Functional inhibition of the p75 receptor using a small interfering RNA

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Abstract

The neurotrophin receptor p75NTR mediates a wide variety of biological effects. Consistent with the function in controlling the survival and neurite formation, p75NTR is expressed during the developmental stages of the nervous system. Importantly, p75NTR is re-expressed in various pathological conditions and is suggested to contribute to the inhibition of neuronal regeneration and the death of the neurons. Here we develop a tool to knock down the expression of p75NTR by employing a small interfering RNA (siRNA). The siRNA for p75NTR effectively reduces the expression of endogenous p75NTR both in Schwann cells and dorsal root ganglion neurons in vitro. NGF-induced cell death in Schwann cells and the neurite retraction in DRG neurons induced by myelin-associated glycoprotein are attenuated by the siRNA. Inhibition of p75NTR in specific pathological conditions by the siRNA may provide a potential therapeutic agent.

Section snippets

Materials and methods

Isolation of Schwann cells. Sciatic nerves were dissected from mice at postnatal day 4, treated with 0.1% collagenase type I (Sigma) and 0.25% trypsin (Gibco-BRL), and incubated with 200 U/ml DNase1 (Takara) for 30 min at 37 °C. The cells were triturated and plated on poly-l-lysine-coated Lab-Tek four-chamber tissue culture slides (Nunc) cultured in DMEM containing 10% fetal calf serum (FCS).

Isolation of DRG neurons. DRGs were removed from mice at postnatal day 9 and dissociated into single cells

siRNA construct

Several groups showed evidence for sequence-specific inhibition of the target gene when the appropriate siRNAs were transfected with somatic mammalian cell lines [18], [19]. siRNAs have been shown to act as guides for the enzymatic complex required for the degradation of the target mRNA [20]. Specific repression of target gene is caused by duplexes of 20–23 bp and the 21 nt duplexes with 2 nt 3 overhangs were shown to be efficient in mediating RNAi in Drosophila[21]. In a recent study in

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