High-efficiency transfection of cultured primary motor neurons to study protein localization, trafficking, and function
© Fallini et al; licensee BioMed Central Ltd. 2010
Received: 26 February 2010
Accepted: 21 April 2010
Published: 21 April 2010
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.
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.
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.
Motor neuron diseases are a group of lethal neurological disorders with different etiologies and clinical variability that have one characteristic in common: the selective degeneration of motor neurons, a small population of cells within the central nervous system with a highly specialized morphology and function . To study the selective vulnerability of motor neurons in these diseases, isolated primary motor neurons, either cultured pure or in co-culture with glia or muscle cells, have been established as an important in vitro model. Studies on cultured spinal cord motor neurons have made important contributions to our understanding of motor neuron neurobiology, such as the identification of neurotrophic factors that regulate survival, regeneration and plasticity [2, 3].
However, functional studies in primary motor neurons have been hampered by the lack of reliable and efficient transfection protocols to enable not only protein overexpression, but also gene knockdown with shRNA constructs. Currently, the only method with acceptable transfection efficiency is transduction with lentiviral vectors, which has some limitations (e.g. size of the insert), and is both time consuming and requires additional safety precautions and special equipment [4, 5]. A representative example for a motor neuron disease where most in vitro studies have been performed in immortalized cell lines is spinal muscular atrophy (SMA). SMA is caused by mutations or gene deletions in the survival of motor neuron (SMN1) gene encoding the ubiquitously expressed SMN protein [6–8]. SMN has a critical housekeeping function in the correct assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) . It is a major conundrum in SMA research how a reduction in levels of the ubiquitously expressed and essential SMN protein in all tissues selectively affects motor neurons. Since SMN-containing granules have been found to be actively transported in processes and growth cones of chick forebrain neurons  and axonal defects have been observed in SMA animal models [11, 12], we and others have proposed an additional role for SMN in the axon that may contribute to the unique vulnerability of motor neurons to low levels of SMN protein [7, 8, 13–15]. However, expression of fluorescent-tagged SMN protein has never been used as a complementary approach to immunofluorescence in motor neurons, and SMN trafficking has never been studied in axons of live primary motor neurons before. To address these limitations of motor neuron cell culture models, we have established magnetofection as a novel tool for the reliable, rapid and efficient non-viral transfection of embryonic primary motor neurons. In the magnetofection method, DNA aggregated with superparamagnetic iron oxide nanoparticles is deposited on target cells by the application of a magnetic field and taken up via endocytosis .
We have carefully optimized the time course, media, and reagent concentration for primary motor neurons to routinely achieve transfection efficiencies higher than 45%. Importantly, survival and morphology of the transfected motor neurons were not altered. Magnetofection allows both efficient overexpression and knockdown of the gene of interest to study the effects of both toxic gain of function and loss of function on the differentiation and survival of primary motor neurons.
Using this method, we have shown for the first time the active transport of the survival of motor neuron (SMN) protein along the axon of live primary motor neurons.
The described method has not been used in motor neurons before and represents a powerful new tool to study the role of proteins implicated in SMA and other motor neuron diseases in the very cell type that is primarily affected by these disorders.
Results and Discussion
Primary motor neurons can be efficiently transfected with magnetic nanobeads
In an effort to overcome fundamental limitations of viral and non-viral gene transfer into primary cultured motor neurons, we have tested transfection with magnetic nanobeads (magnetofection), a method that has been previously used on primary hippocampal neurons . Postmitotic neurons are sensitive to cytotoxicity and difficult to transfect. This is especially true for primary motor neurons, in which the efficiency of conventional transfection techniques (i.e. liposomes, electroporation) is far too low for most applications or the transfection procedures are cytotoxic, inducing morphological changes and cell death. Whereas in hippocampal neurons several transfection methods such as nucleofection , liposomes , Polyethylenimine  and Ca(2+)-phosphate  have been established, currently there is no efficient method for the transfection of primary motor neurons available.
Taken together, these data show that the use of paramagnetic nanobeads represents a new and highly efficient method to transfect primary cultured motor neurons at different stages of differentiation and maturation. On average, two thirds of the cells obtained from one single lumbar spinal cord were used per magnetofection. Using coverslips smaller than 15 mm would further reduce the required cell number. Importantly, this shows that our protocol works with low cell numbers and therefore can be used to perform multiple transfections of motor neurons from single mouse embryos, for example to compare the effect of gene expression in wild type and knockout motor neurons.
Primary motor neurons show normal morphology and survival after magnetofection
In parallel to quantifying transfection efficiencies, we investigated potential direct toxic effects or cell membrane perturbations caused by the magnetic beads, that could cause changes to cell morphology and survival. Magnetic nanoparticles are made of biodegradable iron oxide with a polymer coating and have been shown in other cell types to be non-toxic and not to influence cellular functions [17, 22–24]. Primary motor neurons, however, are especially sensitive to changes to the environment and may be more susceptible to cytotoxicity than other cell types.
Taken together, these data demonstrate that motor neurons can be efficiently transfected without a negative impact on their survival and morphology, and that gene expression persists at a high level several days after transfection, establishing the magnetofection method as a valuable tool for studying motor neuron biology.
Several constructs can be co-transfected efficiently into primary motor neurons
Axonal localization and bi-directional axonal transport of Smn in transfected motor neurons
As of now, the vast majority of studies on SMN function have been performed in non-neuronal cells (i.e. fibroblasts) that do not represent the cell population affected in SMA. Due to the lack of reliable transfection methods, SMN localization and trafficking using fluorescent reporters has yet to be studied in motor neurons.
Since our results have shown that magnetofection has no apparent toxic effect on motor neurons, we attempted to use this method for studying the axonal transport of Smn. Performing successful live-cell imaging experiments demands that the cells remain in a healthy state and that cell morphology and function are preserved during examination under the fluorescence microscope. To study trafficking of Smn-containing granules in the axon of living motor neurons, we transfected an expression construct for Smn tagged with mCherry into primary motor neurons. In order to increase the chances to observe moving granules, cells were starved with medium lacking trophic factors before adding complete glia-conditioned imaging medium supplemented with 100 μM of the cell-permeable cAMP analogue 8-Bromo-cAMP, a paradigm known to stimulate β-actin mRNA granule transport . Using time-lapse video microscopy, we could show for the first time that Smn is actively transported in axons of primary motor neurons, both retro- and anterogradely (Fig. 5B and additional file 1). Smn-containing granules moved at a speed of 2.32 ± 0.7 μm/sec. No differences were observed between particles moving anterogradely toward the growth cone (2.21 ± 0.37 μm/sec; n = 5) or retrogradely toward the cell body (2.46 ± 1.02 μm/sec; n = 4). These results show that magnetofection can meet the technical challenges presented by live cell imaging and allow for analysis of axonal protein transport in primary motor neurons.
Magnetofection can be used to silence gene expression
In this study we have established magnetofection as a novel method for the transfection of primary motor neurons that is efficient, robust and non-toxic. It is cost-effective, easy to perform, and does not require specialized equipment. Our optimized protocol allows for efficient transfection of motor neurons during different stages of maturation, high levels of expression over several days, and the co-expression of at least three proteins in one cell.
To test the application of this promising new method, we have for the first time overexpressed and knocked-down the SMA disease protein Smn in the cell body and axon of motor neurons. We have also shown for the first time the active transport of Smn-containing granules along motor neuron axons anterogradely and retrogradely with a speed consistent with fast axonal transport.
We anticipate that this efficient transfection method for primary motor neurons and its applications described in this study will also have more general applications in neurodegeneration research. We expect that this approach will facilitate the research on pathways regulating motor neuron development and survival as well as molecular mechanisms underlying motor neuron disease.
Embryonic primary motor neuron culture
C57BL6 timed pregnant mice (Charles Rivers) were sacrificed following the protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Emory University. Primary motor neurons from E13.5 mouse embryos were isolated and cultured essentially as described [30, 31], but the magnetic column step was omitted, and 6% Optiprep (Sigma) was used for gradient centrifugation. Glia cells obtained from embryonal spinal cords after motor neuron isolation were grown in MEM, 10%FBS, 10 mM Hepes, 33 mM glucose. Secondary glia cells were incubated with motor neuron culture medium (Neurobasal, 0.5 mM Glutamax, 2% B27; Invitrogen) for one day to obtain glia-conditioned media. Motor neurons were plated on 15 mm coverslips (Assistant cover glasses; Carolina) coated with 0.5 mg/ml poly-ornithine (MW 30-70 KDa; Sigma) and 0.5 mg/ml laminin (Invitrogen), and cultured in glia-conditioned Neurobasal/B27 medium supplemented with 2% horse serum (Sigma), and 10 ng/ml each BDNF, CNTF, and GDNF (Peprotech).
Magnetofection of primary motor neurons
Motor neurons (2 DIV) were transfected using paramagnetic nanobeads (NeuroMag, Oz Biosciences) as follows: complete medium was replaced 1 hour prior to magnetofection with serum-free glia-conditioned Neurobasal/B27 medium. Plasmid DNA (0.5 μg) was incubated with 1.75 μl NeuroMag beads in 100 μl minimal essential medium (MEM) for 15 minutes, and then added drop-wise to the cultures. Cells were incubated on top of a magnetic plate (Oz Biosciences) for 15 minutes and complete medium was restored after 1 hour.
mCherry , Lifeact-GFP  and pEBFP2-Nuc  were kindly provided by Dr. Roger Tsien (UCSD, USA), Dr. Wedlich-Soldner (MPI of Biochemistry, Germany), and Dr. Campbell (University of Alberta, Canada; Addgene plasmid #14893). The pmaxGFP vector was from Lonza. The SMN (V2LMM_6035) and control (RHS4346) shRNA vectors were from Open Biosystems. Green (EGFP) or red (mCherry) fluorescent proteins were fused N- or C-terminal to Smn. A flexible liker [(SGGG)3] was inserted between the fusion partners to facilitate correct protein folding. A point mutation was introduced (A206K) to generate monomeric forms of GFP as described .
Cell staining and imaging
Magnetofected motor neurons were fixed for 15 minutes with 4% paraformaldehyde 24 hours to 5 days after transfection, as indicated. Anti- neurofilament (1:500; 2H3, DHSB), HB9 (1:5000; Abcam), and SMN (1:500; BD) antibodies were incubated overnight at 4°C. Rhodamine-conjugated phalloidin (1:500; Invitrogen), and Cy3-, Cy2- or Cy5-conjugated secondary antibodies were incubated for 1 hour at room temperature. Cells were imaged using a widefield microscope (TE300, Nikon) equipped with a high resolution CCD camera (Quantix, Photometrics). Image stacks were deconvolved (Autodeblur, Media Cybernetics) and analyzed using the Imaris software (Bitplane). Entire 15 mm coverslips were scanned using a motorized confocal microscope (LSM 510, Zeiss). The 2H3 antibody developed by Dr. Jessell was obtained from the Developmental Studies Hybridoma Bank (DSHB) developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242.
Life cell imaging
Motor neurons were plated on poly-ornithine/laminin coated Delta T culture dishes (Bioptechs). Twenty-four hours after transfection, cells were starved in plain Neurobasal medium for 30 minutes and then stimulated for 15 minutes with 100 μM 8-Bromo-cAMP (Calbiochem) in glia-conditioned imaging medium (Hibernate E low fluorescence, Brain Bits) supplemented with 2% B27 and 2% horse serum. Movies were acquired using a widefield epifluorescence microscope (TE2000, Nikon) with a high speed cooled CCD camera (Cascade 512 b, Photometrics).
This work was supported by National Institutes of Health (NIH) grant HD055835 and Spinal Muscular Atrophy Foundation to GJB, and NIH grant HD056130 and Families of SMA (FSMA) to WR. We thank Dr. Roland Wedlich-Soeldner, Dr. Robert Campbell, and Dr. Roger Tsien for plasmids. We are indebted to Dr. Georg Haase and Dr. Thierry Bordet for help with the motor neuron isolation protocol. We also thank Lian Li and Andrew Swanson for excellent technical support and members of the laboratory for critical reading.
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