Changes in synthesis of specific proteins in axotomized dorsal root ganglia

EXPERIMENTAL

Changes

76, 83-93 (1982)

NEUROLOGY

in Synthesis of Specific Proteins in Axotomized Dorsal Root Ganglia MICHAEL

Department

E. HALL’.’

of Physiology

and Biophysics, University Miami, Florida 33010

of Miami

School

Received

10, 1981:

November

3, I981

July

revision

received

of Medicine,

Changes in the relative synthesis rates of a number of specific proteins from rat dorsal root ganglia (DRG) were observed in response to transection of the sciatic nerve. The pattern of these changes was compared to that in rat sympathetic ganglia after transection of the postganglionic nerves. Although the overall pattern of proteins synthesized, and the pattern of changes seen after nerve transection, were quite similar in both ganglia, some specific differences were observed.

INTRODUCTION Dorsal root ganglia, like autonomic ganglia, respond to axon transection with a number of comparable morphologic and biochemical changes (7). Unlike autonomic ganglia, however, the axons of dorsal root ganglia (DRG) bifurcate near their exit from the cell body. One branch of each axon innervates peripheral structures and the other branch, in the dorsal root, innervates the dorsal horn of the spinal cord. The peripheral branch transmits sensory information to the DRG. The pronounced morphologic alterations typical of axon transection are observed in DRG neurons to follow transection of the peripheral, but not the central, axon branch (1). Recently changes in the relative synthesis rates of specific proteins were seen in sympathetic ganglia after axon transection (3). The purpose of this study was to look for similar changes in synthesis of specific DRG proteins after Abbreviations: DRG-dorsal root ganglion; SCG-superior cervial ganglion; NGF-nerve growth factor. ’ This research was supported by National Institutes of Health grant NS-14328. * Present address: Department of Biochemistry (B-126), University of Colorado Health Sciences Center, Denver, Colorado 80262. 83 0014-4886/82/040083-11$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

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transection of the peripheral axon branch, and to see if alterations in protein synthesis can be detected after dorsal-root transection. METHODS Rats, 200 to 300 g in weight, were anesthetized with chloral hydrate, supplemented with ether. The left or right sciatic nerve was exposed and transected at the level of the hip. In other animals, the peripheral branches of the fourth or fifth lumbar DRG were exposed and transected unilaterally where they exit from the vertebral column. In a third group of rats, a spinal laminectomy was carried out. The dura was cut and reflected, exposing the dorsal roots. The fourth lumbar dorsal root was gently lifted with sterile glass hooks and cut unilaterally with fine scissors. In some cases, the dorsal root was ligated and cut for later identification. An additional group of animals was similarly anesthetized and the superior cervical ganglion (SCG) exposed bilaterally. On either the left or the right side, the two principle postganglionic nerves, the internal and external carotid nerves, were cut within 1 mm of the ganglion body. All surgery was carried out under aseptic conditions. Some subjects were treated with penicillin after surgery. Three days after nerve transection, the subjects were again anesthetized. For DRG animals, spinal laminectomy was carried out, and the fourth through sixth lumbar DRG exposed bilaterally. The subjects were then decapitated and the DRG quickly removed. The ventral nerve root, which comprises a significant portion of the DRG, was removed and the cell bodies incubated in an aerated salt solution containing essential amino acids, glucose, vitamins, and 2.5 &i [‘4C]leucine (Schwartz/Mann, 312 mCi/mmol), as described elsewhere (3). The SCG animals were decapitated 3 days after axotomy and both SCGs quickly removed. Ganglia were desheathed in Ringer’s solution and incubated as described for the DRG. After l-h incubation, the ganglia were homogenized separately in small glass grinders, in a homogenization buffer containing sodium dodecyl sulfate (SDS), /3-mercaptoethanol, and NP-40, in 8 M urea (3). Ganglia homogenates were subjected to two-dimensional (2-D) polyacrylamide gel electrophoresis and quantitative autoradiography (16). Briefly, homogenate proteins were first separated by isoelectric focusing in a pH gradient of 4.5 to 8. They were then separated on the basis of molecular weight, by SDS electrophoresis, over a range of 20,000 to 200,000. The resultant 2-D gels were stained with Coomassie brilliant blue R, dried under vacuum, and used to expose x-ray film (Kodak NS-2T) for detection of incorporated [14C]leucine. Autoradiographs of the 2-D gels were used to visualize several hundred of the most abundantly synthesized

PROTEIN

SYNTHESIS

IN AXOTOMIZED

DRG

85

ganglion proteins (see Figs. 1 and 2). Proteins exhibiting an apparent alteration in relative synthesis rate, as determined by differences in autoradiographic intensity, were located on the dried gels using the autoradiograph as a guide. The protein spots were removed from the dried gels with a scalpel, dissolved in scintillation fluid (13) and counted in a Packard

FIG. 1. Top-autoradiographic pattern root ganglion (DRG). Bottom-autoradiographic after transection of the sciatic nerve.

of proteins newly synthesized in a control pattern of proteins from the DRG

dorsal 3 days

86

MICHAEL

E. HALL

1b

PROTEIN

SYNTHESIS

IN AXOTOMIZED

DRG

87

scintillation counter. Other protein spots not exhibiting an apparent change in synthesis were also analyzed. Because the object was to detect changes in specific proteins relative to overall ganglion protein synthesis, changes in absolute synthesis in experimental gels relative to control gels were eliminated by data normalization, as described elsewhere (3). RESULTS Several hundred of the more abundant DRG and SCG proteins were visualized on two-dimensional gel autoradiographs from 10 axotomized and 10 intact ganglia for both DRG and SCG. Three days after transection of the sciatic nerve, comparison of raw and normalized data for DRG proteins not exhibiting an apparent change in relative synthesis indicated that axon transection had induced a mean increase in total protein synthesis of about 30%. In addition, a number of proteins in the fourth and fifth lumbar DRG exhibited changes in relative synthesis rates (Fig. 1, bottom). Actin and tubulin, previously identified on the basis of comigration with purified actin and tubulin (16) exhibited increases in relative synthesis of about 30 and 50%, respectively. In terms of absolute rates of synthesis, actin and tubulin exhibited increases of about 40 and 65%, respectively. Of the other, unidentified proteins exhibiting changes in relative synthesis rate, three proteins (3C2, 2E6, and 2Fl) more than doubled their rate of precursor incorporation (Table 1; Fig. 2). A similar pattern of changes was seen whether axon transection was at the level of the hip or within a few millimeters of the DRG. Gels prepared from four animals with unilateral transection of the L4 peripheral nerve within 5 mm of the DRG revealed marked increases in relative synthesis of proteins 3C2, 2E6, 2FI, and 2D16, and a significant decrease for 1Fl (data not shown). In contrast, transection of dorsal roots induced no significant changes in synthesis, even for those proteins whose synthesis was most markedly altered by cutting the sciatic nerve (Table 1). However, there may have been a trend toward slightly higher rates of synthesis for those proteins increasing most markedly after axotomy. For instance, labeling of protein 2D16 was consistently higher in the dorsal-root-transected DRG than in the contralateral controls. Protein 3C2, which exhibited a dramatic increase in synthesis after sciatic transection, appeared to increase slightly after FIG. 2. Tracing of typical autoradiograph with molecular weights andpH gradient indicated. Proteins referred to in the text can be located using the protein’s number-letter designation. The first number and letter refer to the row and column on the figure, and the last number refers to the particular protein within the designated square.

MICHAEL

88

E. HALL

TABLE

1

Effects of Axotomy on the Dorsal Root Ganglion Mean DPM (+SE) Protein spot lD1 lE8 1Fl lF7 lF8 2Cl 2c20 2Dl 2D8 2D14 2D16 2E1 2E2 2E3 2E4 2E6 2E7 2F1 3C2 3Dl 3El 3E2 3E3 3Fl 3F2 3F7

Control 241 + 53 + 403 + 341 + 22 + 75 + 57 + 512 + 152 + 45 + 18 t 358 + 231 + 70 + 83 t 23 + 50 + 35 + 36+ 42 + 205 + 181 t 152 t 297 + 72 + 34 +

16 14 133 17 5 18 10 16 10 20 6 91 53 67 14 17 20 23 13 8 8s 100 29 99 25 24

DRG axotomy

Change (%I

P

272 t 44 61 + 11 223 + 72

-44

0.01

100 + 23

33

0.002

664 172 71 33 541 392

30

0.01

58 83 51 70

0.01 0.02 0.01 0.01

52 t 29

126

0.05

116 + 109 101 + 36 63 + 23

231 180 50

0.05 0.002 0.02

186 t 105 100 t 60

-34

0.05

t + + t t t

95 25 13 8 179 121

43 + 26

dorsal-root transection, but this increase was slight compared with sciatic transection. The two-dimensional pattern of newly synthesized proteins from the DRG is strikingly similar to the pattern seen in the SCG. Not only is the intact ganglion pattern similar (Fig. 3, top), the pattern of alterations induced by axotomy is also very similar. Of the 200 most abundantly labeled DRG proteins visualized, all 200 could also be identified in the SCG. The only qualitative difference was the presence of a series of minor spots running diagonally from spot 3E5 (Fig. 3 bottom inset) in the DRG, not seen in the SCG. As seen in Table 2, the relative abundances of 22 proteins were compared. Data from the DRG and SCG were normalized to represent labeling relative to 100 pg total protein/gel. For most proteins,

FIG. 3. Top-pattern of newly synthesized proteins from rat sympathetic ganglia. Inset is enlargement of area indicated by box. Bottom-pattern of newly synthesized proteins from control DRG. Inset is enlargement of area indicated by box. The inset shows only area where major differences in sympathetic ganglia and DRG patterns were seen. 89

90

MICHAEL TABLE

E. HALL 2

Comparison of the Superior Cervical (SCG) and Dorsal Root Ganglia (DRG)

Protein spot 1Dl lE8 1Fl 2Cl 2c20 2Dl (actin) 2D8 2D14 2D16 2El (tub.) 2E2 (tub.) 2E4 2E6 2Fl 3C2 3Dl 3El 3E2 3E3 3Fl 3F2 3Fl

Control DRG (Mean DPM)

Control SCG (Mean DPM)

241 53 403 15 57 512 152 45 16 358 231 83 23 35 36 42 205 181 152 291 72 34

236 69 101 146 48 516 90 43 10 458 224 107 16 36 32 48 216 80 30 210 70 46

Change (V&o)

Axotomy

DRG axo.

SCG axo.

DRG = SCG?

-44’ 33*

-51* 10

Yes No

30* 12 58* 83 51* 70*

22* -18* 74; 200* 4 5

Yes No Yes No No No

126’ 231’ 180* 50’

125* 139* 250; 2

Yes ? Yes No

2 -34*

22 57+

No

1

1598

No

a tub., tubulin. * Indicates axotomy differs from control by P < 0.01.

the relative abundances are similar. Significant differences exist for a few, however. Protein 1Fl, for example, was almost four times as heavily labeled in the DRG as in the SCG, whereas protein 2D5 was much less abundant in the DRG (data not shown). Proteins 3E2,3E3, and 3E5 were synthesized at a much greater rate in the DRG. When the responses to axotomy of the DRG and SCG were compared, some similarity was observed. Five of the 22 proteins in Table 2 exhibited significant changes in relative synthesis rate in both the DRG and SCG, and to the same extent. For eight of the 22 proteins, however, the response was not the same. Of seven of these (2C 1,2D8,2D 16,3D 1, 3F7, and tubulin) a significant response to axotomy was seen in one ganglion but not the other. Protein 3E3 was unique in that its synthesis rate increased significantly in the SCG but decreased significantly in DRG after axotomy.

PROTEIN

SYNTHESIS

IN AXOTOMIZED

DRG

91

After transection of the peripheral branch of the L4 DRG, the pattern of newly synthesized proteins in the adjacent L5 DRG was examined. The peripheral denervation after transection of the L4 DRG might be expected to induce collateral sprouting from the axons of L5. Analysis of three sets of gels revealed no evidence of altered synthesis rates of any L5 DRG proteins. DISCUSSION Within 3 days after transection of the sciatic nerve, specific proteins in the fourth and fifth lumbar DRG exhibit significant changes in relative synthesis rates. These proteins include actin and tubulin. Tubulin, unlike actin, also exhibits a significant increase in total protein. The same basic pattern of changes is also seen when the peripheral nerve is transected close to the ganglion body. Transection of the dorsal roots, on the other hand, does not result in significant changes in protein synthesis. This is in keeping with numerous reports where no axon reaction (e.g., chromatolysis) follows dorsal-root transection ( 1,7). It was suggested that the effects of dorsal root transection are “subthreshold” for the induction of an axon reaction (1). It is known that, in other systems, the presence of sustaining axon collaterals may prevent the typical response to axotomy ( 1). The dorsal root branch of the DRG axon is generally smaller in diameter than the peripheral branch (7). Ochs et al (1 I), and Mori et al. (9, lo), showed that the volume and rate of material in axonal flow is greater in the peripheral than in the central branch. However, it is also possible that some qualitative difference exists between the peripheral and central branches, or that the trophic dependencies of the DRG neurons are maintained by the peripheral branch exclusively. Loss of the trophic substance nerve growth factor (NGF) appears responsible for axotomy-induced changes in sympathetic ganglia. Chromatolysis (15) synaptic disjunction (12) and some of the protein changes (4) seen to follow axotomy can be prevented by exogenous NGF, normally supplied by axonal transport. The response of DRG to axotomy could be explained if it is only the peripheral branch that supplies the DRG with trophic substance(s). Asymmetries of transport are known to occur, as in the case of substance P in the nodose ganglion (6). The striking similarity of the protein pattern for the DRG and SCG indicates that most of the proteins so visualized represent the “standard” proteins common to many nervous tissues. Although the similarity is less striking, the pattern of proteins synthesized in the hippocampus and neocortex are also similar to the DRG (Hall, unpublished observation). It is therefore not too surprising that axotomy should induce changes in many

92

MICHAEL

E. HALL

of the same proteins in both structures. Protein 3C2, for example, exhibited a dramatic change in both the SCG and DRG. Protein 3C2 also changed dramatically in the nodose ganglia after transection of the vagus distal to the ganglion (Hall, unpublished observation). The small but significant increase in the synthesis and content of tubulin in the axotomized DRG is in keeping with other reports (5) but is at odds with what is seen in the SCG. It must be noted, however, that alpha and beta tubulin are not well isolated on the gel system used. Other proteins overlap with tubulin to some extent, making exact quantification difficult. No such uncertainty exists for those four proteins whose responses to axotomy differed markedly between the SCG and DRG. In particular, protein 3E3 responds in the opposite manner in the DRG and SCG, decreasing in the former and increasing in the latter. Given the overall marked similarity in protein composition and response to axotomy, this peculiarity stands out. Watson (14) reported that collateral sprouting by the DRG produces changes in RNA synthesis comparable to that seen in the axotomized DRG. No changes in total or specific protein synthesis were observed in the L5 DRG when the adjacent L4 was axotomized, a procedure that might be expected to induce collateral sprouting in L5 (2, 8). As the extent of collateral sprouting induced in L5 was undermined in these experiments, this negative observation must be treated with caution. Nonetheless, it is certain that if collateral sprouting induced a two- or threefold change in the synthesis of a major DRG protein, such a change would have been detected. REFERENCES 1. CRAGG, B. G. 1970. What is the signal for chromatolysis? Brain Res. 23: l-21. 2. GOLDBERGER, M. E., AND M. MURRAY. 1978. Recovery of movement and axonal sprouting obey some of the same laws. Pages 73-97 in CARL COT~AN, Ed., Neuronal Plosricify. Raven Press, New York. 3. HALL, M. E., D. L. WILSON, AND G. STONE. 1978. Changes in synthesis of specific proteins following axotomy: detection with two-dimensional gel electrophoresis. J. Neurobiol.

9: 353-366.

4. HALL, M. E., AND D. L. WILSON. 1982. Nerve growth factor effects on gene expression after nerve damage. In preparation. 5. HEACOCK, A., AND B. AGRANOFF. 1976. Enhanced labeling of a retinal protein during regeneration of optic nerve in goldfish. Proc. N&l. Acad. Sci. U.S.A. 73: 828-832. 6. HOKFELT,

T., 0.

JOHANSSON,

A.

LJUNGDAHL,

J. LUNDBERG,

AND

M.

SCHULTZBERG.

1980. Peptidergic neurons. Nature (London) 284: 515-521. 7. LEIBERMAN, A. 1971. The axon reaction: a review of the principal features of perikaryal responses to axon injury. Int. Rev. Neurobiol. 14: 49-124. 8. LIU, C.-N., AND W. W. CHAMBERS. 1958. Intraspinal sprouting of dorsal root axons. Arch. Neural. Psychiatr. 79: 46-61. 9. MORI, H., Y. KOMIYA, AND M. KUROKAWA. 1977. Slow axonal transport: its asymmetry in two branches of bifurcating axons. Proc. Jup. Acad. 53: 252-256. 10. MORI, H., Y. KOMIYA, AND M. KUROKAWA. 1979. Slowly migrating axonal polypeptides:

PROTEIN

11. 12. 13. 14.

SYNTHESIS

IN AXOTOMIZED

DRG

93

inequalities in their rate and amount of transport between two branches of bifurcating axons. J. Cell. Biol. 82: 114- 184. OCHS, S., J. ERDMAN, R. JERSILD, AND V. MCADOO. 1978. Routing of transported materials in the dorsal root and nerve fiber branches of the dorsal root ganglion. J. Neurobiol. 9: 465-48 1. PURVFS, D., AND A. NJA. 1976. Effect of nerve growth factor on synaptic depression after axotomy. Nature (London) 2601 535-536. WARD, S., D. WILSON, AND J. GILLIAM. 1970. Methods for fractionation and scintillation counting of radioisotope-labeled polyacrylamide gels. Anal. Biochem. 38: 90-97. WATSON, W. E. 1974. Cellular responses to axotomy and to related procedures. Br. Med.

Bull. 15. WEST,

30: 112-l

15.

N. R., AND R. P. BUNGE. 1976. Prevention of the chromatolytic response in rat superior cervical ganglion neurons by nerve growth factor. Sot. Neurosci. Abstr. 2: 1038. 16. WILSON, D. L., M. E. HALL, G. STONE, AND R. RUBIN. 1977. Some improvements in two-dimensional gel electrophoresis of proteins: protein mapping of eukaryotic tissue extracts. Anal. Biochem. 83: 33-44.