the ELAVL4 gene transcript, which encodes the RNA-binding protein HuD. HuD is predominantly expressed in neurons where it controls splicing, translation, localization, and stability of several important neuronal mRNAs [reviewed in Bronicki and Jasmin (2013)]. Overexpression of pre-miR-129-1 inhibited neurite outgrowth and differentiation via HuD silencing in vitro, while its inhibition with an antagomir rescued the phenotype. Importantly, we showed that administration of an ASO inhibitor of miR-129 to SOD1(G93A) mice extends survival and rescues the body weight and grip strength loss. These findings identify miR-129 as a promising therapeutic target that is amenable to ASO modulation for ALS.
Section snippets
Cell lines
HEK293T cells and SH-SY5Y cells, either untransfected or stably transfected with cDNAs encoding wild type SOD1 or the mutant SOD1(G93A) (Carri et al., 1997), SH-SY5Y/miR-129-1, SH-SY5Y/Vec, NSC-34/miR-129-1, and NSC-34/Vec (Babetto et al., 2005), were cultured in DMEM high-glucose medium, 10 % fetal bovine serum (FBS), 2.5 mM l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (all products were purchased from Euroclone) at 37 °C with 5 % CO2. Stably transfected cells were maintained
miR-129-5p is upregulated in human and mouse models of SOD1-linked ALS and in PBMCs of sALS patients
Dysregulated miRNA biogenesis and expression is a key pathogenetic element in ALS pathogenesis. To thoroughly investigate this phenomenon, we analyzed the expression levels of the components in the miRNA processing apparatus in an ALS SOD1-linked cellular model.
We compared the abundance of components in the miRNA biogenesis machinery between human neuroblastoma SH-SY5Y cells stably expressing either the wild type SOD1 (SH-SY5Y/SOD1 cells) or the mutant SOD1(G93A) protein [SH-SY5Y/SOD1(G93A)
Discussion
In the present study, we showed that miR-129-5p is upregulated in familial SOD1-linked ALS and in sALS, where it suppresses HuD expression and impairs neurite formation. Moreover, inhibition of miR-129-5p with an antagomir restored neuritogenesis in vitro and ameliorated survival and neuromuscular function when administered in the CSF of SOD1(G93A) mice in vivo.
Since their discovery, more than 2500 miRNAs have been identified in human cells according to the most recent release of the miRBase
Authors’ contributions
A.L. conceived and performed the experiments and the data analysis for Fig. 1, Fig. 2, Fig. 3 and associated supplementary data and participated in preparing figures and tables. M.N. conducted the in vivo MO studies and provided data for Fig. 4 and participated in the manuscript writing. A.A. conducted the mice studies and provided data thereof. M-D.R. performed the experiment in Fig. S1A. R.A.C. was responsible for miRNA-seq and data analysis. S.V. performed the T-REX analysis and
Ethical approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the
Conflict of interests
AL, MN, SC and SB have deposited a patent n. PCT/EP2020/058571 - PCT 143051 ("Inhibitor of iR-129 and uses therof"). The authors have no other competing interest.
Acknowledgments
We would like to dedicate this paper to the memory of Maria Teresa Carrì and thank her for her precious contribution to the ALS field. We are in debt to the patients and their families for their participation in this project. We also thank the Italian Association for ALS (AISLA) for their continuous support and G. Meister, T. Treiber and N. Treiber for technical assistance. We thank A. Poletti for the kind gift of NSC-34/SOD1 and NSC-34/SOD1(G93A) cells and M.T. Carri’ for the SOD1(G93A) mouse
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- 1
Present address: Istituto Scientifico Ospedale San Raffaele, Centro di Imaging Sperimentale 20132 Milano, Italy.
- 2
Present address: UK Dementia Research Institute at King’s College London, Institute of Psychiatry, Psychology and Neuroscience, King’s College London SE5 9NU London, UK.