An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Podsumowanie

In this report, we describe a simple protocol for studying neurite outgrowth in embryonic rat cortical neurons by co-transfecting with EGFP and the protein of interest.

Streszczenie

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic dysfunction occur in various neurodegenerative diseases and age-related degeneration. Investigation of the mechanisms that regulate neurite outgrowth would not only shed valuable light on brain developmental processes but also on such neurological disorders. Due to the low transfection efficiency, it is currently challenging to study the effect of a specific protein on neurite outgrowth in primary mammalian neurons. Here, we describe a simple method for the investigation of neurite outgrowth by the co-transfection of primary rat cortical neurons with EGFP and a protein of interest (POI). This method allows the identification of POI transfected neurons through the EGFP signal, and thus the effect of the POI on neurite outgrowth can be determined precisely. This EGFP-based assay provides a convenient approach for the investigation of pathways regulating neurite outgrowth.

Wprowadzenie

Neurites, including both axons and dendrites, are the projections from neurons involved in the establishment of the neural networks. The dynamic outgrowth of neurites is essential for neurodevelopment. However, the underlying regulatory mechanisms underneath remain unclear. In particular, neurite damage is often observed in various neurodegenerative diseases and after brain injuries¹. Therefore, investigation of the roles of putative molecules in various neurite outgrowth regulatory pathways would improve our understanding of the process. Moreover, it may reveal novel therapeutic targets for various neurological disorders. Neuronal cell lines are valuable models for studying neuronal processes including neurite outgrowth as they are easy to manipulate and transfect^2,3. However, genetic drift has been reported to occur in some commonly used cell lines, which could lead to variations in their physiological responses⁴. Moreover, differential protein expression has been shown between neuronal cell lines and primary neurons. For instance, PC12, a neuronal cell line derived from rat adrenal gland that is widely used for studying neurite outgrowth^2,3, does not express NMDA receptors⁵. Furthermore, it has been proposed that the reduced responsiveness of the mouse neuroblastoma line neuro-2a to neurotoxins in comparison to primary neurons is due to the lack of expression of certain membrane receptors and ion channels⁶. Therefore, primary neurons are a more desirable and representative model for the investigation of neurite outgrowth. However, the use of primary neurons is hindered by their low transfection efficiency⁷.

Here, we describe a method that involves the co-transfection of the protein of interest (POI) and EGFP to primary rat cortical neurons. The EGFP acts as a morphological marker for the identification of successfully transfected neurons and permits the measurement of neurites. We validated this method by using compounds/molecules that have been reported to modulate neurite outgrowth. Moreover, FE65, a neuronal adaptor protein that has been shown to stimulate neurite outgrowth, was used to illustrate this approach^8,9. This protocol involves (1) the isolation of primary cortical neurons from embryonic day 18 (E18) rat embryos, (2) the co-transfection of neurons with EGFP and the POI (FE65 in this study) and (3) the imaging and analysis of the neurons by using the image processing software ImageJ with the NeuronJ plugin^10,11.

Protokół

All procedures followed were in accordance with the ethical standards of the animal experimentation ethics committee of the Chinese University of Hong Kong.

1. Preparation of Coverslips

1. Place a sterile 18 mm circular coverslip into each well of a 12-well tissue culture plate.
2. Coat the coverslip with 5 µg/mL poly-D-lysine solution in a humidified 37 °C incubator for at least 1 h.
3. Aspirate the poly-D-lysine solution from the tissue culture plate and rinse the coated coverslips once with sterile water.

2. Rat Embryonic Neuron Dissection

1. Sacrifice a timed-pregnant Sprague-Dawley rat at a gestational age of 18 days (E18) by either cervical dislocation or CO₂ asphyxiation.
   NOTE: Please check local regulations for the sacrifice of pregnant rats.
2. Open the abdominal cavity of the pregnant rat with dissecting scissors and transfer the uterus to a 10 cm Petri dish.
3. Open the uterus and the amniotic sac carefully with small dissecting scissors and remove the placenta from the rat embryo using small dissecting scissors. Transfer the whole embryo to a 10 cm Petri dish with pre-chilled phosphate buffered saline supplemented with glucose (PBS-glucose, 10 mM sodium phosphates, 2.68 mM potassium chloride, 140 mM sodium chloride and 3 g/L glucose) using a pair of small forceps.
4. Cut along the sagittal suture of the skull and open it carefully with a pair of small dissecting scissors. Transfer the embryonic brain with a small flat spatula to a 10 cm Petri dish with ice-cold PBS-glucose.
5. Separate the two cerebral hemispheres from the cerebellum and brain stem using two #5 tweezers under a dissection microscope.
   NOTE: Please see reference¹² for the structure of the rat brain.
6. Remove the meninges using the #5 tweezers.
7. Isolate the cortex from the cerebral hemispheres with two straight #5 tweezers.
8. Transfer the isolated cortex to ice-cold PBS-glucose in a 15 mL centrifuge tube.

3. Primary Cortical Neuron Culture

NOTE: All procedures in steps 3 and 4 are performed inside a Class II Biosafety cabinet.

1. Settle the isolated cortex by gravity at 4 °C for 5 min and aspirate the PBS-glucose.
2. Add 1 mL of 0.05% Trypsin-EDTA to the isolated cortex and mix gently by tapping and incubate the tissue in a 37 °C water bath for 10 min to allow enzymatic digestion. Tap the tube gently to mixing every 2 min.
3. Add 4 mL of maintenance medium (e.g., Neurobasal Medium) to the tissue/trypsin mixture.
   NOTE: All the maintenance medium used in this protocol is supplemented with Penicillin-Streptomycin and B-27 supplement¹³.
4. Dissociate the tissue gently by trituration using a 1 mL pipette.
5. Pellet the dissociated cells by centrifugation at 200 x g for 5 min. Aspirate the supernatant.
6. Repeat steps 3.5 to 3.7 twice.
7. Resuspend the cell pellet in 1 mL of maintenance medium.
8. Add 10 µL of 0.4% Trypan Blue solution to 10 µL of cell suspension for counting of viable cells by a hemocytometer.
9. Plate the neurons at a density of 65,000/cm² (viable cells) in 1 mL of maintenance medium per well in a 12-well plate.

4. Cell Transfection and Fixation

1. At 2 days in vitro (DIV2), transfect 0.5 µg of EGFP construct (pEGFP-C1) to neurons together either with or without of 0.5 µg of POI by using 1 µL of transfection reagent (e.g., Lipofectamine 2000). Use manufacturer's instructions.
   NOTE: Mammalian expression constructs were prepared by using an endotoxin free plasmid preparation kit. Treatment with chemicals/molecules (in this manuscript cytochalasin D (Cyto D) and nerve growth factor (NGF) were used) can be done at 6 h after transfection.
2. Aspirate the culture medium 24 h post-transfection and wash the transfected cells once with 37 °C PBS (10 mM sodium phosphates, 2.68 mM potassium chloride, 140 mM sodium chloride).
3. Fix the cells with 4% paraformaldehyde in PBS for 10 min in the dark at room temperature.
4. Wash the fixed cells three times with PBS.
5. Add a minimal amount of fluorescence mounting medium on a microscope glass slide. Carefully transfer the coverslip from the 12-well plate onto the mounting medium with the sample facing the glass slide.
   NOTE: Seal the edge of the coverslip with nail polish if an aqueous mounting medium is used.

5. Measurement of Neurite Outgrowth

1. Use a 40x objective for capturing images using an epi-fluorescent microscope.
2. Capture images from at least 40 intact neurons with EGFP signal per transfection.
3. Open the captured image in ImageJ software with the NeuronJ plugin¹¹ to measure the length of the longest neurite, from the cell body to the tip of the growth cone, of each imaged neuron.
4. Analyze the data obtained with the software to determine the effect of the targeted proteins in neurite outgrowth.

Wyniki

To test this methodology, we used Cyto D and nerve growth factor NGF, which have been shown to inhibit and stimulate neurite outgrowth respectively^14,15,16. The neurite length of neurons transfected with EGFP were measured after treatment with Cyto D or NGF. The transfection efficiency of EGFP to the neurons was 2.7% (1,068 neurons counted). As shown in Figure 1

Dyskusje

As stated before, PC12 and its subclones are widely employed for studying neurite extension because they have excellent transfection efficiency^2,3. In contrast, primary neurons have a low transfection rate, which is a major obstacle for studying neurite outgrowth regulators by transfection⁷. Here, we describe a convenient protocol for quantifying neurite outgrowth in primary neurons. Despite the low overall transfection efficiency, more th...

Materiały

Name	Company	Catalog Number	Comments
#5 tweezers	Regine	5-COB
18 mm Circle Cover Slips	Thermo Scientific	CB00180RA	Sterilize before use
B27 Supplement	Gibco	17504044
Cytochalasin D	Invitrogen	PHZ1063	Dissolved in DMSO
D-(+)-Glucose	Sigma-Aldrich	G8270
Dimethyl Sulfoxide	Sigma-Aldrich	D2650
Dissecting Scissors, 10 cm	World Precision Instruments	14393
Dissecting Scissors, 12.5 cm	World Precision Instruments	15922
EndoFree Plasmid Maxi Kit	QIAGEN	12362
Fluorescence Mounting Medium	Dako	S302380
Lipofectamine 2000 Transfection Reagent	Invitrogen	11668019
Neurobasal Medium	Gibco	21103049
NGF 2.5S Native Mouse Protein	Gibco	13257019
Nugent Utility Forceps, 10 mm, Straight Tip	World Precision Instruments	504489
Paraformaldehyde	Sigma-Aldrich	P6148
pEGFP-C1	Clontech	#6084-1
pCI FE65			Please see references 8 and 15
PBS Tablets	Gibco	18912014
Penicillin-Streptomycin	Gibco	15140122
Poly-D-lysine hydrobromide	Sigma-Aldrich	P7280
Spatula	Sigma-Aldrich	S4147
Trypsin-EDTA (0.05%), phenol red	Gibco	25300062
Trypan Blue Solution, 0.4%	Gibco	15250061