P,P -Irninodipropionitrile Impairs Retrograde Axonal Transport

Transcription

1 Joural of Neurocheristry Rave Press, New York teratioal Society for Neurochemistry P,P -riodipropioitrile mpairs Retrograde Axoal Trasport David J. Fik, David Purkiss, ad Maria Mata Neurology Research Laboratory, Uiversity ofmichiga ad V.A. Medical Ceter, A Arbor, Michiga, USA. Abstract: P,fl-miodipropioitrile (DPN), a eurotoxi, causes redistributio of eurofilamets i axos followed by the developmet of proximal axoal swelligs ad, i chroic itoxicatio, a distal decrease i axoal caliber. The latter chages are caused by a selective impairmet i the slow aterograde axoal trasport of eurofilamet proteis. To assess the role of retrograde axoal trasport i DPN toxicity, we used [3H]N-succiimidyl propioate ([ 3H]NSP) to label covaletly edogeous axoal proteis i sciatic erve of the rat ad measured the accumulatio of radioactively labeled proteis i the cell bodies of motor ad sesory euros over time. DPN was ijected itraeurally 6 h or itraperitoeally 1 day before subepieurial - ijectio of [3H]NSP ito the sciatic erve, ad the aimals were killed 1,2, ad 7 days after [3H]NSP ijectio. Neurotoxicity was assessed by electro microscopic observatio of the erves ofsimilarly treated aimals. Both itraeural ad itraperitoeal ijectio of DPN caused a acute reductio i the amout of labeled proteis trasported back to the cell bodies. The early appearace of these chages suggests that alteratios i retrograde trasport may play a role i the productio of the europathic chages. Key Words: Axoal trasport - Neurotoxicity - p,p - miodipropioitrile-peripheral erve. Fik D. J. et al. &@-miodipropioitrile impairs retrograde axoal trasport. J. Neurochem. 47, (1986). P,P-miodipropioitrile (DPN) is a eurotoxi whose effect o erve cells has led to its use as a model for uderstadig eurofilametous euroal pathology. Withi 24 h after subperieurial ijectio (Griffi et al., 1983) or 4 days after itraperitoeal ijectio of DPN (Papasozomeos et al., 198 l), the axoal cytoskeleto redistributes as the eurofilamets are displaced toward the periphery of the axo ad the microtubules, mitochodria, ad smooth edoplasmic reticulum cluster. This is followed over weeks by the developmet of giat axoal swelligs, cosistig of masses of disordered eurofilamets, ear the erve cell bodies (Chou ad Hartma, 1964, 1965). chroically itoxicated aimals, the axo distal to the swellig ultimately decreases i caliber. Griffi et al. ( 1978) have demostrated that both the proximal axoal swelligs ad the distal axoal shrikage result from a specific ad selective impairmet of the slow aterograde trasport of eurofilamet proteis. Aterograde axoal trasport serves to supply the axo with the structural ad fuctioal macromolecules ecessary for cotiued axoal fuctio. The compoet of slow axoal trasport ivolvig eurofilamets is disrupted i DPN itoxicatio, but other compoets of slow trasport ad rapid aterograde trasport have bee reported to be uaffected i the redistributio ad accumulatio of eurofilamets (Griffi et al., 1978). Retrograde axoal trasport from the axo back toward the cell body is geerally cosidered to provide feedback to the cell body about the state of the axo ad to play a critical role i maitaiig ormal euroal fuctioal itegrity. Retrograde trasport cosists of the turaroud trasport of aterogradely trasported macromolecules ad the movemet back to the cell body of iitially extracellular macromolecules, which are take up by piocytosis ito specific itraaxoal orgaelles (Grafstei ad Forma, 1980; Bisby, 198 1). Studies of retrograde axoal trasport i DPN itoxicatio have yielded coflictig results. The accumulatio of itramuscularly ijected tetaus toxi distal to a ligature o the sciatic erve appears to be uaffected by DPN (Griffi et al., 1978). O the other had, the retrograde trasport of itraeurally i- Received December 27, 1985; accepted March 27, Address correspodece ad reprit requests to Dr. D. J. Fik at Neurology Research Laboratory, (1 27), Fuller Road, A Arbor, M 48105, U.S.A. A prelimiary commuicatio of this work was preseted i ab- stract form at the meetig of the America Academy of Neurology i April Abbreviatios used: DRG, dorsal root gaglia; HRP, horseradish peroxidase; DPN, P,fl-imiodipropioitrile; NSP, N-succiimidyl propioate. 032

2 DPN MPARS RETROGRADE AXONAL TRANSPORT 1033 jected horseradish peroxidase (HRP) from the sciatic erve to the motor euros of the lumbosacral spial cord is delayed i both acute ad chroic DPN itoxicatio (Kuzuhara ad Chou, 198 la,b). Aother measure of retrograde axoal trasport ca be defied usig [3H]N-succiimidyl propioate ([3H]NSP). [3H]NSP is a acylatig reaget that covaletly radioactively labels edogeous axoal proteis i vivo (Fik ad Gaier, 1980a,b). After subepieurial ijectio of [3H]NSP ito the sciatic erve, labeled proteis progressively accumulate i the cell bodies of the sesory ad motor euros that project ito the sciatic erve (Fik et al., 1985a,b, mauscript submitted). The amout of radioactively labeled protei reachig the dorsal root gaglia (DRG) ad vetral hor of the spial cord allows a quatitative estimate of the amout ad the rate of trasport of edogeous proteis from the axo back to the cell body i sesory ad motor euros, respectively. To explore further the effect of DPN o retrograde axoal trasport, we have used the [3H]NSP method to determie the amout ad rate of retrograde axoal trasport i rats followig either itraeural or itraperitoeal ijectio of DPN. We have compared the time of occurrece of chages i retrograde trasport to the time of appearace of the ultrastructural chages characteristic of DPN eurotoxicity. The results suggest that a reductio i amout of retrograde trasport is a early feature of DPN itoxicatio. MATERALS AND METHODS Determiatio of retrograde trasport Male Sprague Dawley rats (body weight g) were used i all experimets. The aimals were aesthetized with chloral hydrate, ad the sciatic erve was exposed i the gluteal regio. [3H]NSP (50 Ci/mmol i bezee, New Eglad Nuclear) was evaporated to dryess uder vacuum o ice ad resuspeded i 3 pl of physiologic salie, ph 7.4. Oe microliter was ijected subepieurially over a 5-mi period usig a 3 1-gauge eedle attached to a Hamilto syrige i a Harvard ifusio pump, ad the woud was closed with woud clips. At selected times after ijectio (1, 2, ad 7 days), the aimals were agai aesthetized with chloral hydrate ad were killed by perfusio through the heart with 0.1 Mphosphate-buffered salie, ph 7.4. The ipsilateral sciatic erve, bilateral L4, L5, ad L6 DRG, ad the spial cord were rapidly removed ad froze o dry ice. The erve was miced ad placed i 5 mm Tris-HC1 salie, ph 7.5. The DRG from each side were pooled i 50 pl of the same buffer. Sectios (200 pm) of the spial cord through the regio of the lumbar elargemet were cut i a cryostat at -8"C, ad the ipsilateral ad cotralateral vetral hor regios were removed with a 750-pm diameter puch. The samples of erve, DRG, ad vetral hor were soicated i a Kotes microultrasoic cell disrupter for 1 mi. The protei i the samples was precipitated twice with 10% trichloroacetic acid ad washed twice with acetoe ad twice with ether, ad the fial pellets were suspeded i 50 pl of sample buffer cotaiig 1% sodium dodecyl sulfate ad 1% P-mercaptoethaol. After beig heated to 95 C for 3 mi, duplicate ali- quots were take for determiatio of radioactivity i a LKB beta couter. The amout of retrograde axoal trasport to the motor ad sesory euroal cell bodies was calculated by determiig the amout of trichloroacetic acid-precipitable radioactivity (ipsilateral to the ijectio mius cotralateral) i the DRG ad vetral hor of spial cord, respectively, at the specified times after [3H]NSP ijectio. These results were expressed both as total dpm ad as a ratio of cell body couts to the amout of radioactivity remaiig i the erve (see Statistical aalysis below). There were five or six aimals i each experimetal ad cotrol group. The umber of couts i the cotralateral DRG raged from 30 to 540 i the cotrols, from 10 to 740 i the itraperitoeal DPN aimals, ad from 0 to 640 i the itraeural DPN aimals. The umber of couts i the cotralateral spial cord puch samples raged from 0 to 230 i the cotrols, from 0 to 210 i the itraperitoeal DPN aimals, ad from 0 to 90 i the itraeural DPN aimals. DPN ijectio The effects of both itraperitoeal ad itraeural ijectio of DPN were assessed. DPN was diluted 1:l with phosphate-buffered salie, ph 7.3. For itraeural ijectio, 5 p1 of DPN was ijected subepieurially ito the sciatic erve usig a 3 1-gauge eedle attached to a Hamilto syrige 6 h before ijectio with [3H]NSP. Cotrol aimals for the itraeural experimets were ijected i a similar maer with 5 p1 of salie 6 h before [3H]NSP ijectio. the itraperitoeal group, aimals were ijected with DPN, 2 g/kg as a sigle itraperitoeal dose, 24 h before ijectio of [3H]NSP. Electro microscopy At specific times after itraperitoeal (1 ad 8 days) or itraeural ( 1 day) ijectio of DPN, the dorsal ad vetral roots ad the sciatic erve were examied ultrastructurally. Uder chloral hydrate aesthesia, the aimals were perfused through the heart with 0.1 M phosphate buffer followed by 3% glutaraldehyde, 0.5% paraformaldehyde, ad 0.1 M phosphate buffer, ph 7.4, for - 1 h. The sciatic erve, roots, ad DRG were dissected out ad kept i fixative for a additioal 2 h at 4"C, postfixed i 2% Os04 i the same buffer, dehydrated, ad embedded i a Polybed-Araldite mixture. Thi sectios were staied with urayl acetate ad examied i a JEOL 100s electro microscope. Statistical aalysis Several aalyses were used to compare retrograde trasport i cotrol ad DPN-treated aimals. Because of the variatio i the amout of ijected radioactivity, aalyses were performed usig the ratio of couts i the cell body to couts remaiig i the erve. These ratios were o the order of Usig aalysis of covariace, we assessed the relatioship betwee the couts i the cell bodies ad those remaiig i the erve i idividual cotrol ad experimetal aimals ad foud there was o statistically sigificat liear relatioship betwee those couts. We performed aalyses of variace o the couts ad o the ratios usig Boferroi's procedure for multiple comparisos to cotrol values for the multiple comparisos beig made. Results from both aalyses led to the same coclusios, but we foud that the aalyses ofcouts accouted for a greater proportio of the variability tha did the aalyses o the ratios. Therefore, we have expressed the data as trichloro- J. Neurochem., Vol. 47, No. 4, 1986

3 034 D. J. FNK ET AL. FG. 1. Sciatic erve 24 h after itraeural ijectio of 5 p of DPN. A May fibers showed redistributio of the cytoskeleto with clusterig of microtubules cetrally ad the eurofilamets peripherally. 6: Some fibers, however, showed evidece of vesicular accumulatio ad retractio of the axolemma from the myeli sheath. A, X10,900; 6, X acetic acid-precipitable couts (ipsilateral mius cotralateral) rather tha as ratios i the figures preseted here. RESULTS The ormal accumulatio of radioactively labeled proteis i the DRG ad the spial cord at 1,2, ad 7 days is show i the cotrol curves of Figs. 5 ad 6, respectively. the DRG, the icrease i couts from 1 to 2 days is statistically sigificat (p < 0.005), as is the icrease from 2 to 7 days (p < 0.005). the spial cord, the accumulated couts are less, ad the apparet icrease from 1 to 2 days is ot statistically sigificat (p = 0.6), whereas the icrease from 2 to 7 days is sigificat (p < 0.001). Beyod 7 days, DRG couts drop sigificatly (p < by 14 days), whereas the spial cord couts plateau at 14 ad 21 days ad do ot drop sigificatly util 28 days after ijectio (Fik et al., 1985b, mauscript submitted). traeural ijectio of DPN causes redistributio of the axoal cytoskeleto, which begis withi hours after the ijectio, is maximal at 6 h, ad the resolves over days (Griffi et al., 1983). our iitial series of experimets, we ijected DPN itraeurally ad 6 h later ijected [3H]NSP subepieurially distal to the site of the DPN ijectio. Aimals were the allowed to survive or 7 days, durig which retrograde axoal trasport occurred. At 1 day after itraeural DPN ijectio, substatial redistributio of eurofilamets, clusterig of tubules ad mitochodria, ad some axoal swellig is apparet (Fig. 1A). some of the fibers, there is also evidece of early stages of degeeratio, maifest by accumulatio of vesicular ad tubulovesicular profiles (Fig. B). traeural ijectio of salie caused a reductio i the amout of radiolabeled material accumulatig at the DRG ad spial cord whe compared with uijected cotrols. This may be due to erve damage from elevated edoeurial pressure caused by the relatively large volume (5 pl) of the ijectio. Noetheless, the icrease ormally foud from 1 to 7 days i labeled proteis at the cell body is still apparet i these salie cotrol aimals. traeural DPN caused a sigificat reductio from the value i salie cotrols i trasport to the DRG after 7 days of trasport (p < ; Fig. 2) ad i trasport to the spial cord after 7 days of trasport (p < 0.005; Fig. 3). The accumulatio i the cell bodies 24 h after [3H]NSP ijectio appeared to be reduced i both DRG ad M s. X a 4- L 3 - E 64 0 N SALNE 0 LPN, FG. 2. Retrograde trasport of labeled protei from the sciatic erve to the cell bodies of DRG euros i aimals ijected with [3H]NSP 6 h after itraeural ijectio of either salie or DPN. jectio of DPN caused a sigificat reductio from the salie cotrol value at 7 days (p i 0.005) but ot at 1 day. Data are mea rt SEM (bars) values. J. Neurochem.. Val. 47, No. 4, 1986

4 DPN MPARS RETROGRADE AXONAL TRANSPORT _ - l a FG. 3. Retrograde trasport of labeled protei from the sciatic erve to the cell bodies of vetral hor euros i aimals ijected with r3h]nsp 6 h after itraeural ijectio of either salie or DPN. jectio of DPN caused a sigificat reductio compared with the salie cotrol value at 7 days (p < 0.02) but ot at 1 day (p = 0.2). Data are mea k SEM (bars) values. spial cord, but those reductios were ot statistically sigificat (Figs. 2 ad 3). These results suggest that, cocurret with toxiiduced cytoskeletal rearragemet, there is disruptio of the late accumulatio of retrogradely trasported radioactive proteis i motor ad sesory euros. To avoid the possible cofoudig variable itroduced by itraeural ijectio ad to determie whether the reductio i retrograde trasport occurs before or after the cytoskeletal rearragemet, we wet o to study aimals after itraperitoeal ijectio of DPN. At 1 day after itraperitoeal ijectio of DPN, the erve, dorsal root, ad vetral root all appear ormal (Fig. 4A-C). By 8 days after itraperitoeal ijectio, there is redistributio of microtubules ad eurofilamets i the erve (Fig. 4D), ad massive swelligs occur i the proximal dorsal ad vetral roots (Fig. 4E ad F), as have bee described above. Despite the fact that the erve ad roots appear ormal at 1 day, retrograde trasport i the sesory euros is reduced to 30% of cotrol values (p < 0.005) at 1 day (Fig. 5). There is o sigificat differece betwee the results of itraeural or itraperitoeal ijectio of DPN with 1 day of trasport or at later times. The differeces i retrograde trasport to the DRG after 2 ad 7 days of trasport are also statistically sigificat (p < 0.005). Trasport i motor euros is affected by itraperitoeal DPN i a maer similar to that of itraeural ijectio. There are o sigificat differeces from cotrol values at 1 ad 2 days, but a sigificat reductio i trasport occurred at 7 days (p < 0.006; Fig. 6). DSCUSSON These studies demostrate that alteratios i retrograde axoal trasport occur early i DPN eurotoxicity. A itraeural ijectio of DPN causes cytoskeletal redistributio withi hours. By 1 day, ot oly is redistributio apparet, but early evidece of degeeratio i some fibers has begu to appear. Griffi et al. (1983) have suggested that the degeerative chages may be a artifact of the itraeural ijectio. that coditio, retrograde trasport to both the motor ad sesory euros is sigificatly reduced at 7 days, ad although the amout of radiolabeled proteis reachig the cell bodies appears to be reduced at 1 day, that reductio is ot statistically sigificat. Are these alteratios i retrograde trasport due to the cytoskeletal disruptio, or are they caused by other eurotoxic effects of DPN? To aswer that questio, we wet o to study aimals after itraperitoeal admiistratio of DPN. At 1 day after itraperitoeal DPN ijectio, there are o apparet chages i cytoskeleto or axoal architecture. Despite the absece of ultrastructural chages, trasport to the sesory euros is reduced both at the early (1 ad 2 days) as well as the late (7 day) itervals, whereas trasport to the motor euros is ot affected at the early itervals but is sigificatly reduced by the late iterval. The effect o the later accumulatio of retrogradely trasported proteis, which accouts for the majority of labeled trasported proteis, is similar i motor ad sesory euros. The reaso why retrograde trasport to the motor euros at the earlier itervals appears to be less affected by DPN is ot clear. The umber of couts that reach the motor euros are a order of magitude less tha those reachig the DRG, ad the SE is much greater relative to the umber of couts, so it is possible that a small but sigificat differece failed to be detected. Alterately, there may be real differeces i the vulerability of rapid retrograde trasport to DPN betwee motor ad sesory euros. Kuzuhara ad Chou (198 lab) foud that, i rats 3 days or 4 weeks after itraperitoeal ijectio of DPN, HRP ijected ito the sciatic erve was delayed i its arrival at the large euros i the spial cord, whereas the labelig of small euros appeared ormal. By 48 h after HRP ijectio, they could detect tubules ad vesicles cotaiig HRP reactio product desely i the axoal swelligs ad also i the euroal somata. Although we were uable to detect a statistically sigificat decrease i trasport to the motor euro perikarya util a loger time for trasport had elapsed, these results are, i geeral, i agreemet with our fidigs that a defect i retrograde trasport to the cell bodies is a characteristic of DPN eurotoxicit y. cotrast, Griffi et al. (1978) were uable to demostrate a defect i retrograde axoal trasport i rats J. Neurochem., Vol. 47, No. 4, 1986

5 1036 D. J. FNK ET AL. FG. 4. Compariso of the ultrastructural appearace of erve, vetral root, ad dorsal root at 1 day (A-C) ad 8 days (D-F) after itraperitoeal ijectio of DPN. At 1 day, the erve appears ormal (A), but by 8 days, clusterig of the microtubules ito discrete groups withi the eurofilamet matrix is apparet (D). Similarly, the proximal vetral root appears ormal 1 day after DPN ijectio (B), but by 8 days, massive swelligs created by the accumulatio of disordered eurofilamets are foud (E). Like the vetral root, the dorsal root appears ormal at 1 day (C) but by 8 days is distorted by the accumulatio of eurofilamets (F). A, X7,ZOO; B, X16,300; C, X 2,200; D, X10,ZOO; E, X16,300; F, X12,ZOO. J. Neitrochem., Vol. 47, No. 4, 1986

6 DPN MPARS RETROGRADE AXONAL TRANSPORT m a 9 x c 8 CONTROL 1 i ; FG. 5. Retrograde trasport of labeled protei from the sciatic erve to the cell bodies of DRG euros i aimals ijected with [3H]NSP 24 h after itraperitoeal ijectio of DPN. The accumulatio of labeled proteis at the cell bodies is reduced sigificatly whe compared with that i uijected aimals at 1 day (p < 0.005), 2 days (p < 0.005), ad 7 days (p < 0.005) after [3H]NSP ijectio. Data are mea * SEM (bars) values. teratios i retrograde trasport may occur i may toxic europathies. Defects i the turaroud trasport of aterogradely trasported radiolabeled proteis have bee demostrated i europathies iduced by hexacarbos, zic pyridiethioe, ad p- bromopheylacetylurea (Sahek ad Medell, 1980, 1981; Jakobse ad Brimijoi, 1981). Acrylamide i doses of 100 mg/kg, isufficiet to cause a cliically apparet europathy, oetheless reduces the retrograde turaroud trasport of labeled proteis i rats (Jakobse ad Sideius, 1983). Also, sigle doses of acrylamide have bee show to reduce the velocity of retrograde trasport of lz5-1abeled tetaus toxi by up to 75% withi hours of admiistratio of the toxi (Miller ad Specer, 1984). The latter two studies have led to speculatio that the alteratios i retrograde trasport may be sigificat i the chai of evets that lead to the europathy. t has log bee suspected, pricipally o the basis of the reactio of euros to axoal ijury, that retrogradely trasported substaces may play a critical role i the cotiued ormal fuctio of the euro (Cragg, 1970). The alteratios i trasport that have bee demostrated i the pathologic states oted above ad the evidece that these chages may be a 10 - after log-term DPN itoxicatio. They foud that, 18 h after itramuscular ijectio of 2S-labeled tetaus toxi, the amout of radioactivity accumulatig distal to a ligature o the sciatic erve was ot sigificatly reduced. There are two major differeces betwee the paradigm they used for measurig axoal trasport ad the oe used i this study. They measured retrograde trasport from the termial to a poit i the axo, whereas we measured trasport from the axo to the cell body. t is possible that the disruptio of retrograde trasport occurs i proximal but ot distal axos, so that trasport from the termial to the axo is uaffected whereas trasport from the axo to the cell body is impaired. A secod possibility-that the retrograde trasport of exogeous macromolecules is affected i a maer differet from the trasport of edogeous axoal proteiscould accout for the differece betwee their results ad ours, but would ot explai the differece betwee their results ad the results of Kuzuhara ad Chou (198 la,b). The results of our study-that a decrease i retrograde axoal trasport is a early feature of DPN itoxicatio-is part of the growig evidece that al- N X a- P - 6- CL 0. u 1 4 z 4- a a FG. 6. Retrograde trasport of labeled protei from the sciatic erve to the cell bodies of vetral hor euros i aimals ijected with [3H]NSP 24 h after itraperitoeal ijectio of DPN. The accumulatio of labeled proteis is reduced sigificatly at 7 days of trasport (p < 0.006). Data are mea? SEM (bars) values. J. Neurochem., Vol. 47, No. 4, 1986

7 1038 D. J, FNK ET AL. very early maifestatio of itoxicatio led support to that otio. Our fidigs that reductios i axoal trasport are a early ad persistet chage after sigle doses of DPN are further evidece that some of those trasported proteis may serve critical sigal fuctios i the biology of the euro. Ackowledgmet: The statistical aalysis was performed by Dr. Judy Bromberg of V.A. HSR&D. We wish to ackowledge the excellet techical assistace i histology ad photography of Julie Staple ad Barbara Rogers ad the secretarial assistace of Caroly White. The graphs were produced by Dr. Ro Craig. This research was supported by V.A. Merit Review grats to Dr. Mata ad to Dr. Fik. REFERENCES Bisby M. A. (198 1) Fuctios of retrograde axoal trasport. Fed. Proc. 41, Chou S. H. ad Hartma H. A. (1964) Axoal lesiosad waltzig sydrome after DPN admiistratio i rats. Acta Neuropathol. (Berl.) 3, Chou S. H. ad Hartma H. A. (1965) Electro microscopy of focal euroaxoal lesios produced by beta,beta -imiodipropioitrile (DPN) i rats. Acta Neuropathol. (Berl.) 4, Clark A. W., Griffi J. W., ad Price D. L. (1980) The axoal pathology i chroic DPN itoxicatio. J. Neuropathol. Exp. Neurol. 39, Crag B. G. (1970) What is the sigal for chromatolysis? Brui Rex 23, Fik D. J. ad Gaier H. (1980~) Retrograde axoal trasport of edogeous proteis i sciatic erve demostrated by covalet labelig i vivo. Sciece 208, Fik D. J. ad Gaier H. (1 980b) Axoal trasport of proteis: a ew view usig i vivo covalet labelig. J. Cell Biol. 85, FikD. J., PurkissD., admatam. (1985u)Rapidretrogradetrasport of proteis i sesory euros. Brui. Res. 345, Fik D. J., Purkiss D., ad Mata M. (19856) Retrograde axoal trasport i motor ad sesory euros of the rat sciatic erve. (Abstr) Soc. Neurosci. Abstr. 11, Grafstei B. ad Forma D. (1980) tracellular trasport i euros. Physiol. Rev. 60, Griffi J. W., Hoffma P. N., Clark A. W., Carroll P. T., ad Price D. L. (1978) Slow axoal trasport of eurofilamet proteis: impairmet by beta,beta -imiodipropioitrile admiistratio. Sciece 202, Griffi J. W., Fahestock K. E., Price D. L., ad Hoffma P. N. ( 1983) Microtubule-eurofilamet segregatio produced by beta,beta -imiodipropioitrile: evidece for the associatio of fast axoal trasport with microtubules. J. Neurosci. 3, Jakobse J. ad Brimijoi S. (1981) Axoal trasport of ezymes ad labeled proteis i experimetal axoopathy iduced by p-bromopheylacetylurea. Brui Rex 229, Jakobse J. ad Sideius P. (1 983) Early ad dose depedet decrease of retrograde axoal trasport i acrylamide itoxicated rats. J. Neurochem. 40, Kuzuhara S. ad Chou S. M. ( a) mpairmet of retrograde axoal trasport of horseradish peroxidase (HPR) i beta,beta - imiodipropioitrile (DPN). Neurology 31, Kuzuhara S. ad Chou S. M. (19816) Retrograde axoal trasport of HRP i DPN iduced axoopathy. J. Neuropathol. Exp. Neurol. 40,356. Miller M. S. ad Specer P. S. (1984) Sigle doses of acrylamide reduce retrograde trasport velocity. J. Neurochem. 43, Papasozomeos S. C., Autilio-Gambetti L., ad Gambetti P. (98 1) Reorgaizatio of axoplasmic orgaelles followig beta,beta - imiodipropioitrile admiistratio. J. Cell Biol. 91, Papasozomeos S. C., Bider L.., Beder P. K., ad Paye M. R. ( 985) Microtubule-associated protei 2 withi axos ofspial motor euros: associatios with microtubules ad eurofilamets i ormal ad beta,beta -imiodipropioitrietreated axos. J. Cell Biol. 100, Sahek Z. ad Medell J. R. (1980) Axoplasmic trasport i zic pyridiethioe europathy: evidece for a abormality i distal turaroud. Brui Res. 86, Sahek Z. ad Medell J. R. (98 ) Acrylamide ad 2,5 hexaedioe europathies: abormal bidirectioal trasport rate i distal axos. Brui Res. 219, J. Neurochem., Vol. 47, Nu. 4, 1986