High-efficiency transfection of cultured primary motor neurons to study protein localization, trafficking, and function

Abstract

Background: Cultured spinal motor neurons are a valuable tool to study basic mechanisms of development, axon growth and pathfinding, and, importantly, to analyze the pathomechanisms underlying motor neuron diseases. However, the application of this cell culture model is limited by the lack of efficient gene transfer techniques which are available for other neurons. To address this problem, we have established magnetofection as a novel method for the simple and efficient transfection of mouse embryonic motor neurons. This technique allows for the study of the effects of gene expression and silencing on the development and survival of motor neurons.

Results: We found that magnetofection, a novel transfection technology based on the delivery of DNA-coated magnetic nanobeads, can be used to transfect primary motor neurons. Therefore, in order to use this method as a new tool for studying the localization and transport of axonal proteins, we optimized conditions and determined parameters for efficient transfection rates of >45% while minimizing toxic effects on survival and morphology. To demonstrate the potential of this method, we have used transfection with plasmids encoding fluorescent fusion-proteins to show for the first time that the spinal muscular atrophy-disease protein Smn is actively transported along axons of live primary motor neurons, supporting an axon-specific role for Smn that is different from its canonical function in mRNA splicing. We were also able to show the suitability of magnetofection for gene knockdown with shRNA-based constructs by significantly reducing Smn levels in both cell bodies and axons, opening new opportunities for the study of the function of axonal proteins in motor neurons.

Conclusions: In this study we have established an optimized magnetofection protocol as a novel transfection method for primary motor neurons that is simple, efficient and non-toxic. We anticipate that this novel approach will have a broad applicability in the study of motor neuron development, axonal trafficking, and molecular mechanisms of motor neuron diseases.

Figure 1

Magnetofection of primary motor neurons is highly efficient. Primary motor neurons (2 DIV) were transfected with pmaxGFP using magnetic nanobeads. Different amounts of DNA and beads and different media were tested to optimize transfection efficiency. Cells were fixed 2 days post transfection and stained using neurofilament (NF) and HB9 antibodies to identify motor neurons. A. Representative image of a motor neuron culture from an entire 15 mm diameter coverslip. B. GFP/HB9⁺ cells were scored and normalized to the number of HB9⁺ cells. Maximum efficiency of transfection (46%) was achieved using 0.5 μg DNA and 1.75 μl beads (mean and SEM, n = 3). C. Neurobasal medium inhibited motor neuron transfection when used during DNA/beads complex formation. MEM is shown as a positive control. Size bars: 1 mm in A, 50 μm in B.

Figure 2

Young and mature motor neuron cultures are efficiently transfected by magnetofection. 1 DIV (top), 2 DIV (middle), and 7 DIV (bottom) motor neurons were transfected with pmaxGFP. Twenty-four hours after transfection cells were fixed and stained with neurofilament antibody to identify neurons (NF, red). DAPI was used to stain nuclei (blue). GFP-positive cells (green) are visible in all tested conditions. Size bars: 100 μm.

Figure 3

Magnetofection does not affect motor neuron morphology and survival. Primary motor neurons (2 DIV) were transfected with Lifeact-GFP (A) or pmaxGFP (B) and fixed 1 day (A), or 3 and 5 days (B) after transfection. A. Motor neuron morphology was evaluated using the F-actin probe Lifeact-GFP and compared to rhodamine phalloidin staining. Cell body, dendrites (left) and growth cone (right) did not show any difference between transfected (top panel) and control (bottom) cells. Size bar: 10 μm. B. Surviving motor neurons were scored based on neurofilament (NF) and HB9 staining (left). Representative images are shown (bottom right). Bars represent mean and SEM from 3 independent experiments. One way repeated measures ANOVA, p > 0.05.

Figure 4

Magnetofection allows high-efficient transfection using multiple constructs. Primary motor neurons were transfected using three different constructs coding for the F-actin binding protein Lifeact fused to GFP, the soluble red fluorescent protein mCherry, and the nuclear localized blue fluorescent protein EBFP2-Nuc. A. All transfected cells expressed all three fluorescent proteins. B. High magnification shows the different subcellular localization of all three proteins in the same cell. Background dots in the red and blue channels are due to remaining autofluorescent beads. Size bars: 200 μm in A and 10 μm in B.

Figure 5

Fluorescent protein-tagged Smn is localized and actively transported in the axons of primary motor neurons after magnetofection. A. Full-length Smn fused to the N- (top) or C-terminus (bottom) of EGFP or mCherry fluorescent proteins (FP) was tested in order to exclude artefacts caused by the fusion partner. Compressed deconvolved 3-D image stacks show Smn-positive gems in the nucleus (arrows) and small granules localized along the axon (arrowheads). DAPI staining (blue) identifies nuclei. B. Live cell imaging of Smn-mCherry granules. Single snapshots of a representative Smn-positive granule moving anterogradely toward the growth cone (star) are shown. The granule moved 50.05 μm with an average speed of 1.80 μm/sec. Size bars: 10 μm in A, 15 μm in B.

Figure 6

Smn knockdown in primary cultured motor neurons after magnetofection with an shRNA construct. Primary motor neurons were transfected with an Smn-specific (top) or control shRNA plasmid (bottom) for 5 days. Transfected cells were identified by EGFP expression (green). Anti-Smn antibody staining (red) was used to evaluate Smn protein levels. Fluorescence intensity was quantified in the cell body and axon using the Imaris software and normalized to the axon volume. Smn protein levels were reduced in both cell body and axon when the Smn-specific shRNA was used, in comparison to the control shRNA construct. Bars represent mean and SEM. One way ANOVA, n = 3; ***p < 0.001, **p < 0.01. Size bar: 10 μm.