1Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, 2Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine
This article is a part ofJoVE Neuroscience. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.Recommend JoVE to Your Librarian
Current Access Through Your IP Address
Current Access Through Your Registered Email Address
Saijilafu, Zhou, F. Q. Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons. J. Vis. Exp. (66), e4141, doi:10.3791/4141 (2012).
It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.
1. Preparation of Coverslips, Culture Medium, and Digestion Enzymes
2. Dissection and Harvest of Adult Mouse DRG Neurons
3. Digestion and Dissociation of Adult Mouse DRG Neurons
4. Genetic Manipulation of Neurons via Electroporation
5. Culturing Adult DRG Neurons for Axon Growth Analysis
6. Fixation, Immuno-staining, and Fluorescence Imaging
7. Representative Results
In the absence of any added extracellular growth factors, the adult DRG neurons usually start to grow axons 48 hr after first plating. The axons often show branched morphologies (Figure 1). In contrast, the re-plated neurons start to extend axons only a few hr after plating, and the axons elongate with much reduced branching (Figure 2). These results suggest that re-plated neurons share similar properties to those of conditioning lesioned neurons. By using this approach, we have recently performed loss-of-function studies to examine the role of axon regeneration associated transcription factor c-Jun in axon growth from adult DRG neurons in vitro. The results showed that electroporation of a group of 4 different siRNAs targeting different regions of c-Jun (ON-TARGETplus) markedly reduced the protein levels of c-Jun in adult DRG neurons 3 days after transfection (Figure 3)9. When the neurons were re-plated and cultured overnight, axon growth from c-Jun knockdown neurons was significantly reduced (Control: 348.37± 16.21mm; si-c-Jun: 262.32±15.69 μm, Figure 3)9. These results indicate that cultured adult DRG neurons provide a useful model system to study axon growth from adult neurons.
Figure 1. Adult mouse DRG neurons cultured at low density for 3 days. The neurons were stained with anti--βIII tubulin antibody. Note that most axons show branched morphologies. Scale bar: 125 μm.
Figure 2. Re-plating adult mouse DRG neurons after 3 days in culture. (A) Adult DRG neurons cultured at high density for 3 days. (B) Re-plated adult DRG neurons were cultured at low density for overnight. Note that most axon show elongated morphologies with little axon branching. Scale bar: 250 μm in A and 125 μm in B.
Figure 3. Role of c-Jun in axon growth from adult DRG neurons in vitro. (A) Western blot analysis of c-Jun in adult mouse DRG neurons after electroporation of c-Jun siRNAs. The result shows markedly reduced level of c-Jun. (B) Control neurons transfected with EGFP grew long axons after re-plating and overnight culture. (C) Co-transfection of c-Jun siRNAs and EGFP impaired axon growth from adult DRG neurons after re-plating and overnight culture. Red: Tuj-1 staining; Green: EGFP. Scale bar: 125 μm. These results have been published in Saijilafu et al.9.
Adult DRG neurons regenerate their axons robustly after peripheral nerve injury in vivo and in vitro, thus providing a useful system to study axon regeneration in adult animals. In vitro culture of adult DRG neurons is becoming a widely used method to investigate the molecular mechanisms by which axon regeneration is regulated. The in vitro procedure of culturing adult mouse DRG neurons presented here enables rapid and effective genetic study of regenerative axon growth. The re-suspension and re-plating procedure is particularly useful for loss-of-function studies because it allows axons re-grow from neurons in which the targeted protein has already been depleted. Moreover, re-plating cultured DRG neurons mimics an in vivo conditioning lesion effect, thus providing a more physiological relevant approach to study axon regeneration in vitro.
The transfection efficiency of adult DRG neurons with the described electroporation approach is about 20-50% for DNA plasmids depending on the sizes of the constructs, which is sufficient for morphological analysis of axon growth. Comparing to electroporation, the viral-mediated gene transfer has much higher efficiency, which is suitable for biochemical analysis using DRG neurons. However, constructing and producing virus for each gene-of-interest is much more labor intensive and time consuming. For siRNA oligos, the transfection efficiency can reach nearly 90% based on the knocking down efficiency of the targeted proteins (see Figure 3A), which makes biochemical analysis of adult DRG neurons possible. However, the effect of siRNAs reduces after longer period due to the degradation of the siRNA oligos.
When neurons are co-transfected with 2 plasmids mixed at 1:1 ratio, the plasmid with smaller size usually has higher transfection efficiency than the plasmid with bigger size. As a result, adjusting the ratio of the two plasmids will change the co-transfection efficiency accordingly. For siRNA transfection, we often co-transfect the neurons with EGFP to label neurons. Because the siRNA oligos are much smaller than EGFP, their transfection efficiency is much higher. Therefore, in our experiments we generally think that all EGFP positive neurons are also positive for siRNAs.
Many genetic profiling studies of adult DRG neurons after peripheral axotomy have identified a large number of regeneration-associated genes (RAGs)10-12, which are believed to underlie the regeneration ability of adult DRG neurons. However, the functions of these RAGs in mediating axon growth of adult DRG neurons have not been well characterized. Here we examined the role of a well-known RAG, c-Jun, in mediating axon growth from adult DRG neurons via RNAi-mediated loss-of-function approach. Such method provides a valuable in vitro tool to investigate the roles of other RAGs in regulation of axon regeneration.
No conflicts of interest declared.
This work was supported by grants to F. Z. from NIH (R01NS064288) and the Craig H. Neilsen Foundation.
|Poly-D-Lysine hydrobromide||Sigma -Aldrich||P6407|
|Fetal bovine serum||Invitrogen||10270-098|
|Glass coverslips (#1)||Electron Microscopy sciences||72196-12|
|24 well cell culture plate||Becton Dickinson||35-3047|
|Sterile, distilled and deionized water||Mediatech||25-055-CV|
|Nucleofector and electroporation Kits for Mouse Neurons||Lonza||VPG-1001|
|ON-TARGETplus siRNA against c-Jun||Dharmacon||L-043776|
|Anti--βIII tubulin antibody (Tuj-1)||Covance||MMS-435P|
|ProLong Gold Antifade mounting solution||Invitrogen||P36930|