Proc. Nati. Acad. Sci. USA Vol. 82, pp. 920-924, February 1985 Neurobiology Giant axonal neuropathy: Acceleration of transport in optic (axonal morphometry/fluorography/2,5-hexanedione/intermediate filaments/i ) SALVATORE MONACO, LUCILA AUTILIO-GAMBETTI, DAVID ZABEL, AND PIERLUIGI GAMBETTI* Division of Neuropathology, Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106 Communicated by George B. Koelle, September 4, 1984

ABSTRACT Giant axonal neuropathies are a group of ac- ane are widely used as solvents and have caused outbreaks quired and inherited human diseases morphologically charac- of polyneuropathies among industrial workers (11, 12) and in terized by accumulation of (NF) in enlarge- individuals intentionally inhaling glue vapors (13). The distal ments of preterminal regions of central and peripheral axons. giant axonopathy produced by these compounds in experi- Slow axonal transport was studied in the optic systems of rats mental and clinical conditions is indistinguishable from that treated with 2,5-hexanedione, a toxic compound that produces of the inherited forms (4). an experimental model of giant axonal neuropathy. The trans- The pathogenetic mechanism of distal axonal accumula- port rate of NF and of two other polypeptides ofMr 64,000 and tion of NF is unknown. Two main hypotheses have been put 62,000 were selectively increased. Other components of the forward, both assuming that NF are transported at decreas- slow axonal transport were not affected. Acceleration of la- ing rates and are eventually blocked (14, 15). However, slow beled NF was also observed when 2,5-hexanedione was given axonal transport has not been studied. after [35S~methionine administration. Morphometric analysis We analyzed the labeled polypeptides migrating along the revealed that the number of NF and the size were de- optic system with the slow component of the axonal trans- creased in regions of optic axons proximal to the enlargements. port in rats treated with 2,5-HxD and in controls. The find- It is suggested that acceleration of NF transport leads to a lon- ings were correlated with morphometric analyses of axons gitudinal redistribution of NF: NF decrease proximally and in- and axonal . This study has been reported in crease distally, forming NF-containing enlargements. Evi- part (16, 17). dence was obtained that polypeptides of Mr 64,000 and 62,000 are cytoskeletal components related to intermediate filaments, MATERIALS AND METHODS normally migrating with the component a of the slow axonal Induction of the Neuropathy. Male Sprague-Dawley rats transport. The 2,5-hexanedione axon may provide insight into (12 weeks old) were continuously given 0.5% 2,5-HxD in the pathogenesis of inherited and acquired giant axonal neuro- drinking water for up to 14 weeks. Age-matched controls re- pathies and offers a model to investigate the relationship be- ceived regular drinking water or similar amounts of the non- tween number of NF and axonal size in central axons. neurotoxic compound 2,4-hexanedione (18). Morphological Studies. Experimental and control rats were Axonal transport involves the migration of highly ordered sacrificed at weekly intervals from the 3rd to 14th week of complexes of proteins at five distinct rates (1). In mammali- treatment. Rats were perfused through the ascending aorta an optic axons two groups of proteins, slow component a with 0.2 M sodium cacodylate buffer, pH 7.4, followed by (SCa) and slow component b (SCb), have transport rates of 5% (wt/vol) glutaraldehyde in 0.1 M sodium cacodylate buff- 0.3 mm/day and 2-3 mm/day, respectively (2). SCa com- er, pH 7.4, with 0.03% CaCl2, at 37°C. Samples were taken prises neurofilaments (NF), tubulin, and -associ- at 5-mm intervals from the sciatic, sural, tibial, and plantar ated proteins, whereas SCb carries over 200 polypeptides, . The primary optic pathway was sampled at 2-mm including , calmodulin, neuronal-specific enolase, and intervals. Tissues were postfixed with 2% osmium tetroxide, creatine kinase (3). dehydrated in graded acetone, and embedded in Spurr resin. The use of experimental models has added new insight to Morphometric analysis of axons and quantitation of microtu- the role that disturbances of the axonal transport play in the bules (MT) and NF were performed in three rats after 11 pathophysiology of human diseases of the central and pe- weeks of intoxication and in three controls. From each rat, ripheral nervous systems. 12 random micrographs of entire cross-sections of one optic Giant axonal neuropathies are a group of diseases com- , 5 mm from the eyeball, were taken at x2700 and prising toxic as well as inherited conditions (4, 5). The patho- printed at x 10,000 final magnification. The area of each logical hallmark of these diseases is the presence of masses axon was determined by using a computer-assisted digitizer. of NF producing focal enlargements in the preterminal re- Axons with the major diameter twice the size of the minor gions of axons. In advanced stages of the neuropathy, the one were discarded. Totals of 6679 and 10,182 axons were enlargements extend proximally and the distal part of the in- measured in control and experimental animals, respectively. volved axons eventually undergoes degeneration in several To determine number of MT and NF per axon, optic nerves central and peripheral axonal pathways. However, in experi- were photographed randomly at x 10,000 and enlarged up to mental models no degeneration has been observed in the pri- x30,000. NF and MT were counted in the entire cross-sec- mary optic pathway despite the presence of numerous en- tion of axons with areas ranging from 0.15 to 0.75 ,Um2; 171 larged axons (6-8). axons from control and 226 from experimental animals were used. Student's t test and two-way analysis of variance were Methyl n-butyl ketone, n-hexane, and their metabolite 2,5- used hexanedione (2,5-HxD) (9, 10) are toxic agents that cause for the statistical analysis of axonal area and number of distal giant axonopathies. Methyl n-butyl ketone and n-hex- MT and NF per axon, respectively. Abbreviations: SCb, slow component b; SCa, slow component a; The publication costs of this article were defrayed in part by page charge NF, neurofilaments; MT, ; IF, intermediate filaments; payment. This article must therefore be hereby marked "advertisement" 2,5-HxD, 2,5-hexanedione. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 920 Downloaded by guest on September 23, 2021 Neurobiology: Monaco et aL Proc. NatL Acad. Sci. USA 82 (1985) 921 Axonal Transport. Fifteen experimental and nine control polypeptides were identified as the NF subunits by their lo- rats were injected intraocularly with 500 gCi (1 Ci = 37 GBq) cations in fluorograms of two-dimensional gel electrophore- of L-[35S]methionine (New England Nuclear) from the 4th to sis (Fig. 2). As assessed from quantitation of radioactivity in the 9th week of treatment and sacrificed 25 or 50 days after the Mr 145,000 NF subunit, the distribution of labeled NF in labeling. In three experimental and three control rats, 2,5-HxD-treated animals (Fig. 3A) was significantly different [35S]methionine was injected 10 days before administration from that of the controls and appeared to be bimodal, sug- of 1% 2,5-HxD in drinking water and sacrificed 25 days after gesting that a fraction of the transported NF moved coher- labeling. Optic nerves and tracts were cut in consecutive 3- ently with SCa, as in controls, while another fraction moved mm segments; the chiasm and superior colliculus were used at a rate approaching that of SCb. No significant difference uncut. Each segment was dissolved in 2% NaDodSO4 in 50 was found in the distributions of radioactivity associated mM Tris, pH 6.8; total radioactivity was determined in an with a Mr 30,000 polypeptide representative of SCb (Fig. aliquot of each sample and the remainder was used for one- 3B). and two-dimensional gel electrophoresis, as previously de- Fifty days after labeling, NF polypeptides were present scribed (19); all slab gels were 10% acrylamide, and 6% Am- only at the level of the last segment of the optic tract and of pholines (LKB) was used in the isoelectric focusing gels. the superior colliculus in experimental animals. In controls Fluorography was carried out as described by Bonner and these polypeptides peaked at the 5th segment, 15 mm from Laskey (20). the eyeball. At both 25 and 50 days after labeling, NF poly- Quantitation of Transported Polypeptides. Radioactivity peptides were more prominent in the most distal optic tract associated with a given polypeptide was assessed either by segment than in the superior colliculus, suggesting that at the determining the amount of radioactivity present in the corre- stages of intoxication we studied impairment of transport sponding gel band or by scanning the fluorogram. Labeled and accumulation of NF occurred predominantly in this re- polypeptides in the gel were located by superimposing the gion of the visual system. corresponding fluorogram; bands were excised, incubated Fluorograms from animals whose intoxication began 10 overnight in NCS solubilizer (Amersham)/0.1 M acetic acid days after intraocular labeling, when radioactive slow trans- (9:1, vol/vol) at 60'C and radioactivity was measured by liq- ported proteins had already migrated into the axons, also uid scintillation counting. The areas of the peaks in the fluor- showed a faster moving fraction of NF; these results indicate ogram scans were integrated with a computer-assisted digi- that 2,5-HxD acts on NF already undergoing transport. tizer, using bands corresponding to the Mr 145,000 NF sub- Characterization of the Mr 64,000 and 62,000 Polypeptides. unit and to a Mr 30,000 polypeptide migrating with SCb. In two-dimensional gel electrophoresis, both the Mr 64,000 Student's t test was used for statistical analysis. and the Mr 62,000 polypeptide were seen as sets of two or Immunoblotting. Triton X-100-insoluble cytoskeleton frac- three isoelectric variants. These polypeptides were recov- tions from rat optic tracts, obtained as previously described ered entirely in cytoskeleton fractions prepared from rat op- (21), were separated by two-dimensional gel electrophoresis tic nerves or spinal cord (Fig. 4A). Since these fractions are and transferred to nitrocellulose (22). The blots were incu- known to consist mostly of IF, the possibility that these bated with a monoclonal antibody to intermediate filaments polypeptides belong to this class of proteins was investigat- (IF) (ref. 23; American Type Culture Collection) and the re- ed. Fig. 4B shows that both these polypeptides react with a action was visualized by the peroxidase-antiperoxidase monoclonal antibody that recognizes an epitope present in method (24). all IF subunits (23). The molecular weights of these polypep- MT Preparation. Rat optic nerves and tracts, 35 days after tides are in the range of those of r factors; therefore, we intraocular labeling, were homogenized with 1 vol of assem- studied their distribution during purification of MT by cycles bly buffer (100 mM 4-morpholineethanesulfonic acid/1 mM of temperature-dependent assembly and disassembly; both EGTA/1 mM GTP/5 mM MgCl2, pH 6.4) and centrifuged at these polypeptides were present only in the initial 4°C pellet, 100,000 x g for 60 min. The supernatant was mixed with a along with NF, and were not detectable in the supernatant twice-cycled MT fraction from rat brain and MT were as- containing the unpolymerized tubulin (data not shown). sembled as described by Shelanski et al. (25).

200 .. P 2!0. _-200 RESULTS 145 145- Clinical and Morphologic Changes. After 4-5 weeks of 68 ~~~~~68 treatment, experimental rats showed progressive signs of pe- II _ I ripheral neuropathy. Histologically, there were focal axonal enlargements in the distal region of the sciatic system, fol- lowed by Wallerian degeneration at later stages, as previous- *1 : -s q -t * ly reported (26). In the optic system, enlarged axons were II first detected in the stratum opticum of the superior collicu- ®.~~~~~~~~~~~~~.6 lus at 5 weeks of intoxication. Later, the enlargements in- creased in number and extended proximally. At 14 weeks, 1 2 34 5 67 SC 123 4586 7 SC the longest period studied, enlargements were present in the FIG. 1. One-dimensional gel electrophoresis fluorograms of con- 2- to 3-mm most distal segment of the optic tracts. Even at secutive segments of the primary optic pathway at 11 weeks of treat- this stage of intoxication, axonal degeneration was not ob- ment, 25 days after intraocular[CSimethionine.injection of Each served. segment was 3 mm long, except for the chiasm (4th segment), which Slow Axonal Transport. Fluorograms of 2,5-HxD-treated was used in its entire 2-mm length. Segment numbers from sclera to animals sacrificed 25 days after labeling showed that poly- superior colliculus (SC) are indicated in the bottom. NF 200, NF peptides of Mr 68,000, 145,000, and 200,000 and two poly- 145, NF 68, tubulin (T), and actin (A) are indicated. In the 2,5-HxD- peptides of approximate Mr treated rat (A), labeled NF polypeptides are seen up to the last seg- 64,000 and 62,000 were trans- ment of the optic tract. Two other polypeptides of M, 64,000 and ported faster and were spread over a longer length of the 62,000 (arrows) show a distribution similar to that of NF. In the optic system than in controls (Fig. 1); they traveled about 9 control (B), the NF and the Mr 64,000 and 62,000 polypeptides are mm more than in controls and partially overlapped with seen only up to the chiasm (4th segment). Both fluorograms were polypeptides of SCb. The Mr 68,000, 145,000, and 200,000 exposed for 10 days. Downloaded by guest on September 23, 2021 922 Neurobiology: Monaco et al. Proc. NatL Acad Sci. USA 82 (1985)

6.7 PH 4.8 6.7 pH 4.8 -200

_b 145

-M. 4 I ,1 681 I *W I I I *T _o~ fNET * _ .:1 9- WIA are qNSE ...... -'A . A * :''_ -.~p14 .. A.

0 _c 68

FIG. 2. Two-dimensional gel electrophoresis fluorograms of optic system at 9 weeks of intoxication, 25 days after labeling. (A-E) Segments 6 and 7 (optic tract); (F) segment 2 (optic nerve). (A) 2,5-HxD-treated rat; (B) control. Note the presence in A of the Mr 68,000 NF subunit (68) and two polypeptides of Mr 64,000 and 62,000 (arrowheads) seen as sets of two or three isoelectric variants, which are not present in the control (B). Other polypeptides, including actin (A), neuronal-specific enolase (NSE), and traces of tubulin (T) are present in both 2,5-HxD-treated and control rats. (C and D) Enlarged areas indicated in A and B, respectively. (E) Enlarged part of the fluorogram shown in A after longer exposure, demonstrating the presence also of the M, 145,000 and 200,000 NF subunits. The M, 200,000 NF subunit that is lightly labeled by [35S]methio- nine and focuses poorly in two-dimensional gel electrophoresis is barely visible. (F) Fluorogram from control optic nerve segment 2, showing that the polypeptides of Mr 64,000 and 62,000 (arrowheads) are normal components of SCa. (A-D) Fourteen-day exposure.

Morphometric Analysis. To determine whether the faster number of MT was not significantly different (Fig. 5). Axons rate of transport resulted in a decreased number of NF in in this region of the optic nerve were markedly reduced in regions of the axon proximal to the axonal enlargements, a size in experimental animals (Table 1; Fig. 5). morphometric analysis was carried out (Table 1). After 11 weeks of treatment the number of NF per axon in experi- DISCUSSION mental animals was almost half that of controls, whereas the The present study shows that administration of 2,5-HxD in- creases the rate of transport of NF and of two other polypep- 501 tides of Mr 64,000 and 62,000 in rat optic axons. It also dem- onstrates that 2,5-HxD produces a longitudinal redistribu- 40 \\ tion of NF along the optic axons; NF are decreased in 301 number proximally and accumulate distally. Although an in- creased rate of the entire slow transport has been reported in 20 i injured axons (27), acceleration in transport rate of a selec- tive group of polypeptides and a decrease in NF number in proximal regions of central axons have not been previously observed. , 4 7 10 1215 18 10 Formation of Distal Axonal Enlargements. Two main hypotheses have been advanced to explain the formation of

s 50 40 30 .**. --I .8 so 68. *4hb. 20 + a3 10 ae

4 7 10 12 15 18 21 Distance from eye, mm

q FIG. 3. (A) Distribution of the radioactivity associated with the .68 Mr 145,000 NF subunit in 2,5-HxD-treated rats (-) and controls (---). In experimental animals the curve appears to be bimodal, with most of the radioactivity present in the optic tract (12-18 mm from the eyeball). In controls, the distribution is unimodal, with most of the radioactivity in the two segments of the optic nerve (1-7 FIG. 4. (A) Region of a two-dimensional gel electrophoresis of a mm from the eyeball). (B) Much less difference was found between cytoskeleton fraction from the optic tract showing the presence of experimental (-) and control (---) animals in the radioactivity as- Mr 64,000 and 62,000 polypeptides (arrows). (B) Nitrocellulose blot sociated with a Mr 30,000 polypeptide, migrating with SCb. Values of the same preparation immunostained with an antibody that recog- are means of three experiments, 25 days after intraocular labeling. nizes an epitope common to all IF (25). Both the Mr 64,000 and the o, Significantly different from control (P < 0.05-0.001). Mr 62,000 polypeptides (arrows) react strongly with this antibody. Downloaded by guest on September 23, 2021 Neurobiology: Monaco et aL Proc. Natl. Acad. Sci. USA 82 (1985) 923

Table 1. Morphometric analysis of axons Treatment NF per axon MT per axon Axon area, AMm2 2,5-HxD 14.0 ± 1.0* 24.6 ± 0.7 0.48 ± 0.006t Control 35.2 ± 1.1 25.2 ± 0.7 0.82 ± 0.06 Results are mean ± SD. *Significantly different from control, P < 0.001. tSignificantly different from control, P < 0.002. NF-containing distal axonal enlargements. Spencer et al. (14) have suggested that six-carbon compounds and their de- rivatives impair enzymes of intermediary metabolism, caus- ing a progressive failure of the energy supply needed for NF transport; hence, NF would stop and accumulate focally. According to Graham et al. (28) 2,5-HxD reacts directly with 6-amino groups of lysine residues of NF proteins to form aromatic pyrrole derivatives; autooxidation ofthese pyrroles would lead to cross-link formation; the high stability and low turnover rate of axonal NF would make them especially vul- nerable to this toxic effect; aggregates of NF, progressively cross-linked during their migration along the axon, would be transported at decreasing rates and eventually blocked at distal nodes of Ranvier. In view of the present findings, all previous hypotheses that toxic giant axonal neuropathies re- sult from a progressive slowdown of NF transport need to be revised. We propose that acceleration of NF transport in the presence of a normal rate of NF synthesis and inser- tion into the transport system would lead to the formation of the NF-containing distal axonal enlargements and to a longi- tudinal redistribution of NF. The distal regions of the axon would receive an increased number of NF at the expense of FIG. 5. Cross-sections of optic nerves, 5 mm from the sclera. (A the proximal regions, resulting in the formation of distal axo- and B) Rat treated with 2,5-HxD for 11 weeks; (C and D) control. (A nal enlargements and a decrease of NF proximally. The pre- and C, x6000; B and D, x30,000.) Axons of the 2,5-HxD-treated rat cise mechanism of formation of the enlargements remains to (A) are visibly reduced in size as compared to control (C). In the 2,5- be established. It has been shown that they initially form at HxD axons (B), NF but not MT are reduced in number as compared the proximal side of the nodes of Ranvier (29). It is likely that to control (D). because of the faster transport rate the increased bulk of NF cannot course through nodes of Ranvier in the distal axonal are not yet morphologically identifiable, NF subunits are regions. Other mechanisms, however, such as saturation or transported at rates 8 times faster than those of adult animals impairment of the catabolic pathway, cannot be ruled out. (33). A marked decrease in the rate of transport coincides The Mr 64,000 and 62,000 Polypeptides. The nature and with the morphological appearance (33) and increase in num- role of these polypeptides, which, along with NF, are trans- ber of axonal NF (34, 35), indicating that the transport rate is ported at an increased rate, is unclear. It is unlikely that related to the molecular form. 2,5-HxD might interfere with these polypeptides are T factors (30, 31), a class of microtu- NF assembly, resulting in a faster transport of unassembled bule-associated proteins transported with SCa (32), since (i) NF subunits and a decrease of morphologically identifiable they are not recovered with tubulin after temperature-depen- NF in the proximal regions of the optic axon as we have dent assembly and disassembly; (ii) after Triton X-100 ex- observed. In this case, one has to postulate that assembly traction they are recovered in cytoskeleton fractions along takes place in the distal part of the axon, since the axonal with other IF; (iii) they immunoreact with a monoclonal anti- enlargements are filled with morphologically identifiable body that recognizes an epitope common to all IF (23). Our NF. Since 2,5-HxD accelerates transport also of those NF data indicate that these polypeptides are related to IF and that are already in the axon at the time of intoxication, it is are normal components of SCa in the optic system. The Mr unlikely that it disassembles NF proximally and allows NF 64,000 polypeptide, the more abundant of the two, seems to assembly distally. be present only in the central , since it is not A direct disruption of the MT-NF axonal meshwork is detectable in cytoskeletal preparations or among the SCa more likely and could also explain the finding that along with components of peripheral nerve (data not shown). NF two other cytoskeletal components of SCa, the Mr The finding that during 2,5-HxD intoxication the polypep- 64,000 and 62,000 polypeptides, are transported at a faster tides of Mr 64,000 and 62,000 migrate at a faster rate along rate, whereas transport of tubulin is not affected. with NF has three possible explanations: (i) they are normal- It has recently been shown that methylation of 2,5-HxD ly associated with NF; (ii) they are components of the NF- affects the location of the axonal enlargements (36, 37). MT meshwork, which is disrupted by 2,5-HxD in such a way Thus, the dimethyl derivative, 3,4-dimethyl-2,5-hexane- that they remain attached to the NF; (iii) 2,5-HxD acts inde- dione, is 20 to 30 times more neurotoxic than 2,5-HxD and pendently on NF and on them, altering their transport rate in produces proximal axonal enlargements (36, 37); the mono- similar ways. methyl derivative, 3-methyl-2,5-hexandione, produces en- Mechanism of Action of 2,5-HxD. The mechanism of 2,5- largements at intermediate locations along the axon (38). HxD action on NF transport remains to be determined. At These findings suggest that all these compounds share a least two hypotheses should be considered: interference common mechanism. with NF assembly and a direct disruption of the NF-MT Other Models of Abnormal Slow Transport with Alteration meshwork. of Axonal Cytoskeleton. To date two models of impairment of During early stages of postnatal development, when NF NF transport and alteration of axonal cytoskeleton have Downloaded by guest on September 23, 2021 924 Neurobiology: Monaco et al. Proc. NatL Acad Sci. USA 82 (1985) been reported. Administration of /3,4'-iminodipropionitrile 14. Spencer, P. S., Sabri, M. I., Schaumburg, H. H. & Moore, results in severe slowdown or block of NF transport (39), C. L. (1979) Ann. Neurol. 5, 501-507. with NF accumulation in the proximal region ofthe axon (40) 15. Graham, D. G. (1980) Chem. Biol. Interact. 32, 339-341. and segregation of NF and MT distal to this region (41). Af- 16. Monaco, S., Crane, R., Autilio-Gambetti, L. & Gambetti, P. (1983) J. Neuropathol. Exp. Neurol. 42, 330 (abstr.). ter local administration of aluminum, transport of NF is im- 17. Monaco, S., Autilio-Gambetti, L., Crane, R. & Gambetti, P. paired and NF accumulate in the proximal region of the axon (1983) Soc. Neurosci. Abstr. 9, 1191. and in the neuronal cell bodies. Transport of NF, however, 18. Spencer, P. S., Bischoff, M. & Schaumburg, H. H. (1978) is maintained in the distal region of the axon, resulting in Toxicol. Appl. Pharmacol. 44, 17-28. lack of NF in axonal segments immediately distal to the 19. Autilio-Gambetti, L., Velasco, M. E., Sipple, J. & Gambetti, block (42). 2,5-HxD, which causes an increased rate of NF P. (1981) J. Neurochem. 37, 1260-1265. transport that leaqs: to a longitudinal rearrangement of NF, 20. Bonner, W. M. & Laskey, R. A. (1974) Eur. J. Biochem. 461, provides a different model of alteration of NF transport and 83-88. axonal cytoskeletbn. 21. Autilio-Gambetti, L., Sipple, J., Sudilovsky, 0. & Gambetti, P. (1982) J. Neurochem. 38, 774-780. In the peripheral axons it has been shown that cross-sec- 22. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. tional axonal area is related to the number of NF (43, 44). Acad. Sci. USA 76, 4350-4354. The "2,5-HxD axon" shows that also in central axons, num- 23. Pruss, R. M., Mirsky, R., Raff, M. C., Thorpe, R., Dowding, ber of NF determines axonal size, and this system provides A. J. & Anderton, B. H. (1981) Cell 27, 419-428. an attractive model to investigate this relationship in the cen- 24. Sternberger, L. A. (1979) in Immunocytochemistry (Wiley, tral nervous system. New York), 2nd Ed., p. 122-127. Experimental and Inherited Distal Giant Axonal Neuro- 25. Shelanski, M. L., Gaskin, F. & Cantor, C. R. (1973) Proc. pathies. Currently, three forms of inherited giant axonal neu- Natl. Acad. Sci. USA 70, 765-768. ropathy are known: autosomal recessive (45) and autosomal 26. Spencer, P. S. & Schaumburg, H. H. (1977) J. Neuropathol. Exp. Neurol. 36, 276-299. dominant (46) with onset during infancy or childhood and a 27. Cancalon, P. (1982) J. Cell Biol. 95, 989-992. recently reported congenital form (47). In the recessive 28. Graham, D. G., Anthony, D. C., Boekelheide, K., Masch- form, IF have been shown to be disorganized not only in mann, N. A., Richards, R. G., Wolfram, J. W. & Shaw, B. R. axons but also in fibroblasts (48), astrocytes, and endothelial (1982) Toxicol. Appl. Pharmacol. 64, 415-422. and Schwann cells (49). Patients with giant axonal neuropa- 29. Jones, H. B. & Cavanagh, J. B. (1983) J. Neurocytol. 64, 415- thy also have kinky hair (45). It is therefore likely that inher- 422. ited giant axonal neuropathies are diseases of all IF. The 30. Weingarten, M. D., Lockwood, A. H., Hwo, S. Y. & morphologic similarity of axonal changes between inherited Kirschner, M. W. (1975) Proc. Natl. Acad. Sci. USA 72, 1858- and 2,5-HxD-induced giant axonal neuropathies is striking. 1862. 31. Penningroth, S. M., Cleveland, D. W. & Kirschner, M. W. Besides NF-containing distal axonal enlargements, these (1976) in Cell Motility, eds. Goldman, R. D., Pollard, T. D. & diseases also show a reduction in number of NF in the seg- Rosenbaum, J. L. (Cold Spring Harbor Laboratory, Cold ments proximal to the enlargements (45). The presence of a Spring Harbor, NY), Vol. 3, pp. 1233-1258, similar longitudinal redistribution of NF strongly suggests 32. Tytell, M., Brady, S. T. & Lasek, R. J. (1984) Proc. Natl. that NF transport is accelerated also in the inherited giant Acad. Sci. USA 81, 1570-1574. axonal neuropathy. Moreover, disorganization of fibroblast 33. Levine, J., Simon, C. & Willard, M. (1982) in Axoplasmic IF has been observed in 2,5-HxD intoxication, suggesting Transport, ed. Weiss, D. G. (Springer, Berlin), pp. 275-278. that the effect of this compound involves other types of IF 34. Komiya, Y. (1980) Brain Res. 183, 477-480. also (50). The elucidation of the effect of on 35. Hoffman, P., Lasek, R. J., Griffin, J. W. & Price, D. L. (1983) precise 2,5-HxD J. Neurosci. 3, 1694-1700. IF may therefore provide important insight in the genetic de- 36. Anthony, D. C., Boekelheide, K. & Graham, D. G. (1983) fect responsible for the inherited forms of giant axonal neu- Toxicol. Appl. Pharmacol. 71, 362-371. ropathy. 37. Anthony, D. C., Boekelheide, K., Anderson, C. W. & Gra- ham, D. G. (1983) Toxicol. Appl. Pharmacol. 71, 372-382. This work was supported by National Institutes of Health Grant 38. Monaco, S., Wongmongkolrit, T., Sayre, L., Autilio-Gam- NS 14509. S.M. is supported in par by a grant from The Standard betti, L. & Gambetti, P. (1984) J. Neuropathol. Exp. Neurol. Oil Company of Ohio. 43, 304 (abstr.). 1. Tytell, M., Black, M. M., Garner, J. A. & Lasek, R. J. 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