Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

Summary

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

Abstract

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 CNS^1,2. 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 growth^5-7.

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 transfection^6,8. 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.

Protocol

1. Preparation of Coverslips, Culture Medium, and Digestion Enzymes

1. The 12-mm round #1 glass coverslips are used for the neuronal culture. The coverslips are cleaned with 10% HCL overnight followed by ultrasonic wash with distilled and deionized water for 3 times (20 min/time). The cleaned coverslips are stored in 70% ethanol for future use. Before each experiments, the coverslips are air dried and placed into the culture plate.
2. To coat the dried coverslips, 100 μl of coating solution containing 100 μg/ml Poly-D-Lysine and 10 μg/ml Laminin working solution is added onto each coverslip and the culture plate is transferred into a 37 °C incubator. A....

Discussion

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-suspens.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MEM	Invitrogen	11090-081
Poly-D-Lysine hydrobromide	Sigma -Aldrich	P6407
Laminin	Invitrogen	23017-015
5-fluoro-2-deoxyuridine	Sigma -Aldrich	F0503
Uridine	Sigma -Aldrich	U3003
Collagenase A	Roche	10103578001
TrypLE Express	Invitrogen	12604-013
Fetal bovine serum	Invitrogen	10270-098
Penicillin-streptomycin (100X)	Invitrogen	15140-122
GlutaMAX-I (100X)	Invitrogen	35050-038
Glass coverslips (#1)	Electron Microscopy sciences	72196-12
24 well cell culture plate	Becton Dickinson	35-3047
1X PBS	Mediatech	21-040-CV
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