Brain Research, 85 (1975) 325-330 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands

R E T R O G R A D E AXONAL TRANSPORT PROTEINS IN P E R I P H E R A L NERVES

J. S J O S T R A N D

AND M.

OF

RAPIDLY

325

MIGRATING

FRIZELL

Institute of Neurobiology, University of Giiteborg, G6teborg (Sweden)

The anterograde axonal transport of axonal constituents has been well established in various systems. In previous studies we have characterized the anterograde transport in the rabbit vagus and hypoglossal nerves during normal conditions, as well as during nerve regenerationS-7,14. Little is known about the fate of axonai proteins once they have reached the axon terminals. While some proteins will replace degraded material, or will be released, part of the material is supposed to return from the terminals by retrograde flow~,8. Further information about the retrograde axonal transport would be important for the understanding of the protein economy of the neurone and the transfer of informational molecules from the terminals to the nerve cell body. Since it is possible that proteins ascending from the growing axon tips or the axon terminals are controlling the metabolism of the nerve cell perikarya, our present work is aimed at obtaining information about the retrograde axonal transport in mature and regenerating peripheral neurones. We have studied the retrograde redistribution of labelled material in ligated normal 8 and regenerating vagus and hypoglossal nerves following application of [3H]leucine to the brain stem of rabbits according to MianPL As this retrograde accumulation occurred in injured nerves, these results concerning retrograde transport cannot be directly extrapolated to intact nerves. Retrograde accumulation in the vagus nerve. The rapidly migrating labelled proteins of the anterograde flow14 were stopped at a ligature applied to the vagus nerve, and the retrograde redistribution of labelled material was studied following the application of a second ligature proximal to the first one (Fig. 1). During the first 8 h after labelling, rapidly migrating proteins accumulated in the 5-mm segment proximal to ligature 1 (Fig. 1). When the second ligature was applied at the end of that time, the radioactivity between the ligatures redistributed to give an accumulation in the 5-ram segment distal to the second ligature within 6 h, as compared with the activity of intermediate segments (Table I). When the retrograde accumulation was studied 16 h after labelling, the accumulation increased with time during the following 3-21 h. To study the influence of the distal accumulation zone on the magnitude of

326

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E E E40 d. "d

F

o x

1(]

25

7~

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from bulb

Fig. 1. The redistribution of rapidly migrating proteins between double ligatures in the right vagus nerve of the rabbit 14 h after labelling of the dorsal nucleus of the vagus with 30 #1 of [3H]leucine (1/~Ci//fl). Distal ligature (1) was applied simultaneously to labelling and proximal ligature (2) 8 h later, at 6 h before sacrifice. Arrows indicate sites of ligation. Hatched columns represent activity of 5-mm nerve segment excised proximal to the second ligature at 6 h before sacrifice. The diagram shows one representative experiment from 3 experiments with varying distances between ligatures.

TABLE I REDISTRIBUTION OF RAPIDLY MIGRATING PROTEINS BETWEEN DOUBLE LIGATURE 1N THE VAGUS NERVE

The dorsal nucleus of the vagus nerve was labelled with 30 #1 of [aH]leucine (1/~Ci//~l) and the distal part of the right cervical vagus nerve was ligated. Eight or 16 h later a second ligature was applied, 22.5-35.0 m m proximal to the first ligature and the animals (3 in each group) were killed after various times (3-21 h). The radioactivity of the 5-mm segments at distal and proximal ends of the isolated nerve segment is expressed in per cent of the total radioactivity between ligatures. The radioactivity per 5 m m of the nerve segments intermediate to the proximal and distal 5-mm segments is also expressed in per cent of total radioactivity. The mean ± S.D. is calculated for the different groups. The data for the 16 h animals are taken from ref. 8.

Time (h)

8 16 16 16 16

+ + + + ÷

6 3 6 6* 21

Total radioactivity between ligatures (disint./min)

Per cent of total radioactivity Distal

Proximal

Intermediate

14,562 11,853 7641 20,755 5633

74.8 70.5 59.8 72.2 68.0

7.9 8.5 11.6 6.1 16.3

3.1 4.8 6.3 4.3 4.7

± 4518 + 4046 ± 2498 _L 7704 _~ 745

± 4.6 ± 7.1 -~ 6.2 ± 2.5 ~ 2.0

£ 0.4 ~ 1.3 -Sz 2.0 dz 0.8 ~_~2.2

± ± ± ± i

0,5 0.5 1.6 0.3 1.4

* A third ligature was applied 5 m m proximal to the first, distal ligature at 16 h after labelling,

327

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E d. 0 x

10'

5mm

Fig. 2. The accumulation of [3H]leucine-labelled proteins proximal and distal to ligatures applied to the right (R) or left (L) hypoglossal nerve, 15 m m from the bulb. The hypoglossal nuclei were labelled with 30 ,ul of [3H]leucine (1/zCi//~l) 14 h before sacrifice. The left nerve was ligated at the time of labelling, the right 6 h before sacrifice. The arrows indicate sites of ligature. The diagram shows one of 3 such experiments, and the segments proximal to ligature are to the left. Calculation of the retrograde accumulation during 6 h as a percentage of the amount of labelled proteins transported in the anterograde direction during 8 h gives a value of 28.1 4- 6.4 (mean i S.D., n = 4).

the retrograde accumulation, a third ligature was applied in some experiments 5 mm proximal to ligature 1 to trap this activity. In these experiments, material accumulated both distal to ligature 2 and proximal to ligature 3, indicating a redistribution between ligature 2 and 3. This retrograde accumulation, however, was only about half of that found in the ordinary double ligature experiments at 6 h (Table I). The activity accumulating proximal to ligature 1 after 16 h in doubly ligated nerves accordingly contributed to about half of the retrograde accumulation, the other half being supplied from the intermediate segments. Retrograde accumulation in the hypoglossal nerve. When a ligature was applied to the right hypoglossal nerve 8 h after labelling of the hypoglossal nucleus, TCAinsoluble radioactivity accumulated in the 5-mm nerve segment distal to the ligature within 6 h (Fig. 2), indicating rapid retrograde transport of labelled proteins from the distal part of the nerve, including the hypoglossal terminals in the tongue, brought there with the rapid anterograde transport. The retrograde accumulation studied 16 h following labelling increased with time during the following 21 hS. These experiments suggest that as much as 50~o of the rapidly migrating proteins which had reached the distal part of the right hypoglossal nerve after 16 h return, and accumulate distal to a ligature within 21 h. In the vagus nerve, however, the retrograde accumulation was 20 ~ of the anterograde in the double ligature experiments (Table I).

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E

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20

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~'--1

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Fig. 3. The redistribution of rapidly migrating proteins between the crush zone (x) and a ligature (arrow) in the right vagus nerve of the rabbit 22 h after labelling of the dorsal nucleus of the vagus with 30/~1 of [aH]leucine (1/~Ci/#l). Crush was performed one week before labelling, and a ligature was applied 16 h after labelling. The hatched column represents the radioactivity of a 5-ram segment excised at the time of ligation. The diagram shows one of 4 such experiments. The retrograde accumulation in the 5-mm segment distal to the ligature was 20.8 ± 1.3 (mean S.D., n -- 4) per cent of the total radioactivity between the ligature and the crush zone. The retrograde accumulation of choline acetyltransferase (ChAc) and acetylcholinesterase (ACHE) in doubly ligated nerve was about 50 % of the anterograde for both enzymes 4. The magnitude o f the retrograde axonal transport as calculated from the present results indicates that this transport could be an important mechanism for the return of axonal proteins to the nerve cell body, as previously suggested from redistribution of labelled proteins in ligated nerves o f chicks 2 and frogs a. Although the design of the present experiments provided no possibility for an accurate calculation of the rate of retrograde transport, the rapid redistribution of the labelled proteins indicated that at least part of this material was transported with a rapid retrograde transport at a rate of several mm/h. In the vagus nerve the minimal and maximal transport rates would be 30 ram/21 h (34 mm/day) and 35 mm/3 h (280 ram/day), if all material transported in the retrograde direction used the distal ligature as a starting point. The corresponding rates in the hypoglossal nerve would be 25 ram/21 h (29 ram/day) and 25 mm/3 h (200 mm/day). Since the retrograde transport is probably established when the ligatures used for the retrograde accumulation are applied, and it cannot be excluded that part of the material turns its transport direction en route, these maximal rates are almost certainly overestimated. The retrograde axonal transport of exogenous proteins in the hypoglossal nerve of the rabbit has been calculated as 120 mm/day 10.

329

I-

CRUSH

CONTROL

20

16-

E E

LO o.

-d

1_.-

5 mm

Fig. 4. The accumulation of [3H]leucine-labelled proteins proximal and distal to ligatures (arrows) applied to crushed (right) and contralateral (left) hypoglossal nerves, 10-15 mm from the bulb, 6 h before sacrifice, 16 h after labelling of the hypoglossal nucleus with 30 pl of [aH]leucine (1 #Ci//~l). The right hypoglossal nerve was crushed (x) one week before labelling. The diagram shows one of 4 such experiments, and the segments proximal to the ligatures are to the left. The retrograde and anterograde accumulation in the 5-mm segments distal and proximal to the ligature on the regenerating side was 176 ± 14 and 158 4- 19 (mean S.D., n = 3) per cent of the corresponding contralateral segment, respectively.

In our previous w o r k 4 the retrograde transport rate o f C h A c was calculated to be a b o u t 50 and 60 m m / d a y and that o f A C h E to be a b o u t 70 and 120 m m / d a y in the hypoglossal and vagus nerves respectively. In frog sciatic nerve the retrograde transport rate o f labelled proteins was calculated to be 60 m m / d a y at 18 °C (see ref. 3), and the rate for retrograde transport o f A C h E in dog peroneal nerve was calculated to be 134 m m / d a y 11. Little is k n o w n about the composition o f the material transported by the retrograde axonal transport. Microphotographical studies on cultured axons have shown that granules and pinocytotic vesicles are transported in the retrograde direction in vitro g,18. Since the bulk o f rapidly migrating labelled proteins are k n o w n to be associated with particulate fractions and A C h E is a m e m b r a n e - b o u n d enzyme, it is likely that part o f the material transported in the retrograde direction is membrane-associat-

330 ed. As indicated by previous studies ~, soluble proteins like C h A c might also be transp o r t e d in the r e t r o g r a d e direction. Retrograde accumulation during nerre regeneration. Previous studies o f the a n t e r o g r a d e axonal t r a n s p o r t in the r a b b i t vagus and hypoglossal nerves have demo n s t r a t e d a c c u m u l a t i o n o f rapidly m i g r a t i n g p r o t e i n s in a n d below the crush zone, respectively, one week after nerve crush 7. [n o r d e r to elucidate the r e t r o g r a d e t r a n s p o r t d u r i n g this phase o f axon regeneration, the r e t r o g r a d e r e d i s t r i b u t i o n o f labelled m a t e r i a l was followed distal to a ligature applied to the p r o x i m a l part o f the regenerating vagus nerve. A t 6 h following ligation, an a c c u m u l a t i o n in the 5-mm segment distal to the ligature was observed (Fig. 3). This r e t r o g r a d e a c c u m u l a t i o n was a b o u t 21 ~o (see legend to Fig. 3) o f the total r a d i o a c t i v i t y between the ligature a n d the crush zone, c o m p a r e d with a value o f a b o u t 12 °~,~in d o u b l y ligated n o r m a l nerves (Table I). Similarly, in the regenerating h y p o g l o s s a l nerve an increased retrog r a d e a c c u m u l a t i o n c o m p a r e d with that on the c o n t r a l a t e r a l side was found one week after nerve crush (Fig. 4). These d a t a indicate that an active r e t r o g r a d e t r a n s p o r t o f a x o n a l p r o t e i n s is present in regenerating axons. A n interesting possibility is that the r e t r o g r a d e t r a n s p o r t is involved in the m e m b r a n e movements at the g r o w t h cone as p o s t u l a t e d by Bray 1.

t BRAY, D., Model for membrane movements in the neural growth cone, Nature (Lond.), 244

(1973) 93-95. 2 BRAY,J. J., KON, C. M., AND BRECKENRIDGE, B. M., Reversed polarity of rapid axonal transport in chicken motoneurons, Bra& Research, 33 (1971) 560-564. 3 EDSTR6M, A., AND HANSON, M., Retrograde axonal transport of proteins in vitro in frog sciatic nerves, Brain Research, 61 (1973) 311-320. 4 FONNUM, F., FRIZELL, M., AND SJOSTRAND, J., Transport, turnover and distribution of choline acetyltransferase and acetylcholinesterase in the vagus and hypoglossal nerves of the rabbit, J. Neurochem., 21 (1973) 1109-1120. 5 FRIZELL, M., AND SJ()STRAND, J., Transport of proteins, glycoproteins and cholinergic enzymes in regenerating hypoglossal neurons, J. Neurochem., 22 (1974) 845-850. 6 FRIZELL, M., AND SJ()STRAND, J., The axonal transport of [SH]fucose-labelled glycoproteins in normal and regenerating peripheral nerves, Brain Research, 78 (1974) 109-123. 7 FRIZELL, M., AND SJOSTRAND, J., The axonal transport of slowly migrating [3H]leucine-labelled proteins and the regeneration rate in regenerating hypoglossal and vagus nerves of the rabbit, Brain Research, 81 (1974) 267-283. 8 FRIZELL, M., AND SJ()STRAND, J., Retrograde axonal transport of rapidly migrating proteins in the vagus and hypoglossal nerves of the rabbit, J. Neurochem., 23 (1974) 651-657. 9 HUGrtES,A., The growth of embryonic neurites. A study on cultures of chick neural tissue, J. Anat. (Load.), 87 (1953) 150-162. 10 KRISTENSSON,K., OLSSON, Y., AND SJ/JSTRAND,J., Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve, Brain Research, 32 (1971) 399-406. 11 LUSlfiSKA,L., AND NIEMIERKO,S,, Velocity and intensity of bidirectional migration of AChE in transected nerves, Brain Research, 27 (1971) 329-342. 12 MIANI, N., Analysis of the somato-axonal movement of phospholipids in the vagus and hypoglossal nerves, J. Neurochem., 10 (1963) 859-874. 13 POMERAT,C. M., HENDELMAN,W. J., RAmORN, C. W., AND MASSEY,J. F., Dynamic activities of nervous tissue in vitro. In H. HYD~N (Ed.), The Neuron, Elsevier, Amsterdam, 1967, pp. 119-178. 14 SJOSTRAND,J., Fast and slow components of axoplasmic transport in the hypoglossal and vagus nerves of the rabbit, Brain Research, 18 (1970) 461-467.

Retrograde axonal transport of rapidly migrating proteins in peripheral nerves.

Brain Research, 85 (1975) 325-330 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands R E T R O G R A D E AXONAL TRANSPOR...
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