Correspondence to: Robert A. Lazzarini, Brookdale Center for Developmental and Molecular Biology, Box 1126, Mount Sinai School of Medicine, New York, NY 10029., rlazzar{at}smtplink.mssm.edu (E-mail), (212) 241-4272 (phone), (212) 860-9279 (fax)
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Abstract |
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Neurofilaments are central determinants of the diameter of myelinated axons. It is less clear whether neurofilaments serve other functional roles such as maintaining the structural integrity of axons over time. Here we show that an age-dependent axonal atrophy develops in the lumbar ventral roots of mice with a null mutation in the mid-sized neurofilament subunit (NF-M) but not in animals with a null mutation in the heavy neurofilament subunit (NF-H). Mice with null mutations in both genes develop atrophy in ventral and dorsal roots as well as a hind limb paralysis with aging. The atrophic process is not accompanied by significant axonal loss or anterior horn cell pathology. In the NF-Mnull mutant atrophic ventral root, axons show an age-related depletion of neurofilaments and an increased ratio of microtubules/neurofilaments. By contrast, the preserved dorsal root axons of NF-Mnull mutant animals do not show a similar depletion of neurofilaments. Thus, the lack of an NF-M subunit renders some axons selectively vulnerable to an age-dependent atrophic process. These studies argue that neurofilaments are necessary for the structural maintenance of some populations of axons during aging and that the NF-M subunit is especially critical.
Key Words: aging, axonal atrophy, neurofilament proteins, neuronal cytoskeleton, knockout mice
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Introduction |
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NEUROFILAMENTS (NFs)1 are the most prominent cytoskeletal elements in large myelinated axons. Three proteins, known as the light (NF-L), mid-sized (NF-M), and heavy (NF-H) NF subunits are the principal constituents of NFs. Each subunit is the product of a separate gene (
The NF proteins are members of the family of intermediate filament proteins ( helical rod domain of ~310 amino acids with variable NH2-terminal and COOH-terminal regions. The COOH-terminal domains of NFs, however, differ from other intermediate filament proteins in being greatly extended. These extensions contain a glutamic acid rich region of unknown significance and in NF-M and NF-H a series of lysine-serine-proline (KSP) repeats (
helical rod domains. By contrast, the phosphorylated COOH-terminal tail sequences of NF-M and NF-H are found in filament sidearms that extend away from the core filaments (
Although much is known about NF structure and assembly many questions remain concerning NF function. NFs are most prominent in large axons (
More recently several animal models including a Japanese quail (Quiver) with a spontaneous mutation in the NF-L gene (Yamasaki et al., 1992;
Recently, we and others have generated mice lacking the NF-M (
To determine if axonal stability or other pathological effects develop with aging in mice lacking selective NF subunits we examined 2-yr-old NF-M, NF-H, and NF-M/Hnull mutant mice along with 2-yr-old wild-type controls. Here we show that an age-dependent axonal atrophy develops in the lumbar roots of NF-M and NF-M/Hdeficient mice but not in animals deficient only in the NF-H subunit. Thus the lack of an NF-M subunit renders some axons selectively vulnerable to an age-related atrophic process.
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Materials and Methods |
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Generation of Mice with NF-M and NF-Hnull Mutations
As described elsewhere, mice with singly disrupted NF-M (
Measurement of Axonal Diameters
Axonal diameters were measured as described previously (
Electron Microscopy
Tissues were processed for electron microscopy by standard methods as described previously (
NFs and microtubules (MTs) were counted in cross-sectional images of axons photographed at a magnification of 20,000 and enlarged an additional 2.5-fold during printing. NF densities were determined as described previously (
Preparation of Specimens for Immunoperoxidase Staining
Control and mutant mice were perfused with buffered 4% paraformaldehyde and 50-µm-thick sections of spinal cord were cut with a vibratome. Immunoperoxidase staining was performed with the monoclonal antibodies SMI-31 or SMI-32 (Sternberger Monoclonals Inc.) or with a rabbit antiNF-L polyclonal antiserum provided by Dr. Virginia Lee (University of Pennsylvania, Philadelphia, PA). Primary antibodies were diluted 1:1,000 in PGBA (0.12 M phosphate buffered saline, 0.1% gelatin, 1% BSA, 0.05% sodium azide) and were visualized with species-specific biotinylated secondary antibodies (Amersham Pharmacia Biotech) followed by peroxidase conjugated streptavidin (Jackson ImmunoResearch Laboratories Inc.). Peroxidase reactions were developed with diaminobenzadine. Preparations were examined and photographed with a Zeiss Axiophot microscope.
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Results |
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Ventral Root Axons Atrophy with Aging in Mice Lacking a Mid-sized Neurofilament Subunit
Previously we produced mice with null mutations in the NF-M (
At the light microscopic level no abnormalities were noted in any of the control animals. No abnormalities were noted in any of the NF null mutant animals in the brain, spinal cord, or optic nerves except that as in 4-mo-old NF-null mutants, the myelinated axons in all regions were visibly smaller.
By contrast, many of the ventral lumbar roots in 2-yr-old NF-M and essentially all of the lumbar ventral roots in the 2-yr-old NF-M/Hnull animals showed pathological changes. Examples of lumbar ventral roots from wild-type, NF-M, NF-H, and NF-M/Hnull mutant animals are shown in Figure 1. Myelinated axons in the NF-M and NF-M/H mice were frequently irregular in shape and appeared shrunken and collapsed, resulting in axonal profiles that were dramatically smaller than wild-type axons. In the NF-M/Hnull mutants occasional dystrophic axons with accumulations of cellular organelles and multilamellar membranous profiles could also be seen. Occasionally, giant ballooned axons could also be seen in NF-M/Hdeficient roots. More rarely, degenerating profiles could also be seen in the NF-Mnull mutants. However, such changes occurred in <1% of the axonal populations in either mutant, although they were never observed in the controls. There was no evidence in either mutant of macrophage infiltration or other features of Wallerian degeneration. Accompanying the axonal shrinkage and collapse there was frequently an expansion of the endoneurial space.
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Similar changes were seen in 9 of 20 ventral roots examined in NF-Mnull mutant animals and examples of atrophic roots could be seen at all lumbar levels examined (L3 through L5). All 19 roots examined in the NF-M/H showed dramatic shrinkage of axonal diameter. By contrast none of 15 ventral roots from the three 2-yr-old wild-type animals and none of 18 ventral roots from the 2-yr-old NF-Hnull mutants showed any changes similar to those in the NF-M and NF-M/H animals. Thus, lumbar ventral roots in NF-M and NF-M/Hdeficient animals but not NF-Hdeficient animals develop an axonal atrophy with aging.
Interestingly, in these same animals the pattern of selectivity was different in the lumbar dorsal roots. Examples of dorsal roots from 2-yr-old wild-type, NF-M, NF-H, and NF-M/Hdeficient animals are shown in Figure 1. Whereas lumbar dorsal root axons in the NF-M/Hdeficient animals exhibited similar changes to those in the ventral roots, none of 16 lumbar dorsal roots from the 2-yr-old NF-Mdeficient animals showed any obvious changes. Dorsal roots were also unremarkable in appearance in the NF-Hnull mutant animals. Thus, dorsal root axons appear to be less sensitive to the loss of the NF-M subunit, although removal of both the NF-M and NF-H subunits renders these axons vulnerable to the atrophic process.
Previously we found that in 4-mo-old NF-Mnull mutant animals axonal diameters in the ventral roots are decreased by ~20% (
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Examination of the frequency distribution of axons in the ventral roots (Figure 2 A) shows the dramatic depletion of large axons in the NF-M and NF-M/Hnull mutant roots. Whereas >70% of axons in the NF-Hnull mutant and control were >5 µm, few (<3%) were >5 µm in the NF-M or NF-M/Hnull mutants. By contrast, in the dorsal roots a different pattern was seen (Figure 2 B). Although both the NF-M and NF-Hnull mutant roots contain fewer large diameter axons than the control, the distribution of axonal sizes in the NF-M root appeared more similar to the NF-Hnull mutant and wild-type roots than to the NF-M/H. In contrast to the NF-M/Hnull mutant in which <5% of dorsal root axons were >2.5 µm, >35% of axons in the wild-type, NF-M, and NF-H mutant roots were >2.5 µm. Table 1 shows that, as expected from this distribution, average axonal diameter and area are also relatively preserved in the NF-M compared to the NF-M/Hnull mutant dorsal roots.
To determine if axons were being lost in the NF-M or NF-M/Hnull mutants the number of axons remaining in the ventral roots of 2-yr-old mutant and control roots were counted. There was no significant difference in axonal counts in the L3, L4, or L5 ventral roots or the L4 dorsal root between wild-type, NF-M, and NF-M/Hnull mutant animals (Figure 2 C). In the NF-M mutant there also did not appear to be a significant difference in the number of surviving axons when comparing roots that were clearly atrophic with roots that were less affected by the process (data not shown). Thus, we conclude that permanent axonal loss is not a major feature of the pathological process and that the depletion of large axons in the ventral roots of NF-M and NF-M/Hnull mutants and the dorsal roots of NF-M/Hnull mutants is the result of an atrophic process.
Neurofilaments Are Depleted and the Ratio of Microtubules/Neurofilaments Is Increased in Ventral Root Axons of NF-Mdeficient Animals
To look for an ultrastructural basis for the atrophic collapse of axons in the aging NF-M and NF-M/Hnull mutants we examined electron micrographs of lumbar ventral roots from mutant and control animals as well as NF-Hnull mutant animals. Previously we found that NFs were depleted in ventral root axons of 4-mo-old NF-Mnull mutants, although the filaments were otherwise of normal configuration (
NFs were plentiful in the control and NF-Hnull mutant axons (Figure 3). Also as expected, axons in the 2-yr-old NF-M/H animals were essentially devoid of NFs. Axons in atrophic roots of old NF-Mnull mutant animals contained relatively normal appearing NFs. However, NF numbers appeared even more dramatically depleted than in axons of young NF-Mnull mutants. To quantify the effect on NF number in the old NF-Mnull mutant, NFs were counted in the internodal regions of axons over a range of sizes and NF counts were plotted against axonal area. As shown in Figure 4 A, axons in the null mutant consistently contained vastly fewer NFs than axons in controls with the mutant axons having only ~20% as many NFs as a comparably sized wild-type axons.
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NF densities were determined directly as described in Figure 4 B (also see Table 2). NF density was reduced from 180/µm2 in 2-yr-old control axons to 62/µm2 in the 2-yr-old mutant (P < 0.0001). Thus, compared to 4-mo-old NF-Mnull mutants, NFs are even further depleted in axons of old NF-Mnull mutant animals (34% of control in 2-yr-old vs. 43% in 4-mo-old, P < 0.0001 for 1 yr vs. 2 yr).
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By contrast, these same axons contained relatively more MTs. Axons in the NF-Mnull mutant contained nearly double the number of MTs found in comparably sized wild-type axons (Figure 5 A), increasing the average ratio of MTs to NFs from 0.18 ± 0.9 (SD) in wild-type to 1.57 ± 1.13 in the mutant axons (P < 0.0001, see Figure 5 B and Table 2). By comparison, MT to NF ratios in 4-mo-old NF-Mnull mutants increase from 0.22 in wild-type to 0.83 in mutant axons (
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Thus, aging in the NF-Mnull mutant is associated with a loss of NFs from axons that already possess a depleted NF content and is accompanied by a major reorganization of the axoplasm towards a MT-based cytoskeleton. It has long been known that NF number correlates better with axonal diameter than MT number, particularly in larger axons (
Relative Preservation of Neurofilament Numbers in Unaffected Dorsal Root Axons of NF-Mdeficient Animals
We also examined wild-type and NF-Mmutant dorsal root axons from 2-yr-old animals to determine if the depletion of NFs was a selective effect seen only in vulnerable ventral root axons. Interestingly, NF depletion in the dorsal roots did not occur to the same extent as in ventral roots. NF densities were 92/µm2 in 2-yr-old NF-Mnull mutant roots compared to 162/µm2 in the 2-yr-old controls (P < 0.0001) and the ratio of MTs/NFs was 0.51 ± 0.39 in mutant and 0.15 ± 0.08 in 2-yr-old control (P < 0.0001). As shown in Figure 4 C (see also Table 2), whereas little difference exists between NF densities in control dorsal and ventral root axons, NFs are significantly more depleted in mutant ventral than dorsal roots (P < 0.0001). We also measured NF densities in dorsal root axons of 4-mo-old and 1-yr-old NF-Mdeficient animals and found NF densities in these axons to be 89/µm2 and 104/µm2, respectively. Thus, NFs are significantly less depleted in dorsal compared to ventral root axons and dorsal root axons do not undergo the age-related decrease in NF densities seen in the ventral root axons.
Neurofilament Depletion without Atrophic Changes in Ventral Roots of One-Year-Old NF-Mnull Mutant Animals
To determine the time course of the axonal atrophy in the ventral roots we examined six 1-yr-old NF-M and four 1-yr-old NF-M/Hnull mutants. Figure 6 shows a comparison of the appearance of ventral root axons from 4-mo-, 1-yr-, and 2-yr-old wild-type and mutant animals. As expected, myelinated axons in 1-yr-old NF-M and NF-M/H animals appeared smaller than 1-yr-old control (Figure 6, DF). However, the axons appeared relatively normal in shape and we did not observe any definite pathological changes like those seen in 2-yr-old ventral roots (Figure 6, GI) in any of 42 ventral roots collected from 1-yr-old NF-M or in 24 roots from the NF-M/H animals. Thus, the atrophy is predominantly occurring after 1 yr of age. Quantitative longitudinal data on L5 ventral roots from wild-type, NF-M, and NF-M/Hnull mutant animals is presented in Table 3. Most remarkably, these data show that whereas a significant expansion of axonal caliber occurs between 4 mo and 1 yr of age in wild-type animals, axons in the NF-Mnull mutant expand only slightly and axons in NF-M/Hnull mutant roots do not expand at all. Wild-type as well as NF-M and NF-M/Hmutant axons then all undergo varying degrees of age related atrophy between one and two years of age. Interestingly, on a percentage basis the atrophy in the L5 roots between 1 and 2 yr of age in the NF-M and NF-M/Hnull mutants is actually slightly less than wild-type. However, the lower base from which the mutants start at one year causes these smaller percentage changes to have a significant absolute effect on axonal caliber at 2 yr.
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Despite the relatively maintained axonal calibers at 1 yr of age, ultrastructural analysis of ventral root axons from 1-yr-old NF-M animals revealed that the depletion of NFs had already occurred at this age. NF density (see Table 2) was 61/µm2 in 1-yr-old ventral root axons compared to the 62/µm2 noted above that was observed in 2-yr-old animals (P = 0.68). Thus, the NF depletion appears to be established before gross atrophic changes occur.
Lack of Pathology in Anterior Horn Cells or Muscle in Aging NF-M and NF-M/Hnull Mutant Mice
The lumbar ventral roots contain axons that arise from motor neurons in the lumbar spinal cord. To determine if changes in anterior horn cells might be responsible for the axonal atrophy, spinal cord sections from the lumbar and cervical levels were examined to assess anterior horn cell morphology. Light microscopy revealed no evidence for anterior horn cell degeneration in either region in 2-yr-old NF-M or NF-M/Hnull mutants (Figure 7, AC). Examination of lumbar spinal cord sections by electron microscopy also found no perikaryal, dendritic, or axonal abnormalities in the mutants (data not shown).
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To determine if NF-L or NF-H might be accumulating in anterior horn cell perikarya we immunostained spinal cord sections from NF-M and NF-M/Hnull mutants as well as controls with an antiNF-L antibody or with the antibodies SMI-31 or SMI-32, which detect phosphorylated (SMI-31) or unphosphorylated epitopes (SMI-32) on the NF-M and NF-H subunits (
To determine if the axonal atrophy was producing changes in muscle we examined toludine bluestained sections from the tibialis anterior muscles of 2-yr-old mutant and control animals. Consistent with the lack of axonal loss noted above, muscle fibers in the NF-M and NF-M/Hnull mutants appeared normal without any evidence of group atrophy or other changes suggestive of functional denervation (data not shown).
Hind Limb Paralysis Develops in Two-Year-Old NF-M/Hnull Mutant Animals
In contrast to the lack of neurological findings in young NF-deficient animals, four of five NF-M/H animals that have lived to 2 yr of age have developed a grossly apparent hind limb paralysis (Figure 8). Thus, the axonal atrophy in the lumbar roots appears to be functionally significant even though no significant axonal loss or muscle atrophy is occurring.
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Discussion |
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NFs have long been suspected to be critical determinants of axonal diameter based on the close correlation between NF number and axonal caliber (
The most important function of NF-L may well be its ability to stimulate filament formation. Axonal NFs are absent in both Japanese quail (Quiver) with a nonsense mutation in the NF-L gene (
Previous studies have also pointed to an essential role for NF-M in driving the formation or maintenance of normal numbers of NFs (
The process is best termed atrophic since it represents an abnormal collapse of axonal structure that is not seen with normal aging. The net effect is the near complete elimination of all large myelinated axons in affected nerves. The process is selective in affecting peripheral nervous system but not central nervous system axons, and in the peripheral nervous system in affecting axons in ventral but not dorsal roots in the NF-Mnull mutant. Among ventral roots in the NF-Mnull mutant the process is also selective in not affecting all ventral roots equally. The lack of any significant reduction in axonal number in either ventral or dorsal roots and the rarity of degenerating profiles suggests that the process does not result in permanent axonal loss and that the loss of large myelinated axons is due to their shrinkage to become small myelinated axons.
Some degree of perikaryal and axonal atrophy is considered a normal aspect of aging (
The ultrastructural correlate of the collapsed axonal caliber was a depletion of axonal NFs. NF densities in ventral root axons of 4-mo-old NF-Mnull mutants are reduced to 43% of the wild-type level although the filaments are otherwise of normal configuration (
By contrast, these same axons appeared to contain more MTs. NF-Mmutant axons contained nearly double the number of MTs found in comparably sized control axons resulting in myelinated axons in the NF-Mnull mutant in which MTs generally outnumbered NFs. The prominence of MTs in NF-deficient axons could reflect a true increase in MT content per axon, perhaps as a reaction to the loss of NFs. Alternatively, axons might contain their normal complement of MTs but MTs might appear increased due to a concentration effect caused by a decreasing axonal caliber. We have investigated previously these possibilities in 4-mo-old NF-M/Hnull mutant animals (
The relative roles of MTs and NFs in determining axonal diameter have long been discussed (
We do not know what underlies the basis for the selective vulnerability of ventral root axons in the NF-Mnull mutant animals. The lumbar ventral roots contain axons that arise from motor neurons in the lumbar spinal cord, whereas the lumbar dorsal roots are composed of axons emerging from the sensory neurons of the dorsal root ganglia. Overexpression of either normal or mutant NF proteins in transgenic mice can cause a motor neuron disease that resembles the human disease amyotrophic lateral sclerosis ( helical rod domain of NF-L. However, the NF-Mnull mutant differs fundamentally from the NF-L(Pro) mutation in lacking perikaryal aggregates of NFs.
The most conspicuous difference between the dorsal and ventral root axons in old NF-M animals was the ultrastructural finding that NFs are less depleted and the ratio of MTs/NFs remains closer to normal in the dorsal root axons. Interestingly, dorsal roots do atrophy in NF-M/Hdeficient axons where NFs are essentially absent. This finding may again point to a NF-based cytoskeleton being more resistant to collapse during aging than a MT-dominated one. Besides the differences in NF number, relative differences in the stresses that the dorsal and ventral roots are normally subjected to might potentiate the selective vulnerability. Additionally, levels of or posttranslational modifications to the NF-H subunit may differ in dorsal and ventral roots and leave axons with L/H filaments in the ventral roots more vulnerable to degeneration. It is well known that different neuronal populations can express different NF profiles, especially with regard to phosphorylation (
It is clear, however, that NF loss cannot be directly correlated with axonal collapse since axons in NF-M/Hnull mutants lack NFs throughout life, yet axonal collapse occurs only in old animals. NF loss is also already present by 1 yr of age in the NF-Mnull mutant although the gross collapse of axonal calibers was only seen in 2-yr-old animals. This argues that the collapse is not simply a direct result of a depleted NF number but rather a depleted NF content renders these axons more susceptible to atrophy.
The reduced NF densities must reflect fewer NFs being transported into or a decreased stability of axonal filaments that are formed. As noted above, some perikaryal and axonal atrophy is considered a normal aspect of aging (
The process described here is functionally significant with old NF-M/H animals developing a gross hind limb paralysis. We have not observed any gross paralysis in old NF-Mnull mutants. However, we have tested a group of 1-yr-old NF-Mnull mutants in the rotarod test and in preliminary studies found that their performance appears to be impaired (our unpublished observations). Thus, we suspect that future studies will document motor impairments in NF-Mnull mutants as well. One expected consequence of a reduced axonal diameter is a reduced nerve conduction velocity and this has been demonstrated to occur in the NF-Ldeficient quail (
Collectively, these results indicate that the NF-M subunit plays a previously unknown role in maintaining axonal structure with aging. These findings may have implications for neurodegenerative diseases. For example, a depleted NF content might render other neuronal populations more susceptible to excitotoxic or other insults thought to be involved in human neurodegenerative diseases. Reports have described decreased levels of NF-L mRNA beyond that seen in normal aging in Alzheimer's disease brain (
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Acknowledgements |
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We thank Dr. Ron Gordon for assistance with electron microscopy, Dr. Paul Wen for photographic assistance, and Dr. Virginia Lee for gift of antibodies.
This work was supported by the Amyotrophic Lateral Sclerosis Association (G.A. Elder), and National Institute on Aging grant P50 AG 05138 (R.A. Lazzarini).
Submitted: December 3, 1998; Revised: June 3, 1999; Accepted: June 3, 1999.
1.used in this paper: MT, microtubule; NF, neurofilament; NF-H, heavy neurofilament; NF-L, light neurofilament; NF-M, mid-sized neurofilament
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References |
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Berthold, C.-H. (1978) Morphology of normal peripheral axons. In Waxman S.G., ed. Physiology and Pathobiology of Axons. New York, Raven Press, Ltd., 3-63.
Carden, M.J., Trojanowski, J.Q., Schleapfer, W.W., Lee, V.M.-Y. (1987) Two-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation. J. Neurosci. 7:3489-3504[Abstract].
Ching, G., Liem, R. (1993) Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments. J. Cell Biol. 122:1323-1335[Abstract].
Elder, G.A., Friedrich, V.L., Bosco, P., Kang, C., Gourov, A., Tu, P.-H., Lee, V.M.-Y., Lazzarini, R.A. (1998a) Absence of the mid-sized neurofilament subunit decreases axonal calibers, levels of light neurofilament (NF-L), and neurofilament content. J. Cell Biol. 141:727-739
Elder, G.A., Friedrich, V.L., Kang, C., Bosco, P., Gourov, A., Tu, P.-H., Zhang, B., Lee, V.M.-Y., Lazzarini, R.A. (1998b) Requirement of heavy neurofilament subunit in the development of axons with large calibers. J. Cell Biol. 143:195-205
Friedrich, V.L., Jr., Mugnaini, E. (1981) Preparation of neural tissues for electron microscopy. In Heimer L., Robards M., eds. A Handbook of Neuroanatomical Tract Tracing Techniques. New York, Plenum Publishing Corp, 377-406.
Hirokawa, N., Glicksman, M.A., Willard, M.B. (1984) Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton. J. Cell Biol. 98:1523-1536[Abstract].
Hoffman, P.N., Griffin, J.W., Price, D.L. (1984) Control of axonal caliber by neurofilament transport. J. Cell Biol. 99:705-714[Abstract].
Hoffman, P.N., Griffin, J.W., Gold, B.G., Price, D.L. (1985a) Slowing of neurofilament transport and the radial growth of developing nerve fibers. J. Neurosci. 5:2920-2929[Abstract].
Hoffman, P.N., Thompson, G.W., Griffin, J.W., 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[Abstract].
Hoffman, P.N., Koo, E.H., Muma, N.A., Griffin, J.W., Price, D.L. (1988) Role of neurofilaments in the control of axonal caliber in myelinated nerve fibers. In Lasek R.J., Black M.M., eds. Intrinsic Determinants of Neuronal Form and Function. Vol. 37. New York, Alan R. Liss, 389-402.
Julien, J.-P., Meyer, D., Flavell, D., Hurst, J., Grosveld, F. (1986) Cloning and developmental expression of the murine neurofilament gene family. Mol. Brain Res. 1:243-250.
Lee, M., Xu, Z., Wong, P., Cleveland, D. (1993) Neurofilaments are obligate heteropolymers in vivo. J. Cell Biol. 122:1337-1350[Abstract].
Lee, V.M.-Y., Otvos, L., Jr., Carden, M., Hollosi, M., Dietzschold, B., Lazzarini, R.A. (1988) Identification of the major multiphosphorylation site in mammalian neurofilaments. Proc. Natl. Acad. Sci. USA. 85:1998-2002[Abstract].
Lees, J.F., Shneidman, P.S., Skuntz, S.F., Carden, M.J., Lazzarini, R.A. (1988) The structure and organization of the human heavy neurofilament subunit (NF-H) and the gene encoding it. EMBO (Eur. Mol. Biol. Org.) J. 7:1947-1955[Abstract].
Myers, M.W., Lazzarini, R.A., Lee, V.M.-Y., Schlaepfer, W.W., 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. EMBO (Eur. Mol. Biol. Org.) J. 6:1617-1626[Abstract].
Nakagawa, T., Chen, J., Zhang, Z., Kanai, Y., Hirokawa, N. (1995) The distinct functions of the carboxyl-terminal tail domain of NF-M upon neurofilament assembly: cross-bridge formation and longitudinal elongation of filaments. J. Cell Biol. 129:411-429[Abstract].
Ohara, O., Gahara, Y., Miyake, T., Teraoka, H., Kitamura, T. (1993) Neurofilament deficiency in quail caused by nonsense mutation in neurofilament-L gene. J. Cell Biol. 121:387-395[Abstract].
Peters, A., Vaughn, D. (1981) Central nervous system. In Johnson J.J., ed. Aging and Cell Structure. New York, Plenum Publishing Corp, 1-34.
Rao, M.V., Houseweart, M.K., Williamson, T.L., Crawford, T.O., Folmer, J., Cleveland, D.W. (1998) Neurofilament-dependent radial growth of motor axons and axonal organization of neurofilaments does not require the neurofilament heavy subunit (NF-H) or its phosphorylation. J. Cell Biol. 143:171-181
Spencer, P., Ochoa, J. (1981) The mammalian peripheral nervous system in old age. In Aging and Cell Structure. J.J. Johnson, editor. Plenum Publishing Corp., New York. 35103.
Tu, P.-H., Elder, G., Lazzarini, R.A., Nelson, D., Trojanowski, J.Q., Lee, V.M.-Y. (1995) Overexpression of the human NFM subunit in transgenic mice modifies the level of endogenous NFL and the phosphorylation state of NFH subunits. J. Cell Biol. 129:1629-1640[Abstract].
Vickers, J.C., Morrison, J.H., Friedrich, V.L., Elder, G.A., Perl, D.P., Katz, R.N., Lazzarini, R.A. (1994) Age-associated and cell type specific neurofibrillary pathology in transgenic mice expressing the human midsized neurofilament subunit. J. Neurosci. 14:5603-5612[Abstract].
Zhu, Q., Lindenbaum, M., Levavassiur, F., Jacomy, H., Julien, J.-P. (1998) Disruption of the NF-H gene increases axonal microtubule content and velocity of neurofilament transport: relief of axonopathy resulting from the toxin ß,ß'-imminodipropionitrile. J. Cell Biol. 143:183-193