Targeted silencing of TrkA expression in rat forebrain neurons via the p75 receptor.
Basal forebrain neurons express the neurotrophin receptors, p75NTR and tyrosine kinase receptor A (TrkA). We tested the hypothesis that impairment of memory in rats could be achieved by RNA interference (RNAi) -induced silencing of TrkA specifically within these neurons. A novel fusogenic, karyophilic immunoporter (fkAb(p75)-ipr) was constructed from the antibody, MC192 (monoclonal antibody to the rat neurotrophin receptor p75NTR, Ab(p75)), poly-l-lysine together with the hemagglutinin 2 and VP1 nuclear localization peptides of influenza and SV40 virus, respectively. Plasmid DNA constructs containing short hairpin sequences inhibitory to tyrosine kinase receptor A expression (TrkAi) and the gene encoding cGFP (green fluorescent protein from coral fish) was produced. These TrkAi plasmids were mixed with the immunoporter, forming the immunogene, TrkAi-fkAb(p75). A control TrkAsc complexed with fkAb(p75) (TrkAsc-fkAb(p75)) immunogene was constructed from a scrambled sequence (TrkAsc) and fkAb(p75)-ipr. Rats were infused using an osmotic mini-pump into the third ventricle with either TrkAi-fkAb(p75) or TrkAsc-fkAb(p75). Naive rats were also included as additional controls. After 7 days, examination of gene expression on forebrain sections of some rats revealed cGFP expression in TrkA neurons. Fifteen to 19 days after infusion, rats were tested in a Morris water maze apparatus. Animals that received TrkAi-fkAb(p75) showed significantly impaired spatial memory learning ability compared with naive or TrkAsc-fkAb(p75)-treated rats. Western blot and immunofluorescence analysis showed that TrkA protein levels and numbers of TrkA positive neurons were reduced by 60% and 55% respectively in TrkAi-fkAb(p75)-infused rats compared with infused controls or naive animals. We conclude that p75-receptor-mediated RNAi-induced silencing of genes offers a novel and powerful way to study the function of specific endogenous genes within distinct neuronal subpopulations of the brain.
Berhanu
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Department of Human Physiology and Centre for Neuroscience, Flinders University of South Australia, Adelaide, SA 5001, Australia. degu0008@flinders.edu.au
Rush
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RA
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism; D001522:Behavior, Animal; D002452:Cell Count / Q000379:methods; D005786:Gene Expression Regulation / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018782:Maze Learning; D008568:Memory / Q000187:drug effects / Q000502:physiology; D009419:Nerve Tissue Proteins; D009474:Neurons / Q000378:metabolism*; D016548:Prosencephalon / Q000166:cytology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011930:Reaction Time / Q000187:drug effects; D020917:Receptor, trkA / Q000276:immunology / Q000378:metabolism*; D017978:Receptors, Growth Factor; D017475:Receptors, Nerve Growth Factor / Q000276:immunology / Q000502:physiology*; D013997:Time Factors; D014162:Transfection
D000906:Antibodies; D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering; D017978:Receptors, Growth Factor; D017475:Receptors, Nerve Growth Factor; C068313:Ngfr protein, rat; D049452:Green Fluorescent Proteins; D020917:Receptor, trkA