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Neurofilaments are intrinsic determinants of axonal caliber
Micron,Vol. 24, No. 6, pp. 677~83, 1993 Copyright© 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0968-4328/93 $24.00
Pergamon
Neurofilaments are Intrinsic Determinants of Axonal Caliber N A N C Y A. M U M A * a n d P A U L N. H O F F M A N t
*Department of Pharmacology, Loyola University Chicago Stritch School of Medicine, 2160 South First Avenue, Maywood, IL 60153, U.S.A. t Departments of Ophthalmology and Neurology, 515 Pathology Building, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21205, U.S.A.
Abstract--Neurofilaments(NFs) are intrinsic determinants ofaxonal caliber in large-caliber myelinated nerve fibers. Axonal caliber is influenced by the number of NFs in the axon (axonal NF content) and the spacing between adjacent NFs (interfilament distance). Axonal NF content depends on the level ofNF gene expression, the amount of NF protein entering the axon, and the velocity at which NF protein moves within the axon. In this review we discuss possible factors affecting both axonal NF content and interfilament distance; these include the ratio of NF subunits, the level of phosphorylation of these subunits, interactions with target cells, and interactions with ensheathing glial cells. Key words: Neurofilament, axon, myelinated nerve.
CONTENTS I. II. III. IV. V.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The composition of axonal neurofilaments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurofilament metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interfilament spacing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors that influence axonal neurofilament content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Neurofilament gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The entry of neurofilaments into the axonal transport system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The velocity of neurofilament transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. I N T R O D U C T I O N I n this review we will c o n s i d e r evidence suggesting that n e u r o f i l a m e n t s ( N F s ) , the p r i n c i p a l i n t e r m e d i a t e filam e n t s (IFs) of m a t u r e n e u r o n s , are i m p o r t a n t determin a n t s of a x o n a l caliber in large-caliber m y e l i n a t e d nerve fibers. N a t u r e has evolved two strategies to achieve fast impulse c o n d u c t i o n velocities in nerve fibers. O n e strategy, e m p l o y e d by i n v e r t e b r a t e species, is the f o r m a t i o n of so-called g i a n t a x o n s with d i a m e t e r s of several h u n d r e d ~tms. T h e o t h e r m e c h a n i s m , f o u n d exclusively in vertebrates, is the f o r m a t i o n o f myelin sheaths a r o u n d axons. M y e l i n a t i o n allows a x o n s with d i a m e t e r s two o r d e r s of m a g n i t u d e less t h a n those of g i a n t axons to achieve c o n d u c t i o n velocities c o m p a r a b l e to those of giant axons. Since the c o n d u c t i o n velocity o f m y e l i n a t e d nerve fibers is directly p r o p o r t i o n a l to a x o n a l d i a m e t e r (Gasser a n d G r u n d f e s t , 1939), the factors which d e t e r m i n e a x o n a l caliber have a p r o f o u n d influence o n this i m p o r t a n t p h y s i o l o g i c a l p r o p e r t y o f neurons. T h e first hint t h a t N F c o n t e n t influences a x o n a l caliber c a m e from m o r p h o l o g i c a l studies d e m o n s t r a t i n g t h a t
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N F s are the m o s t n u m e r o u s cytoskeletal elements in large-caliber m y e l i n a t e d a x o n s a n d t h a t the n u m b e r s of N F s correlates closely with a x o n a l cross-sectional areas (Friede a n d S a m o r a j s k i , 1970; Weiss a n d M a y r , 1971). A m o r e d y n a m i c d e m o n s t r a t i o n of the r e l a t i o n s h i p between a x o n a l caliber a n d N F c o n t e n t c a m e from studies e x a m i n i n g the spatial a n d t e m p o r a l e v o l u t i o n of the r e d u c t i o n s in a x o n a l caliber which o c c u r in the p r o x i m a l s t u m p s of injured nerve fibers (Cragg a n d T h o m a s , 1961). M o r p h o m e t r i c analyses d e m o n s t r a t e d t h a t these reductions in a x o n a l cross-sectional a r e a correlate with p r o p o r tional decreases in a x o n a l N F c o n t e n t which start p r o x i m a l l y n e a r the n e u r o n cell b o d y (soma) a n d p r o c e e d s o m a t o f u g a l l y a l o n g nerve fibers at the rate of N F t r a n s p o r t (Hoffman et al., 1984). In contrast, the n u m b e r of m i c r o t u b u l e s in these axons r e m a i n s relatively u n c h a n g e d ; the density of m i c r o t u b u l e s increases in p r o p o r t i o n to the r e d u c t i o n in cross-sectional a r e a (Hoffman et al., 1984). D i r e c t evidence for the role of N F s in the c o n t r o l of a x o n a l caliber in m y e l i n a t e d fibers comes from recent studies of a x o n a l m o r p h o l o g y in a m u t a n t strain of quail 677
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(quiver) whose neurons are devoid of NFs. Although myelinated axons are present in these animals, the cytoskeletons of these axons are comprised exclusively of microtubules. All of the myelinated axons in quiver quail are much thinner than the large-caliber myelinated axons in control animals (Yamasaki et al., 1991, 1992). Similarly, myelinated axons are devoid of NFs and are much thinner than comparable axons in control animals in a line of transgenic mice expressing a transgene construct (i.e. in which the carboxy-terminal tail of NF-H is conjugated to/3-galactosidase) which prevents NFs from entering axons. NFs accumulate in neuron cell bodies. (J. Eyer, personal communication). NF content does not appear to be a major determinant of axonal caliber in either unmyelinated axons, which are uniformly thin and largely devoid of NFs, or in small-caliber myelinated fibers, which contain relatively few NFs compared to large-caliber myelinated axons (Berthold, 1978). The cross-sectional areas of small-caliber myelinated axons, such as those in the optic nerve, do not correlate closely with axonal NF content (Berthold, 1978; Nixon and Logvinenko, 1986). Furthermore, alterations in axonal NF content in optic nerve axons induced by intoxication with an agent which impairs NF transport in axons (/~,/~'iminodipropionitrile (IDPN)) (Griffin et al., 1978), do not correlate with proportional changes in axonal crosssectional areas (Parhad et al., 1987).
II. THE COMPOSITION OF AXONAL NEUROFILAMENTS The subunit composition of NFs appears to be related to their proposed function as intrinsic determinants of axonal caliber. Neurofilaments are normally comprised of three protein subunits, NF-L, NF-M and NF-H, with apparent molecular weights of 68, 145 and 200kDa, respectively (Hoffman and Lasek, 1975). Immunohistochemical analyses demonstrate that NF-L is associated with the core of the NF, while NF-H, and to a lesser extent NF-M, are associated with NF sidearms (Hirokawa et al., 1984; Hisanaga and Hirokawa, 1988). Although NF-L, alone is capable of self-assembly into filaments in a cellfree system (Geisler and Weber, 1981 ), recent transfection experiments in which genes encoding NF subunits were introduced into a line of cultured cells lacking endogenous IFs suggest that at least two subunits (NF-L and NF-M) are required for filament assembly in vivo (M. K. Lee, personal communication). This is consistent with the observation that filaments comprised of NF-L and NF-M (i.e. lacking NF-H) are present in the developing neurons of embryonic and neonatal animals (Shaw and Weber, 1982; Carden et al., 1987; Schlaepfer and Bruce, 1990). Each neurofilament subunit is encoded by a separate gene (Lewis and Cowan, 1985; Robinson et al., 1986; Julien et al., 1986, 1987; Myers et al., 1987; Napolitano et al., 1987; Zopf et al., 1987; Dautigny et al., 1988; Shneidman et al., 1988; Lieberburg et al., 1989). Like all other IF proteins, each NF subunit contains a highly-
conserved coiled-coil rod domain (Geisler et al., 1983). NF-M and NF-H are unique among the IF proteins in that they contain extended carboxy-terminal tail domains. The carboxy-terminal tail domains of NF-M and NF-H contain multiple repeats (up to 50 for NF-H) of the K-S-P (lysine-serine-proline) sequence; these serine residues are major sites ofNF phosphorylation (Myers et al., 1987; Julien et al., 1988). The presence of highlycharged phosphate groups associated with NF sidearms may promote the repulsion of adjacent NFs and contribute to their function as intrinsic determinants of axonal caliber.
III. NEUROFILAMENT METABOLISM The genes encoding the NF proteins are transcribed and translated in the neuron cell body. The NF proteins then enter the axon where they move somatofugally at rates of several mm/day in the SCa component of slow axonal transport (Hoffman and Lasek, 1975; Lasek and Hoffman, 1976). The observation that essentially all of the NF protein in the axon is associated with NF polymers (Morris and Lasek, 1982) favors a model in which the NF proteins are assembled into polymers in the cell body and undergo transport in their polymeric form (Lasek, 1986). An alternative model proposes that the NF subunits are transported as either protofilaments or unassembled subunits (Nixon and Logvinenko, 1986). The proposal that axons contain a substantial population of stationary NFs has been controversial. This hypothesis is based on the observation that pulse-labeled NF proteins are retained along optic nerve axons after passage of the SCa wave (Nixon and Logvinenko, 1986). Recent data indicating that some of the pulse-labeled NF protein in this system is transported at rates much slower than the SCa wave provides an alternative explanation for this finding (Lasek et al., 1992). Relatively little is known about the mechanism of NF transport. The rate of NF transport differs in various classes of neurons. For example, the rate is an order of magnitude faster in peripheral sensory and motor fibers than optic nerve axons (McQuarrie et al., 1986). The rate of NF transport also declines with age (Hoffman et al., 1983) and with increasing distance along peripheral nerve fibers (Hoffman et al., 1985a). The available evidence suggests that there is relatively little turnover of transported NFs until they reach the axon terminals. The proposal that axonally-transported NF proteins are normally degraded by calcium-activated proteolysis as they enter the axon terminals (Lasek and Hoffman, 1976) is supported by the observation that the injection of leupeptin, an inhibitor of calcium-activated proteolysis, into goldfish brain results in the abnormal accumulation of NFs in axon terminals (Roots, 1983). This concept is also supported by the demonstration that pulse-labeled NF proteins disappear shortly after entering the axon terminals (Paggi and Lasek, 1987; Garner, 1988).
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undergoing axonal transport primarily reflects the level of N F gene expression.
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@ Fig. 1. Regulationof axonal caliber.Axonalcaliberis dependent on axonal neurofilament (NF) content and interfilament distance. AxonalNF content is regulatedby (1) the levelofN F gene expression, (2) entry of filaments into the slow axonal transport system and (3) the rate of transport of NFs in the axon. Factors such as the levelof NF phosphorylation,the ratio of NF subunit proteins,neuron-target interactionand neuron-glialinteraction may influenceaxonal NF content and interfilament distance. IV. I N T E R F I L A M E N T S P A C I N G The caliber of myelinated axons is affected by the N F content of axons and by the spacing between adjacent NFs. Interfilament distance varies among various classes of neurons and within different regions of the same axon; regions with small calibers (i.e. nodes of Ranvier) have decreased interfilament spacing compared to internodal regions (Price et al., 1990). Clues about the regulation of interfilament spacing have come from experimental models in which interfilament distance is altered. In these models factors such as N F subunit ratios or N F phosphorylation have been associated with alterations in interfilament distance. Interfilament spacing decreased in peripheral fibers of a transgene model which overexpressed NF-L; axonal caliber is slightly reduced in these axons (Monteiro et al., 1990). In control mice which were grafted with nerve segments from trembler mice, reductions in interfilament distance are associated with decreases in N F phosphorylation (deWaegh et al., 1992). V. FACTORS T H A T I N F L U E N C E AXONAL NEUROFILAMENT CONTENT The factors which influence axonal N F content are better defined than those which control interfilament spacing. We will now consider evidence indicating that the N F content of axons is influenced by the level of N F synthesis (gene expression), the amount of N F protein entering the axonal transport system and the N F transport velocity (Fig. 1). The amount of protein
Axonal N F content correlates closely with the level of N F gene expression in the neuron cell body. In general, mature neurons containing relatively low levels of N F mRNAs give rise to either unmyelinated axons (e.g. small sensory neurons) (Hoffman et al., 1987) or small-caliber myelinated fibers (e.g. retinal ganglion cell neurons) (Hoffman et al., 1993). Conversely, mature neurons with relatively high levels of N F expression such as ventral motor neurons (Muma et al., 1990; Tetzlaff et al., 1991) and large sensory neurons (Hoffman et al., 1987) give rise to large-caliber myclinated axons. These differences in axonal N F content also correlate with differences in the relative amounts of N F protein undergoing transport in these axons. Relatively little pulse-labeled N F protein is transported in either unmyelinated fibers (McLean et al., 1983) or optic nerve fibers (Black and Lasek, 1980), compared to large-caliber peripheral sensory (Mori et al., 1979) and motor fibers (Hoffman and Lasek, 1975; McQuarrie et al., 1986). Perhaps the best illustration of the relationship between axonal N F content and the level of N F expression is found in systems where changes in the level of N F expression correlate with comparable alterations in axonal N F content. Such changes have been studied extensively during postnatal development and after axonal injury (axotomy). Reductions in neurofilament expression after axotomy (Hoffman et al., 1987; Wong and Oblinger, 1987; Greenberg and Lasek, 1988; Goldstein et al., 1988; Muma et al., 1990; Tetzlaff et al., 1991) correlate with a decrease in the amount of pulse-labeled N F protein transported in the proximal stumps of injured nerve fibers (Hoffman et al., 1985b) and reductions in the N F content and caliber of axons in the proximal stump (Hoffman et al., 1984). Conversely, increased neurofilament expression in peripheral sensory neurons during early post-natal development correlates with increases in the neurofilament content and caliber of axons (Fig. 2) (Muma et al., 1991). Neurofilament expression is low in embryonic neurons, whose axons lack NFs. Expression increases dramatically during early postnatal development in neurons giving rise to myelinated axons; this correlates with the onset of radial growth of axons. As seen in the axotomy model, the abundance of mRNA encoding neurofilaments correlates closely with neurofilament protein levels. Neurofilament expression remains low in mature neurons with unmyelinated axons. Changes in neurofilament gene expression correlate selectively with radial growth; the expression of tubulin and actin, the other major cytoskeletal proteins in axons, declines as radial growth occurs during early post-natal development (Muma et al., 1991). The level of N F gene expression appears to be influenced by interactions between neurons and their targets. Reduced neurofilament gene expression after axotomy appears to reflect the loss of neuron-target
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Fig. 2. Neurofilament gene expression during early post-natal development affects axonal caliber. RNA (Northern) blots (A) demonstrate selective increases in low molecular weight neurofilament subunit (NF-L) and the high molecular weight neurofilament subunit (NF-H) mRNA levels at 5 (b), 11 (c) and 28 (d) days of age in rat dorsal root ganglia. In contrast, levels of mRNAs encoding fl-tubulin (Tub) and actin (Act) decrease during this period. Electron micrographs of dorsal root nerve fibers demonstrate a progressive, age-related increase in the calibers of myelinated axons at 5 (B), 11 (C) and 28 (D) days of age. All micrographs are shown at the same magnification. Scale marker indicates 2 p.m.
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interactions, while the return of neurofilament expression to pre-axotomy levels depends on the reconnection of regenerating axons with targets (Hoffman et al., 1984, 1987; M u m a et al., 1990). Trophic factors produced by target cells may play a role in this process. In the case of axotomized sensory neurons, exogenous nerve growth factor appears to reduce the severity of reductions in N F expression (Verge et al., 1990) and axonal caliber (Gold et al., 1991).
B. The entry of neurofilaments into the axonal transport system The amount of neurofilament protein entering the axonal transport system from the cell body affects the axonal neurofilament content and thus axonal caliber. Axons with reduced calibers are seen in models in which the amount of neurofilament protein entering the axon has been decreased. As mentioned earlier, axonal caliber (of myelinated fibers) is greatly reduced in mice expressing a transgene (i.e. in which the carboxy-terminal portion of N F - H is conjugated to fl-galactosidase) which prevents N F s from entering the axon. Distal axonal caliber is also reduced in select neuronal populations in aluminum treated rabbits. In these neuronal groups, the amount of neurofilament protein entering the slow axonal transport system is reduced (without alterations in the rate of transport of those neurofilaments which do enter the slow axonal transport system) (Bizzi et al., 1984; Troncoso et al., 1985). NFs which do not enter the axonal transport system accumulate in swollen neuron cell bodies (Fig. 3).
Fig. 3. Entry of filamentsinto the axonal transport systemaffects axonal caliber. Photomicrographs of neurons in spinal cords of control (A) and aluminum (B) treated rabbits demonstrate accumulations of neurofilaments (NFs) in swollen cell bodies induced by aluminum salts. In the aluminum treated animals the distribution of NFs within the neurons is altered; fewer neurofilamentsenter the slow axonal transport systemand distal axonal caliber is reduced. Scale marker indicates 50 lam.
C. The velocity of neurofilament transport N F content in any region of an axon is influenced by the rate at which N F s enter that region via axonal transport and the rates at which N F s leave via axonal transport and turnover. Therefore, if we assume that turnover is relatively low, the N F content of a local region of the axon (e.g. an internodal segment, which is the portion of a myelinated axon located between adjacent nodes) is largely determined by the difference between the rates at which N F s enter and leave that segment. The most dramatic example of this mechanism is illustrated after systemic intoxication with I D P N , a neurotoxin which impairs N F transport along the axon (Griffin et al., 1978), but does not affect either the level of N F synthesis (Parhad et al., 1988) or the entry of newly-synthesized N F s into the axon (Griffin et al., 1978). NFs enter the first internodal segment of the axon, presumably at their normal rate, but leave that internodal segment at a greatly reduced rate (i.e. at velocities approaching zero). This leads to the local accumulation of N F s and to the formation of giant axonal swellings filled with N F s (Fig. 4) (Chou and Hartmann, 1964, 1965; Clark et al., 1980). A similar mechanism may contribute to the increase in axonal N F content which occurs during the maturation of developing nerve fibers (Friede and Samorajski, 1970; Berthold, 1978; M u m a et al., 1991). In these axons the
Fig. 4. The axonal transport velocity of neurofilament (NFs) affectsaxonal caliber. Systemicintoxication with IDPN impairs the transport of NFs within axons (without alteringthe transport of NFs from the cell body into the axon) and leads to the formation of proximal axonal swellings. This electron micrograph of a paranodal region illustrates that these swellingsreflect the accumulation of axonal NFs. Scale marker indicates 1 lam.
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velocity of N F t r a n s p o r t progressively declines with increasing distance a l o n g the nerve (Hoffman et al., 1985a). This allows N F s to enter each i n t e r n o d a l segment slightly faster t h a n they leave, leading to a net a c c u m u l a t i o n over time. These examples suggest that local reductions in the velocity of N F t r a n s p o r t result in increases in a x o n a l N F content. Conversely, increases in the velocity of N F t r a n s p o r t i n d u c e d by systemic i n t o x i c a t i o n with 2,5h e x a n e d i o n e (2,5-HD) result in a decrease in a x o n a l N F c o n t e n t in the p r o x i m a l a x o n ( M o n a c o et al., 1989b). A l t h o u g h relatively little is k n o w n a b o u t the mecha n i s m of N F t r a n s p o r t , alterations in the velocity of N F t r a n s p o r t correlate with changes in either the ratio of N F s u b u n i t proteins or the levels of p h o s p h o r y l a t i o n of these proteins. I n n e o n a t a l optic nerves, a r e d u c t i o n the velocity of N F t r a n s p o r t correlates with the a d d i t i o n of N F - H to a x o n a l N F s ; prior to that time the N F s in these a x o n s are comprised almost entirely of N F - L a n d N F - M (Willard a n d Simon, 1983). After chronic i n t o x i c a t i o n with 2,5h e x a n e d i o n e , increases in the rate of N F t r a n s p o r t correlate with reductions in the p h o s p h o r y l a t i o n of N F - H ( W a t s o n et al., 1991). C h a n g e s in the rate of N F t r a n s p o r t associated with myelin f o r m a t i o n also correlate with alterations in the level of p h o s p h o r y l a t i o n of N F - M (deWaegh et al., 1992).
VI. C O N C L U S I O N S N e u r o f i l a m e n t s are intrinsic d e t e r m i n a n t s of axonal caliber in large caliber m y e l i n a t e d fibers. A x o n a l caliber is influenced by b o t h the n u m b e r of N F s (axonal N F c o n t e n t ) a n d the spacing between adjacent N F s (interfilam e n t distance). A x o n a l n e u r o f i l a m e n t c o n t e n t depends o n the level of n e u r o f i l a m e n t gene expression, the a m o u n t of N F p r o t e i n entering the axon, a n d the velocity at which this N F p r o t e i n moves within the axon. The caliber of m y e l i n a t e d axons can be regulated by factors which influence a n y of these three m e c h a n i s m s or by those which affect interfilament distance.
REFERENCES Berthold, C. H., 1978. Morphology of normal peripheral axons. In: Physiology and Pathobiology of Axons, Waxman, S. G., (ed.). Raven, New York, pp. 3-63. Black, M. M. and Lasek, R. J., 1980. Slow components of axonal transport: two cytoskeletal networks. J. Cell Biol. 86, 616-623. Bizzi, A., Crane, R., Autilio-Gambetti, L. and Gambetti, P., 1984. Aluminum effect on slow axonal transport: a novel impairment of neurofilament transport. J. Neurosci., 4, 722 731. Carden, M. J., Trojanowski, J. Q., Schlaepfer, W. W. and Lee, V. M. Y., 1987. Two-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns. J. Neurosci. 7, 3489-3504. Chou, S.-M. and Hartmann, H. A., 1964. Axonal lesions and waltzing syndrome after IDPN administration in rats. Acta Neuropath. 3, 428-450. Chou, S.-M. and Hartmann, H. A., 1965. Electron microscopy of focal neuroaxonal lesions produced by fl,fl-iminodipropionitrile(IDPN) in rats. Acta Neuropath. 4, 59t~603.
Clark, A. W., Griffin,J. W. and Price, D. L., 1980.The axonal pathology in chronic IDPN intoxication. J. Neuropath. exp. Neurol. 39, 42 55. Cragg, B. G. and Thomas P. K., 1961. Changes in conduction velocity and fiber size proximal to peripheral nerve lesions.J. Physiol., Lond. 157, 315-327. deWaegh, S. M., Lee, V. M.-Y. and Brady, S. T., 1992. Local modulation of neurofilament phosphorylation, axonal caliber, and slow axonal transport by myelinating Schwann cells. Cell 68, 451-463. Dautigny, A., Pham-Dinh, D., Roussel,C., Felix,J. M., Nussbaum, J. L. and Jolles, P., 1988. The large neurofilament subunit (NF-H) of the rat: cDNA cloning and in situ detection. Biochem. biophys. Res. Commun. 154(3), 1099-1106. Friede, R. L. and Samorajski T., 1970. Axonal caliber related to neurofilaments and microtubules in sciatic nerve fibers of rats and mice. Anat. Rec. 167, 379-388. Garner, J. A., 1988. Differentialturnover of tubulin and neurofilament proteins in central nervous system neuron terminals. Brain Res. 458, 309-318. Gasser, H. S. and Grundfest, H., 1939. Axon diameters in relation to the spike dimensions and the conduction velocity in mammalian A fibers. Am. J. Physiol. 127, 393M14. Geisler, N. and Weber, K., 1981. Self-assemblyin vitro of the 68,000 molecular weight component of the mammalian neurofilament triplet proteins into intermediate-sizefilaments. J. molec. Biol. 151, 565-571. Geisler, N., Kaufmann, E. Fischer, S. Plaessmann, U. and Weber K., 1983, Neurofilament architecture combines structural principles of intermediate filaments with carboxy-terminal extensions increasing in size between triplet proteins. EMBO J. 2, 1295 1302. Gold, B. G., Mobley, W. C. and Matheson S. F., 1991. Regulation of axonal caliber, neurofilament content, and nuclear localization in mature sensory neurons by nerve growth factor. J. Neurosci. 11, 943455. Goldstein, M. E., Weiss, S. R., Lazzarini, R. A., Shneidman, P. S.,Lees, J. F. and Schlaepfer W. W., 1988. RNA levels of all three neurofilament proteins decline following nerve transection. Molec. Brain Res. 3, 287-292. Greenberg, S. G. and Lasek, R. J., 1988. Neurofilament protein synthesis in DRG neurons decreases more after peripheral axotomy than after central axotomy. J. Neurosci. 8, 1739 1746. Griffin,J. W., Hoffman, P. N., Clark, A. W., Carroll, P. T. and Price, D. L., 1978. Slow axonal transport of neurofilament proteins: impairment by iminodipropionitrile administration. Science 202, 633 635. Hirokawa, N., Glicksman, M. A. and Willard, M. B., 1984. Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton. J. Cell Biol. 98, 1523-1536. Hisanaga, S.-I. and Hirokawa, N., 1988. Structure of the peripheral domains of neurofilaments revealed by low angle rotary shadowing. J. molec. Biol. 202, 297 305. Hoffman, P. N. and Lasek, R. J., 1975. The slow component of axonal transport: Identification of the major structural polypeptides of the axon and their generality among mammalian neurons. J. Cell Biol. 66, 351 366. Hoffman, P. N., Lasek, R. J., Griffin, J. W. and Price, D. L., 1983. Slowing of the axonal transport of neurofilament proteins during development. J. Neurosci. 3, 1694-1700. Hoffman, P. N., Griffin, J. W. and Price D. L., 1984. Control of axonal caliber by neurofilament transport. J. Cell Biol. 99, 705-714. Hoffman, P. N., Griffin, J. W., Gold, B. S. and Price, D. L., 1985a. Slowing of neurofilament transport and the radial growth of developing nerve fibers. J. Neurosci. 5, 2920~2929. Hoffman, P. N., Thompson, G. W., Griffin,J. W. and Price D. L., 1985b. Changes in neurofilament transport coincide temporally with alterations in the caliber of axons in regenerating motor fibers.J. Cell Biol. 101, 1332 1340. Hoffman, P. N., Cleveland,D. W., Griffin,J. W., Landes, P. W., Cowan, N. J. and Price, D. L., 1987. Neurofilament gene expression:A major determinant of axonal caliber. Proc. natn. Acad. Sci. U.S.A. 84, 3472 3476. Hoffman, P. N., Pollock, S. C. and Striph, G. G., 1993. Altered gene expression after optic nerve transection: reduced neurofilament expression as a general response to axonal injury. Expl Neurol. 119, 32 36. Julien, J.-P., Meyer, D., Flavell, D., Hurst, J. and Grosveld, F., 1986. Cloning and developmental expression of the murine neurofilament gene family. Molec. Brain Res. 1, 243 250. Julien, J.-P., Grosveld, F., Yazdanbaksh, K., Flavell, D., Meijer, D. and Mushynski, W., 1987. The structure of a human neurofilament gene
Neurofilaments and Axonal Caliber (NF-L): a unique exon-intron organization in the intermediate filament gene family. Biochim. biophys. Acta 909, 10-20. Julien, J.-P., Cote, F., Beaudet, L., Sidky, M., Flavell, D., Grosveld, F. and Mushynski, W., 1988. Sequence and structure of the mouse gene coding for the largest neurofilament subunit. Gene 68, 307-314. Lasek, R. J. and Hoffman, P. N., 1976. The neuronal cytoskeleton, axonal transport and axonal growth. Cold Spring Harbor Conf. Cell Proliferation 3, 1021-1049. Lasek, R. J., 1986. Polymer sliding in axons. J. Cell Sci. 5(Suppl.), 161-179. Lasek, R. J., Paggi, P. and Katz, M. J., 1992. Slow axonal transport mechanisms move neurofilaments relentlessly in mouse optic axons. J. Cell Biol. 117, 607-616. Lewis, S. A. and Cowan, N. J., 1985. Genetics, evolution, and expression of the 68,000-mol-wt neurofilament protein: isolation of a cloned cDNA probe, d. Cell Biol. 100, 843-850. Lieberburg, I., Spinner, N., Snyder, S., Anderson, J., Goldgaber, D., Smulowitz, M., Carroll, Z., Emanuel, B., Breitner, J. and Rubin, L., 1989. Cloning of a cDNA encoding the rat high molecular weight neurofilament peptide (NF-H): developmental and tissue expression in the rat, and mapping of its human homologue to chromosomes 1 and 22. Proc. natn. dead. Sci. U.S.A. 86, 1-6. McLean, W. G., McKay, A. L. and Sjostrand J., 1983. Electrophoretic analysis of axonally transported proteins in rabbit vagus nerve. J. Neurobiol. 14, 227-236. McQuarrie, I. G., Brady, S. T. and Lasek, R. J., 1986. Diversity in the axonal transport of structural proteins: major differences between optic and spinal axons in rat. J. Neurosci. 6, 1593-1605. Monaco, S., Autilio-Gambetti, L., Lasek, R. J., Katz, M. J. and Gambetti P., 1989a. Experimental increase of neurofilament transport rate: decreases in neurofilament number and in axon diameter. J. Neuropath. Exp. Neurol. 48, 23-32. Monaco, S., Jacob, J., Jenich, H., Patton, A., Autilio-Gambetti, L. and Gambetti, P., 1989b. Axonal transport of neurofilament is accelerated in peripheral nerve during 2,5-hexanedione intoxication. Brain Res. 491, 328-334. Monteiro, M. J., Hoffman, P. N., Gearhart, J. D. and Cleveland, D. W., t990. Expression of NF-L in both neuronal and nonneuronal cells of transgenic mice: increased neurofilament density in axons without affecting caliber. J. Cell Biol. 111, 1543-1557. Mori, H., Komiya, Y. and Kurokawa M., 1979. Slowly migrating axonal proteins. Inequalities in their rate and amount of transport between two branches of bifurcating axons. J. Cell Biol. 82, 174-184. Morris, J. R. and Lasek, R. J., 1982. Stable polymers of the axonal cytoskeleton: the axoplasmic ghost. J. Cell Biol. 92, 192-198. Muma, N. A., Hoffman, P. N., Slunt, H. H., Applegate, M. D., Lieberburg, I. and Price, D. L., 1990. Alterations in levels ofmRNAs coding for neurofilament protein subunits during regeneration. Expl Neurol. 107, 230-235. Muma, N. A, Slunt, H. H. and Hoffman P. N., 1991. Postnatal increase in neurofilament gene expression and the radial growth of axons. J. Neurocytol. 20, 844-854. Myers, M. W., Lazzarini, R. A., Lee, V. M. Y., Schlaepfer, W. W. and Nelson, D. L., 1987. The human mid-size neurofilament subunit: a repeated protein sequence and the relationship of its gene to the intermediate filament gene family. E M B O J. 6, 1617-1626. Napolitano, E. W., Chin, S. S. M., Colman, D. R. and Liem, R. K. H., 1987. Complete amino acid sequence and in vitro expression of rat NF-M, the middle molecular weight neurofilament protein. J. Neurosci. 7(8), 2590-2599. Nixon, R. A. and Logvinenko, K. B., 1986. Multiple fates of newly
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synthesized neurofilament proteins: evidence for a stationary neurofilament network distributed nonuniformly along axons of retinal ganglion cell neurons. J. Cell Biol. 102, 647-659. Paggi, P. and Lasek R. J., 1987. Axonal transport of cytoskeletal proteins in oculomotor axons and their residence times in axon terminals. J. Neurosci. 7, 2397-2411. Parhad, I. M., Clark, A. W. and Griffin, J. W., 1987. Effect of changes in neurofilament content on caliber of small axons: the iminodipropionitrile model. J. Neurosci. 7, 2256-2263. Parhad, I. M., Swedberg, E. A., Hoar, D. I., Krekoski, C. A. and Clark, A. W., 1988. Neurofilament gene expression following flff-iminodipropionitrile (IDPN) intoxication. Molec. Brain Res. 4, 293-301. Price, R. L., Lasek, R. J. and Katz, M. J., 1990. Internal axonal cytoarchitecture is shaped locally by external compressive forces. Brain Res. 530, 205-214. Robinson, P. A., Wion, D. and Anderton, B. H., 1986. Isolation of a eDNA for the rat heavy neurofilament polypeptide (NF-H). FEBS Left. 209(2), 203-205. Roots, B. I., 1983. Neurofilament accumulation induced in synapses by leupeptin. Science 221, 971-972. Schlaepfer, W. W. and Bruce, J., 1990. Simultaneous up-regulation of neurofilament proteins during the postnatal development of the rat nervous system. J. Neurosci. Res. 25, 39-49. Shneidman, P. A., Carden, M. J., Lees, J. F. and Lazzarini, R. A., 1988. The structure of the largest murine neurofilament protein (NF-H) as revealed by eDNA and genomic sequences. Molec. Brain Res. 4, 217-231. Shaw, G. and Weber K., 1982. Differential expression of neurofilament triplet proteins in brain development. Nature 298, 277-279. Tetzlaff, W., Alexander, S. W., Miller, F. D. and Bisby M. A., 1991. Response of facial and rubrospinal neurons to axotomy: changes in mRNA expression for cytoskeletal proteins and GAP-43. J. Neurosci. 11, 2528-2544. Troncoso, J. C., Hoffman, P. N., Griffin, J. W., Hess-Kozlow, K. M. and Price D. L., 1985. Aluminum intoxication: a disorder of neurofilament transport in motor neurons. Brain Res. 342, 172-175. Verge, V. M. K., Tetzlaff, W., Bisby, M. A. and Richardson, P. M., 1990. Influence of nerve growth factor on neurofilament gene expression in mature primary sensory neurons. J. Neurosci. 10, 2018-2025. Watson, D. F., Fittro, K. P., Hoffman, P. N. and Griffin J. W., 1991. Phosphorylation-related immunoreactivity and the rate of transport of neurofilaments in chronic 2,5-hexanedione intoxication. Brain Res. 539, 103-109. Weiss, P. A. and Mayr, R., 1971. Organelles of neuroplasmic ("axonal") flow: Neurofilaments. Proc. natn. Acad. Sci. U.S.A. 68, 846-850. Willard, M. and Simon C., 1983. Modulations of neurofilament axonal transport during the development of rabbit retinal ganglion cells. Cell 35, 551-559. Wong, J. and Oblinger M. M., 1987. Changes in neurofilament gene expression occur after axotomy of dorsal root ganglion neurons: an in situ hybridization study. Metab. Brain Dis. 2, 291-303. Yamasaki, H., Itakura, C. and Mizutani, M., 1991. Hereditary hypotrophic axonopathy with neurofilament deficiency in a mutant strain of the Japanese quail. Acta Neuropath. 82, 427-434. Yamasaki, H., Bennett, G. S., Itakura, C. and Mizutani, M., 1992. Defective expression of neurofilament protein subunits in hereditary hypotrophic axonopathy of quail. Lab. Invest. 66, 734-743. Zopf, D., Hermans-Borgmeyer, I., Gundelfinger, E. D. and Betz, H., 1987. Identification of gene products expressed in the developing chick visual system: characterization of a middle-molecular-weight neurofilament cDNA. Genes Dev. 1, 699-708.
Pergamon
Neurofilaments are Intrinsic Determinants of Axonal Caliber N A N C Y A. M U M A * a n d P A U L N. H O F F M A N t
*Department of Pharmacology, Loyola University Chicago Stritch School of Medicine, 2160 South First Avenue, Maywood, IL 60153, U.S.A. t Departments of Ophthalmology and Neurology, 515 Pathology Building, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21205, U.S.A.
Abstract--Neurofilaments(NFs) are intrinsic determinants ofaxonal caliber in large-caliber myelinated nerve fibers. Axonal caliber is influenced by the number of NFs in the axon (axonal NF content) and the spacing between adjacent NFs (interfilament distance). Axonal NF content depends on the level ofNF gene expression, the amount of NF protein entering the axon, and the velocity at which NF protein moves within the axon. In this review we discuss possible factors affecting both axonal NF content and interfilament distance; these include the ratio of NF subunits, the level of phosphorylation of these subunits, interactions with target cells, and interactions with ensheathing glial cells. Key words: Neurofilament, axon, myelinated nerve.
CONTENTS I. II. III. IV. V.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The composition of axonal neurofilaments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurofilament metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interfilament spacing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors that influence axonal neurofilament content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Neurofilament gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The entry of neurofilaments into the axonal transport system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The velocity of neurofilament transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. I N T R O D U C T I O N I n this review we will c o n s i d e r evidence suggesting that n e u r o f i l a m e n t s ( N F s ) , the p r i n c i p a l i n t e r m e d i a t e filam e n t s (IFs) of m a t u r e n e u r o n s , are i m p o r t a n t determin a n t s of a x o n a l caliber in large-caliber m y e l i n a t e d nerve fibers. N a t u r e has evolved two strategies to achieve fast impulse c o n d u c t i o n velocities in nerve fibers. O n e strategy, e m p l o y e d by i n v e r t e b r a t e species, is the f o r m a t i o n of so-called g i a n t a x o n s with d i a m e t e r s of several h u n d r e d ~tms. T h e o t h e r m e c h a n i s m , f o u n d exclusively in vertebrates, is the f o r m a t i o n o f myelin sheaths a r o u n d axons. M y e l i n a t i o n allows a x o n s with d i a m e t e r s two o r d e r s of m a g n i t u d e less t h a n those of g i a n t axons to achieve c o n d u c t i o n velocities c o m p a r a b l e to those of giant axons. Since the c o n d u c t i o n velocity o f m y e l i n a t e d nerve fibers is directly p r o p o r t i o n a l to a x o n a l d i a m e t e r (Gasser a n d G r u n d f e s t , 1939), the factors which d e t e r m i n e a x o n a l caliber have a p r o f o u n d influence o n this i m p o r t a n t p h y s i o l o g i c a l p r o p e r t y o f neurons. T h e first hint t h a t N F c o n t e n t influences a x o n a l caliber c a m e from m o r p h o l o g i c a l studies d e m o n s t r a t i n g t h a t
677 678 678 679 679 679 681 681 682 682
N F s are the m o s t n u m e r o u s cytoskeletal elements in large-caliber m y e l i n a t e d a x o n s a n d t h a t the n u m b e r s of N F s correlates closely with a x o n a l cross-sectional areas (Friede a n d S a m o r a j s k i , 1970; Weiss a n d M a y r , 1971). A m o r e d y n a m i c d e m o n s t r a t i o n of the r e l a t i o n s h i p between a x o n a l caliber a n d N F c o n t e n t c a m e from studies e x a m i n i n g the spatial a n d t e m p o r a l e v o l u t i o n of the r e d u c t i o n s in a x o n a l caliber which o c c u r in the p r o x i m a l s t u m p s of injured nerve fibers (Cragg a n d T h o m a s , 1961). M o r p h o m e t r i c analyses d e m o n s t r a t e d t h a t these reductions in a x o n a l cross-sectional a r e a correlate with p r o p o r tional decreases in a x o n a l N F c o n t e n t which start p r o x i m a l l y n e a r the n e u r o n cell b o d y (soma) a n d p r o c e e d s o m a t o f u g a l l y a l o n g nerve fibers at the rate of N F t r a n s p o r t (Hoffman et al., 1984). In contrast, the n u m b e r of m i c r o t u b u l e s in these axons r e m a i n s relatively u n c h a n g e d ; the density of m i c r o t u b u l e s increases in p r o p o r t i o n to the r e d u c t i o n in cross-sectional a r e a (Hoffman et al., 1984). D i r e c t evidence for the role of N F s in the c o n t r o l of a x o n a l caliber in m y e l i n a t e d fibers comes from recent studies of a x o n a l m o r p h o l o g y in a m u t a n t strain of quail 677
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N.A. Mumaand P. N. Hoffman
(quiver) whose neurons are devoid of NFs. Although myelinated axons are present in these animals, the cytoskeletons of these axons are comprised exclusively of microtubules. All of the myelinated axons in quiver quail are much thinner than the large-caliber myelinated axons in control animals (Yamasaki et al., 1991, 1992). Similarly, myelinated axons are devoid of NFs and are much thinner than comparable axons in control animals in a line of transgenic mice expressing a transgene construct (i.e. in which the carboxy-terminal tail of NF-H is conjugated to/3-galactosidase) which prevents NFs from entering axons. NFs accumulate in neuron cell bodies. (J. Eyer, personal communication). NF content does not appear to be a major determinant of axonal caliber in either unmyelinated axons, which are uniformly thin and largely devoid of NFs, or in small-caliber myelinated fibers, which contain relatively few NFs compared to large-caliber myelinated axons (Berthold, 1978). The cross-sectional areas of small-caliber myelinated axons, such as those in the optic nerve, do not correlate closely with axonal NF content (Berthold, 1978; Nixon and Logvinenko, 1986). Furthermore, alterations in axonal NF content in optic nerve axons induced by intoxication with an agent which impairs NF transport in axons (/~,/~'iminodipropionitrile (IDPN)) (Griffin et al., 1978), do not correlate with proportional changes in axonal crosssectional areas (Parhad et al., 1987).
II. THE COMPOSITION OF AXONAL NEUROFILAMENTS The subunit composition of NFs appears to be related to their proposed function as intrinsic determinants of axonal caliber. Neurofilaments are normally comprised of three protein subunits, NF-L, NF-M and NF-H, with apparent molecular weights of 68, 145 and 200kDa, respectively (Hoffman and Lasek, 1975). Immunohistochemical analyses demonstrate that NF-L is associated with the core of the NF, while NF-H, and to a lesser extent NF-M, are associated with NF sidearms (Hirokawa et al., 1984; Hisanaga and Hirokawa, 1988). Although NF-L, alone is capable of self-assembly into filaments in a cellfree system (Geisler and Weber, 1981 ), recent transfection experiments in which genes encoding NF subunits were introduced into a line of cultured cells lacking endogenous IFs suggest that at least two subunits (NF-L and NF-M) are required for filament assembly in vivo (M. K. Lee, personal communication). This is consistent with the observation that filaments comprised of NF-L and NF-M (i.e. lacking NF-H) are present in the developing neurons of embryonic and neonatal animals (Shaw and Weber, 1982; Carden et al., 1987; Schlaepfer and Bruce, 1990). Each neurofilament subunit is encoded by a separate gene (Lewis and Cowan, 1985; Robinson et al., 1986; Julien et al., 1986, 1987; Myers et al., 1987; Napolitano et al., 1987; Zopf et al., 1987; Dautigny et al., 1988; Shneidman et al., 1988; Lieberburg et al., 1989). Like all other IF proteins, each NF subunit contains a highly-
conserved coiled-coil rod domain (Geisler et al., 1983). NF-M and NF-H are unique among the IF proteins in that they contain extended carboxy-terminal tail domains. The carboxy-terminal tail domains of NF-M and NF-H contain multiple repeats (up to 50 for NF-H) of the K-S-P (lysine-serine-proline) sequence; these serine residues are major sites ofNF phosphorylation (Myers et al., 1987; Julien et al., 1988). The presence of highlycharged phosphate groups associated with NF sidearms may promote the repulsion of adjacent NFs and contribute to their function as intrinsic determinants of axonal caliber.
III. NEUROFILAMENT METABOLISM The genes encoding the NF proteins are transcribed and translated in the neuron cell body. The NF proteins then enter the axon where they move somatofugally at rates of several mm/day in the SCa component of slow axonal transport (Hoffman and Lasek, 1975; Lasek and Hoffman, 1976). The observation that essentially all of the NF protein in the axon is associated with NF polymers (Morris and Lasek, 1982) favors a model in which the NF proteins are assembled into polymers in the cell body and undergo transport in their polymeric form (Lasek, 1986). An alternative model proposes that the NF subunits are transported as either protofilaments or unassembled subunits (Nixon and Logvinenko, 1986). The proposal that axons contain a substantial population of stationary NFs has been controversial. This hypothesis is based on the observation that pulse-labeled NF proteins are retained along optic nerve axons after passage of the SCa wave (Nixon and Logvinenko, 1986). Recent data indicating that some of the pulse-labeled NF protein in this system is transported at rates much slower than the SCa wave provides an alternative explanation for this finding (Lasek et al., 1992). Relatively little is known about the mechanism of NF transport. The rate of NF transport differs in various classes of neurons. For example, the rate is an order of magnitude faster in peripheral sensory and motor fibers than optic nerve axons (McQuarrie et al., 1986). The rate of NF transport also declines with age (Hoffman et al., 1983) and with increasing distance along peripheral nerve fibers (Hoffman et al., 1985a). The available evidence suggests that there is relatively little turnover of transported NFs until they reach the axon terminals. The proposal that axonally-transported NF proteins are normally degraded by calcium-activated proteolysis as they enter the axon terminals (Lasek and Hoffman, 1976) is supported by the observation that the injection of leupeptin, an inhibitor of calcium-activated proteolysis, into goldfish brain results in the abnormal accumulation of NFs in axon terminals (Roots, 1983). This concept is also supported by the demonstration that pulse-labeled NF proteins disappear shortly after entering the axon terminals (Paggi and Lasek, 1987; Garner, 1988).
Neurofllaments and Axonal Caliber
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undergoing axonal transport primarily reflects the level of N F gene expression.
Neuron-Target Interactions
V A. Neurofilament gene expression
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@ Fig. 1. Regulationof axonal caliber.Axonalcaliberis dependent on axonal neurofilament (NF) content and interfilament distance. AxonalNF content is regulatedby (1) the levelofN F gene expression, (2) entry of filaments into the slow axonal transport system and (3) the rate of transport of NFs in the axon. Factors such as the levelof NF phosphorylation,the ratio of NF subunit proteins,neuron-target interactionand neuron-glialinteraction may influenceaxonal NF content and interfilament distance. IV. I N T E R F I L A M E N T S P A C I N G The caliber of myelinated axons is affected by the N F content of axons and by the spacing between adjacent NFs. Interfilament distance varies among various classes of neurons and within different regions of the same axon; regions with small calibers (i.e. nodes of Ranvier) have decreased interfilament spacing compared to internodal regions (Price et al., 1990). Clues about the regulation of interfilament spacing have come from experimental models in which interfilament distance is altered. In these models factors such as N F subunit ratios or N F phosphorylation have been associated with alterations in interfilament distance. Interfilament spacing decreased in peripheral fibers of a transgene model which overexpressed NF-L; axonal caliber is slightly reduced in these axons (Monteiro et al., 1990). In control mice which were grafted with nerve segments from trembler mice, reductions in interfilament distance are associated with decreases in N F phosphorylation (deWaegh et al., 1992). V. FACTORS T H A T I N F L U E N C E AXONAL NEUROFILAMENT CONTENT The factors which influence axonal N F content are better defined than those which control interfilament spacing. We will now consider evidence indicating that the N F content of axons is influenced by the level of N F synthesis (gene expression), the amount of N F protein entering the axonal transport system and the N F transport velocity (Fig. 1). The amount of protein
Axonal N F content correlates closely with the level of N F gene expression in the neuron cell body. In general, mature neurons containing relatively low levels of N F mRNAs give rise to either unmyelinated axons (e.g. small sensory neurons) (Hoffman et al., 1987) or small-caliber myelinated fibers (e.g. retinal ganglion cell neurons) (Hoffman et al., 1993). Conversely, mature neurons with relatively high levels of N F expression such as ventral motor neurons (Muma et al., 1990; Tetzlaff et al., 1991) and large sensory neurons (Hoffman et al., 1987) give rise to large-caliber myclinated axons. These differences in axonal N F content also correlate with differences in the relative amounts of N F protein undergoing transport in these axons. Relatively little pulse-labeled N F protein is transported in either unmyelinated fibers (McLean et al., 1983) or optic nerve fibers (Black and Lasek, 1980), compared to large-caliber peripheral sensory (Mori et al., 1979) and motor fibers (Hoffman and Lasek, 1975; McQuarrie et al., 1986). Perhaps the best illustration of the relationship between axonal N F content and the level of N F expression is found in systems where changes in the level of N F expression correlate with comparable alterations in axonal N F content. Such changes have been studied extensively during postnatal development and after axonal injury (axotomy). Reductions in neurofilament expression after axotomy (Hoffman et al., 1987; Wong and Oblinger, 1987; Greenberg and Lasek, 1988; Goldstein et al., 1988; Muma et al., 1990; Tetzlaff et al., 1991) correlate with a decrease in the amount of pulse-labeled N F protein transported in the proximal stumps of injured nerve fibers (Hoffman et al., 1985b) and reductions in the N F content and caliber of axons in the proximal stump (Hoffman et al., 1984). Conversely, increased neurofilament expression in peripheral sensory neurons during early post-natal development correlates with increases in the neurofilament content and caliber of axons (Fig. 2) (Muma et al., 1991). Neurofilament expression is low in embryonic neurons, whose axons lack NFs. Expression increases dramatically during early postnatal development in neurons giving rise to myelinated axons; this correlates with the onset of radial growth of axons. As seen in the axotomy model, the abundance of mRNA encoding neurofilaments correlates closely with neurofilament protein levels. Neurofilament expression remains low in mature neurons with unmyelinated axons. Changes in neurofilament gene expression correlate selectively with radial growth; the expression of tubulin and actin, the other major cytoskeletal proteins in axons, declines as radial growth occurs during early post-natal development (Muma et al., 1991). The level of N F gene expression appears to be influenced by interactions between neurons and their targets. Reduced neurofilament gene expression after axotomy appears to reflect the loss of neuron-target
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A b
c
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NF-L
NF-H
Tub
Act
Fig. 2. Neurofilament gene expression during early post-natal development affects axonal caliber. RNA (Northern) blots (A) demonstrate selective increases in low molecular weight neurofilament subunit (NF-L) and the high molecular weight neurofilament subunit (NF-H) mRNA levels at 5 (b), 11 (c) and 28 (d) days of age in rat dorsal root ganglia. In contrast, levels of mRNAs encoding fl-tubulin (Tub) and actin (Act) decrease during this period. Electron micrographs of dorsal root nerve fibers demonstrate a progressive, age-related increase in the calibers of myelinated axons at 5 (B), 11 (C) and 28 (D) days of age. All micrographs are shown at the same magnification. Scale marker indicates 2 p.m.
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interactions, while the return of neurofilament expression to pre-axotomy levels depends on the reconnection of regenerating axons with targets (Hoffman et al., 1984, 1987; M u m a et al., 1990). Trophic factors produced by target cells may play a role in this process. In the case of axotomized sensory neurons, exogenous nerve growth factor appears to reduce the severity of reductions in N F expression (Verge et al., 1990) and axonal caliber (Gold et al., 1991).
B. The entry of neurofilaments into the axonal transport system The amount of neurofilament protein entering the axonal transport system from the cell body affects the axonal neurofilament content and thus axonal caliber. Axons with reduced calibers are seen in models in which the amount of neurofilament protein entering the axon has been decreased. As mentioned earlier, axonal caliber (of myelinated fibers) is greatly reduced in mice expressing a transgene (i.e. in which the carboxy-terminal portion of N F - H is conjugated to fl-galactosidase) which prevents N F s from entering the axon. Distal axonal caliber is also reduced in select neuronal populations in aluminum treated rabbits. In these neuronal groups, the amount of neurofilament protein entering the slow axonal transport system is reduced (without alterations in the rate of transport of those neurofilaments which do enter the slow axonal transport system) (Bizzi et al., 1984; Troncoso et al., 1985). NFs which do not enter the axonal transport system accumulate in swollen neuron cell bodies (Fig. 3).
Fig. 3. Entry of filamentsinto the axonal transport systemaffects axonal caliber. Photomicrographs of neurons in spinal cords of control (A) and aluminum (B) treated rabbits demonstrate accumulations of neurofilaments (NFs) in swollen cell bodies induced by aluminum salts. In the aluminum treated animals the distribution of NFs within the neurons is altered; fewer neurofilamentsenter the slow axonal transport systemand distal axonal caliber is reduced. Scale marker indicates 50 lam.
C. The velocity of neurofilament transport N F content in any region of an axon is influenced by the rate at which N F s enter that region via axonal transport and the rates at which N F s leave via axonal transport and turnover. Therefore, if we assume that turnover is relatively low, the N F content of a local region of the axon (e.g. an internodal segment, which is the portion of a myelinated axon located between adjacent nodes) is largely determined by the difference between the rates at which N F s enter and leave that segment. The most dramatic example of this mechanism is illustrated after systemic intoxication with I D P N , a neurotoxin which impairs N F transport along the axon (Griffin et al., 1978), but does not affect either the level of N F synthesis (Parhad et al., 1988) or the entry of newly-synthesized N F s into the axon (Griffin et al., 1978). NFs enter the first internodal segment of the axon, presumably at their normal rate, but leave that internodal segment at a greatly reduced rate (i.e. at velocities approaching zero). This leads to the local accumulation of N F s and to the formation of giant axonal swellings filled with N F s (Fig. 4) (Chou and Hartmann, 1964, 1965; Clark et al., 1980). A similar mechanism may contribute to the increase in axonal N F content which occurs during the maturation of developing nerve fibers (Friede and Samorajski, 1970; Berthold, 1978; M u m a et al., 1991). In these axons the
Fig. 4. The axonal transport velocity of neurofilament (NFs) affectsaxonal caliber. Systemicintoxication with IDPN impairs the transport of NFs within axons (without alteringthe transport of NFs from the cell body into the axon) and leads to the formation of proximal axonal swellings. This electron micrograph of a paranodal region illustrates that these swellingsreflect the accumulation of axonal NFs. Scale marker indicates 1 lam.
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velocity of N F t r a n s p o r t progressively declines with increasing distance a l o n g the nerve (Hoffman et al., 1985a). This allows N F s to enter each i n t e r n o d a l segment slightly faster t h a n they leave, leading to a net a c c u m u l a t i o n over time. These examples suggest that local reductions in the velocity of N F t r a n s p o r t result in increases in a x o n a l N F content. Conversely, increases in the velocity of N F t r a n s p o r t i n d u c e d by systemic i n t o x i c a t i o n with 2,5h e x a n e d i o n e (2,5-HD) result in a decrease in a x o n a l N F c o n t e n t in the p r o x i m a l a x o n ( M o n a c o et al., 1989b). A l t h o u g h relatively little is k n o w n a b o u t the mecha n i s m of N F t r a n s p o r t , alterations in the velocity of N F t r a n s p o r t correlate with changes in either the ratio of N F s u b u n i t proteins or the levels of p h o s p h o r y l a t i o n of these proteins. I n n e o n a t a l optic nerves, a r e d u c t i o n the velocity of N F t r a n s p o r t correlates with the a d d i t i o n of N F - H to a x o n a l N F s ; prior to that time the N F s in these a x o n s are comprised almost entirely of N F - L a n d N F - M (Willard a n d Simon, 1983). After chronic i n t o x i c a t i o n with 2,5h e x a n e d i o n e , increases in the rate of N F t r a n s p o r t correlate with reductions in the p h o s p h o r y l a t i o n of N F - H ( W a t s o n et al., 1991). C h a n g e s in the rate of N F t r a n s p o r t associated with myelin f o r m a t i o n also correlate with alterations in the level of p h o s p h o r y l a t i o n of N F - M (deWaegh et al., 1992).
VI. C O N C L U S I O N S N e u r o f i l a m e n t s are intrinsic d e t e r m i n a n t s of axonal caliber in large caliber m y e l i n a t e d fibers. A x o n a l caliber is influenced by b o t h the n u m b e r of N F s (axonal N F c o n t e n t ) a n d the spacing between adjacent N F s (interfilam e n t distance). A x o n a l n e u r o f i l a m e n t c o n t e n t depends o n the level of n e u r o f i l a m e n t gene expression, the a m o u n t of N F p r o t e i n entering the axon, a n d the velocity at which this N F p r o t e i n moves within the axon. The caliber of m y e l i n a t e d axons can be regulated by factors which influence a n y of these three m e c h a n i s m s or by those which affect interfilament distance.
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