title: D-amino acid oxidase knockdown in the mouse cerebellum reduces NR2A mRNA.
abstract: Virus mediated RNA-interference (RNAi) is a powerful approach to study genes in vivo. Here we report a method using lentivirus-delivered RNAi to knockdown the glial enzyme, D-amino acid oxidase (DAO), in the mouse cerebellum. After initial characterisation in vitro, we achieved a 40-50% reduction of DAO mRNA in the cerebellum 7 and 28 days after a single injection of lentivirus encoding a DAO-specific, short-hairpin RNA. Injections also decreased DAO immunoreactivity (-33%). The major substrate for DAO is D-serine, an N-methyl-D-aspartate receptor (NMDAR) co-agonist. Thus, we also measured whether DAO knockdown impacted on d-serine, or expression of NMDAR subunits, and found that DAO RNAi led to increased cerebellar D-serine levels (+77%), and decreased NMDAR subunit NR2A mRNA (-22%), but did not affect NR1 or NR2C mRNAs. These data demonstrate the utility of lentiviruses to deliver RNAi to glial cells within the cerebellum, and confirm the role of DAO in D-serine metabolism. They also provide a tool to investigate DAO, an enzyme currently of considerable interest in the pathophysiology and therapy of schizophrenia.
authors.affiliation: Department of Psychiatry, University of Oxford, Warneford Hospital, Oxford OX3 7JX, UK. phil.burnet@psych.ox.ac.uk
authors.forename: Philip W J
authors.initials: PW
authors.lastname: Burnet
authors.forename: Patrick N
authors.initials: PN
authors.lastname: Anderson
authors.forename: Li
authors.initials: L
authors.lastname: Chen
authors.forename: Natalia
authors.initials: N
authors.lastname: Nikiforova
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Harrison
authors.forename: Matthew J A
authors.initials: MJ
authors.lastname: Wood
mesh_terms: D000818:Animals; D002460:Cell Line; D002531:Cerebellum / Q000201:enzymology*; D003605:D-Amino-Acid Oxidase / Q000235:genetics* / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D008297:Male; D001523:Mental Disorders / Q000503:physiopathology; D051379:Mice; D008810:Mice, Inbred C57BL; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics* / Q000378:metabolism; D012694:Serine / Q000378:metabolism
chemical_list: C120997:NR2A NMDA receptor; D021122:Protein Subunits; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; D049452:Green Fluorescent Proteins; D012694:Serine; C485958:Dao1 protein, mouse; D003605:D-Amino-Acid Oxidase