Abstract
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 (fkAbp75-ipr) was constructed from the antibody, MC192 (monoclonal antibody to the rat neurotrophin receptor p75NTR, Abp75), 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-fkAbp75. A control TrkAsc complexed with fkAbp75 (TrkAsc-fkAbp75) immunogene was constructed from a scrambled sequence (TrkAsc) and fkAbp75-ipr. Rats were infused using an osmotic mini-pump into the third ventricle with either TrkAi-fkAbp75 or TrkAsc-fkAbp75. 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-fkAbp75 showed significantly impaired spatial memory learning ability compared with naive or TrkAsc-fkAbp75-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-fkAbp75-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.
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