Abstract
The study of signal transduction in dopamine (DA)-containing neurons as well as the development of new therapeutic approaches for Parkinson's disease requires the selective expression of transgenes in such neurons. Here we describe optimization of the use of the NTS-polyplex, a gene carrier system taking advantage of neurotensin receptor internalization, to transfect mouse DA neurons in primary culture. The plasmids DsRed2 (4.7 kbp) and VGLUT2-Venus (11 kbp) were used to compare the ability of this carrier system to transfect plasmids of different sizes. We examined the impact of age of the neurons (1, 3, 5 and 8 days after seeding), of culture media used during the transfection (Neurobasal with B27 vs. conditioned medium) and of three molar ratios of plasmid DNA to carrier. While the NTS-polyplex successfully transfected both plasmids in a control N1E-115 cell line, only the pDsRed2 plasmid could be transfected in primary cultured DA neurons. We achieved 20% transfection efficiency of pDsRed2 in DA neurons, with 80% cell viability. The transfection was demonstrated pharmacologically to be dependent on activation of neurotensin receptors and to be selective for DA neurons. The presence of conditioned medium for transfection was found to be required to insure cell viability. Highest transfection efficiency was achieved in the most mature neurons. In contrast, transfection with the VGLUT2-Venus plasmid produced cell damage, most likely due to the high molar ratios required, as evidenced by a 15% cell viability of DA neurons at the three molar ratios tested (1∶36, 1∶39 and 1∶42). We conclude that, when used at molar ratios lower than 1∶33, the NTS-polyplex can selectively transfect mature cultured DA neurons with only low levels of toxicity. Our results provide evidence that the NTS-polyplex has good potential for targeted gene delivery in cultured DA neurons, an in vitro system of great use for the screening of new therapeutic approaches for Parkinson's disease.
Highlights
Selective gene transfer to dopamine (DA) neurons has been of much interest to study signal transduction in this cell population, which is otherwise critical for a number of physiological functions such as motor control or motivation
A retardation gel assay was performed with a constant concentration (6 nM) of plasmid DNA and increasing concentrations of karyophilic peptide (KP)
We found that 6 mM of KP was optimal for pEGFP-N1 (Fig. 1A) and pDsRed2 (Fig. 1C) and 7 mM for pVGLUT2-Venus (Fig. 1E)
Summary
Selective gene transfer to dopamine (DA) neurons has been of much interest to study signal transduction in this cell population, which is otherwise critical for a number of physiological functions such as motor control or motivation. The transfection efficiency of NTS-polyplex is lower than that of viral vectors, but it was recently improved by coupling the vector with two short viral peptides, the hemagglutinin-derived HA2 FP and the Vp1 SV40 KP [4] These viral peptides are thought to rescue the NTSpolyplex from acidic endosomes and to target the pDNA to the cell nucleus respectively, enhancing transfection efficiency and prolonging transgene expression [1,4]. For routine gene transfer to DA neurons in vitro, the NTS-polyplex could be of much interest since its production is typically less labour intensive than viral vector construction
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