RESUMEN
The neuronal RNA-binding protein (RBP) HuD plays an important role in brain development, synaptic plasticity and neurodegenerative diseases such as Parkinson's (PD) and Alzheimer's (AD). Bioinformatics analysis of the human SOD1 mRNA 3' untranslated region (3'UTR) demonstrated the presence of HuD binding adenine-uridine (AU)-rich instability-conferring elements (AREs). Using differentiated SH-SY5Y cells along with brain tissues from sporadic amyotrophic lateral sclerosis (sALS) patients, we assessed HuD-dependent regulation of SOD1 mRNA. In vitro binding and mRNA decay assays demonstrate that HuD specifically binds to SOD1 ARE motifs promoting mRNA stabilization. In SH-SY5Y cells, overexpression of full-length HuD increased SOD1 mRNA and protein levels while a dominant negative form of the RBP downregulated its expression. HuD regulation of SOD1 mRNA was also found to be oxidative stress (OS)-dependent, as shown by the increased HuD binding and upregulation of this mRNA after H2O2 exposure. This treatment also induced a shift in alternative polyadenylation (APA) site usage in SOD1 3'UTR, increasing the levels of a long variant bearing HuD binding sites. The requirement of HuD for SOD1 upregulation during oxidative damage was validated using a specific siRNA that downregulated HuD protein levels to 36% and prevented upregulation of SOD1 and 91 additional genes. In the motor cortex from sALS patients, we found increases in SOD1 and HuD mRNAs and proteins, accompanied by greater HuD binding to this mRNA as confirmed by RNA-immunoprecipitation (RIP) assays. Altogether, our results suggest a role of HuD in the post-transcriptional regulation of SOD1 expression during ALS pathogenesis.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteína 4 Similar a ELAV/genética , Regulación de la Expresión Génica/genética , Corteza Motora/metabolismo , Neuroblastoma/metabolismo , Neuronas/metabolismo , Estrés Oxidativo/genética , Superóxido Dismutasa-1/genética , Esclerosis Amiotrófica Lateral/metabolismo , Línea Celular Tumoral , Proteína 4 Similar a ELAV/metabolismo , Humanos , ARN Mensajero/metabolismo , Superóxido Dismutasa-1/metabolismoRESUMEN
Neuromuscular disorders such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy are neurodegenerative genetic diseases characterized primarily by muscle weakness and wasting. Until recently there were no effective therapies for these conditions, but antisense oligonucleotides, a new class of synthetic single stranded molecules of nucleic acids, have demonstrated promising experimental results and are at different stages of regulatory approval. The antisense oligonucleotides can modulate the protein expression via targeting hnRNAs or mRNAs and inducing interference with splicing, mRNA degradation, or arrest of translation, finally, resulting in rescue or reduction of the target protein expression. Different classes of antisense oligonucleotides are being tested in several clinical trials, and limitations of their clinical efficacy and toxicity have been reported for some of these compounds, while more encouraging results have supported the development of others. New generation antisense oligonucleotides are also being tested in preclinical models together with specific delivery systems that could allow some of the limitations of current antisense oligonucleotides to be overcome, to improve the cell penetration, to achieve more robust target engagement, and hopefully also be associated with acceptable toxicity. This review article describes the chemical properties and molecular mechanisms of action of the antisense oligonucleotides and the therapeutic implications these compounds have in neuromuscular diseases. Current strategies and carrier systems available for the oligonucleotides delivery will be also described to provide an overview on the past, present and future of these appealing molecules.
Asunto(s)
Terapia Genética , Enfermedades Neuromusculares/genética , Enfermedades Neuromusculares/terapia , Oligonucleótidos Antisentido/uso terapéutico , Animales , Transporte Biológico , Péptidos de Penetración Celular/metabolismo , Ensayos Clínicos como Asunto , Evaluación Preclínica de Medicamentos , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Humanos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/terapia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Enfermedades Neuromusculares/diagnóstico , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/metabolismo , Empalme del ARN , ARN Mensajero/genéticaRESUMEN
The use of splice-switching antisense therapy is highly promising, with a wealth of pre-clinical data and numerous clinical trials ongoing. Nevertheless, its potential to treat a variety of disorders has yet to be realized. The main obstacle impeding the clinical translation of this approach is the relatively poor delivery of antisense oligonucleotides to target tissues after systemic delivery. We are a group of researchers closely involved in the development of these therapies and would like to communicate our discussions concerning the validity of standard methodologies currently used in their pre-clinical development, the gaps in current knowledge and the pertinent challenges facing the field. We therefore make recommendations in order to focus future research efforts and facilitate a wider application of therapeutic antisense oligonucleotides.