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Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference Deepak R. Thakker*, Francois Natt†, Dieter Hu¨ sken†, Rainer Maier*, Matthias Mu¨ ller‡, Herman van der Putten*, Daniel Hoyer*, and John F. Cryan*§ *Neuroscience Research, †Functional Genomics, and ‡Models of Disease Center, Novartis Institutes for Biomedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland Edited by Floyd E. Bloom, The Scripps Research Institute, La Jolla, CA, and approved October 18, 2004 (received for review August 23, 2004) Gene expression analysis implicates an increasing number of novel the cleavage products of dsRNA in mammalian cells elude the IFN genes in the brain as potential targets for the treatment of response and produce sequence-specific gene silencing in these cells neurological and psychiatric disorders. Frequently, these genes are (6, 7). siRNAs have been used successfully to investigate gene ubiquitously expressed in the brain and, thus, may contribute to a function in neuronal cultures; however, their delivery into the pathophysiological state through actions in several brain nuclei. mammalian brain to achieve the same knockdown in vivo still Current strategies employing genetically modified animals for in remains a challenge (8, 9). Recent studies describe the use of viral vivo validation of such targets are time-consuming and often vectors to attain stable RNAi in the brain, albeit only in specific limited by developmental adaptations. Somatic gene manipulation brain regions (10–13). This method may be pertinent for pheno- using viral-mediated RNA interference (RNAi) has emerged re- typing of genes expressed in a few distinct loci, but not of genes that cently, although restricting the target validation to specific brain are broadly expressed and frequently contribute to a pathophysi- nuclei. We investigated whether nonviral infusion of short inter- ological state by influencing a complex circuitry involving neurons fering RNA (siRNA) into the ventricular system would enable a from more than one brain region (14–18). Application of a region- sequence-specific gene knockdown. The temporality and extent of ally selective approach also would be difficult for investigating the siRNA-induced down-regulation were analyzed by targeting a function of novel genes with lack of knowledge of their mRNA- transgene, EGFP, in mice overexpressing EGFP. Extensive knock- processing events or of brain regions relevant to their (patho)phys- down of EGFP was observed, especially in regions adjacent or iological actions. Therefore, to facilitate the functional assessment dorsoventrally and mediolaterally distant to the infusion site of such ubiquitously expressed genes and also to enable a rapid and (dorsal third ventricle), with lesser knockdown in more distal regionally unbiased phenotyping of novel genes, we assessed regions. We challenged our RNAi approach to generate a specific whether siRNA could be delivered efficiently to produce gene knockdown of an endogenous gene, encoding the dopamine silencing in the whole brain. transporter (DAT) in regions (ventral midbrain) far distal to the We first used mice overexpressing EGFP under the control of infusion site. DAT-siRNA infusion in adult mice produced a signif- -actin promoter (19) as an in vivo model to test the gene-silencing icant down-regulation of DAT mRNA and protein in the brain and efficiency of our nonviral RNAi method. EGFP expression in these also elicited a temporal hyperlocomotor response similar to that mice is ubiquitous and nearly uniform throughout the brain, thus (but delayed) obtained upon infusion of GBR-12909, a pharmaco- enabling the determination of the extent of siRNA-induced knock- logically selective DAT inhibitor. Application of this nonviral RNAi down of target (EGFP) mRNA and protein levels in the brain. The approach may accelerate target validation for neuropsychiatric specificity, temporality, and magnitude of EGFP knockdown were disorders that involve a complex interplay of gene(s) from various evaluated after infusion of an EGFP-targeting siRNA in the dorsal brain regions. third ventricle. Further, we tested the validity of this RNAi ap- proach by targeting an endogenous gene that codes for the dopa- in vivo ͉ target validation ͉ EGFP ͉ dopamine transporter mine transporter (DAT) within the ventral midbrain neurons, far distal from the siRNA infusion site (20). DAT controls the tem- he burgeoning use of microarray analyses to detect potential poral and spatial activity of dopamine released into the synapse by Ttarget genes relevant to neuropsychiatric disorders necessitates facilitating a rapid uptake of the neurotransmitter into presynpatic the validation of such targets in vivo (1–3). The approach of terminals. DAT is, therefore, a key regulator of dopamine actions genetically modifying animals (knockouts or transgenics) for target on locomotion, emotion, and cognition, and it is implicated in the validation often is limited by developmental adaptations and ge- etiology of hyperkinetic disorders, such as attention-deficit hyper- netic compensation that may mask the establishment of a clear activity disorder (21–24). Here, we compare the behavioral as well phenotype. Further, such methodology is laborious and time- as molecular consequences of infusing the DAT-siRNA vs. those of consuming and not applicable for high-throughput in vivo validation a selective pharmacological DAT inhibitor in the brain. of the large number of hits generated from modern microarray and Methods proteomic target-identification strategies. Additional approaches using ribozymes and antisense oligodeoxynucleotides for somatic Animals. Male EGFP-expressing mice were generated on a ͞ gene manipulation also suffer from drawbacks of eliciting nonspe- BALB c background as described in ref. 19. Briefly, the plasmids cific actions. RNA interference (RNAi) recently has emerged as a potentially superior alternative to the traditional approaches for assessing gene function in adult animals (4). This paper was submitted directly (Track II) to the PNAS office. RNAi is a cellular surveillance mechanism that responds to Abbreviations: RNAi, RNA interference; siRNA, short interfering RNA; scrRNA, scrambled sequence RNA; mmRNA, mismatch siRNA; TH, tyrosine hydroxylase; SERT, serotonin trans- double-stranded RNA (dsRNA) by destroying cytoplasmic mRNAs porter; DAT, dopamine transporter; GABAA, GABA type A; GABAA␣2, GABAA receptor containing sequences homologous to the dsRNA trigger (5). subunit 2. dsRNAs longer than 30 nt introduced in mammalian cells can §To whom correspondence should be addressed. E-mail: johnf.cryan@pharma. induce an undesirable IFN response to produce cell death (6, 7). novartis.com. Conversely, short interfering RNAs (siRNAs; 21–23 nt) that mimic © 2004 by The National Academy of Sciences of the USA 17270–17275 ͉ PNAS ͉ December 7, 2004 ͉ vol. 101 ͉ no. 49 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0406214101 Downloaded by guest on September 28, 2021 pEGFP-N1 (Clontech) and pRAY-2 (25), and a 5.9-kbp KpnI baseline, where day 0 is the day of implanting the cannula- genomic subclone of -actin were assembled to produce a con- minipump assembly in mice. Thereafter, mice were assessed for struct, wherein a promoterless EGFP gene was inserted at the ATG activity levels on days 3, 6, 9, 12, and 14. of -actin and driven from the -actin promoter. Homologous recombination was performed in BALB͞c-I embryonic stem cells. Processing of the Brain for mRNA and Protein Analysis. Mice involved Modification of the -actin locus was confirmed by PCR and in the EGFP-siRNA study were decapitated on day 8 or 15, Southern blotting. Male BALB͞c mice (21–29 g; Iffa Credo) were depending on the model of osmotic minipump implanted. For the used for all other experiments. All mice were housed two per cage DAT-siRNA experiment, mice were decapitated Ϸ24 h after their before surgery and one per cage thereafter in a humidity- and final locomotor activity trial. Brains were removed, and serial temperature-controlled room with a 12-h͞12-h light͞dark cycle coronal sections of 10-m thickness were obtained for each brain (lights on at 6 a.m.). Food pellets and tap water were available ad region at the following anteroposterior coordinates in mm relative libitum, except during behavioral testing. Animals were acclima- to bregma (28): 3.56 (olfactory bulb), 2.46 (prefrontal cortex), 1.18 tized to these housing conditions for at least 1 week after their (caudate putamen and nucleus accumbens), Ϫ0.22 (globus palli- arrival before starting any procedure. Animal experimentation was dus), Ϫ0.46 (to confirm the site of injection), Ϫ1.58 (cerebral performed during the light cycle, in accordance with the Veterinary cortex, hippocampus, habenula, thalamus, hypothalamus, and Authority of Basel-Stadt, Switzerland. amygdala), Ϫ3.08 (substantia nigra and ventral tegmental area), Ϫ4.36 (periaqueductal gray and dorsal raphe), Ϫ5.34 (locus coer- Drug͞siRNA Administration in Mouse Brain. We used an in vitro uleus), and Ϫ7.2 (spinal trigeminal nucleus). Sections were thaw- validated siRNA, targeting EGFP (antisense, 5Ј-AUGAACU- mounted onto poly(L-lysine)-coated slides, coded for a blind anal- UCAGGGUCAGCUTG-3Ј, and sense, 5Ј-AGCUGACCCU- ysis of mRNA and protein, and stored at Ϫ80°C until use in Ͻ1 GAAGUUCAUCT-3Ј) (6), and a siRNA of scrambled sequence week. (scrRNA) (antisense, 5Ј-UCGUCAUAACGUUCAUAGGCG-3Ј, and sense, 5Ј-CCUAUGAACGUUAUGACGATT-3Ј). They In Situ