A New Tool for Targeted Antisense Knockdown in Brain

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DESCRIPTION (provided by applicant): The goal of this project is to develop a new tool for down regulating the expression of specific proteins in selectively targeted cells in the adult brain. The approach will use antibody-targeted antisense PNA (peptide nucleic acid) to selectively knock down expression of the nerve growth factor receptor TrkA within targeted p75NTR-expressing cells in the basal forebrain. Our long-term goal is to use the method to test the hypothesis that sustained reductions in TrkA, specifically within cholinergic neurons in the medial septum and nucleus basalis magnocellularis, are responsible for age-related declines in basal forebrain cholinergic function and cognitive performance. Once developed, this tool could be used to selectively knock down a wide range of proteins within these cholinergic neurons (e.g., neurotransmitter receptors, vesicular transporters, ion channels, etc...), as well as in p75NTR-expressing neurons within other parts of the brain and periphery, in order to assess their functional role in neurodegenerative diseases and age-related cognitive decline. Similar methods could be used to regulate proteins in other (non-p75NTR-expressing) cell types that are amenable to antibody targeting. Studies will use established methods to conjugate fluorescently-tagged PNA to an antibody raised against mouse p75NTR (mouse-192-IgG). Conjugates will be characterized by TOF mass spectrometry and by in vitro binding assays. PNAs will be delivered intraventricularly directly into the rat brain. Uptake of the PNA by cholinergic neurons in the basal forebrain will be evaluated by fluorescence microscopy, combined with immunohistochemical detection of choline acetyltransferase (ChAT), p75NTR, and TrkA protein. Studies will then evaluate the effects of antibody-PNA conjugates on cell viability using standard Nissl stain, as well as immunohistochemical detection of glial fibrillary acidic protein, and histochemical detection of microglial infiltration. Effects on TrkA expression, NGF binding, and cholinergic function will be evaluated by Western blot, by retrograde transport of 125I-NGF from the hippocampus to the medial septum, and by measuring ChAT activity in the septum and hippocampus. This will be the first time that a targeted antisense knockdown approach will have been developed and used to selectively regulate gene expression within an identified subpopulation of neurons in the adult brain.