Giant Axonal Neuropathy

Proc. Nati. Acad. Sci. USA Vol. 82, pp. 920-924, February 1985 Neurobiology Giant axonal neuropathy: Acceleration of neurofilament transport in optic axons (axonal morphometry/fluorography/2,5-hexanedione/intermediate filaments/i proteins) 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 neurofilaments (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 axon 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 cytoskeleton. 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 microtubule-associ- at 5-mm intervals from the sciatic, sural, tibial, and plantar ated proteins, whereas SCb carries over 200 polypeptides, nerves. The primary optic pathway was sampled at 2-mm including actin, 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- nerve, 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, microtubules; 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.

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