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Selective silencing of alpha-synuclein in aminergic neurons In vivo by intranasal delivery of targeted small interfering RNA or antisense oligonucleotides Relevance to Parkinson's disease
|Autor:||Recasens, Ariadna; Galofré, Mireia ; Ferrés-Coy, Albert ; Bortolozzi, Analía ; Vila, M.|
|Fecha de publicación:||13-nov-2013|
|Resumen:||Increased levels of α-synuclein are involved in the pathogenesis of Parkinson’s disease (PD), thereby providing a rationale for potential therapeutic strategies aimed at reducing neuronal α-synuclein burden in this disease. Here, we explored the feasibility and safety of reducing α-synuclein expression levels in vivo selectively in PD-vulnerable neuronal populations, including substantia nigra pars compacta (SNpc), ventral tegmental area (VTA), locus coeruleus (LC) and dorsal raphe nucleus (DR). We assessed first the ability of various small interfering RNA (siRNA) and antisense oligonucleotide (ASO) sequences directed against α-synuclein to downregulate endogenous or overexpressed α-synuclein mRNA levels in BE-M17 neuroblastoma cells by real-time PCR. Based on this assay, siRNA and ASO sequences able to produce a 75- 5% downregulation of α-synuclein without affecting β- and γ-synuclein were selected for further analyses in vivo. We confirmed the ability of the selected sequences to downregulate endogenous α-synuclein mRNA levels by local stereotactic infusion into the SNpc of wild-type mice followed by in situ hybridization analyses in this region. Selected siRNA/ASO sequences were then chemically modified to enhance their biostability and conjugated to a cellspecific ligand, indatraline (a non-selective monoamine transporter inhibitor), to promote their selective delivery into aminergic neurons. To confirm the cell-specific delivery of targeted siRNA/ASO sequences, these molecules were fluorescently labeled with Alexa488 and administered intracerebroventricularly or intranasally into mice. One hour after administration, fluorescently labeled molecules were selectively observed within SNpc, LC and DR neurons but not in other brain regions or in animals administered with fluorescent oligonucleotides without the targeting ligand. Targeted siRNA/ASO molecules were then administered intranasally into adult mice at a dose of 30 µg/day (2.1 nmol/day) for 4 consecutive days and animals were euthanized 24h, 3d and 7d after the last administration. In these animals, intranasal administration of targeted siRNA/ASO molecules produced a cell-specific time-dependent downregulation of endogenous α-synuclein, both at mRNA and protein levels, in SNpc/VTA, LC and DR but not other brain regions (including the olfactory bulb). Knockdown of α-synuclein in these animals was not associated with neurodegeneration in affected areas. These results set the stage for testing these molecules as potential disease-modifying agents in animal models of PD linked to pathogenic increases in α-synuclein|
|Descripción:||Póster presentado en Neuroscience 2013, organizado por la Society for Neuroscience, del 9 al 13 de noviembre de 2013 en San Diego, California (Estados Unidos)|
A. Recasens et al.
|Aparece en las colecciones:||(IIBB) Comunicaciones congresos|
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