Neurotoxicity and axonal transport

243

TIPS - J u n e 1984

excitability with changes of CO2 or HCO.C. It should be noted, however, that such an explanation would not account for the different mechanisms of potentiation of benzodiazepine binding by EDA and GABA. Whatever the details, however, it is likely that the ability of EDA to mimic so closely the effects of GABA, will be of some value in undeistanding the activation and physiological and pathological modulation of GABA receptors. Reading list 1 Anderson, E. G., Haas, H. and Hosli. L. (1973) Brain Res. 49, 471--475 2 Blaxler, T. J. and Cottrell, G. A. (1982) J.

Physiol. (London) 330, 46p 3 Bokisch, A. J., Bold. J. M.. Perkins, M. N . Roberts, C.J.. Stone, T . W . and Walker. R. J. (1982) Br. 1. Pharmacol. 76, 297P 4 Bowery, N. G., Hill, D. R , Hudson, A. L.. Perkins, M. N. and Stone, T. W. (1982,) Br. J Pharmacol. 76, 47P 5 Hill, D. R. Br. 1. Pharmacol. 79. 268P 6 Hughes, P. R., Morgan, P. F. and Stone. T. W. {1982) Br. I. Pharmacol. 77, 691-4~)5 7 Lloyd, H. G. E., Perkins, M. N. and Stone. T. W. (1982)J. Neurochem. 38, !168--1169 8 lloyd, H. G. E., Perkins, M. N., Gaitonde. I'.4. K. and Stone, T. W. (1982) J. Neurochem. 38, 1118-1122 9 Morgan, P. F. and Stone, T. W. (1982) J Neurochem. 39, 1446-1451 10 Morgan, P. F. and Stone, T. W. (1983) Br. J. ,9harmacol. 79, 973-978

11 Perkins. M. N.. Bo,a'eD. N. ( ; . ltdl. D. R. and Sttu~e. 1". W. ( I t ~ l ) Neurm, t. tett. Z~. 3ZS--327 12 Perkins. M. N. and Sione. T. V¢ (191,~1).4nh. Int. Pharmacody n. Fher. 246. 205-214 13 Perkins. M. N. and Stone. T. W (1982) Br. J Pharmacol. 75.93-99 14 Phillis, i. W (ltF/7) ('an. J Ncurol..~ict ,1. 151-195

T. W. Stone gradtmted from the .~ch~,:d O[ Pharmtu'r in London and then went to Aberdeen as a Ph D. Student and then Lecturer. tie t.~ ,',rrenth" Reader in Neuroscwnce.~ at St (;eorge'x H , wttal .th'dtcal School m London. M N. Perkins graduated from Chelsea College. London and moved to St Georee'.l Medical .%ch,:~l for h~s P h D. work. He t~" currer~tlv .~ Re~ear, h FeUow.

I

Neurotoxicity and axonal transport Graham McLean Department o f Pharmacology and Therapeutics, Universt~' o f Liverpool, P.O. Box 14Z Liverpool L69 3BA\ UK.

The continuous transport of materials in both directions within nerve axons is essential for normal nerve cell function. A variety of drugs and chemicals have a selective toxicity on the nervous system. Recent work indicates that ttronal transport may be a target in the toxic action of some of those substances and provides some illumination on the mechanisms of axonal transport itself.

The mature of exomal trmslmrt The characteristics and mechanism of axonal transport have been fully reviewed recently L2. I shall provide here only a brief overview of our present state of knowledge so that the reader may understand the complexity of the alterations which may occur when axonal transport is impaired. Within the axoplasm of central and peripheral nerves there exists continuous traffic, in both directions, of a wide range of substances including proteins, membranous vesicles, neurotransmitters, lipids, mitochondria and RNA. It is assumed that the purpose of this process is to provide the necessary constituents to the axon from their sites of synthesis or asseml:ly in the nerve cell body, or, in the case of retrograde axonal transport, to return redundant cellular materials to the cell body for future re-use. In addition, axonal transport may serve a s a form of communication signal between cell body and the dist~mt nerve terminal. Axonal transport of protein, the most widely studied material, occurs in the anterograde direction (i.e. from cell

The mechanism of axonal transport ~s still poorly understood. A larg~.~ body ~f pharmacological data. mainly with the anti-mitotic agents colchicine and ~i~blastine, as well as morphological evidence, points to axonal microtubules as serving an important role. This has led to the hypothesis that the energy necessary for axonal transport derives from the breakdown of ATP by microtubule-associated ATPases, iargely b~ analogy with the role of dynein ATPast: in the sliding motion of cilia and flagella However. the majority of the ATPasc activity associated with brain micn-tubules is membrane-bound. Th.,s coulc. of course, bc involved in the fast axonal transport of membrane vesicles, but it should be remembered that not tdi axonally transported material is transported vdthin a vesicle and, indeed, transport of completely non-ph)~iolo~cal particles is knov,'n to occur. An alternative, more passive, role for microtubules has been suggested: that they act as the directional environment for "microstreams' of low viscosity within the highly viscous axoplasm. It is en~fsaged in both of the above theories that the different rates of transport of different organeUes reflect their relative affinities for, and hence amount of time spent in association with, the most rapidly moving components of the system. Slow axonal transport may alternatively be considered as a distinct process involving the composite movement of at least two major components of the axoplasm, namely, the microtrabectdar network and its associated proteins, and the tubulin-neurofilament complex. Two other suggestions for mechanisms are relevant to this article. Injection of agents which disrupt the structure of actin inhibits fast axonal transport; this indicates the involvement of actin microfilaments, although the exact l~x-ationof

body to terminal) at a variety of velocities~ ranging from 0.5 to 410 ~am day- t. Within this range, the proteins in any one nerve fall into as many as six main groups. These groups are defined according to the times of arrival at nerve terminals, or the emergence into axons, of radiolabelled protein, following the application of radioactive amino acid to nerve cell bodies (Fig. l). The fastest group, i.e. fast axonal transport, includes mainly small vesicular organelles, and both membranous and soluble material. Two of the slowest groups contain, respectively, elements of the axoplasmic or microtrabecular matrix, i.e. actin, clathrin and myosin-like proteins, and the subunit proteins of microtubules (orand 13-tubulin) and, where present, of neurofilaments. In the retrograde direction both fast (up to 300 mm day -t) and slow (3--6 nun day -~) components have been detected. The material transported includes large multivesicular and raultilaraellar organelles, as well as senun albumin. The protein composition of anterograde and retrograde transport is, however, very similar. © Iq~l..~

Saeno~Ptibli~rs B.V.. ~

01o5- 6147,'84,~1~200

244 their contribution is unclear. Also, the extensive axonal network of smooth endoplasmic reticulum (SER) or axoplasmic reticulum can be shown by autoradiography to be a site of a large proportion of fast transported proteins. Although the reticulum as a whole is probably not transported rapidly, there may be a dynamic interaction between fast transported vesicles and the reticulum membranes which could aot as guiding, or storage, channels. What are the censequem~es of inhibition ef axenal transport? Despite the obvious importance of axonal transport for the provision of axonal material, there is no clearly defined picture of what the consequences of inhibition of transport are for the nerve cell or the tissue which it innervates. Axonal transport in mammalian nerve can be chronically disrupted without producing nerve fibre degeneration or cessation of impulse propagation. Postsynaptic responses to inhibited axonai transport can therefore differ from those of axotomy, in which electrical conduction is, of course, simultaneously impaired3. Yet in some cases the, effects are identical. These differences occur largely because the commonly used experimental 'tool', which consists of "inhibiting' axonal transport with, for example, colchicine, and then observing the results, is extremely blunt. Very few studies have been undertaken in which the extent of graded or selective inhibition of axonal transport has been monitored alongside the physiological consequences. Recent findings suggest that agents which may be used to inhibit ~xonal transport experimentally may show a degree of selectivity against the transport of certain proteins 4. If one also considers that any neurotoxic agent may conceivably impair the tr;~sport and supply of only one minor component of the axon then it is clear that the symptoms of a disorder of axonal transport can be many and varied, and are certainly not immediately recognizable as such with our present level of understanding.

T I P S - Jw~e 1984 Acrylamide A number of studies agree that there is a reduction in the rate of fast axonai transport after ingestion of acrylamide by experimental animals, but the effects in most cases were said to be slight and unrelated to the course of the neuropathy. Of more interest is the observation that fast transported proteins are displaced during transport towards the periphery of the axon, as part of a structural disorganization of the axoplasm, involving focal accumulations of neurofilaments and SER (Ref. 5). The abnormalities in SER are particularly marked in cerebellar Purkinje cells

where SEE-containing protrusions can be seen on the cell surface ¢'. The relationship between SER abnormalities and axonal transport is still not understood. One explanation for the accumulation of SER in the distal axon and the associated distal swellings may be the fact that retrograde axonal transport is reduced in amount, and possibly also rate, as is the retrograde transport of exogenous nerve growth factor. Slow axonal transport is also altered in more severe neuropathy; this may be related to the accumulation of neurofilaments, which are components of the slowest phases of slow transport.

vagus nerve

" •injection of

~ 1 0 0 ~ l C i L-(3H)-Ieucine

or L-(35SJ-methionine

30 03

c-

6

E

E

eJ

~.~

~

20 10 '

{p

,op
e-

E ,,-)

I

l i

40 20

I

~

I

i ~

i

o control -5x10-6M "2x10-5M =lxlO-4M hexachlorophene

.."= -~AI~

~'. . . .

-

'._,L

-

:

-IB

mm from nodose ganglion

Nemtltnk mlmancts whidl affect

axma aamCen A large number of substances have been used experimentally to inhibit axonal transport (see Rcf. 1). l/ere we shall consider mainly those agents which a~e of interest in neurotoxicology, i.e. those w l ~ have a reputation for neurotoxicity on human exposure.

Fig. 1. Some metho~ of wu~asuringaxonal transport in the rabbit vagus nerve. Proteins are radiolabelled by application of radiolabefled amino acid to the nodose ganglion (top). Below, a wave offast transported radiolabelkd protein is formd in ",henerve 4 h after radiolabelling. At that lime, the nerves are cut into 2.5 mm pieces and their nulioactil.ily daermined. Nerves may be ligated at B and nerve trunks incubated for up to 24 h in vitro in the presence or absence o f drugs. Shown (centre) is the effect of/ncreasing concentrat/ons of Iv,xachlorophene on the consequent aLx'umu~on at the ligature of fast transported proteins. Values are means of 4 nerves ± S.D. In the bottom section t~e radiolabelled proteins in 5 evn pieces of nerve, 72 h after radMabelling, have been separated by SDS-polyacrylamide gel electrophoresis and visualized by fluorography. Radiolabelled s~bwly transported proteins including actin (,4) and tubulin (T), are present at that time.

TIPS - June 1984 OrganophosphonL~ compounds Distal axonopathies can be induced by exposure to organophosphorus esters. such as paraoxan, tri-orthocresylphosphate and di-isopropylphosphofluoridate. The neuropathy is associated with a proliferation of SER but other morphological characteristics clearly distinguish it from acrylamide neuropathy. In organophosphorus neurotoxicity there is no known alteration in fast or slow axonal transport of proteins except in the rat optic system after local application 7. Hexacarbons This neuropathy is characterized by an intense proliferation of neurofilaments which appear whorled, particularly above nodes of Ranvier in myelinated axons. Unlike acrylamide neuropathy, the neurofilaments appear separate from the other organelles, which they appear to push aside. This dense packing of neurofilaments might be expected to lead to a disturbance in axonal transport; fast transport is, in fact, impaired especially through the distal axons s, and a reduced rate of retrograde transport also occurs. Zinc pyridinethione The antibacterial-antifungal agent zinc pyridinethione induces a neuropathy which includes proliferation in axon terminals of branched tubulovesicular profiles. The most consistent alteration in axonal transport which has been demonstrated is an effect on retrograde transport, defined as a deficiency in 'turnaround', i.e. the ability of fast anterograde transported proteins to return from the nerve terminal 9. Other substances The effects of the above agents on axonal transport have been studied in some detail and, perhaps more importantly, relatively recently, when knowledge of the characteristics of axonal transport has allowed detailed interpretation. Other substances have been examined less fully, but the results are worth mentioning. Axonal transport has been implicated in the neurotox/city of methyl mercury. Fast transport is inhibited in vitro but apparently accelerated in vivo. The binding of methyl mercury of sulphydryl groups is consistent with the kno~m inhibitory effects of certain other suiphydryl-binding agents on axonal transport. Despite the', neurotoxicity associated with the Vinca alkaloids vinblastine and vincristine and their known interaction with axonal microtubules and inhibition

245 of axonal transport hi vitro, there have been few attempts to link their neurotoxicity in t,ivo with ~ failure of axonal transport. Only minor, inconsistent alterations have been reported in the few studies performed. A number of retinotoxic agents including chloroquine, thioridazine, clioquinol and chlorpromazine inhibit fast transport in vitro, at concentrations which may be reached in the eye as a result of accumulation of the drugs in retinal pigment epithelia "~. Chloroquine does not, however, inhibit axonal transport in periphera~ nerve after prolonged ingestion. The antiseptic agent hexachlorophene similarly inhibits fast axonal transport in vitro, at relatively low concentrations (see Fig. 1). In all the above in-vitro work, the agents which affected axonal transport did so by a mechanism unrelated to any alterations in protein synthesis. An increase in ethanol-treated rats in the radiolabelling of synaptosomai proteins, after intraventricular injection of radioisotope, suggests that ethanol, at an anaesthetic dose, may slightly increase the capacity for fast axonal transport of certain proteins tt. The transport of acetylcholinesterase in peripheral nerve is similarly increased ~2. The significance of the effect is not known.

f3-f31-1minodipropionitrile and p-bromophenylacetylurea While neither of these agents strictly falls into the category of environmental toxin, their toxic effects on axonal transport have nevertheless generated great interest. 13-13t-iminodipropi,~nitrile (IDPN) pr~ duces a condition of proximal neur,> pathy, although ir~ some cases both proximal and distal axonal swellings occur. IDPN experimental neuropathy is one of the first examples of an impairment of transport of an individual protein or group of proteins, in this~case the neurofilament polypeptides. This selective inhibition of neurofilament slow transport is almost certainly related to the morphological observation that in IDPN-treated nerves there is a redistribution of neurofilan~ents to the periphery of the axon. In the interior, in which microtubules and actin filaments remain intact, fast transport proceeds normally t2. Whatever the role of axonal neurofilaments may be, both those results and the fact that fast transport proceeds at normal rates in axons: which possess no neurofilaments, indicate that those organelles are not intimately involved in the axonal transport process.

p-Bromophenylacetylurea (BPAU) produces a motor and sen~,ry, distal axonopathy. Its effects on axonal transport have been studied in some detail in relation to the development of the neuropathy. The changes fimnd are vet)' similar to those produced in experimental diabetes, namely an early reduction in retrograde transport, or turnaround from nerve terminals, with a reduced rate of slow transport associated with a more severe neuropathy t4. The results suggest that multiple abnormalities in axonal transport may underlie the distal axonopathies and provide a sound argument for the investigation of a full range of axonal transport parameters for any one agent. is there a common defect in all the distal axonopathies?

The above results with BPAU have led to the speculation that a deficit in turnaround of proteins on to the retrograde transport system may underEe all the commonly observed distal axonopathies Is. This is an attractive proposition in cases where the pathology, consists of an accumulation in the nerve terminals of tubulomembraneous material, which would be retained in the nerve endings and prevent normal function. Other biochemical alterations are also common to a number of neuropathies. including increases in lysosomal and proteolytic enzyme acti,4ties. Alterations in neuronal energy metabolism also occur in a number of neuropathies in which axonal transport is impaired. Indeed, the h.,,T~othesishas been presented that a defect in ener~' metabolism underlies the majority, of toxic and metabolic neuropathies '6. Against this argument, however, lies the fact that the toxic neuropathies each have clearly defined morphological characteristics: such differences in axonal pathology ~vould suggest that more subtle primary defects are involved. The case for axonal transport studies in neurotoxicology There has been considerable discussion linked with the above arguments as to whether the aherauons in axonal transport in toxic neuropathies are primary. or secondary lesions. In many ways this is unimportant: it is probably wrong to consider "retrograde axonal transport' or 'fast a~xonai transport' as entities. They are merely the exper/mental signs of the continuous deliver}' from and to nerve cell bodies of a large number of unknown, but presumabb' essential, molecules. Abnormalities in

246

synthesis or normal delivery, turnover, or deposition of those molecules can be determined readily by axonal transport techniques in combination with electrophoretic separation of proteins in one or two dimensions (see Fig. 1). Subtle alterations in the axonal transport of individual molecular forms of acetylcholinesterase in acrylamide neuropathy have recently been found ~7 and confirm the observations, mentioned above, with IDPN and colchicine, that axonal transport of individual proteins may be affected. Even fairly gross alterations in transport may occur at very early stages of the toxic neufopathies well before any symptoms such as muscle weakness or alterations in nerve conduction velocity can be detected. Detailed analysis of axonally transported proteins can therefore provide a useful tool for the screening of potentially neurotoxic agents.

n~ung I Grafstein, B. and Forman. D. S. (1980) Physiol. Rev. 60, 1167-1283 2 Weiss. D. G. (ed.) (1982) Axoplasmdc zrans-

TIPS - June 1984 port, Springer-Verlag, Berlin 3 Wan, K. K. and Boegman, R. J. (1981) Exp. Neurol. 74. 439-446 4 Komiya, Y. and Kurokawa, M. (1980) Brain Res. 190, 505-516 5 Chretien, M., Patey, G., Souyri, F. and Droz, B. (1981) Brabl Res. 205, 15-28 6 Cavanagh, J. B. and Gysbers, M. F. (1983) J. Neurocyml. 12,413-437 7 Reichert, B. L. and Abou-Donia, M. B. (1980) MoL Pharmacol. 17, 56.60 8 Mendell, J. R., Sahenk, Z., Saida, K., Weiss, H. S., Savage, R. and Couri, D. (1.o77) Brain Res. 133, 107-118 9 Sahenk, Z. an~ Mendell, J. R. (1979) J. Ne~opathol. Exp. NeuroL 38, 532-550 10 McLean, W. G. and Sj6strand, J. (1977[ in Mechanisms, ttegulation and Special Functions of Protein S~ntheses in the Brain (Roberts, S., Lajtha, A. and Gispen, W. H., eds), pp. 123-128, Elsevier/North-Holland. Amsterdam 11 Israel, M. A., Kuriyama, K. and Yoshikawa, K. (1975) Neuropharmacology 14, 445-451! 12 Bosch, E. P., Pelham, R D., Rasool, C. G.. Chatterjee, A.. Lash. R. W., Brown, L., Munsat, T. L. and Braclley, W. G. (1979) Muscle Nerv. 2, 133-144 13 Griffin, J. W., FahnestocL K. E., Price, D L. and Hoffman, P. N. (1983) J. Neurosci. 3, 55%566 14 lakobsen, J. and Brimijoin, S. (1981) B~ain Res. 229, 103-122

15 Brimijoin, W. S. (1982) Mayo Clin. Proc. 57, 707-714 16 Spencer, P. S., Sabri, M. i., Schaumburg, H. H. and Moore, C. L. (1979) Ann. Neurol. 5, 501-507 17 Courand, J. Y., Di Gianlberdino, L., Chretien, M., Souyri, F. and Fardeau, M. Musc!e Nerv. 5, 302-312

W. G. McLean received his B.Sc. degreefrom the Department of Pharmacology, University of Glasgow, and his Ph.D. from the Department of Pharmacology, University of Bristol. From 1973 until 1975 he held a Research Fellowship at the Institute of Neurobiology, University of G,#teborg, Sweden. He has been a Lecturer in the Department of Pharmacology and Therapeutics, University of Liverpool, since 1980.

Books . nerican perspectives on dementia Banbury Report 15, Biological oI~Alz_heim~'s Disease e, lised by R. Katzman, Cold Spring Harbor Laboratory, 1983. US $55.00 (J~Jt.00 outside US) (xiv + 495 pages) £gBN 0 ~ 7 9 213 8 1here is a fast increasing interest in n~edical problems associated with the elderly population and particularly in Alzheimefs disease, the major cause of dementia. This reflects the increasing social and economic problem that care of the elderly represents. Another view, which is especially relevant to the phar~ , is the enormous potential market for psychogeriatric drugs which some pharmaceutical companies see as justifying a substantial research investmerit. It may be, however, that the enthusiasm to identify and characterize a single n e u w c h e m i ~ lesion which might lend itself to replacement therapy, in the same way as L-dopa is used to treat Parkinson's disease, has coloured the neurochemical view ~z Alzheimer's disease.

Biological Aspects of Aizheimer's Disease is typical in this respect; the neurochemical emphasis is primarily on the loss of the cholinergic projection to the cortex with little mention of the neuronal deficits associated with other transmitters (including noradrenaline, serotonin and somatostatin) which have been found in the disease. This tray represent a North American bias; considering the European contributions to Alzheimer's disease research, it is surprising that only three Brihsh groups, and no mainland Europeans, are represented. While not being comprehensive in authorship or interpretation, the approach of this collection of about 40 research reports is fairly comprehensive. The 'human dimension' of Aizheimer's disease is discussed with reference to particular case histories; genetic factors and behaviour are also considered.. But the majority of the repor~ are c~ncerned with the neurophysiology of the disease with an emphast~ on biochemcal changes, although there are also some interesting and important new nemJxoanatomical studies included. The book ends with two chapters and a discussion centring on the disappointing effect; of

various therapeutic approaches to increasing cholinergic function. This should not be thought of as a review volume; little attempt at an integration of these research reports is apparent. The relatively short publication delay fol!owing an Autumn 1982 conference is a positive point, although a year can be a long time in such a fast moving field. The editor has included the comments and questions following each paper; these are set down verbatim and as such provide an interesting view of the difficulties scientists experience in verbal communication. However, a summary of these discussions by the editor ~ould have been far more useful to the reader. In s~n~nary then, this volume attempts to present a state-of-the-art of (American) Alzheimer's disease research. It is useful as such, but the TIPS reader may find that other pubfications provide more integrated and more readable assessments of current research in this topical field. GAVIN P. REYNOLDS

The author is at the MR(: Neurochem/ca/Pharmacology Unit, Brain Tissue i~q¢, Hills Road,

Cambridge, CB2 2QH, UK.