Changes in protein phosphorylation and protein expression levels in DRG during neuronal regeneration

 

Abstract

One of the most striking features of neurons in the mature peripheral nerve system is their ability to survive and to regenerate their axons following axonal injury. In this study the phosphorylation and protein expression changes after peripheral nerve crush were examined in DRG, using western blot an mass spectrometric analysis.

Because no difference in phosphorylation was shown, using western blot after one-dimensional gel electrophoresis, it was decided not to immunoblot the two-dimensional gels, but only to examine changes in protein expression.

After silverstaining of the two-dimensional gels, the six most different and abundant spots were punched out of the gels and analyzed with mass spectrometry. We found that Neurofilament triplet M protein, Neurofilament light polypeptide, Heatshock cognate protein 70, Albumine and Peroxiredoxin 1 were downregulated five and ten days after a crush. We could not identify a protein, that was downregulated after five days, and upregulated after ten days.

These five identified proteins are only a small part of the total amount of proteins with an altered expression level during regeneration. Much research will be necessary to overview all events occurring  while a DRG is regenerating.

Introduction

One of the most striking features of neurons in the mature peripheral nerve system is their ability to survive and to regenerate their axons following axonal injury. It has been increasingly evident that the succes of axonal regeneration is dependent on the intrinsic as well as extrinsic growth properties of the axotomized neuron.

The environment in which PNS axons regenerate consists of Schwann cells and their basal laminae, fibroblasts, collagen, degenerating myelin and phagocytic cells. After nerve injury, the distal axonal and myelin segment undergoes dissolution and absorption by the surrounding cellular environment, a process called Wallerian degeneration. Then, the remaining Schwann cells divide and align longitudinally within basal lamina tubes.

Growth comes from regenerating axons extend along the Schwann cell bands, growing along the Schwann cell membranes and basal laminae. Such biological and morphological changes of Schwann cells are thought to be controlled by injury-induced molecules that are expressed by neurons and Schwann cells themselves.

Especially, at around seven days after nerve injury, many nerve regeneration-related factors reach peak levels and the growth cones of regenerating axons begin to move over the Schwann cell surface (Kubo et al., 2002). Neurons can be switched into a growth mode after peripheral nerve crush or transsection (axotomy). All functions, including sensory transduction, motor control and autonomic regulation are shut down and the growth system is activated.

The regrowth of mature peripheral neurons is an important topic of research. Especially the growth activation system has been studied intensively and many injury regulated genes and proteins are identified by different techniques (Snider et al., 2002). 

In different systems phosphorylation of proteins is induced by damage in order to initiate the process of recovery (Kim et al., 2002).  Therefore it is worth investigating phosphorylation changes and changes in protein expression in regeneration of mature peripheral nerve axons.

It was shown that regeneration in mature nerve cells of cyclin D1 knock-out mice alters the phosphorylation level of proteins in a damaged axon (Snider et al., 2002). This has not yet been studied in dorsal root ganglia (DRG).

DRG are the most favorable neurons in experimental research, because they are easy to obtain from an adult animal and it is proved by Smith and Skene (1997) that in DRG a growth program is activated after a crush (Snider et al., 2002).

Many protein expression changes are detected using cDNA micro-array. Different expression of more than 500 genes in injured peripheral nerve was observed. The cDNA-library derived from the distal stumps of post-injured sciatic nerve, which was enriched in non-myelinating Schwann cells (Kubo et al., 2002).

However, because these studies used only cDNA micro-array, in the following experiment changes in protein expression are examined. Protein samples made from isolated DRG were analyzed with mass spectrometry in stead of cDNA micro-array, in order to compare the results of this study with the results of studies using cDNA-micro-array.

The meaning of this study is to look at the phosphorylation and protein expression changes after peripheral nerve crush were examined in DRG, using western blot an mass spectrometric analysis.

Experimental procedures

Protein extraction – Three DRG of each animal (table 1), five and ten days after crush were mixed with 400 ml lysisbuffer containing 8 M Urea, 4% Chaps, 20 mM Tris, 65 mM DTT and pharmacia’s IPG buffer (www.bio.vu.nl). The DRG in the mixture were pulverized with an electrical potter for five minutes and then with a glasspotter for approximately ten minutes. The mixtures with the pulverized DRG were centrifuged for ten minutes until a pellet appears.

Tekstvak: Table 1. Samples
Sample number	Days after crush	Animal	Sample type
1	5	F	Control DRG
2	5	F	Crush DRG
3	5	T	Crush DRG
4	5	T	Control DRG
5	10	T	Control DRG
6	10	F	Control DRG
7	10	F	Crush DRG
8	10	T	Crush DRG

One- dimensional gelelectro-phoresis and immunoblot analysis –  20 ml of the supernatant of the DRG mixtures was mixed with 40 ml SDS sample buffer (www.bio.vu.nl) and these samples were boiled. 15 μL of each sample was loaded onto two 10% polyacrylamide gels, which ran for 1 hour. After electrophoresis one gel was semi-dry-western-blotted and the other one was wet-western-blotted (Fig. 1). Semi-dry western blot was run for an our at a very high voltage, while wet western blot was run overnight at a low voltage. 1x CAPSO/Tris 9.5 electroblot transfer buffer was used with both blots. Semi-dry western blot requires little buffer, while wet western blot requires 2.5 l of the buffer. During the blots, the proteins were transferred from the gel into nitrocellulose membranes.

Blotting paper

Gel

Membrane

Blotting paper

 

Fig. 1 Schematic representation of a Western-blot

The blotted gels were stained with Coomassie Blue to visualize if the proteins were transported to the membrane (Fig. 3).

The blots were incubated overnight at room temperature with anti-phosphotyrosine clone 4G10 (Upstate Biotechnology®) at a 1:2000 dilution in 2X TBS-Tween 20. After incubation with this first antigen, the membrane was coated with BSA to make sure that the second antigen would not bind to the membrane itself. The protein-antibody complexes were visualized with horseradish peroxidase-rabbit anti-mouse immunoglobulin G conjungate (Dako®) at a 1:2000 dilution (Fig. 2).  This second antigen was incubated for 90 minutes at room temperature.  After the incubations with the antigens, the membrane was washed twice. The blot was incubated in the ECL plus kit (Amersham Bioscience®) solution and exposed to x-ray film.

Tekstvak:  
Fig. 2 Antigen interaction of immunoblotting

 

 

 

 

 

 

Two-dimensional gelelectrophoresis - The amount of two DRG was extracted from the samples and diluted with lysisbuffer to a volume of 350 μL. These diluted samples were loaded onto IPG-strips with a 3-10 pH range and run in accordance with the following program (table 2).

Table 2. Run-program first dimension

Step

Voltage

Step duration (hours)

Volt-hours

Gradient type

Rehydration

30

12

360

Step-n-hold

1

500

1

500

Step-n-hold

2

1000

1

1000

Step-n-hold

3

8000

8

64000

Step-n-hold

Total

 

22

65860

 

The rehydration step helps the proteins diffuse in the gelstrip. The slow steps from 30 V to 8000 V makes sure that the salts move to the 10 pH end of the strips, where they have less disturbance in the reaction. In this first dimension the separation of the proteins was based on a difference in pH.

The IPG-strips with the run samples were equilibrated twice, each time for 15 minutes in 2.4 ml equilibrationbuffer. Equilibrationbuffer 1 contains DTT , while equilibrationbuffer 2 contains iodoacetamide to remove excess DTT.

After equilibration the proteins were negatively charged and the strips were put on a 11% DALT slab gel and fixed with agarose. In this second dimension the separation was based on differences in molecular weight.

The run gels were silverstained to visualize proteins as spots in accordance to the following program (table 3).

Table 3. Protocol Silver Staining

Step

Time (minutes)

Chemicals

Fixation

45-60

5%HAc/50% meOH

Wash steps

2x15

50% meOH followed by distilled water

Sensitization

1.5-2.0

0.02% sodiumthiosulphate

Wash steps

1x2 followed by 1x1

Distilled water

Silver Staining

30-45

0.15% silver nitrate

Wash steps

1x2 followed by 1x1

Distilled water

Develop

Until desired intensity is achieved

37% formaldehyde in 2% sodium carbonate

Stop

60

5% HAc

Store

forever

1% HAc/10% meOH


Mass Spectrometric Analysis- The spots on the gels, of the crushed DRG and the control DRG, were compared. The six most different and abundant spots (fig. 5) were punched out of the gels and transferred to 50% acetonitril in order to be decolorized. After the decolorization, the spots were dehydrated in 100% acetonitril, then they were centrifuged in a Speedvac Concentrator. In order to digest the proteins, 15 ml trypsin was added and incubated overnight. The peptides were purified on a column and sequenced in a Q-tof micromass massspectrometer.

Results

One dimensional gelelectrophoresis and immunoblot analysis

After the gels were stained with Commassie Blue they were washed thoroughly and scanned by a Bio-rad scanner (Fig. 3).

 

     

Fig. 3     Blotted gels. A Semi-dry blotted gel. B Wet blotted gel. The black bands represent the proteins that remained on the gel after blotting.

The purpose of the staining of the gels after blotting was to visualize the differences between the two membranes, in order to determine in which blot the transfer of proteins was most succesful. As shown in Fig. 3, there is no significant difference between the protein transfer, since the thickness of the bands is approximately equal in both gels.

The Westernblot was washed and an X-ray was made (Fig. 4).

Figure 4. revised scan of a two-second exposed X-ray.

 

The X-ray shows no difference in protein phosphorylation between the controls and the crushed DRG. Therefore there was no need to blot the two-dimensional gels, and only changes in protein expression were examined.

Two-dimensional gelelectrophoresis

After the gels were run, they were silverstained (fig 5). The gels were scanned and the controlgels were compared with the crushgels to visualize the difference between the spots (fig. 5). The six most different and abundant spots that were punched out of the gels are marked below (fig. 5).

 


A Scan of sample 6 (control after ten days)                            B Scan of sample 7 (crush after ten days)        

C Spot 1, 2, 3 sample 6 (control after ten days)

 

D Spot 1, 2, 3 sample 5 (control after ten days)

 

E Spot 1, 2, 3 sample 1 (control after five days)

 
 


F Spot 1, 2, 3 sample 7 (crush after ten days)

 

G Spot 1, 2, 3 sample 8 (crush after ten days)

 

H Spot 1, 2, 3 sample 3 (crush after five days)

 
 

I Spot 4 sample 6 (control after ten days)

 

J Spot 4  sample 5 (control after ten days)

 

K Spot 4  sample 1 (control after five days)

 


L Spot 4  sample 7 (crush after ten days)

 

M Spot 4  sample 8 (crush after ten days)

 

N Spot 4  sample 3 (crush  after five days)

 


O Spot 5  sample 6 (control after ten days)

 

P Spot 5  sample 5 (control after ten days)

 

Q Spot 5 sample 1 (control after five days)

 

R Spot 5  sample 7 (crush after ten days)

 

S Spot 5  sample 8 (crush after ten days)

 

T Spot 5  sample 3 (crush after five days)

 
 


U Spot 6  sample 6 (control after ten days)

 

V Spot 6  sample 5 (control after ten days)

 

W Spot 6  sample 1 (control after five days)

 

6

 

 

 

 

 


X Spot 6  sample 7 (crush after ten days)

 

Y Spot 6  sample 8 (crush after ten days)

 

Z Spot 6  sample 3 (crush after five days)

 


 

 

 

In figures 5 C – H is shown that protein 1, 2 and 3 are downregulated in animals with a crush. In figures 5 I – N is shown that protein 4 is downregulated in animals with a crush. In figures 5 O – T is shown that protein 5 is downregulated five days after a crush, while ten days after the crush the protein is upregulated. In figures 5 U – Z is shown that protein 6 is downregulated in animals with a crush.

Mass Spectrometric Analysis

The results of the mass spectrometry are compared with an online database (www.matrix science.com) and the found proteins are listed below (table 4).

Table 4. results mass spectrometric analysis
Spotnumber

Protein

Protein description

Mass (Kda)

PI value (pH)

expression

1

gi|128150

Neurofilament triplet M protein

95734

4.77

Downregulation

2

gi|13929098

Neurofilament, light polypeptide

61298

4.63

Downregulation

3

gi|13242237

Heatshock cognate protein 70

71055

5.37

Downregulation

4

gi|19705431

Albumine

68674

6.09

Downregulation

5

unidentified

unidentified

unidentified

unidentified

Upregulation

6

gi|16923958

Peroxiredoxin 1

22095

8.27

Downregulation

Discussion

This study has identified a number of proteins of which the expression level changes after a crush.

A one-dimensional gel was run to examine the changes in phosphorylation of the proteins in DRG after a crush. These gels were blotted (semi-dry and wet western blot) and the semi-dry blot was incubated with anti-phosphotyrosin and a second antigen which could be detected with X-ray. Phosphotyrosin was used because there can be made specific antibodies against it, whereas they cannot be made against phosphoserin and phosphothreonin. Low background was expected by using phosphotyrosin.

After blotting the gels were stained with Coomassie to visualize if the transfer of proteins to the membrane was successful. As shown in figure 3, there is no significant difference between the proteintransfer of the two gels. Therefore, there was no need to incubate the second (wet blotted) membrane with anti-phosphotyrosin.

An X-ray film was made of the incubated (semi-dry) blot (fig.4). The X-ray film was very dark, due to a high background. The cause of this high background was that the blot, after the incubation with the second antigen, was washed only twice in stead of the required four times. The blot was washed only twice because the interaction between the first and the second antigen was thought to be weak. However the proteinbands were clearly visible, so it is recommended to wash four times. No difference was shown between phosphorylation levels in controls and crushes. It is recommended to redo the experiment with a large one-dimensional gel, in order to make a better distinction between the phosphorylation levels. Because no difference in phosphorylation was shown in this experiment, it was decided not to immunoblot the two-dimensional gels, but only to examine changes in protein expression.

Two-dimensional gels were run to examine the changes in protein expression in DRG after a crush. As can be seen in figures 5A and 5B, there was a blank region on the right side of the gels. This phenomenon is probably caused by a high salt concentration in chemicals used for lysisbuffer. However, these chemicals are necessary for good unfolding and unclotting the proteins, in order to run a proper gel.

On most gels one can see vertical black smear. This is due to pollution from the pellet of the samples. To prevent this in the future, it is recommended to centrifuge the samples much longer and to be very careful while transferring the supernatant of the samples.

Two gels failed to give a proper result, namely the gels of sample 2 and 4. On the first dimension of sample 2, too little sample was loaded, which caused a very light gel after staining. While preparing the second dimension of sample 4, the agarose, used to fix the IPG-strip, got between the strip and the gel. This caused vertical black stripes on the gel, which made it impossible to distinguish spots.

The gels of sample 6 and 7 showed the best results, so these gels were used to choose six spots to punch out for mass spectrometric analysis (fig. 5). After the selection of the spots on gels 6 en 7, these spots were compared with the same spots on the other four gels. Only when the spots were present on all gels, they were punched out.

During the comparison of the spots, it was discovered that the protein that represents spot 5 was downregulated in crushed DRG after five days, whereas after ten days the protein was upregulated.  Unfortunately, this protein could not be identified  in this experiment. It is recommended to repeat the two-dimensional gel electrophoresis in order to try to identify this protein again.

Protein 1, 2, 3, 4 and 6 are all downregulated both after five and ten days after crush, as shown in figure 5. With mass spectrometric analysis protein 1 – 6 (except protein 5) were identified (table 4).

Neurofilament triplet M protein (spot 1) and Neurofilament light polypeptide (spot 2) are both part of the cytoskeleton. In the early stages of neuronal growth the cell contains very few neurofilament and therefore it is obvious that these proteins are downregulated shortly after a crush (Karp, 2002)

Heat shock cognate protein 70 (spot 3) belongs to the family of heat shock proteins, also called stress proteins. They are induced when a cell undergoes various types of environmental stresses. HSPs are also present in cells under perfectly normal conditions. They act like “chaperones,” making sure that the cell’s proteins are in the right shape and in the right place at the right time (Karp, 2002). HSPs are induced a few hours after a crush, but they are downregulated after five and ten days.

Albumine (spot 4), a plasma protein, is normally not present in DRG, but only in the blood circulation. Probably the DRG were not properly dissected and there were fragments of blood in the samples, so it is recommended to dissect more carefully in the future.

Peroxiredoxin 1 (spot 6) belongs to peroxyredoxins, a novel family of anti-oxidant proteins that specifically prevent enzymes from metal-catalyzed oxidation. Peroxyredoxins are capable of neutralizing the harmful effects of waste products (www.link.springer.de, www.inmr.com.au). Because after a crush many normal processes in the cell are reduced, less waste products will be formed, so peroxyredoxins are downregulated.   

These five identified proteins are only a small part of the total amount of proteins with an altered expression level during regeneration. Much research will be necessary to overview all events occurring  while a DRG is regenerating.

References

Karp G., Cell and molecular biology, John Wiley & Sons, Inc., third edition 2002, 95 – 98, 364 – 365.

Kim H., Song E. and Lee K. (2002) Proteomic analysis of protein phosphorylations in heat shock response and thermotolerance, in Journal of Biological Chemistry 26, 23193 – 23207.

Kubo T., Yamashita T.,Yamaguchi A., Hosokawa K. and Tohyama M. (2002) Analysis of genes induced in peripheral nerve after axotomy using cDNA micro-array’s, in Journal of Neurochemistry 82, 1129 – 1136.

Snider W., Zhou F., Zhong J. and Markus A. (2002) Signaling the pathway to regeneration, in Neuron 35, 13 – 16

http://www.bio.vu.nl/vakgroepen/mnb/frs1/proteomics.html, 19 – 11 - 2002

http://www.inmr.com.au/inmr_team16.html, 19 – 11 - 2002

http://www.link.springer.de/link/service/journals/00441/contents/99/00115/s004419900115ch002.html, 19 – 11 - 2002

http://www.matrixscience.com, 18 – 11 - 2002