Journnl q/ Nturochi,miair~. 1976. Vol. 27. pp. 19I-lY6. Pergamon Prebs. Printed
in
Great Britain.
RETROGRADE AXONAL TRANSPORT OF RAPIDLY MIGRATING LABELLED PROTEINS AND GLYCOPROTEINS IN REGENERATING PERIPHERAL NERVES M. FRIZELL, W. G. MCLEAN'and J. SJ~STRAND Institute of Neurobiology and Department of Ophthalmology, University of Goteborg. Goteborg. Sweden (Recriaed 9 October 1975. Accepted 5 January 1976)
Abstract-The redistribution of rapidly migrating [3H]leucine-labelled proteins and [3H]fucose-labelled glycoproteins was studied in ligated regenerating hypoglossal and vagus nerves of the rabbit. When regenerating and contralateral hypoglossal nerves were ligated 16 h after labelling of the nerve cell bodies. rapidly migrating proteins and glycoproteins accumulated distal to the ligatures indicating a rapid retrograde transport from the peripheral parts of the nerves within 6 h. The retrograde accumulation of both proteins and glycoproteins was greater on the regenerating side than on the contralateral side at both 1 and 5 weeks after a nerve crush. Labelled proteins and glycoproteins also accumulated proximal to the ligatures, indicating a delayed rapid anterograde phase of axonal transport. The accumulation of this phase was also greater on the regenerating side 1 week after a nerve crush for both labelled proteins and glycoproteins. One week after a crush of the cervical vagus nerve. rapidly migrating proteins and glycoproteins redistributed between he crush zone and a proximal ligature applied 1 6 h after labelling of the nerve cell bodies. A retrograde accumulation occurred distal to the ligature within 6 h, indicating a rapid retrograde transport from the crush zone.
BESIDES the well-established anterograde axonal trans- 1974b,c,d). A changed retrograde axonal transport of port system, an increasing number of reports have exogenous protein was also demonstrated in regenerdocumentcd the existence of a retrograde axonal ating hypoglossal nerve of the rabbit (KRISTENSSON 1972). Studies of retrograde axonal transport system in both central and peripheral & SJOSTRAND, neurons of various species (LUBINSKA et al., 1963; transport in regenerating nerves are of special interest LASEK,1967; KRISTENSSON, 1970; BRAYet al., 1971; since it has been postulated that trophic substances LA VAIL& LA VAIL,1972, 1974). The reversed polar- carried by this transport can reach and influence the ity of radioactively labelled proteins of the rapid metabolism of the nerve cell body, e.g. induction of & OLSSON, anterograde phase of axonal transport, shifting its chromatolysis (CRAGG,1970; KRISTENSSON transport, direction at natural or artificial nerve end-, 1974). The aim of the present study was to examine ings has been used to study the retrograde axonal the retrograde axonal transport of labelled proteins transport of endogenous proteins in various nerves and glycoproteins during the outgrowth and early (BRAYet a/., 1971; EDSTROM& HANSSON,1973; FRI- maturation period in regenerating hypoglossal nerve ZELL & SJOSTRAND, 1 9 7 4 ~ ;ABE et a/., 1974). The and during the latent phase in regenerating vagus retrograde axonal transport of rapidly migrating nerves. The study of retrograde axonal transport of [3H]leucine-labelled proteins was previously demon- labelled glycoproteins was considered to be of special strated in normal hypoglossal and vagus nerves of interest since these are incorporated into axonal the rabbit (FRIZELL & SJOSTRAND, 1 9 7 4 ~ SJOSTRAND ; membranes and retrograde transport might be in& FRIZELL,1975). Exogenous protein tracers have volved in the membrane movements at growth cones also proved to be valuable tools in the evaluation (BRAY,1973). of retrograde axonal transport, expecially as a neuroanatomical tracer (KRISTENSSON, 1970; LA VAL & METHODS LA VAIL,1972, 1974; ELLISON& CLARK,1975). Operative procedure. Twenty-two albino rabbits, Marked metabolic changes are induced in the regenerating neuron including an increased synthesis 1.5-2 kg, were used. The right hypoglossal nerve was of proteins and RNA (BRATTGARD et a/., 1957). These exposed under pentobarbitone anaesthesia and crushed changes are also reflected in the anterograde axonal with 3-0 silk thread against a glass rod 3&35mm from the bulb. The ligature was left loosely tied around the transport of labelled proteins and glycoproteins as nerve to permit regeneration while indicating the site of recently demonstrated in the regenerating hypoglossal crush. In some experiments the right cervical vagus nerve and vagus nerves of the rabbit (FRIZELL & SJOSTRAND, was crushcd in the same way 35 40mm distal to the nodose ganglion. One or five weeks later the fourth venDepartment of Pharmacology, School tricle was exposed under pentobarbitone anaesthesia and of Pharmacy, Liverpool Polytechnic, Byrom St., Liverpool the hypoglossal and dorsal motor nuclei of the vagus nerve were labcllcd with 30 pCi of [3H]lcucine (~-[4,5~H]leucine. L3 3AF England. 191
* Present address:
M. FRIZELL, W. G. MCLEANand J. SJOSTRAND
192
specific activity 38 Ci, mmol. concn. 1 pCi/pl; The Radiochemical Centre, Amershani. England) or 120 pCi of l specific activity 2.8 Ci/mmol, [3H]fucose ( ~ [ -3H]fucose, concn. 4 pCi!pl; The Radiochemical Centre. Amersham, England) applied continuously to the bulb during 30 min according to M I A N 1 ( I 963). Sixteen hours later the animals were reoperated and right hypoglossal nerves were ligated with a 3-0 silk thread 5-10 mm proximal to the previous crush lesion and left nerves were ligated at the same level to serve as controls. In some Experiments the right cervical vagus nerve was ligated just below the nodose ganglion. 35--40mm proximal to the previous crush lesion. 16 h after 1;ibelling. The animals were killed 6 h after ligation, 22 h after labelling. and appropriate parts of the nerves were immediately dissected out and divided into 5 mm pieces for analysis of radioactivity. Defclniiticirion of radioacfitirj.. The individual nerve pieces were placed in 2 ml 0 1 ice-cold IOU, trichloroacetic acid (TCA)(wiv) for 24 h to extract the TCA-soluble fraction and then washed once with 10'" TCA and solubilized in 0.5 ml of Soluene-100 (Packard Instrument Company, Inc.). The radioactivity of the TCA-insoluble material was counted in a Packard Tri-Carb liquid scintillation spectrometer and was corrected for quenching. The activity was expressed as d.p.m. per 5 mm of nerve segment or as ";) distribution of radioactivity along the nerve
i
radioactivity of 5- mm segment tow1 radioactivity of nerve RESULTS
Hypoglo.ssu1 rierw
I
n
FIG.1. The redistribution of [3H]leucine-labelled proteins (a. 1 week; b. 5 weeks) in rcgenerating and contralateral (dashed columns) hypoglossal nerves. The hypoglossal nuclei were labelled with 30pCi or ['Hlleucine and both regenerating and contralateral nerves were ligated 16 h later. The animals were killed after another 6 h. The crush zone ( x ) and the sites of ligation (arrows) are indicated. The drawings show one representative experiment out of 5 or 6 for each regeneration time. The proximal ends of the nerves are to the left.
At both 1 and 5 weeks after a nerve crush there was an accumulation of both [3H]leucine and [3H]fucose-labelled material confined to the 5 mm segment distal to the ligature applied 5-10 mm proxi- cantly increased o n the regenerating side as compared mal tb the crush zone, indicating a retrograde axonal to the contralateral side after both 1 and 5 weeks. transport of labelled material from the distal parts The accumulation of labelled proteins and glycoproof the regenerating and contralateral nerves (Fig. I). teins distal to the ligature on the regenerating side In the I-week but not in the 5-week experiments an in per cent of that on the contralateral side was signiaccumulation of labelled material also occurred in the ficantly greater after 1 week than after 5 weeks of most distal segment of the regenerating nerves, 15 mm regeneration (Table 1). An accumulation of labelled from the crush zone, indicating an increase in the proteins and glycoproteins also occurred in the 5 mm anterograde rapid phase of axonal transport in the segment proximal to the ligature in both regenerating sprouts of the regenerating axons (Fig. la). It has pre- and contralateral nerves (Fig. 1) indicating a delayed viously been demonstrated (FRIZELL & SJOSTRAND, rapid anterograde phase of axonal transport as pre1974di that rapidly migrating [3H]leucine-labelled & SJOSTRAND, 1974~).The viously reported (FRIZELL proteins Rere transported beyond the crush zone in accumulation of this phase was significantly greater regenerating hypoglossal nerve 1 week after a nerve on the regenerating side after 1 week of regeneration, crush, accumulating i n the distal parts of the nerve indicating an increased delayed anterograde axonal and not at the crush zone. The progression of this transport of labelled proteins and glycoproteins at accumulation zone indicated a regeneration rate of that time (Table 2). 4 - 5 mm;day after a latent period of less than 3 days. Vngus n e r w After 5 weeks of regeneration when the sprouting axons should have reached the tongue (GUTMANN et Rapidly migrating [3H]leucine-labelled proteins d..1942: B R A T T ~ R etD a/., 1957: KRISTENSSON& and [3H]fucose-labelled glycoproteins accumulated SJOSTRAND. 1972) there was no evidence of any ac- within 6 h distal to the ligature applied proximal to cumulation of labelled material distal to the crush the crush zone 16 h after labelling (Fig. 2) indicating zone. a retrograde axonal transport of labelled proteins and The amount of both [3H]leucine and [3H]fucoseglycoproteins from the crush zone after I week of labelled material. a s reflected by radioactivity in the regeneration (Fig. 2). An accumulation of labelled 5 mm segment distal to the ligatures, was signifi- proteins and glycoproteins also occurred at the crush
Retrograde transport in regcnerating nerves TARLt
193
1. T H t RETROGRADE ACCUMULATION OF LABELLED PROTEINS AND GLYCOPROTEINS IN REGENERATING HYPOGLOSSAL NERVES
T i m after crush (weeks)
[3H]leucine label (d.p.m.) Regenerating side Contralateral side
1 1
1342 1443 2195 3961 5325 1163
1 1 1 1
(185) (185) (160) (207) (150) (149) (173
5 5 5 5 5 5
6003 2671 4082 3714 2443 902
725 779 1364 1911 3551 776
[3H]fucose label (d.p.m.) Regenerating side Contralateral side
12967 16696 11220 12562 15183
6716 8194 6562 5653 9856
-
F 23)
(143) (124) (156) (138) (128) (154)
(193) (204) (171) (224) (154)
-
(189 f 27) 4179 2144 2603 2672 1896 584
8508 6854 10730 6961 9178
(137) (129) (128) (136) (127)
6220 5273 8371 5104 7176
~
(141 & 13)
~
(131 f 4)
The hypoglossal nuclei were labelled with 30pCi of [3H]leucine or 120pCi of [3H]fucosc 1 or 5 weeks after a unilateral crush of the hypoglossal nerve. Both hypoglossal nerves were ligated 16 h after labelling and killed 6 h later. The radioactivity in the 5 mm segment distal to the ligature is shown. The radioactivity of this segment on the regenerating side is also expressed as a percentage of the corresponding contralateral segment within brackets and mean f S.D. is indicated. The difference in radioactivity between regenerating and contralateral sides is indicated. The difference in radioactivity between regenerating and contralateral sides is significant for both 1 and 5 weeks groups for both [3H]leucine and [3H]fucose label (P < 0.05, Wilcoxon test). The difference in percent of contralateral between I and 5 weeks groups is also significant at the same level.
zone as previously demonstrated (FRIZELL& SJOSTKAND, 1974c, d) indicating a latent period of more than 1 week before the outgrowth of new axons in the regenerating vagus nerve. The retrograde accumulation of labelled proteins and glycoproteins was 20 and 27:/, respectively of the total radioactivity between the crush zone and the ligature (legend to Fig. 2). The corresponding value for [3H]leucine-labelled T A B L2~ THE DELAYED
proteins in normal doubly-ligated vagus nerves had previously been calculated to be 12% (FRIZELL& SJOSTRAND, 1974a). DISCUSSION
The existence of a retrograde azonal transport system in intact peripheral nerve fibres has been documented by microphotographical studies demonstrat-
ANTFROGRADE ACCUMULATION OF LABELLED PROTEIXS AND GLYCOPROTEINS IN KEGENERATING
HYPOGLOSSAL NERVES
Time aftcr crush (weeks)
[3H]leucine label (d.p.m.) Regenerating side Contralateral side
1735 2037 3337 6885 7508 2101
(168 6939 3539 4608 4680 4043 1505
1 I40 1418 1837 5338 4518 870
(152) (143) (178) (129) (166) (241)
(117
6135 3628 3584 4137 4269 1020 22)
13879 16583 12344 11577 16161
7980 9497 8024 6215 9307
(175) (175) (154) (186) (174) (173 & 12)
40)
(113) (97) (128) (113) (95) (154)
[3H]fucose label (d.p.m.) Regenerating side Contralateral side
6512 6073 9585 5867 9094
6887 (105) 6998 (115) 10682 (111) 7462 (127) 11156 (122) (116
-
*
9)
(See legend Table I). The radioactivity in the 5 m m segment proximal to the ligature is shown. The radioactivity of this segmcnt on the regenerating side is also expressed as a percentage of the corresponding segment of the contralateral nerve within brackets, and mean & S.D. is indicated. The difference in radioactivity between regenerating and contralateral side is significant for both one week groups, but only for [3H]fucose label in the 5 weeks group ( P < 0.05, Wilcoxon test). The difference in percent of contralateral between 1 and 5 weeks groups is also significant at the same level. v
1'.
27 i
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I94
M. FRIZELL, W. G. MCLEANand J. SJOSTRAND
use the natural nerve ending or an applied ligature as a point of reversal of their transport direction. It a cannot, however. be excluded that part of the material has turned its transport direction en route, although this is less likely as pointed out by BRAYet al. (1971) and ABE ct ( I / . (1974). The ligation procedure itself may induce n retrograde axonal transport normally not existing. e.g. through electrophoretic forces. Persistent injury discharge, however, has not been denoms 10 strated in motor (ADRIAN. 1930) or sensory nerve fibres (WALLet al., 1974). A retrograde transport of organelles similar to that observed in intact nerve fibres has also been shown in injured nerve fibres and fibres separated from their cell bodies (ZELENA, 1968; SMITH.1971 : CCKWER & SMITH,1974). The continuous accumulation of AChE (LLJBINSKA& NIEMIERKO, & SJOSTRAND, 1971) and labelled proteins (FRIZELL 19740) distal to a nerve ligature up to 21 h, contradicts the objection that retrograde accumulation in ligated nerves should be a passive process due to displacement of the axoplasm by the ligature. The blocking effect of mitosis inhibition on the retrograde accumulation of labelled proteins in ligated nerves (EDSTROM& HANSSON,1973; ABE er a/., 1974) also FIG 2. The redistribution of [ 'Hlleucine labelled proteins indicates that the retrograde accumulation observed (a) and [3H]fucose-lahelled glycoproteins (b) in cervical distal to a ligature is due to a retrograde axonal transvagus nerve after 1 week of regeneration. The dorsal motor port system depending on a transport mechanism nuclei of the vagus nerve wzre labelled with 30pCi of similar to that of anterograde axonal transport. The [.3H]leucine or 120pCi of ['Hlfucose and the cervical retrograde accumulation of labelled proteins and glyvagus nerve a-as ligated 35-40 mm proximal to the crush coproteins reported here is therefore likely to reprone 16 h later. The animals were killed after another 6 h. resent the trapping of labelled material brought there The crush zone ( x ) and the site of ligation (arrow) are indiby retrograde axonal transport from the distal parts cated. The profiles show the percentage distribution of of the nerve. radioactivity between crush zone and ligature and repSince rapidly migrating proteins accumulate distal resent means f s . ~of . four experiments (a) and three exto and not at the crush zone in rabbit hypoglossal periments (b) with the same distance between crush zone nerves after 1 week of regeneration (Fig. l a ; FRIZELL and ligature. The range of total radioactivity between the crush zone and the ligature was 3059- 11.266 d.p.m. (a) and & SJOSTRAND, 1974d), the retrograde pile-up of 14.216-36.434 d.p.m. (b).The retrograde accumulation (seg- labelled material distal to the ligature on the regenerment A ) was 20 I", (mean i s.D.. n = 6) and 27 5 2"" ating side could not have been significantly influenced (mean s.D.. n = 5 ) for [3H]leucine-labelled proteins and by anterograde accumulation at the crush zone after [3H]fucose-lahelled glycoproteins respectively. The corre1 week of regeneration. Previously we demonstrated sponding values for the anterograde accumulation (segthe retrograde axonal transport of rapidly migrating ment B) was 61 10 and 51 i 3"6. In doubly-ligated nor[3H]leucine-labelled proteins in normal hypoglossal mal vagus nerves the retrograde and anterograde accumuand vagus nerves of the rabbit (FRIZELL & SJOSTRAND, lation of ['H]leucine labelled proteins 12 i 2",, and The results of the present study indicate that 60 & 6"/,,(mean s.11..n = 3 ) respectively (data from FRI- 1974~). a similar transport takes place in these nerves during ZELL & SJOS~RAND. 19740). regeneration. The increased accumulation of labelled ing the transport of granules and pinocytotic vesicles proteins and glycoproteins on the regenerating side in the retrograde direction (HUGHES,1953: POMERAT as compared to the contralateral, however, shows that et ul., 1967). Retrograde axonal transport of exo- the retrograde transport of these materials is ingenous protein tracers occurs in both normal and creased during the outgrowth and subsequent early regenerating hypoglossal nerves of the rabbit (KRIS- maturation period of the regenerating hypoglossal TENSSON, 1970; KRISTENSSON & SJOSTRAND, 1972), in nerve. normal vagus nerves of guinea pig and cat (ELLISON During these phases of regeneration the metabo& CLARKE, 1975) and in injured vagus nerves of cat lism of the hypoglossal perikarya is characterized by et ul., and monkey (DE VITO et a/., 1974). The retrograde an increased synthesis of proteins (BRATTG~RD axonal transport of endogenous labelled proteins, 1957) and the anterograde axonal transport of slowly sqnthesized in the nerve cell body, can be studied after migrating labelled prdteins and rapidly migrating ligation of the nerve to trap labelled proteins moving labelled glycoproteins is increased (FRIZELL& SJOSin the retrograde direction. These proteins probably TRAND. 1974h, c, d). These phases of axonal proteins
Retrograde transport in regenerating nerves
consist of structural proteins that might take part in the renewal of the axon by incorporation into membranes at the growth cone (BRAY,1973; LENTZ,1972). In the present study an increased delayed rapid anterograde axonal transport of labelled proteins and glycoproteins in the hypoglossal nerve was indicated by the increased accumulation of these materials proximal to the ligature during 16-22 h after labelling on the regenerating side after one week of regeneration. Previously we found a decreased axonal transport of rapidly migrating labelled proteins in the hypoglossal & SJOSnerve after 1 week of regeneration (FRIZELL TRAND, 19746). These proteins accumulated at a ligature during the 20 h period after labelling, and probably represent an earlier anterograde phase of transport than that observed in the present work between 16 and 22 h after labelling. Thus, while the increased retrograde transport of labelled glycoproteins in the hypoglossal nerve might be secondary to an increased rapid anterograde transport after 1 week of regeneration, this would not be the case for the retrograde transport of labelled proteins. If the major point of reversal of the transport direction of the labelled material is at the tips of the axons, it must be considered that the shorter distance between the ligature and the point of reversal in the growing axons as compared to the longer axons on the contralateral side might be one explanation for the increased retrograde accumulation observed on the regenerating side after 1 week of regeneration. It is unlikely, however, that this is the main explanation for the increased retrograde accumulation in the 1-week experiments, since this increase, although less marked, is still present after 5 weeks of regeneration, at a time when the axons on the regenerating and the contralateral sides should be of equal length. The changes in retrograde axonal transport of labelled glycoproteins are of special interest since glycoproteins are known to be incorporated into membranes and might take part in the renewal of the axonal membrane ( B ~ N N F Tet T ul., 1973). After 5 wecks of regeneration, i.e. at the beginning of the maturation period, when the tips of the regenerating hypoglossal nerve fibres should have reached the tongue, the in& SJOSTRAND,1 9 4 7 ~ ) crease in anterograde (FKIZELL as well as the retrograde axonal transport and uptake of Evans blue-labelled albumin in regenerating hypoglossal neurons occur during the early maturation & SJOSTRAND,1972). Thus the period (KRISTENSSON rctrograde axonal transport of both endogenous and exogenous proteins seems to be increased during this phase of regeneration. The present study also demonstrated that both labelled proteins and glycoproteins are transported in a retrograde direction in the mainly unmyelinated fibres of the dorsal motor nucleus of the vagus nerve after 1 week of regeneration. In contrast to the hypoglossal nerve the transport was studied during the latent period before the outgrowth of sprouts had started. Thus the labelled material used the crush
195
zone as a point of turning its transport direction, and was actually not transported into the regenerating fibres. The retrograde accumulation of [3H]leucine labelled proteins was increased as compared to that in normal doubly-ligated nerves (FRIZELL& SJOSTRAND,1974~).In conclusion, the present results indicate that a rapid retrograde axonal transport of labelled proteins and glycoproteins occurs in ligated regenerating hypoglossal and vagus nerves of the rabbit and that this transport is greater than that of the normal nerve. The retrograde transport system might be relevant as a mediator of signal substances from the regenerating nerve fibres, possibly influencing the metabolism of the regenerating nerve cell bodies.
REFERENCES M. (1974) FEBS Lett. 47. Am T., HAGAT. & KUROKAWA 212-27 5. ADRIAN E. D. (1930) Proc. R. Soc. Ser. B. 106, 59fS618. BENNETT G., DI GIAMBERARDINO L., KOENIGH. L. & DROZ B. (1973) Brain Res. 60. 129-146. S. O., EIXTROMJ. E. & HYDENH. (1957) J . BRATTGARD Neurochem. 1. 316-325. BRAYD. (1973) Nature, Lond. 244. 93-95. B. M. (1971) Brairi BRAYJ. J., KONC. M. & BRECKENRIDGE Res. 33. 56CL564. COOPERP. C. & SMITHR. S. (1974) J . Physiol., Lond. 242. 77-97. CRAGG B. G. (1970) Brain Res. 23. 1-21. DE VITO J. L., CLAUSING K. W. & SMITH0. A. (1974) Brain Res. 82, 269-271. EDSTRBM A. & HANSONM. (1973) Brain Res. 61, 31 1-320. ELLISONJ. P. & CLARKG. M. (1975) J . comp. New-ol. 161. 103-1 14. FRIZELL M. & SJ~STRANI) J. (1974a) J. Neurochen~.23. 651-657. FRIZELL M. & SJOSTRAND J. (19746) J . Neurocheni. 22. 845-850. FRIZELL M. & SJOSTRAND J. (1 974c) Brain Res. 78. 109-1 23. FRIZELL M. & SIBSTRANDJ. (1974d) Brairi Res. 81. 267-283. GUTMANN E., GUTMANN L., MEDAWAR P. B. & YOUNG J. Z. (1942) J . exp. B i d . 19. 1444. HUGHESA. (1953) J . Anat. 87. 150-162. KRISTENSSON K. (1970) Acta neuropath,. Berl. 16. 293-300. KRISTENSSON K. & OLSON Y. (1974) Brain Res. 79. 10 I -1 09. KRISTENSSON K. & SJOSTRAND J. (1972) Brain Res. 45. 175-1 81. LASEKR. J. (1967) Nature, Lond. 216. 1212-1214. LA VAILJ. H. & LA VAIL M. M. (1972) Science, N . Y 176. 1416-141 7. LA VAIL J. H. & LA VAILM. M. (1974) J . comp. Neurol. 157. 303-358. LENTZT. L. (1972) J . Cell Biol. 52. 719-732. S. (1971) Brain Res. 27. LUBINSKAL. & NIEMIERKO 329-342. LUBINSKA L., NIEMIERKO S., ODERFELD B., SWARCL. & ZELENA J. (1963) Acta Biol. exp., Vars. 23. 239-247. MIANIN. (1963) J . Neurochem. 18. 859-874.
196
M. FRIZELL, W. G. MCLEANand J. SJOSTRAND
W. J., RAIBORNC. W. & POMERATC. M., HENDELMAN MASEY J. F. (1967) in The .Veuron (HYDENH., ed.) pp. 119-178. Elsevier. Amsterdam. SJ~STRAN J. D & FRIZELL M. (1075) Bruin Rrs. 85. 325-330. SMITHR. S. (1971) Cjsrohios. 3. 259-262.
WALLP. D.. WAXMAN S. & BASBALM A. I. (1974) Expl. Neurol. 45. 576589. ZELENA J. (1968) 2. Zrllfbrsch. mikrosk. Anut. 92. 186-192.
in
Great Britain.
RETROGRADE AXONAL TRANSPORT OF RAPIDLY MIGRATING LABELLED PROTEINS AND GLYCOPROTEINS IN REGENERATING PERIPHERAL NERVES M. FRIZELL, W. G. MCLEAN'and J. SJ~STRAND Institute of Neurobiology and Department of Ophthalmology, University of Goteborg. Goteborg. Sweden (Recriaed 9 October 1975. Accepted 5 January 1976)
Abstract-The redistribution of rapidly migrating [3H]leucine-labelled proteins and [3H]fucose-labelled glycoproteins was studied in ligated regenerating hypoglossal and vagus nerves of the rabbit. When regenerating and contralateral hypoglossal nerves were ligated 16 h after labelling of the nerve cell bodies. rapidly migrating proteins and glycoproteins accumulated distal to the ligatures indicating a rapid retrograde transport from the peripheral parts of the nerves within 6 h. The retrograde accumulation of both proteins and glycoproteins was greater on the regenerating side than on the contralateral side at both 1 and 5 weeks after a nerve crush. Labelled proteins and glycoproteins also accumulated proximal to the ligatures, indicating a delayed rapid anterograde phase of axonal transport. The accumulation of this phase was also greater on the regenerating side 1 week after a nerve crush for both labelled proteins and glycoproteins. One week after a crush of the cervical vagus nerve. rapidly migrating proteins and glycoproteins redistributed between he crush zone and a proximal ligature applied 1 6 h after labelling of the nerve cell bodies. A retrograde accumulation occurred distal to the ligature within 6 h, indicating a rapid retrograde transport from the crush zone.
BESIDES the well-established anterograde axonal trans- 1974b,c,d). A changed retrograde axonal transport of port system, an increasing number of reports have exogenous protein was also demonstrated in regenerdocumentcd the existence of a retrograde axonal ating hypoglossal nerve of the rabbit (KRISTENSSON 1972). Studies of retrograde axonal transport system in both central and peripheral & SJOSTRAND, neurons of various species (LUBINSKA et al., 1963; transport in regenerating nerves are of special interest LASEK,1967; KRISTENSSON, 1970; BRAYet al., 1971; since it has been postulated that trophic substances LA VAIL& LA VAIL,1972, 1974). The reversed polar- carried by this transport can reach and influence the ity of radioactively labelled proteins of the rapid metabolism of the nerve cell body, e.g. induction of & OLSSON, anterograde phase of axonal transport, shifting its chromatolysis (CRAGG,1970; KRISTENSSON transport, direction at natural or artificial nerve end-, 1974). The aim of the present study was to examine ings has been used to study the retrograde axonal the retrograde axonal transport of labelled proteins transport of endogenous proteins in various nerves and glycoproteins during the outgrowth and early (BRAYet a/., 1971; EDSTROM& HANSSON,1973; FRI- maturation period in regenerating hypoglossal nerve ZELL & SJOSTRAND, 1 9 7 4 ~ ;ABE et a/., 1974). The and during the latent phase in regenerating vagus retrograde axonal transport of rapidly migrating nerves. The study of retrograde axonal transport of [3H]leucine-labelled proteins was previously demon- labelled glycoproteins was considered to be of special strated in normal hypoglossal and vagus nerves of interest since these are incorporated into axonal the rabbit (FRIZELL & SJOSTRAND, 1 9 7 4 ~ SJOSTRAND ; membranes and retrograde transport might be in& FRIZELL,1975). Exogenous protein tracers have volved in the membrane movements at growth cones also proved to be valuable tools in the evaluation (BRAY,1973). of retrograde axonal transport, expecially as a neuroanatomical tracer (KRISTENSSON, 1970; LA VAL & METHODS LA VAIL,1972, 1974; ELLISON& CLARK,1975). Operative procedure. Twenty-two albino rabbits, Marked metabolic changes are induced in the regenerating neuron including an increased synthesis 1.5-2 kg, were used. The right hypoglossal nerve was of proteins and RNA (BRATTGARD et a/., 1957). These exposed under pentobarbitone anaesthesia and crushed changes are also reflected in the anterograde axonal with 3-0 silk thread against a glass rod 3&35mm from the bulb. The ligature was left loosely tied around the transport of labelled proteins and glycoproteins as nerve to permit regeneration while indicating the site of recently demonstrated in the regenerating hypoglossal crush. In some experiments the right cervical vagus nerve and vagus nerves of the rabbit (FRIZELL & SJOSTRAND, was crushcd in the same way 35 40mm distal to the nodose ganglion. One or five weeks later the fourth venDepartment of Pharmacology, School tricle was exposed under pentobarbitone anaesthesia and of Pharmacy, Liverpool Polytechnic, Byrom St., Liverpool the hypoglossal and dorsal motor nuclei of the vagus nerve were labcllcd with 30 pCi of [3H]lcucine (~-[4,5~H]leucine. L3 3AF England. 191
* Present address:
M. FRIZELL, W. G. MCLEANand J. SJOSTRAND
192
specific activity 38 Ci, mmol. concn. 1 pCi/pl; The Radiochemical Centre, Amershani. England) or 120 pCi of l specific activity 2.8 Ci/mmol, [3H]fucose ( ~ [ -3H]fucose, concn. 4 pCi!pl; The Radiochemical Centre. Amersham, England) applied continuously to the bulb during 30 min according to M I A N 1 ( I 963). Sixteen hours later the animals were reoperated and right hypoglossal nerves were ligated with a 3-0 silk thread 5-10 mm proximal to the previous crush lesion and left nerves were ligated at the same level to serve as controls. In some Experiments the right cervical vagus nerve was ligated just below the nodose ganglion. 35--40mm proximal to the previous crush lesion. 16 h after 1;ibelling. The animals were killed 6 h after ligation, 22 h after labelling. and appropriate parts of the nerves were immediately dissected out and divided into 5 mm pieces for analysis of radioactivity. Defclniiticirion of radioacfitirj.. The individual nerve pieces were placed in 2 ml 0 1 ice-cold IOU, trichloroacetic acid (TCA)(wiv) for 24 h to extract the TCA-soluble fraction and then washed once with 10'" TCA and solubilized in 0.5 ml of Soluene-100 (Packard Instrument Company, Inc.). The radioactivity of the TCA-insoluble material was counted in a Packard Tri-Carb liquid scintillation spectrometer and was corrected for quenching. The activity was expressed as d.p.m. per 5 mm of nerve segment or as ";) distribution of radioactivity along the nerve
i
radioactivity of 5- mm segment tow1 radioactivity of nerve RESULTS
Hypoglo.ssu1 rierw
I
n
FIG.1. The redistribution of [3H]leucine-labelled proteins (a. 1 week; b. 5 weeks) in rcgenerating and contralateral (dashed columns) hypoglossal nerves. The hypoglossal nuclei were labelled with 30pCi or ['Hlleucine and both regenerating and contralateral nerves were ligated 16 h later. The animals were killed after another 6 h. The crush zone ( x ) and the sites of ligation (arrows) are indicated. The drawings show one representative experiment out of 5 or 6 for each regeneration time. The proximal ends of the nerves are to the left.
At both 1 and 5 weeks after a nerve crush there was an accumulation of both [3H]leucine and [3H]fucose-labelled material confined to the 5 mm segment distal to the ligature applied 5-10 mm proxi- cantly increased o n the regenerating side as compared mal tb the crush zone, indicating a retrograde axonal to the contralateral side after both 1 and 5 weeks. transport of labelled material from the distal parts The accumulation of labelled proteins and glycoproof the regenerating and contralateral nerves (Fig. I). teins distal to the ligature on the regenerating side In the I-week but not in the 5-week experiments an in per cent of that on the contralateral side was signiaccumulation of labelled material also occurred in the ficantly greater after 1 week than after 5 weeks of most distal segment of the regenerating nerves, 15 mm regeneration (Table 1). An accumulation of labelled from the crush zone, indicating an increase in the proteins and glycoproteins also occurred in the 5 mm anterograde rapid phase of axonal transport in the segment proximal to the ligature in both regenerating sprouts of the regenerating axons (Fig. la). It has pre- and contralateral nerves (Fig. 1) indicating a delayed viously been demonstrated (FRIZELL & SJOSTRAND, rapid anterograde phase of axonal transport as pre1974di that rapidly migrating [3H]leucine-labelled & SJOSTRAND, 1974~).The viously reported (FRIZELL proteins Rere transported beyond the crush zone in accumulation of this phase was significantly greater regenerating hypoglossal nerve 1 week after a nerve on the regenerating side after 1 week of regeneration, crush, accumulating i n the distal parts of the nerve indicating an increased delayed anterograde axonal and not at the crush zone. The progression of this transport of labelled proteins and glycoproteins at accumulation zone indicated a regeneration rate of that time (Table 2). 4 - 5 mm;day after a latent period of less than 3 days. Vngus n e r w After 5 weeks of regeneration when the sprouting axons should have reached the tongue (GUTMANN et Rapidly migrating [3H]leucine-labelled proteins d..1942: B R A T T ~ R etD a/., 1957: KRISTENSSON& and [3H]fucose-labelled glycoproteins accumulated SJOSTRAND. 1972) there was no evidence of any ac- within 6 h distal to the ligature applied proximal to cumulation of labelled material distal to the crush the crush zone 16 h after labelling (Fig. 2) indicating zone. a retrograde axonal transport of labelled proteins and The amount of both [3H]leucine and [3H]fucoseglycoproteins from the crush zone after I week of labelled material. a s reflected by radioactivity in the regeneration (Fig. 2). An accumulation of labelled 5 mm segment distal to the ligatures, was signifi- proteins and glycoproteins also occurred at the crush
Retrograde transport in regcnerating nerves TARLt
193
1. T H t RETROGRADE ACCUMULATION OF LABELLED PROTEINS AND GLYCOPROTEINS IN REGENERATING HYPOGLOSSAL NERVES
T i m after crush (weeks)
[3H]leucine label (d.p.m.) Regenerating side Contralateral side
1 1
1342 1443 2195 3961 5325 1163
1 1 1 1
(185) (185) (160) (207) (150) (149) (173
5 5 5 5 5 5
6003 2671 4082 3714 2443 902
725 779 1364 1911 3551 776
[3H]fucose label (d.p.m.) Regenerating side Contralateral side
12967 16696 11220 12562 15183
6716 8194 6562 5653 9856
-
F 23)
(143) (124) (156) (138) (128) (154)
(193) (204) (171) (224) (154)
-
(189 f 27) 4179 2144 2603 2672 1896 584
8508 6854 10730 6961 9178
(137) (129) (128) (136) (127)
6220 5273 8371 5104 7176
~
(141 & 13)
~
(131 f 4)
The hypoglossal nuclei were labelled with 30pCi of [3H]leucine or 120pCi of [3H]fucosc 1 or 5 weeks after a unilateral crush of the hypoglossal nerve. Both hypoglossal nerves were ligated 16 h after labelling and killed 6 h later. The radioactivity in the 5 mm segment distal to the ligature is shown. The radioactivity of this segment on the regenerating side is also expressed as a percentage of the corresponding contralateral segment within brackets and mean f S.D. is indicated. The difference in radioactivity between regenerating and contralateral sides is indicated. The difference in radioactivity between regenerating and contralateral sides is significant for both 1 and 5 weeks groups for both [3H]leucine and [3H]fucose label (P < 0.05, Wilcoxon test). The difference in percent of contralateral between I and 5 weeks groups is also significant at the same level.
zone as previously demonstrated (FRIZELL& SJOSTKAND, 1974c, d) indicating a latent period of more than 1 week before the outgrowth of new axons in the regenerating vagus nerve. The retrograde accumulation of labelled proteins and glycoproteins was 20 and 27:/, respectively of the total radioactivity between the crush zone and the ligature (legend to Fig. 2). The corresponding value for [3H]leucine-labelled T A B L2~ THE DELAYED
proteins in normal doubly-ligated vagus nerves had previously been calculated to be 12% (FRIZELL& SJOSTRAND, 1974a). DISCUSSION
The existence of a retrograde azonal transport system in intact peripheral nerve fibres has been documented by microphotographical studies demonstrat-
ANTFROGRADE ACCUMULATION OF LABELLED PROTEIXS AND GLYCOPROTEINS IN KEGENERATING
HYPOGLOSSAL NERVES
Time aftcr crush (weeks)
[3H]leucine label (d.p.m.) Regenerating side Contralateral side
1735 2037 3337 6885 7508 2101
(168 6939 3539 4608 4680 4043 1505
1 I40 1418 1837 5338 4518 870
(152) (143) (178) (129) (166) (241)
(117
6135 3628 3584 4137 4269 1020 22)
13879 16583 12344 11577 16161
7980 9497 8024 6215 9307
(175) (175) (154) (186) (174) (173 & 12)
40)
(113) (97) (128) (113) (95) (154)
[3H]fucose label (d.p.m.) Regenerating side Contralateral side
6512 6073 9585 5867 9094
6887 (105) 6998 (115) 10682 (111) 7462 (127) 11156 (122) (116
-
*
9)
(See legend Table I). The radioactivity in the 5 m m segment proximal to the ligature is shown. The radioactivity of this segmcnt on the regenerating side is also expressed as a percentage of the corresponding segment of the contralateral nerve within brackets, and mean & S.D. is indicated. The difference in radioactivity between regenerating and contralateral side is significant for both one week groups, but only for [3H]fucose label in the 5 weeks group ( P < 0.05, Wilcoxon test). The difference in percent of contralateral between 1 and 5 weeks groups is also significant at the same level. v
1'.
27 i
\I
I94
M. FRIZELL, W. G. MCLEANand J. SJOSTRAND
use the natural nerve ending or an applied ligature as a point of reversal of their transport direction. It a cannot, however. be excluded that part of the material has turned its transport direction en route, although this is less likely as pointed out by BRAYet al. (1971) and ABE ct ( I / . (1974). The ligation procedure itself may induce n retrograde axonal transport normally not existing. e.g. through electrophoretic forces. Persistent injury discharge, however, has not been denoms 10 strated in motor (ADRIAN. 1930) or sensory nerve fibres (WALLet al., 1974). A retrograde transport of organelles similar to that observed in intact nerve fibres has also been shown in injured nerve fibres and fibres separated from their cell bodies (ZELENA, 1968; SMITH.1971 : CCKWER & SMITH,1974). The continuous accumulation of AChE (LLJBINSKA& NIEMIERKO, & SJOSTRAND, 1971) and labelled proteins (FRIZELL 19740) distal to a nerve ligature up to 21 h, contradicts the objection that retrograde accumulation in ligated nerves should be a passive process due to displacement of the axoplasm by the ligature. The blocking effect of mitosis inhibition on the retrograde accumulation of labelled proteins in ligated nerves (EDSTROM& HANSSON,1973; ABE er a/., 1974) also FIG 2. The redistribution of [ 'Hlleucine labelled proteins indicates that the retrograde accumulation observed (a) and [3H]fucose-lahelled glycoproteins (b) in cervical distal to a ligature is due to a retrograde axonal transvagus nerve after 1 week of regeneration. The dorsal motor port system depending on a transport mechanism nuclei of the vagus nerve wzre labelled with 30pCi of similar to that of anterograde axonal transport. The [.3H]leucine or 120pCi of ['Hlfucose and the cervical retrograde accumulation of labelled proteins and glyvagus nerve a-as ligated 35-40 mm proximal to the crush coproteins reported here is therefore likely to reprone 16 h later. The animals were killed after another 6 h. resent the trapping of labelled material brought there The crush zone ( x ) and the site of ligation (arrow) are indiby retrograde axonal transport from the distal parts cated. The profiles show the percentage distribution of of the nerve. radioactivity between crush zone and ligature and repSince rapidly migrating proteins accumulate distal resent means f s . ~of . four experiments (a) and three exto and not at the crush zone in rabbit hypoglossal periments (b) with the same distance between crush zone nerves after 1 week of regeneration (Fig. l a ; FRIZELL and ligature. The range of total radioactivity between the crush zone and the ligature was 3059- 11.266 d.p.m. (a) and & SJOSTRAND, 1974d), the retrograde pile-up of 14.216-36.434 d.p.m. (b).The retrograde accumulation (seg- labelled material distal to the ligature on the regenerment A ) was 20 I", (mean i s.D.. n = 6) and 27 5 2"" ating side could not have been significantly influenced (mean s.D.. n = 5 ) for [3H]leucine-labelled proteins and by anterograde accumulation at the crush zone after [3H]fucose-lahelled glycoproteins respectively. The corre1 week of regeneration. Previously we demonstrated sponding values for the anterograde accumulation (segthe retrograde axonal transport of rapidly migrating ment B) was 61 10 and 51 i 3"6. In doubly-ligated nor[3H]leucine-labelled proteins in normal hypoglossal mal vagus nerves the retrograde and anterograde accumuand vagus nerves of the rabbit (FRIZELL & SJOSTRAND, lation of ['H]leucine labelled proteins 12 i 2",, and The results of the present study indicate that 60 & 6"/,,(mean s.11..n = 3 ) respectively (data from FRI- 1974~). a similar transport takes place in these nerves during ZELL & SJOS~RAND. 19740). regeneration. The increased accumulation of labelled ing the transport of granules and pinocytotic vesicles proteins and glycoproteins on the regenerating side in the retrograde direction (HUGHES,1953: POMERAT as compared to the contralateral, however, shows that et ul., 1967). Retrograde axonal transport of exo- the retrograde transport of these materials is ingenous protein tracers occurs in both normal and creased during the outgrowth and subsequent early regenerating hypoglossal nerves of the rabbit (KRIS- maturation period of the regenerating hypoglossal TENSSON, 1970; KRISTENSSON & SJOSTRAND, 1972), in nerve. normal vagus nerves of guinea pig and cat (ELLISON During these phases of regeneration the metabo& CLARKE, 1975) and in injured vagus nerves of cat lism of the hypoglossal perikarya is characterized by et ul., and monkey (DE VITO et a/., 1974). The retrograde an increased synthesis of proteins (BRATTG~RD axonal transport of endogenous labelled proteins, 1957) and the anterograde axonal transport of slowly sqnthesized in the nerve cell body, can be studied after migrating labelled prdteins and rapidly migrating ligation of the nerve to trap labelled proteins moving labelled glycoproteins is increased (FRIZELL& SJOSin the retrograde direction. These proteins probably TRAND. 1974h, c, d). These phases of axonal proteins
Retrograde transport in regenerating nerves
consist of structural proteins that might take part in the renewal of the axon by incorporation into membranes at the growth cone (BRAY,1973; LENTZ,1972). In the present study an increased delayed rapid anterograde axonal transport of labelled proteins and glycoproteins in the hypoglossal nerve was indicated by the increased accumulation of these materials proximal to the ligature during 16-22 h after labelling on the regenerating side after one week of regeneration. Previously we found a decreased axonal transport of rapidly migrating labelled proteins in the hypoglossal & SJOSnerve after 1 week of regeneration (FRIZELL TRAND, 19746). These proteins accumulated at a ligature during the 20 h period after labelling, and probably represent an earlier anterograde phase of transport than that observed in the present work between 16 and 22 h after labelling. Thus, while the increased retrograde transport of labelled glycoproteins in the hypoglossal nerve might be secondary to an increased rapid anterograde transport after 1 week of regeneration, this would not be the case for the retrograde transport of labelled proteins. If the major point of reversal of the transport direction of the labelled material is at the tips of the axons, it must be considered that the shorter distance between the ligature and the point of reversal in the growing axons as compared to the longer axons on the contralateral side might be one explanation for the increased retrograde accumulation observed on the regenerating side after 1 week of regeneration. It is unlikely, however, that this is the main explanation for the increased retrograde accumulation in the 1-week experiments, since this increase, although less marked, is still present after 5 weeks of regeneration, at a time when the axons on the regenerating and the contralateral sides should be of equal length. The changes in retrograde axonal transport of labelled glycoproteins are of special interest since glycoproteins are known to be incorporated into membranes and might take part in the renewal of the axonal membrane ( B ~ N N F Tet T ul., 1973). After 5 wecks of regeneration, i.e. at the beginning of the maturation period, when the tips of the regenerating hypoglossal nerve fibres should have reached the tongue, the in& SJOSTRAND,1 9 4 7 ~ ) crease in anterograde (FKIZELL as well as the retrograde axonal transport and uptake of Evans blue-labelled albumin in regenerating hypoglossal neurons occur during the early maturation & SJOSTRAND,1972). Thus the period (KRISTENSSON rctrograde axonal transport of both endogenous and exogenous proteins seems to be increased during this phase of regeneration. The present study also demonstrated that both labelled proteins and glycoproteins are transported in a retrograde direction in the mainly unmyelinated fibres of the dorsal motor nucleus of the vagus nerve after 1 week of regeneration. In contrast to the hypoglossal nerve the transport was studied during the latent period before the outgrowth of sprouts had started. Thus the labelled material used the crush
195
zone as a point of turning its transport direction, and was actually not transported into the regenerating fibres. The retrograde accumulation of [3H]leucine labelled proteins was increased as compared to that in normal doubly-ligated nerves (FRIZELL& SJOSTRAND,1974~).In conclusion, the present results indicate that a rapid retrograde axonal transport of labelled proteins and glycoproteins occurs in ligated regenerating hypoglossal and vagus nerves of the rabbit and that this transport is greater than that of the normal nerve. The retrograde transport system might be relevant as a mediator of signal substances from the regenerating nerve fibres, possibly influencing the metabolism of the regenerating nerve cell bodies.
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