title: Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference.
abstract: Gene expression analysis implicates an increasing number of novel genes in the brain as potential targets for the treatment of neurological and psychiatric disorders. Frequently, these genes are ubiquitously expressed in the brain and, thus, may contribute to a pathophysiological state through actions in several brain nuclei. Current strategies employing genetically modified animals for in vivo validation of such targets are time-consuming and often limited by developmental adaptations. Somatic gene manipulation using viral-mediated RNA interference (RNAi) has emerged recently, although restricting the target validation to specific brain nuclei. We investigated whether nonviral infusion of short interfering RNA (siRNA) into the ventricular system would enable a sequence-specific gene knockdown. The temporality and extent of siRNA-induced down-regulation were analyzed by targeting a transgene, EGFP, in mice overexpressing EGFP. Extensive knockdown of EGFP was observed, especially in regions adjacent or dorsoventrally and mediolaterally distant to the infusion site (dorsal third ventricle), with lesser knockdown in more distal regions. We challenged our RNAi approach to generate a specific knockdown of an endogenous gene, encoding the dopamine transporter (DAT) in regions (ventral midbrain) far distal to the infusion site. DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein in the brain and also elicited a temporal hyperlocomotor response similar to that (but delayed) obtained upon infusion of GBR-12909, a pharmacologically selective DAT inhibitor. Application of this nonviral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.
authors.affiliation: Neuroscience Research, Novartis Institutes for Biomedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland.
authors.forename: Deepak R
authors.initials: DR
authors.lastname: Thakker
authors.forename: Francois
authors.initials: F
authors.lastname: Natt
authors.forename: Dieter
authors.initials: D
authors.lastname: Hüsken
authors.forename: Rainer
authors.initials: R
authors.lastname: Maier
authors.forename: Matthias
authors.initials: M
authors.lastname: Müller
authors.forename: Herman
authors.initials: H
authors.lastname: van der Putten
authors.forename: Daniel
authors.initials: D
authors.lastname: Hoyer
authors.forename: John F
authors.initials: JF
authors.lastname: Cryan
mesh_terms: D000818:Animals; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D002552:Cerebral Ventricles; D050483:Dopamine Plasma Membrane Transport Proteins; D015536:Down-Regulation / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000235:genetics*; D008722:Methods; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009043:Motor Activity / Q000187:drug effects*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000037:antagonists & inhibitors; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000494:pharmacology
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C493215:Slc6a3 protein, mouse; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins