Original Research Article
miR-129-5p: A key factor and therapeutic target in amyotrophic lateral sclerosis

https://doi.org/10.1016/j.pneurobio.2020.101803Get rights and content

Highlights

  • miR-129-5p is increased in different models of SOD1-linked ALS and in peripheral blood cells of sporadic ALS patients.

  • miR-129-5p upregulation causes the downregulation of one of its targets: the RNA-binding protein ELAVL4/HuD.

  • ASO targeting miR-129-5p in SOD1(G93A) mice results in significant improve in survival and in neuromuscular phenotype.

  • Overexpression of miR-129 inhibits neurite outgrowth in vitro, while its inhibition with antagomir rescues the phenotype in vivo.

Abstract

Amyotrophic lateral sclerosis (ALS) is a relentless and fatal neurological disease characterized by the selective degeneration of motor neurons. No effective therapy is available for this disease. Several lines of evidence indicate that alteration of RNA metabolism, including microRNA (miRNA) processing, is a relevant pathogenetic factor and a possible therapeutic target for ALS.

Here, we showed that the abundance of components in the miRNA processing machinery is altered in a SOD1-linked cellular model, suggesting consequent dysregulation of miRNA biogenesis. Indeed, high-throughput sequencing of the small RNA fraction showed that among the altered miRNAs, miR-129-5p was increased in different models of SOD1-linked ALS and in peripheral blood cells of sporadic ALS patients. We demonstrated that miR-129-5p upregulation causes the downregulation of one of its targets: the RNA-binding protein ELAVL4/HuD. ELAVL4/HuD is predominantly expressed in neurons, where it controls several key neuronal mRNAs. 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.

Remarkably, we showed that administration of an antisense oligonucleotide (ASO) inhibitor of miR-129-5p to an ALS animal model, SOD1 (G93A) mice, result in a significant increase in survival and improved the neuromuscular phenotype in treated mice. These results identify miR-129-5p as a therapeutic target that is amenable to ASO modulation for the treatment of ALS patients.

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

References (97)

  • A. Freischmidt

    Serum microRNAs in sporadic amyotrophic lateral sclerosis

    Neurobiol. Aging

    (2015)
  • K.Y. Kim

    A phosphomimetic mutant TDP-43 (S409/410E) induces Drosha instability and cytotoxicity in Neuro 2A cells

    Biochem. Biophys. Res. Commun.

    (2015)
  • S.T. Lee

    miR-206 regulates brain-derived neurotrophic factor in Alzheimer disease model

    Ann. Neurol.

    (2012)
  • C. Liang

    MicroRNA-153 negatively regulates the expression of amyloid precursor protein and amyloid precursor-like protein 2

    Brain Res.

    (2012)
  • E. Majounie

    Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study

    Lancet Neurol.

    (2012)
  • S. Marcuzzo

    Altered miRNA expression is associated with neuronal fate in G93A-SOD1 ependymal stem progenitor cells

    Exp. Neurol.

    (2014)
  • M. Miyachi

    Circulating muscle-specific microRNA, miR-206, as a potential diagnostic marker for rhabdomyosarcoma

    Biochem. Biophys. Res. Commun.

    (2010)
  • K.M. Schoch et al.

    Antisense oligonucleotides: translation from mouse models to human neurodegenerative diseases

    Neuron

    (2017)
  • E.R. Strickland et al.

    MicroRNA dysregulation following spinal cord contusion: implications for neural plasticity and repair

    Neuroscience

    (2011)
  • A.X. Sun et al.

    MicroRNAs: regulators of neuronal fate

    Curr. Opin. Cell Biol.

    (2013)
  • T. Tebaldi

    HuD is a neural translation enhancer acting on mTORC1-Responsive genes and counteracted by the Y3 small non-coding RNA

    Mol. Cell

    (2018)
  • Y. Wu et al.

    Hoxc10 and Hoxd10 regulate mouse columnar, divisional and motor pool identity of lumbar motoneurons

    Development

    (2008)
  • W. Akamatsu

    The RNA-binding protein HuD regulates neuronal cell identity and maturation

    Proc. Natl. Acad. Sci. U.S.A.

    (2005)
  • B. Akten

    Interaction of survival of motor neuron (SMN) and HuD proteins with mRNA cpg15 rescues motor neuron axonal deficits

    Proc. Natl. Acad. Sci. U.S.A.

    (2011)
  • M. Amadio

    nELAV proteins alteration in Alzheimer’s disease brain: a novel putative target for amyloid-beta reverberating on AbetaPP processing

    J. Alzheimers Dis.

    (2009)
  • G.E. Aranda-Abreu et al.

    Embryonic lethal abnormal vision-like RNA-binding proteins regulate neurite outgrowth and tau expression in PC12 cells

    J. Neurosci.

    (1999)
  • M. Ballarino

    TAF15 is important for cellular proliferation and regulates the expression of a subset of cell cycle genes through miRNAs

    Oncogene

    (2013)
  • M. Benigni

    Identification of miRNAs as potential biomarkers in cerebrospinal fluid from amyotrophic lateral sclerosis patients

    Neuromol. Med.

    (2016)
  • L.M. Bronicki et al.

    Emerging complexity of the HuD/ELAVl4 gene; implications for neuronal development, function, and dysfunction

    RNA (New York, N.Y.)

    (2013)
  • B.R. Brooks et al.

    El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis

    Amyotroph. Lateral Scler. Other Motor Neuron Disord.

    (2000)
  • E. Buratti

    Nuclear factor TDP-43 can affect selected microRNA levels

    FEBS J.

    (2010)
  • O. Butovsky

    Modulating inflammatory monocytes with a unique microRNA gene signature ameliorates murine ALS

    J. Clin. Invest.

    (2012)
  • O. Butovsky

    Targeting miR-155 restores abnormal microglia and attenuates disease in SOD1 mice

    Ann. Neurol.

    (2015)
  • O. Butovsky et al.

    Targeting miR-155 restores abnormal microglia and attenuates disease in SOD1 mice

    Ann. Neurol.

    (2015)
  • D. Campos-Melo et al.

    Altered microRNA expression profile in amyotrophic lateral sclerosis: a role in the regulation of NFL mRNA levels

    Mol. Brain

    (2013)
  • L. Chen

    Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms

    Nature

    (2000)
  • F. Cloutier et al.

    MicroRNAs as potential circulating biomarkers for amyotrophic lateral sclerosis

    J. Mol. Neurosci.

    (2015)
  • E. Dalla Bella

    Amyotrophic lateral sclerosis causes small fiber pathology

    Eur. J. Neurol.

    (2016)
  • D. Damiani

    Dicer inactivation leads to progressive functional and structural degeneration of the mouse retina

    J. Neurosci.

    (2008)
  • T.H. Davis

    Conditional loss of Dicer disrupts cellular and tissue morphogenesis in the cortex and hippocampus

    J. Neurosci.

    (2008)
  • B. De Felice

    miR-338-3p is over-expressed in blood, CFS, serum and spinal cord from sporadic amyotrophic lateral sclerosis patients

    Neurogenetics

    (2014)
  • D. De Pietri Tonelli

    miRNAs are essential for survival and differentiation of newborn neurons but not for expansion of neural progenitors during early neurogenesis in the mouse embryonic neocortex

    Development

    (2008)
  • E.M. DeBoer

    Prenatal deletion of the RNA-binding protein HuD disrupts postnatal cortical circuit maturation and behavior

    J. Neurosci.

    (2014)
  • A.L. DeStefano

    Replication of association between ELAVL4 and Parkinson disease: the GenePD study

    Hum. Genet.

    (2008)
  • A. Emde

    Dysregulated miRNA biogenesis downstream of cellular stress and ALS-causing mutations: a new mechanism for ALS

    EMBO J.

    (2015)
  • C. Fallini

    The survival of motor neuron (SMN) protein interacts with the mRNA-binding protein HuD and regulates localization of poly(A) mRNA in primary motor neuron axons

    J. Neurosci.

    (2011)
  • C. Fallini et al.

    The ALS disease protein TDP-43 is actively transported in motor neuron axons and regulates axon outgrowth

    Hum. Mol. Genet.

    (2012)
  • M.J. Fogarty et al.

    Motor areas show altered dendritic structure in an amyotrophic lateral sclerosis mouse model

    Front. Neurosci.

    (2017)
  • Cited by (33)

    View all citing articles on Scopus
    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.

    View full text