Cessation of axonal transport of acetylcholinesterase after administration of cycloheximide

Cessation of axonal transport of acetylcholinesterase after administration of cycloheximide

Brain Research, 124 (1977) 379-384 379 © Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands Cessation of axonal transport...

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Brain Research, 124 (1977) 379-384

379

© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands

Cessation of axonal transport of acetylcholinesterase after administration of cycloheximide

MILENA BURE~OV/~and STANISLAVTU~EK Institute of Physiology, Czechoslovak Academy of Sciences, 14220 Prague (Czechoslovakia)

(Accepted December 17th, 1976)

Inhibitors of protein synthesis have been successfully used as tools in studies of axonal transport. The observation that no labelled proteins appeared in axons after synthesis of proteins in perikarya had been inhibited served as a proof of the perikaryal origin of labelled proteins occurring in axons following the administration of labelled amino acids into the vicinity of nerve cell bodiesa,4,s,9.1a-15,17,1s. If, in experiments with pulse labelling of neuronal proteins, the inhibitors were applied only after the synthesis of labelled proteins in the perikarya had been completed, fast transport of labelled proteins in the axons was found to continue notwithstanding the inhibition of further protein synthesis in the cell bodies3,S,15. In the present study the effect of inhibition of protein synthesis on fast axonal transport was investigated using the technique of placing ligatures on the nerves (with subsequent measurement of accumulation of acetylcholinesterase (AchE) proximally to the ligatures) rather than the technique of pulse labelling. The technique of ligatures enables us to make observations in the course of more prolonged time periods. Thanks to this it proved possible to obtain information about the length of time during which the proximodistal transport of AChE continues after the synthesis of new proteins had been inhibited. White male rats (Wistar), weighing 100-110 g, were lightly anesthetized with ether and their left peroneal nerve was ligated with a very thin silk thread and left in situ. The distance between the ligature and the bodies of corresponding motoneurons in the spinal cord was about 65 mm. After 5 or 10 h (or at various other time intervals in preliminary experiments not reported here) the animals were sacrificed. A nerve segment of approximately 4 mm in length immediately proximal to the ligature was taken out and used for measurement of the activity of ACHE. The corresponding segment from the contralateral (intact) peroneal nerve was used as control. The length of the segments was measured while they were freely lying in a thin layer of physiological saline solution. In experiments with the inhibition of protein synthesis, rats were intraperitoneally injected with cycloheximide (CHX, supplied by Sigma Chemical Co.~ 2 mg/kg body weight) immediately after the nerve had been ligated; unless stated otherwise, the injection was repeated after 4 h. The inhibition of new protein synthesis at various time intervals after the injections of CHX was evaluated according to the

380 TABLE I

Comparison of the action of cycloheximide ( CHX) on protein synthesis in the brain, liver and gastrocnemius muscle Values are means of duplicate measurements performed on 3 rats weighing 102-106 g. The first received no C H X , the second received CHX in a single dose of 3 m g / k g and the third in a single dose of 15 m g / k g i.p. They were killed 270 rain after the start of the experiment. Twenty minutes before killing each rat was injected with [U-14C]leucine (10/~Ci, 48 nmole) i.p. Relative specific radioactivity of proteins was calculated as the ratio (disint./min in proteins/100 m g of proteins)/(disint./min in trichloroacetic acid-soluble fraction/l g tissue).

Organ

Dose of CHX (mg/kg)

Disint./min in triDisint./min in chloroacetic acidproteins/lO0 mg soluble fraction~1 g of proteins tissue

Relative ~pecific radioactivity of proteins

Relative specific radioactivity of proteins in CHXtreated animals as per cent of control values

Brain

----

32,790 104,390 35,270

29,628 168,980 31,020

0.904 1.619 0.880

----

Liver Muscle

3 3 3

50,850 123,460 73,580

10,915 17,640 2349

0.215 0.143 0.032

23.8 8.8 3.6

Brain Liver Muscle

15 15 15

85,080 344,640 88,780

9579 12,1 04 669

0.113 0.035 0.008

12.5 2.2 0.9

Liver Muscle Brain

changes of the relative specific radioactivity of proteins found 20 min after an i.p. injection of [U-14C]leucine (Isokommerz, Berlin; 24-48 nmole, 5-10 #Ci). Proteins from the brain or other organs were isolated by precipitation in trichloroacetic acid; the precipitate was washed with hot (90 °C) trichloroacetic acid, extracted with a mixture of chloroform, ethanol and ether (1:2:2) and with ether alone, dissolved in 1 N NaOH and its radioactivity was measured by liquid scintillation technique. The relative specific radioactivity of proteins was calculated as the ratio (radioactivity incorporated into 100 mg of proteins)/(radioactivity in trichloroacetic acid-soluble pool/1 g of tissue weight). The amount of protein was measured using the modified 1° procedure proposed by Lowry at al. 5. The activity of AChE was determined using 3 mM [1-14C]acetylcholine (The Radiochemical Centre, Amersham) as substrate 19. The inhibition of protein synthesis produced by CHX in the brain was lower than in other organs (Table I). The difference was presumably due to the interference of the hematoencephalic barrier. The doses of CHX adopted after preliminary experiments (2 mg/kg i.p. at time 0 and 4 h) lowered the synthesis of proteins in the brain (evaluated by their relative specific radioactivity 20 min after the injection of [U-14C] leucine) to 7-37 ~ of control values (Fig. 1). It was assumed that the synthesis of proteins in the spinal cord and its motoneurons was inhibited to a similar degree. The activity of AChE in control (non-ligated) nerves was not significantly changed by the administration of CHX. It was (mean -4- S.E.M.) 285 -_-~ 14 and 243

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CHX CHX Fig. I. Effect of cycloheximide (CHX) on the synthesis of proteins in the brain. Rats were injected with 2 mg CHX/kg body wt. i.p. at time 0 and 4 h. They were killed at time intervals indicated on the abscissa. Twenty minutes before killing, they were injected i.p. with [U-14C]leucine (24 nmoles, 5/tCi). Data on the ordinate indicate the specific radioactivity of brain proteins (mean ~: S.E.M.) calculated as the ratio (radioactivity incorporated into 100 mg of proteins)/(radioactivity in trichloroacetic acidsoluble pool/1 g brain weight). The number of observations is given at individual points. 4- 25 pmole A C h / m m nerve/h in the control (untreated) groups for the 5 h and 10 h experiments, respectively, and 262 ± 12 and 245 -k- 12 pmole A C h / m m nerve/h in the CHX-treated groups in the 5 h and l0 h experiments, respectively. As expected, the activity of AChE in the 4 m m segments of the nerve immediately proximal to the ligature was found to increase. In control rats, the increase was approximately linear during the first l0 h after ligation (Fig. 2). After 5 h, the activity in the 4 m m segment above the ligature was 49 ~ higher and after 10 h 108 ~o higher than in the corresponding segment of the contralateral (non-ligated) nerve. The amount of surplus AChE accumulating above the ligature during the first 5 h corresponded to the content of enzyme in 1.80 m m of control nerve; after 10 h it corresponded to the content of enzyme in 4.19 m m of control nerve. In CHX-treated rats the accumulation of AChE proximal to the ligature 5 h after the ligation did not differ from that found in control rats (Fig. 2). After 10 h, however, the activity of AChE found in the nerve segment immediately above the ligature was approximately the same as after 5 h. Consequently, the accumulation of

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Fig. 2. Effect of cycloheximide (CHX) on the accumulation of acetylcholinesterase(ACHE) proximally to a ligature made on rat peroneal nerve at time 0. CHX (2 mg/kg body wt.) was injected i.p. at time 0 and 4 h. The accumulation of the enzyme (ordinate) vs. time (abscissa) is expressed in three ways. A: per cent increase in the segment above the ligature as compared to the corresponding contralateral nerve whose activity is taken as 100 ~ ; B: the length of control nerve which contained the same amount of enzyme as that which accumulated above the ligature in excess of the normal enzyme content; C: in terms of absolute activity of the accumulated (surplus) enzyme (pmoles acetylcholine hydrolyzed per min). Empty circles, control rats; solid circles, rats injected with CHX. Individual points are means i S.E.M. ; in some points the S.E.M. was too small to be shown on the scale used. The number of observations is indicated by figures in A. The activity of AChE in non-ligated control nerves (expressed in pmole ACh hydrolyzed/min/mm) was 285 ± 14 and 243 ± 25 in control groups for the 5 h and 10 h experiments, respectively, and 262 ± 12 and 245 ± 12 in the CHX-treated groups in the 5 h and 10 h experiments, respectively. A C h E p r o x i m a l to the ligature did n o t continue during the time p e r i o d o f 5-10 h after the ligation o f the nerve a n d first injection o f C H X . The o b s e r v a t i o n that the a c c u m u l a t i o n o f A C h E p r o x i m a l to a ligature on the nerve is n o t affected by C H X d u r i n g the first 5 h after its a d m i n i s t r a t i o n accords well with findings that the p r o x i m o d i s t a l t r a n s p o r t o f proteins in the axons continues after the synthesis o f p r o t e i n s in the bodies o f the nerve cells h a d been arrested3,8,11,1L N o effect o f the inhibitors o f p r o t e i n synthesis on a x o n a l t r a n s p o r t d u r i n g short time p e r i o d s after their a d m i n i s t r a t i o n indicates that, at least for the fast axonal t r a n s p o r t , there is no need for a ' v i s a t e r g o ' p r o d u c e d by newly synthesized proteins which m i g h t be t h o u g h t to p r o p e l the c o n t e n t o f the axons in the p r o x i m o d i s t a l direction. N o evidence is available, however, r e g a r d i n g the question as to h o w long does the p r o x i m o d i s t a l a x o n a l t r a n s p o r t continue after the synthesis o f proteins had been inhibited. In the present experiments, the a c c u m u l a t i o n o f A C h E clearly s t o p p e d after 5 h. The a c c u m u l a t i o n o f A C h E p r o x i m a l to a ligature is generally r e g a r d e d as a measure o f its p r o x i m o d i s t a l t r a n s p o r t (see review by Lubifiska 6) a n d the d i s t o p r o x i m a l trans p o r t o f ACHE, occurring simultaneously ~1, does n o t interfere with itT, 16. The most likely e x p l a n a t i o n o f the cessation o f a c c u m u l a t i o n o f A C h E after 5 h is that by this time no A C h E was available for t r a n s p o r t in the central nerve stump. The cessation o f A C h E t r a n s p o r t occurred at a time when the activity o f A C h E p e r m m o f c o n t r o l (intact) nerves was n o t significantly changed. The A C h E m o v i n g p r o x i m o d i s t a l l y m a y represent a very low p r o p o r t i o n o f the t o t a l A C h E content in the nerve; an estimate o f 10 has been m a d e for canine 7 a n d feline ~6 nerves, a n d 5 ~o for a r a b b i t nerve 2°. The absence o f a significant change o f t o t a l A C h E activity in c o n t r o l nerves therefore does n o t rule

383 out the assumption that the mobile proportion of AChE was exhausted in CHX-treated animals. It has been shown conclusively that AChE moves with the fast transport; its rate of transport was estimated as 260 mm/day in dogs 7, 431 mm/day in cats x6 and 480 mm/day in rabbits 2°. Direct estimates of the rate of AChE transport in rat nerves are not available because of technical difficulty in estimating the proportion of mobile enzyme in small animals. The rate of fast transport in rat nerves measuredin experiments with the incorporation of labelled amino acids into neuronal proteins was found to be 411 mm/day 1~ or 428 mm/day 2. Assuming that in the present experiments the rate of the proximodistal transport of AChE was 420 mm/day, i.e. 17.5 mm/h, it can be calculated that the enzyme which, at time 0, had just entered the axon, needed 3.7 h to pass the distance of 65 mm separating it from the ligature. After that the increase in AChE activity continued for only 1.3 h. The enzyme accumulating during this time period is likely to have come from the perikaryal storage compartment. If the rate of transport of AChE were lower than 420 mm/day, the period during which we have to assume a supply of enzyme from the perikarya into the axons would be even shorter. It seems evident that, after the synthesis of new proteins had been inhibited, the transport of AChE already located in the axons proceeds without obstruction. Similarly, Banks et al. 1 observed in experiments in vitro that CHX did not interfere with the transport of the noradrenaline which, at the start of the experiment, has already been in the axons. On the other hand, the capability of the perikarya to support further transport of AChE appears to be very limited. There could be several reasons for this: (a) the perikaryal store of AChE is extremely small; (b) the transport stops, not because of lack of ACHE, but because of lack of some component of the transporting machinery, e.g., of the assumed transporting filament; and (c) the gating mechanism responsible for the release of AChE from the perikaryon into the axon is itself rapidly impaired under conditions of inhibited protein synthesis. Presently available experimental observations cannot differentiate between these possibilities. To summarize, during the first 5 h after the administration of CHX there was no difference between the accumulation of AChE proximally to ligatures on peroneal nerves in experimental and control rats. Between 5 and 10 h, however, the accumulation of AChE was arrested in the CHX-injected animals, while it continued in controls. It is suggested that the transport of the AChE which is already present in the axons at the time of administration of CHX is not affected by the drug, but that the ability of perikaryal stores of AChE in spinal motoneurons to supply further AChE for transport in the axons is extremely limited under conditions of inhibition of new protein synthesis. Thanks are expressed to Dr. Jifina Zelen~ for valuable comments on an earlier version of the manuscript.

1 Banks, P., Mayor, D. and Mraz, P., Metabolic aspects of the synthesis and intra-axonal transport of noradrenaline storage vesicles, J. PhysioL (Lond.), 229 (1973) 383-394. 2 Bisby, M. Y., Orthograde and retrograde axonal transport of labelled protein in motoneurons, Exp. NeuroL, 50 (1976) 628-640.

384 3 Edstr6m, A. and Mattsson, H., Fast axonal transport in vitro in the sciatic system of the frog, J. Neuroehem., 19 (1972) 205-221. 4 Edstr6m, A. and Mattsson, H., Rapid axonal transport in vitro in the sciatic system of the frog of fucose-, glucosamine- and sulphate-containing material, J. Neurochem., 19 (1972) 1717-1729. 5 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 6 Lubifiska, L., On axoplasmic flow, Int. Rev. Neurobiol., 17 (1975) 241-296. 7 Lubifiska, L. and Niemierko, S., Velocity and intensity of bidirectional migration of acetylcholinesterase in transected nerves, Brain Research, 27 (1971) 329-342. 8 McEwen, B. S. and Grafstein, B., Fast and slow components in axonal transport of protein, .L Cell Biol., 38 (1968) 494-508. 9 McLean, W. G., Frizell, M. and Sj6strand, J., Axonal transport of labelled proteins in sensory fibres of rabbit vagus nerve in vitro, J. Neurochem., 25 (1975) 695-698. 10 Miller, G. L , Protein determination for large numbers of samples, Analyt. Chem., 31 (1959) 964. 11 Niemierko, S. and Kowalska, K., The effect of cycloheximide on the activity of lactate dehydrogchase in transected peripheral nerves of the dog and of the rat, Comp. Bioehem. PhysioL, 48 (1974) 211-223. 12 Ochs, S., Rate of fast axoplasmic transport in mammalian nerve fibres, J. Physiol. (Lond.), 227 (1972) 627-645. 13 Ocbs, S. and Johnson, J., Fast and slow phases of axoplasmic flow in ventral root nerve fibres, J. Neurochem., 16 (1969) 845-853. 14 Ochs, J., Johnson, J. and Ng, M.-H., Protein incorporation and axoplasmic flow in motoneuron fibres following intra-cord injection of labelled leucine, J. Neuroehem., 14 (1967) 317-331. 15 Ochs, S., Sabri, M. I. and Ranish, N., Somal site of synthesis of fast transported materials in mammalian nerve fibres, J; Neurobiol., 1 (1970) 329-344. 16 Ranish, N. and Ochs, S., Fast axoplasmic transport of acetylcholinesterase in mammalian nerve fibres, J. Neurochem., 19 (1972) 2641-2649. 17 Sj/Sstrand, J., Rapid axoplasmic transport of labelled proteins in the vagus and hypoglossal nerves of the rabbit, Exp. Brain Res., 8 (1969) 105-112. 18 Sj6strand, J. and Karlsson, J.-O., Axoplasmic transport in the optic nerve and tract of the rabbit: a biochemical and radioautographic study, J. Neuroehem., 16 (1969) 833-844. 19 Tu~.ek, S., Transport and changes of activity of choline acetyltransferase in the peripheral stump of an interrupted nerve, Brain Research, 82 (1974) 249-261. 20 Tu~ek, S., Transport of choline aeetyltransferase and acetylcholinesterase in the central stump and isolated segments of a peripheral nerve, Brain Research, 86 (1975) 259-270. 21 Zelen~, J. and Lubifiska, L., Early changes of acetylcholinesterase activity near the lesion in crushed nerves, Physiol. bohemoslov., 11 (1962) 261-268.