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.T he burgeoning use of microarray analyses to detect potential target genes relevant to neuropsychiatric disorders necessitates the validation of such targets in vivo (1-3). The approach of genetically modifying animals (knockouts or transgenics) for target validation often is limited by developmental adaptations and genetic compensation that may mask the establishment of a clear phenotype. Further, such methodology is laborious and timeconsuming and not applicable for high-throughput in vivo validation of the large number of hits generated from modern microarray and proteomic target-identification strategies. Additional approaches using ribozymes and antisense oligodeoxynucleotides for somatic gene manipulation also suffer from drawbacks of eliciting nonspecific actions. RNA interference (RNAi) recently has emerged as a potentially superior alternative to the traditional approaches for assessing gene function in adult animals (4).RNAi is a cellular surveillance mechanism that responds to double-stranded RNA (dsRNA) by destroying cytoplasmic mRNAs containing sequences homologous to the dsRNA trigger (5). dsRNAs longer than 30 nt introduced in mammalian cells can induce an undesirable IFN response to produce cell death (6, 7). Conversely, short interfer...

siRNA-mediated knockdown of the serotonin transporter in the adult mouse brain

“…NM_010484), and its efficiency in reducing the mRNA levels from 100% to the value in parentheses, along with the corresponding coefficient of variation, is indicated as follows: nt 429-449 (27%, 4. 20 A maximally effective dose of siRNA was used that was well tolerated with no signs of neurotoxicity (hind-limb paralysis, vocalization, food intake or neuroanatomical damage) following i.c.v. application for 2 weeks.…”

“…Implantation of the cannula for infusion into the dorsal third ventricle (AP: À0.5 mm; ML: 0 mm, DV: À3 mm, relative to bregma), 26 from the subcutaneously -implanted minipump, was performed as described previously. 20 Behavioral assessment Forced swim test was conducted as described previously, 27,28 14 days after implanting the cannulaminipump assembly. Briefly, mice were individually placed into plexiglas cylinders (24 cm high  21 cm internal diameter) filled with water (25.470.71C) to a depth of 15 cm.…”

“…20 RNAi is a cellular surveillance mechanism that destroys the endogenous mRNAs containing sequences complimentary to a double-stranded RNA (dsRNA) trigger. 21 Short interfering RNAs (siRNAs) mimic the cleavage products of dsRNA and produce sequence-specific gene-knockdown in mammalian cells, without eliciting the lethal dsRNA-interferon response.…”

“…infused siRNAs can efficiently produce a widespread downregulation of gene expression in the adult mouse brain. 20 Here, we compare the neurochemical as well as the behavioral consequences of infusing a SERTtargeting siRNA vs the SSRI, citalopram, in the brain.…”

RNAi: a potential therapy for the dominantly inherited nucleotide repeat diseases

“…Nonetheless, implanted pumps have been used to deliver chemically synthesized siRNAs for short-term knockdown of the painrelated cation channel P2X 3 47 or the D2 receptor. 48 Direct delivery of viruses expressing shRNAs can also be used for inhibition of gene expression 49,50 or disease targets. [51][52][53][54][55] Both a benefit and a concern from viralmediated delivery is that expression lasts from months to years.…”