Neurofilament Gene Expression: a Major Determinant of Axonal Caliber (Cdna/Mrna/Actin/Tubulin) PAUL N

Home , Neurofilament

Proc. Nati. Acad. Sci. USA Vol. 84, pp. 3472-3476, May 1987 Neurobiology Neurofilament gene expression: A major determinant of axonal caliber (cDNA/mRNA/actin/tubulin) PAUL N. HOFFMAN*tt, DON W. CLEVELAND§, JOHN W. GRIFFINt¶, PHILLIP W. LANDES*, NICHOLAS J. COWAN"I, AND DONALD L. PRICE*¶** Departments of *Ophthalmology, tNeurology, §Biological Chemistry, INeuroscience, and **Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; and ItDepartment of Biochemistry, New York University School of Medicine, New York, NY 10016 Communicated by Keith R. Porter, December 18, 1986 (received for review October 23, 1986)

ABSTRACT Within the wide spectrum of axonal diame- theless, it should be noted that the close relationship between ters occurring in mammalian nerve fibers, each class of axonal caliber and NF content present in normal fibers is neurons has a relatively restricted range ofaxonal calibers. The altered in a variety of pathological situations-e.g., NF control of caliber has functional significance because diameter density is markedly increased in giant axonal neuropathy (9), is the principal determinant of conduction velocity in myelin- hexacarbon (10), iminodipropionitrile (IDPN) (11), and alu- ated nerve fibers. Previous observations support the hypothesis minum intoxication (12), and in internodes that have under- that neurofilaments (NF) are major intrinsic determinants of gone segmental demyelination (13). axonal caliber in large myelinated nerve fibers. Following We propose that NF gene expression, at least in part, interruption of axons (axotomy) by crushing or cutting a specifies axonal caliber in large myelinated nerve fibers. peripheral nerve, caliber is reduced in the proximal axonal Because axonal caliber is the principal determinant of con- stumps, which extend from the cell bodies to the site of duction velocity in myelinated fibers (14-16), the control of axotomy. (The distal axonal stumps, which are disconnected caliber has important functional implications. For example, from the cell bodies, degenerate and are replaced by the in the simplest neuronal circuit, the monosynaptic reflex arc, outgrowth of regenerating axonal sprouts arising from the the latency (duration) of the reflex changes when axonal proximal stump.) This reduction in axonal caliber in the caliber is altered (17). Moreover, conduction velocity de- proximal stumps is associated with a selective diminution in the clines in diseases associated with reductions in axonal caliber amount of NF protein undergoing slow axonal transport in (e.g., Charcot-Marie-Tooth disease) (18). Thus, according to these axons, with a decrease in axonal NF content, and with this hypothesis, axonal caliber, a feature of neuronal mor- reduced conduction velocity. The present report demonstrates phology with important physiological consequences, is spec- that changes in axonal caliber after axotomy correlate with a ified by NF gene expression. selective alteration in NF gene expression. Hybridization with In this study we further tested the hypothesis that NF gene specific cDNAs was used to measure levels of mRNA encoding expression specifies axonal caliber in large myelinated nerve the 68-kDa neurofilament protein (NF68), ,3-tubulin, and actin fibers. A simple maneuver, axotomy, was used to induce a in lumbar sensory neurons ofrat at various times after crushing reduction in axonal caliber in nerve fibers. This highly the sciatic nerve. Between 4 and 42 days after axotomy by nerve reproducible model system allows investigation ofthe nature crush, the levels of NF68 mRNA were reduced 2- to 3-fold. At and time course of changes in the axonal cytoskeleton (1, 2). the same times, the levels of tubulin and actin mRNAs were Crushing the sciatic nerve is followed by a reduction in the increased several-fold. These findings support the hypothesis relative amount (but not the velocity) of pulse-labeled NF that the expression of a single set of neuron-specific genes protein undergoing axonal transport in rat lumbar motor (encoding NF) directly determines axonal caliber, a feature of neurons (2). Decreased transport of NF proteins in motor neuronal morphology with important consequences for phys- fibers is associated with a concomitant reduction in axonal iology and behavior. caliber that begins near the cell body (soma) and proceeds anterogradely at the rate of slow axonal transport (1); we The synthesis and axonal transport of neurofilament (NF) have termed this process "somatofugal atrophy." Quantita- proteins are thought to play a major role in the control of tive ultrastructural studies showed that NF density is un- axonal caliber in large myelinated nerve fibers (1, 2). This changed in atrophic axons, but NF numbers are reduced in proportion to the diminution in axonal cross-sectional area concept is based on several observations: in normal nerve (1). fibers NF are the most numerous cytoskeletal elements, NF A central but heretofore unresolved question is whether density remains constant over a wide range of calibers, and this sequence of events reflects reduced NF gene expression, NF number correlates closely with axonal area (1, 3-5). The or, alternatively, changes in the "gating" of newly-synthe- relatively constant density of axonal NF is closely related to sized proteins into the axon. To examine this question, we the presence of interfilament cross-bridges that appear to studied the response of sensory neurons in rat dorsal root determine the spacing between adjacent NF (4, 6-8). The ganglia (DRG) at various times after axotomy. The DRG observation that the 200-kDa NF protein (NF200) is directly contains neurons with a wide range of axonal calibers, associated with these cross-bridges (8) raises the possibility including the la sensory fibers, which are among the largest that cross-bridge formation is an intrinsic property of NF. myelinated axons in the mammalian nervous system (19). Thus, the volume of axoplasm occupied by the three-dimen- Each sensory neuron gives rise to a single axonal stem sional network of interconnected NF correlates closely with process, which bifurcates within the ganglion into a central the number of NF profiles per axonal cross-section. Never- process in the dorsal root and spinal cord and a peripheral

The publication costs of this article were defrayed in part by page charge Abbreviations: NF, neurofilament(s); DRG, dorsal root ganglia; L, payment. This article must therefore be hereby marked "advertisement" lumbar. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 3472 Downloaded by guest on September 29, 2021 Neurobiology: Hoffman et aL Proc. Natl. Acad. Sci. USA 84 (1987) 3473 process in the peripheral nerve (19). Axotomy of the periph- RESULTS eral process leads to reductions in axonal caliber (20) and conduction velocity (21, 22) in both the peripheral and central Axonal Atrophy in Lumbar Sensory Fibers. To determine processes. These reductions in the time course ofaxonal atrophy in lumbar sensory neurons, caliber correlate with con- we performed quantitative morphometric analyses of comitant decreases in the amounts of pulse-labeled NF axotomized and control axons. As shown in Fig. lA, when protein undergoing axonal transport in these processes (23). the percentile of axons (percent of axons with areas equal to To monitor any alterations in the levels of NF gene or less than a given value) is plotted as a function of expression in response to axotomy of peripheral processes, cross-sectional area, the distributions are nearly superimpos- we measured NF mRNA levels by RNA blotting and in situ able in peripheral and central branches of control axbns. In hybridization techniques. The results of these studies were axotomized neurons, reductions in caliber near the DRG correlated with morphometric analyses of affected axons. In occurred earlier in peripheral than in central branches; concert, these approaches showed that decrease in NF reductions were first detected in the peripheral branch at 4 mRNA preceded reductions in axonal caliber. These results days after crush, and in the central branch at 14 days (Fig. 1 support the hypothesis that NFgene expression is an intrinsic B and C). As presented in Table 1, maximal reductions in modulator of axonal NF content and thus of axonal caliber. axonal areas (to 50% ofcontrol values) were demonstrated by 14 days in the peripheral branch and by 28 days in the central branch. The central branch did not show further reductions MATERIALS AND METHODS in caliber after 28 days (data not shown). To examine in greater detail the spatial distribution of Morphometric Analyses of Sensory Fibers. Sciatic nerves of atrophy in the central branch, caliber was measured at anesthetized (chloral hydrate; 400 mg/kg i.p.) 7-week-old proximal, middle, and distal levels of the L5 dorsal root at 4 male Sprague-Dawley rats were crushed twice for 30 sec at weeks after axotomy. Reductions in axonal caliber were the junction of the L4 and L5 spinal nerves using a no. 7 greatest near the cell bodies: mean axonal cross-sectional Dumontjeweler's forceps. Previous studies have shown that areas were 52%, 79%, and 84% of control values in segments this method completely interrupts all axons in the nerve (1, located near the DRG, in the middle ofthe root, and adjacent 2). At 4, 7, 14, 21, 28, and 35 days after axotomy, animals to the spinal cord, respectively. Thus, it was clear that lumbar were anesthetized and perfused through the ascending aorta sensory neurons responded to axotomy by a decrease in with 5% glutaraldehyde in 0.1 M Sorenson's phosphate buffer axonal caliber that began near the cell body and advanced (pH 7.3). Segments were obtained from the proximal, middle, distally (somatofugal atrophy). and distal regions of L5 dorsal roots, which extended 0-3, LeVels of NF mRNAs Following Axotomy. To determine 12-15, and 24-27 mm from the spinal cord, respectively, and whether the decrease in axonal caliber was accompanied by from the peripheral branches of these sensory axons in the altered gene expression of NF proteins, levels of mRNA mixed spinal roots (adjacent to the ganglion). Control seg- encoding NF68 were examined in total RNA isolated from ments were obtained from unoperated, contralateral nerves. DRG at various times after axotomy. Equal amounts ofRNA After processing, tissue was sectioned, stained, and photo- from each sample were analyzed electrophoretically, trans- graphed. Axonal areas were measured as previously de- ferred to nitrocellulose membranes, and NF68 mRNAs were scribed (1) in 200 randomly selected axons in the central and detected by hybridization to a 32P-labeled cloned DNA probe. peripheral projections of individual L5 DRG at each time The resultant autoradiogram (shown in Fig. 2) demonstrated period. The percentile of axons (percent of axons with areas that both of the two known mRNAs (with lengths of 2.5 kb equal to or less than a given value) was plotted as a function A ofcross-sectional area to compare areas at various times after axotoMy. 7! C P RNA Blots. Between 2 and 70 days after bilateral sciatic nerve crush, animals were killed by anesthesia, and L4 and 5C L5 DRG were removed. Unoperated control animals of the same starting age were similarly dissected. Eight to 12 ganglia 25 FIG. 1. (A) Axonal cross- (from two or three animals, respectively) were pooled. An sectional area is compared advantage of this system is that all nerve cell bodies are 10 20 40 in the central (C)(Am2)and peripheral harvested by DRG removal, free of contamination by other (P) branches ofcontrol DRG cell (a populations ofneurons. RNA isolated from these ganglia (24) z axons. The percentile of axons was separated on agarose-formaldehyde gels and transferred 0 (percent of axons with areas x equal to or less than a given to nitrocellulose membranes for hybridization with 32P- 4! I value) is plotted as a function of labeled cDNA (25). The NF68 cDNA was previously de- cross-sectional area. Areas were scribed (26); actin and /3-tubulin cDNAs were clones pAl and 0 comparable in peripheral and U A' pT2, respectively (27). Labeled hybrids were detected -i central branches. (B) Axonal ar- autoradiographically and quantitated densitometrically using ea is compared in peripheral a computer-based system (Loats Associates, Westminster, z3 10 20 30 40 branches at various times after MD). axotomy. Area was reduced at 4 IC C days after axotomy and contin- In Situ Hybridization. Two weeks after the sciatic nerves of wU IL ued to undergo further reduc- 7-week-old rats were crushed unilaterally, animals were 7! S tions at 7 and 14 days; no further anesthetized and perfused through the ascending aorta with * 4D decrements were seen at 21 0.9% saline followed by 4% paraformaldehyde. L5 DRG were 5c 7 days. (C) Axonal area is com- removed, embedded in paraffin, and sectioned (6 Anm). Tissue 0o2821 pared in central branches at var- sections were hybridized with 35S-labeled cDNA according to 25 ious times after axotomy. The published methods (28). The density of silver grains over L first decrease in area was detected at 14 days after axotomy, and neurons (number per unit area) was analyzed using a com- 10 20 30 40 additional reductions were dem- puter-based system (Loats Associates). AXONAL AREA onstrated at 21 and 28 days. Downloaded by guest on September 29, 2021 3474 Neurobiology: Hoffman et al. Proc. Natl. Acad Sci. USA 84 (1987) Table 1. Areas of axons (Aum) in the central and peripheral was selective for NF, levels of actin and f-tubulin mRNAs branches of axotomized sensory neurons were also examined in axotomized ganglia using duplicate RNA blots. Inspection of these autoradiograms (Fig. 3) Time after Mean cross-sectional area clearly demonstrates that at 2 weeks after axotomy, when crush, days Central Peripheral NF68 mRNA was reduced approximately 3-fold, levels of 0 (control) 12.40 ± 0.12 12.32 ± 0.12 actin and tubulin mRNAs were increased -2- and -4-fold, 4 12.49 11.95 respectively. Thus, there was no evidence for a generalized 7 13.19 9.55 decrease in mRNA levels in axotomized ganglia. On the 14 10.38 5.98 contrary, the reduction in mRNA was selective for NF68. 21 9.28 6.65 Measurement of NF68 mRNA Levels in Individual Neurons 28 6.29 Using in Situ Hybridization. To test whether NF68 mRNA levels were reduced in individual axotomized neurons, we Data for each postoperative time period represent mean areas of 200 randomly selected axons in the central and peripheral projections used in situ hybridization of labeled NF68 cDNA sequences ofindividual L5 DRG. Control data were obtained from unoperated, to tissue sections of axotomized and control ganglia. An contralateral ganglia. example of such an experiment is illustrated in Fig. 4. Clearly, the levels ofNF68 mRNA were reduced in individual neurons after axotomy. Moreover, this qualitative finding and 4.0 kb) (26) that are encoded by a single NF68 gene were was confirmed by quantitation of grain densities. As dem- present in sensory neurons, although the smaller RNA onstrated in Fig. 5, 2 weeks after axotomy there were species was clearly more prominent (Fig. 2, control lane). significantly greater numbers of grains per unit area over These RNA blots revealed that there was a 2- to 3-fold control neurons than over axotomized neurons. This was true decrease in NF68 mRNAs in IDRG between 4 and 42 days for both large and small neurons (P < 0.001, Student's t test), after axotomy. At intermediate times after axotomy (e.g., 14 althoulgh the levels of NF68 mRNA were normally greater days), the reduction was greater for the 2.5-kb mRNA than over large than small neurons (P < 0.001). For example, at 2 for the 4.0-kb NF68 mRNA (Fig. 3). By 70 days, when nerve weeks after crush, grain densities declined to 30% and 44% regeneration was completed, levels ofNF68 mRNA returned of control values in large and small neurons, respectively to normal (Fig. 2, 70-day lane). (Fig. 5), indicating a reduction in NF68 mRNA comparable in These blots demonstrated that NF68 mRNA accounted for magnitude to that measured on RNA blots (i.e., reductions of a smaller proportion oftotal RNA in axotomized ganglia than 56-70%o and 65%, respectively). The close correspondence in controls. This could result from several mechanisms. First, between the results of in situ and blot analyses suggests that levels of NF mRNA could be selectively decreased in the total amount of RNA in these neurons (primarily ribo- axotomized neurons. Second, it is possible that the expres- somal RNA) was not substantially altered after axotomy. sion of all mRNAs is reduced in these neurons (i.e., the loss of NF mRNA is the result of a nonspecific decrease in all DISCUSSION mRNAs). Third, total cellular RNA, :90% of which is ribosomal, could increase without any change in the amount Factors Regulating NF Expression. In lumbar sensory fibers of NF mRNA in individual neurons (i.e., NF mRNA would somatofugal axonal atrophy occurs in response to axotomy, represent a smaller proportion of total cellular RNA). In and decreased NF transport in these neurons presumably is order to distinguish among these possibilities, two additional related to the decline in perikaryal mRNA content for the experiments to be described were performed: (i0 blots were major NF protein (NF68). This reduction in NF mRNA is hybridized with cDNA probes encoding other cytoskeletal selective; the same ganglia show corresponding elevation in proteins (actin and P-tubulin); and (it) levels of NF68 mRNA levels of mRNAs for actin and tubulin, the other major in individual neurons were measured using in situ hybridiza- proteins of the axonal cytoskeleton. These findings are tion. consistent with results showing that the incorporation of labeled amino acids into NF protein by DRG in vitro is Levels of Other Cytoskeletal mRNAs Following Axotoiny. selectively reduced after axotomy with a time course similar To determine whether the reduction in mRNA after axotomy to that of the reductions in mRNA levels found in this study C 2 4 14 28 42 70 kb (29). Together, they suggest that alterations in NF mRNA levels are mirrored by concomitant changes in rates of NF synthesis. Thus, our results support the hypothesis that NF __ 4.0 gene expression is a major determinant of axonal caliber in large myelinated nerve fibers. HiW A 2.5 Somatofugal atrophy, the morphological correlate of decreased NF gene expression, is initiated when a neuron is disconnected from its targets, and recovery of caliber correlates with reinnervation of targets (1, 30). This decrease in caliber does not depend on degeneration of the distal axon. For example, all sensory axons undergo somatofugal atrophy early in the course of exposure to acrylamide (31), a neurotoxin which, after more prolonged exposure, induces FIG. 2. This RNA blot shows reduced expression of NF68 in distal axonal degeneration in long, large-caliber fibers. In axotomized DRG. The left-hand lane contains RNA from the ganglia addition, somatofugal atrophy occurs in motor fibers after of unoperated control animals (C); the postcrush intervals (in days) intramuscular injection of botulinum toxin (B. G. Gold, are indicated above each lane. NF68 cDNA hybridizes with two J.W.G., A. Pestronk, P.N.H., E. F. Stanley, and D.L.P., mRNA transcripts (2.5 and 4.0 kb) (26). The clear band below the unpublished work), which blocks muscle activity by inhibit- 2.5-kb message corresponds to 18 S ribosomal RNA. Levels of mRNAs were reduced between 4 and 42 days after axotomy, and ing release of quanta of the neurotransmitter acetylcholine returned to control levels at 70 days after crush. Ten micrograms of from motor nerve terminals (32). These observations suggest RNA was analyzed on each gel slot; the autoradiogram was exposed that interactions between neurons and their targets play an for 1 day. important role in maintaining NF gene expression. Downloaded by guest on September 29, 2021 Neurobiology: Hoffman et aL Proc. Natl. Acad. Sci. USA 84 (1987) 3475

r c c

FIG. 3. The expression of NF68, (B tubulin, and actin genes were compared in axotomized (r) and control (c) ganglia 2 weeks after axotomy. Levels of mRNAs were measured using duplicate _1_J_ad pairs of samples. NF68 mRNA was re-

Z. duced in axotomized ganglia as compared with controls. In contrast, tubulin and actin mRNAs were greater in axotomized ganglia than in controls. Ten micrograms of RNA was analyzed on each gel slot; autoradiograms were exposed NF68 Tubulin Actin for 1 day.

We propose that the signal generated by these interactions produced by target cells, transferred to axon terminals, and is carried from the axon terminals to its site of action in carried retrograde to cell bodies; or (it) an axonally trans- neuronal perikarya by retrograde axonal transport. This ported molecule (i.e., produced in the neuron) that is mod- signal could be either of the following: (i) a molecule ified as it enters axon terminals and then returns (as a signal) to perikarya. In either case, production of this signal would reflect normal axon-target cell interactions. ~ ~ W Increased Expression of Actin and Tubulin in Axotomized Ganglia. The increased expression of actin and tubulin in i%;4~~ ~ ~ axotomized ganglia is consistent with previous observations Aev showing that levels of tubulin mRNA are increased in goldfish retina after crushing the optic nerve (33), and that the synthesis of these proteins is selectively increased during axonal outgrowth (elongation) in cultured neurons (34). Although the increased expression of tubulin in axotomized '4nfe neurons appears to be inconsistent with the previous observation that tubulin immunoreactivity is reduced in the proximal stumps of axotomized motor fibers (2), this apparent discrepancy may reflect alterations in either the turnover or distribution of axonal tubulin after axotomy; the velocity of J tubulin transport increases nearly two-fold after axotomy (2), resulting in increased delivery of tubulin to the tips of may to alter- ,.4', _ regenerating fibers. In addition, axotomy lead ations in the amount of newly-synthesized tubulin entering the axon from the perikaryon. In any case, because tubulin and actin are present in satellite and root sheath cells as well as neurons, in situ hybridization studies will be needed to document that these mRNA levels are increased in neurons. Time Course of Axonal Atrophy in Central and Peripheral Branches of Spinal Sensory Neurons. Known differences in ~~ ~ ~ ~ ~ ~ ~ ~ *- R /o St~~~~l '4 I'd 45 .0

0 0 30 0

b- ox ;~~ ~ > '- C 15

¢ airs s Ho ary

Large Small neurons neurons FIG. 5. Density of autoradiographic silver grains after in situ B k -f*e- , I hybridization with NF68 cDNA. DRG contain sensory neurons with either large or small perikaryal areas (>600 and <600 pm2, respectively). In control ganglia, grain densities were greater over large than over small neurons. Two weeks after axotomy, levels of NF68 FIG. 4. In situ hybridization of NF68 cDNA with control (A) and mRNA were reduced in both neuronal populations. SD, vertical lines axotomized sensory neurons 2 weeks after unilateral axotomy (B). In above the bars; n, number of neurons analyzed in axotomized and control ganglia, the density of silver grains was greater over large control ganglia, 10 and 16, respectively, for large neurons, and 23 and neurons than over small neurons. After axotomy, grain densities 52, respectively, for small neurons. Neuronal areas and grain declined over both large and small neurons. Autoradiograms were densities were measured using a computer-based system developed exposed for 5 days. Bars, 10 /.&m. by Loats Associates. Downloaded by guest on September 29, 2021 3476 Neurobiology: Hoffman et al. Proc. Natl. Acad. Sci. USA 84 (1987) the kinetics of NF transport in the central and peripheral 5. Berthold, C. H. (1978) in Physiology and Pathobiology of branches of sensory neurons account for differences in the Axons, ed. Waxman, S. G. (Raven, New York), pp. 3-63. of axonal atrophy. NF proteins and other slow- 6. Ellisman, M. H. & Porter, K. R. (1980) J. Cell Biol. 87, timing 464-479. component constituents are transported at approximately 7. Hirokawa, N. (1982) J. Cell Biol. 94, 129-142. twice the rate in the peripheral branch as in the central branch 8. Hirokawa, N., Glicksman, M. A. & Willard, M. B. (1984) J. (35). In the peripheral branch the earliest postcrush changes Cell Biol. 98, 1523-1536. in axonal caliber follow a time course comparable to the 9. Asbury, A. K., Gale, M. K., Cox, S. C., Baringer, J. R. & decrease in NF mRNA levels in the perikaryon. Axonal Berg, B. 0. (1972) Acta Neuropathol. (Berlin) 20, 237-247. caliber is reduced in the peripheral branch within 4 days after 10. Spencer, P. S. & Schaumberg, H. H. (1977) J. Neuropathol. axotomy, and the full reduction in caliber (mean axonal Exp. Neurol. 36, 276-299. cross-sectional area is reduced by 50%) is found by 21 days. 11. Clark, A. W., Griffin, J. W. & Price, D. L. (1980) J. In the central branch, at a similar distance from the ganglion, Neuropathol. Exp. Neurol. 39, 42-55. 12. Troncoso, J. C., Price, D. L., Griffin, J. W. & Parhad, I. M. axonal atrophy does not occur until 14 days, and the full (1982) Ann. Neurol. 12, 278-283. reduction in caliber is not apparent until 28 days. Thus, the 13. Parhad, I. M. & Swedberg, E. A. (1986) Peripher. Neuropathy onset of somatofugal atrophy, as previously described in Assoc. Am. Abstr. p. 44. motor axons (1), and its rate ofprogression in both the central 14. Hursh, J. B. (1939) Am. J. Physiol. 127, 131-139. and peripheral processes correlate with the kinetics of NF 15. Minwegen, P. & Friede, R. L. (1984) Brain Res. 297, 105-113. transport in these branches. 16. Gillespi, M. J. & Stein, R. B. (1983) Brain Res. 259, 41-56. NF Expression as a Major Determinant of Axonal Caliber. 17. Farel, P. B. (1978) Brain Res. 158, 331-341. The expression of NF genes is the primary step in the 18. Nukada, H. & Dyck, P. J. (1984) Ann. Neurol. 16, 238-241. sequence of events leading to the appearance of NF in axons. 19. Lieberman, A. R. (1976) in The Peripheral Nerve, ed. Landon, D. N. (Wiley, New York), pp. 188-278. Each of the three NF subunit polypeptides (200, 145, and 68 20. Dyck, P. J., Lais, A., Karnes, J., Sparks, M. & Dyck, P. J. B. kDa) (36) is encoded by a separate gene (26). Transcription of (1985) Brain Res. 340, 19-36. these genes yields NF mRNAs, which are translated exclu- 21. Czeh, G., Kudo, N. & Kuno, M. (1977) J. Physiol. (London) sively in perikarya and proximal dendrites. Shortly after they 270, 165-180. are synthesized, these proteins are assembled into stable 22. Hoffer, J. A., Stein, R. B. & Gordon, T. (1979) Brain Res. 178, heteropolymers. This assembly also appears to occur exclu- 347-361. sively in perikarya (37). Since axons do not contain ribo- 23. Lasek, R. J., Oblinger, M. M. & Drake, P. F. (1983) Cold somes (38), NF must be delivered to axons where they Spring Harbor Symp. Quant. Biol. 48, 731-744. undergo somatofugal translocation via slow axonal transport 24. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J. (1979) Biochemistry 18, 5294-5299. (36). The absence of unassembled NF subunits in axons 25. Shank, P. R., Hughes, S. H., Kung, H.-J., Majors, J. E., precludes local assembly (39) (i.e., NF proteins are trans- Quintrell, N., Guntaka, R. V., Bishop, J. M. & Varmus, H. E. ported in the form of NF organelles). Normally, there is (1978) Cell 15, 1383-1395. relatively little turnover of axonal NF-except at the axon 26. Lewis, S. A. & Cowan, N. J. (1985) J. Cell Biol. 100, 843-850. terminals (40, 41). NF are degraded as they enter the axon 27. Cleveland, D. W., Lopata, M. A., MacDonald, R. J., Cowan, terminals, presumably through the action of calcium-activat- N. J., Rutter, W. J. & Kirschner, M. W. (1980) Cell 20, ed proteases (40, 42). [Although this study emphasized the 95-105. role of NF gene expression in the control of axonal caliber, 28. Lewis, S. A. & Cowan, N. J. (1985) J. Neurochem. 45, it should be noted that axonal NF content is also influenced 913-919. 29. Greenberg, S. G. (1986) Dissertation (Case Western Reserve by local changes in the kinetics of NF transport (43, 44).] Univ., Cleveland, OH). Levels of NF mRNA represent the net outcome of the 30. Kuno, M., Miyata, Y. & Munoz-Martinez, E. J. (1974) J. competing processes of synthesis (transcription) and RNA Physiol. (London) 242, 273-288. turnover. Although we might anticipate that NF mRNA 31. Gold, B. G., Griffin, J. W. & Price, D. L. (1985) J. Neurosci. levels in these neurons are largely under transcriptional 5, 1755-1768. control, technical limitations preclude the measurement of 32. Simpson, L. L. (1981) Pharmacol. Rev. 33, 155-188. mRNA synthesis directly. In any event, our data suggest that 33. Neumann, D., Scherson, T., Ginzburg, I., Littauer, U. Z. & either transcription of NF mRNA, or cytoplasmic stabiliza- Schwartz, M. (1983) FEBS Lett. 162, 270-276. tion of that mRNA, is a primary regulator of axonal caliber 34. Fine, R. E. & Bray, D. (1971) Nature (London) New Biol. 234, 115-118. in large myelinated nerve fibers. 35. Mori, H., Komiya, Y. & Kurokawa, M. (1979) J. Cell Biol. 82, We thank Kenneth Fahnestock, Richard Altschuler, Donald Price, 174-184. Jr., and Mary Ellen Dorman for their excellent technical assistance 36. Hoffman, P. N. & Lasek, R. J. (1975) J. Cell Biol. 66, 351-366. and Margaret Lopata and Kevin Sullivan for their helpful sugges- 37. Black, M. M. (1987) in Intrinsic Determinants of Neuronal tions. Aspects of this work were supported by National Institutes of Form, ed. Lasek, R. J. (Liss, New York), in press. Health Grants NS-20164, GM-29513, NS-14784, NS-22849, NS- 38. Lasek, R. J., Dabrowski, C. & Nordlander, R. (1973) Nature 10580, AG-05146, and NS-15721 and a gift from the Robert L. and (London) New Biol. 244, 162-165. Clara G. Patterson Trust. P.N.H. is an Alfred P. Sloan Foundation 39. Momis, J. R. & Lasek, R. J. (1982) J. Cell Biol, 92, 192-198. Fellow and the recipient of a Research Career Development Award 40. Lasek, R. J. & Hoffman, P. N. (1976) Cold Spring Harbor from the National Institutes of Health (NS-00896). D.W.C. is the Conf. Cell Proliferation 3, 1021-1049. recipient of a Research Career Development Award from the 41. Lasek, R. J. & Black, M. M. (1977) in Mechanisms, Regula- National Institutes of Health (HD-00504). tion and Special Functions ofProtein Synthesis in the Brain, 1. Hoffman, P. N., Griffin, J. W. & Price, D. L. (1984) J. Cell ed. Roberts, E. (Elsevier/North-Holland Press, New York), Biol. 99, 705-714. pp. 161-169. 2. Hoffman, P. N., Thompson, G. W., Griffin, J. W. & Price, 42. Roots, B. I. (1983) Science 221, 971-972. D. L. (1985) J. Cell Biol. 101, 1332-1340. 43. Hoffman, P. N., Griffin, J. W., Gold, B. G. & Price, D. L. 3. Friede, R. L. & Samorajski, T. (1970) Anat. Rec. 167, (1985) J. Neurosci. 5, 2920-2929. 379-387. 44. Hoffman, P. N., Koo, E. H., Muma, N. A., Griffin, J. W. & 4. Weiss, P. A. & Mayr, R. (1971) Proc. Natl. Acad. Sci. USA Price, D. L. (1987) in Intrinsic Determinants of Neuronal 68, 846-850. Form, ed. Lasek, R. J. (Liss, New York), in press. Downloaded by guest on September 29, 2021