§
* Ludwig Institute for Cancer Research, Division of Cellular and Molecular Medicine, § Department of Neuroscience, University
of California at San Diego, La Jolla, California 92093; and
Department of Neurology, School of Medicine, ¶ School of Hygiene,
Johns Hopkins Medical Institutes, Baltimore, Maryland 21205
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Abstract |
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Neurofilaments are essential for establishment and maintenance of axonal diameter of large myelinated axons, a property that determines the velocity of electrical signal conduction. One prominent model for how neurofilaments specify axonal growth is that the 660-amino acid, heavily phosphorylated tail domain of neurofilament heavy subunit (NF-H) is responsible for neurofilament-dependent structuring of axoplasm through intra-axonal crossbridging between adjacent neurofilaments or to other axonal structures. To test such a role, homologous recombination was used to generate NF-H-null mice. In peripheral motor and sensory axons, absence of NF-H does not significantly affect the number of neurofilaments or axonal elongation or targeting, but it does affect the efficiency of survival of motor and sensory axons. Loss of NF-H caused only a slight reduction in nearest neighbor spacing of neurofilaments and did not affect neurofilament distribution in either large- or small-diameter motor axons. Since postnatal growth of motor axon caliber continues largely unabated in the absence of NF-H, neither interactions mediated by NF-H nor the extensive phosphorylation of it within myelinated axonal segments are essential features of this growth.
Key words: neurofilaments; radial growth; axoplasm; motor neurons; sensory neurons ![]() |
Introduction |
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Aseries of preceding efforts (Friede and Samorajski,
1970; Hoffman et al., 1987
; Cleveland et al., 1991
;
Lee and Cleveland, 1994
) have proven that neurofilaments are essential elements for establishing the correct diameters (and hence volume) of large myelinated
motor and sensory axons. These axons elongate during development with diameters of ~1-2 µm, but after stable
synapse formation in early postnatal life and concomitant
with myelination, they grow markedly in diameter. This
growth continues at a slow rate throughout adulthood, ultimately yielding axons that in humans reach diameters of
up to 14 µm (Kawamura et al., 1981
), corresponding to a
>100-fold increase from their initial volumes. Establishment of axon caliber is of importance for normal functioning of the nervous system since caliber is a principal determinant of the conduction velocity at which nerve impulses
are propagated along the axon (Gasser and Grundfest,
1939
; Arbuthnott et al., 1980
; Sakaguchi et al., 1993
).
Overwhelming evidence has demonstrated that neurofilamentsassembled as obligate heteropolymers (Ching
and Liem, 1993
; Lee et al., 1993
) of three polypeptide subunits, light neurofilament (NF-L; 68 kD),1 mid-sized neurofilament (NF-M; 95 kD), and heavy neurofilament (NF-H; 115 kD)
are essential for establishing the caliber
of large myelinated axons. The initial suggestion of this
arose from the linear relationship between neurofilament
number and axonal cross-sectional area during the phase
of rapid growth in diameter (Friede and Samorajski, 1970
)
and during regrowth after axonal injury (Hoffman et al.,
1987
). The importance of neurofilaments in specifying normal axonal caliber was proven unequivocally by analysis of a recessive mutation (quv) in a Japanese quail that
lacks neurofilaments as the result of a premature translation terminator in the NF-L gene. Radial growth of axons
fails completely in these animals (Yamasaki et al., 1992
;
Ohara et al., 1993
), with a consequent reduction in axonal
conduction velocity and generalized quivering (Sakaguchi
et al., 1993
). This requirement for neurofilaments has been
confirmed in mice both by expression of a NF-H-
-galactosidase fusion protein that completely inhibits neurofilament transport into axons (Eyer and Peterson, 1994
) and
by targeted deletion of the NF-L gene (Zhu et al., 1997
).
In both cases, loss of axonal neurofilaments results in failure of normal radial growth. Moreover, since neurofilaments are obligate heteropolymers of NF-L and substoichiometric levels of NF-M or NF-H (Ching and Liem, 1993
;
Lee et al., 1993
), absence of NF-M caused by gene disruption leads to markedly fewer axonal neurofilaments and a
suppression of radial growth (Elder et al., 1998
).
Although it is clear that neurofilaments are essential for
specifying axonal diameter, the mechanism through which
neurofilaments mediate increases in axonal size remains
unsettled. The linear correlation between neurofilament
number and axonal cross-sectional area initially suggested
that the axon expanded or contracted to maintain a constant density of neurofilaments (Hoffman et al., 1987).
That radial growth was not simply a function of the number of neurofilaments was proven by elevation of wild-type NF-L levels in transgenic mice. This revealed that a
two- to threefold increase in the number of neurofilaments
(with an elevated proportion of the NF-L subunit) actually
decreases axonal diameter slightly (Monteiro et al., 1990
;
Xu et al., 1993
). This led to one attractive and plausible
model for how neurofilaments mediate initial growth and
then maintain it: the long, COOH-terminal tail domains of NF-M and NF-H, which extend from the core of the 10-nm-diameter filament (Hirokawa et al., 1984
; Hisanaga
and Hirokawa, 1988
; Troncoso et al., 1990
), support axonal growth by crossbridging between adjacent neurofilaments or to other axonal constituents, thereby forming a
three-dimensional lattice that determines volume. Consistent with this, transgenic methods that increased filament number (by producing higher levels of NF-L) combined
with higher levels of NF-M or NF-H yielded increased axonal volumes by 30-45% (Xu et al., 1996
).
Superimposed on contributions to radial growth provided by the number and subunit composition of neurofilaments, it is also now clear that the general relationship of
neurofilament content and caliber is apparently regulated
by the relative degree of phosphorylation of NF-M and
NF-H. The tail domain of NF-H is largely composed of a
motif repeated between 43 and 51 times in mammals (Julien et al., 1988; Lees et al., 1988
) containing a central KSP tripeptide, the serine of which is nearly stoichiometrically phosphorylated in myelinated axonal segments
(Julien and Mushynski, 1982
; Carden et al., 1985
; Lee et
al., 1988
). It has been proposed that phosphorylation of
NF-H, and to a lesser extent NF-M, increases the total
negative charge and lateral extension of their side arms
(Glicksman et al., 1987
; Myers et al., 1987
), thereby mediating the increased neurofilament spacing found in myelinated segments and/or increased crossbridging to other
axonal components such as microtubules (Hirokawa,
1982
). Phosphorylation of both NF-H and NF-M tail domains is strongly correlated with radial growth, but the
more extensive repeat domain in NF-H (which in mice
contains 51 KSP repeats versus only 4 for murine NF-M)
has focused most attention on NF-H. In the normal setting, unmyelinated initial axonal segments contain dephosphorylated NF-H and display higher filament density and
much smaller diameters than the adjacent myelinated segments (Hsieh et al., 1994
; Nixon et al., 1994
). Consistent
with this is the finding that a primary defect in myelination (in the Trembler mouse) decreases phosphorylation of
NF-H, increases neurofilament density, and inhibits normal radial growth of axons (de Waegh et al., 1992
). Similarly, deletion of the peripheral myelin-associated glycoprotein, which has been proposed to signal from the
myelinating Schwann cell to the axon, results in reduced neurofilament phosphorylation, decreased neurofilament
spacing, and reduced axonal calibers (Yin et al., 1998
).
These examples show the direct relationship between neurofilament phosphorylation and axonal diameter within
myelinated axonal segments.
That NF-H is a primary component of radial growth has
been supported by strong correlative evidence: increases
in the level of NF-H mRNA is most pronounced during
the earliest phase of radial growth (0-4 wk postnatal)
(Schlaepfer and Bruce, 1990), suggesting its importance in
the process. Doubling NF-M content in transgenic mice
yields a 50% reduction in axonal NF-H and strongly inhibits radial growth, despite a constant level of NF-L (Wong
et al., 1995
). Furthermore, modest increases in NF-H
mildly enhance radial growth in transgenic mice (although
higher levels severely retard growth by slowing transport
and trapping neurofilaments in neuronal cell bodies)
(Marszalek et al., 1996
). These three examples lend support to the idea that NF-H levels modulate radial growth
of axons.
To examine directly the role of NF-H and its phosphorylation in structuring axoplasm and on neurofilament-dependent radial growth of large myelinated axons, we now report the use of homologous recombination to produce mice devoid of NF-H and to document the consequences of chronic absence of NF-H (and its phosphorylation) on elongation and survival of both large and small motor and sensory neurons, organization of neurofilaments and other organelles in those axons, and establishment and maintenance of axonal caliber.
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Materials and Methods |
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Production and Screening of Mice with an NF-H Gene Disruption
A targeting vector for disrupting the mouse NF-H gene using homologous
recombination was constructed (from a mouse NF-H gene cloned from a
129 SVJ library) by inserting a 1.6-kb KpnI-SacI fragment of NF-H (which
lies 1.5-kb 5' to NF-H transcriptional initiation site; see Fig. 1 A) adjacent
to the phosphoglycerol kinase-promoted neomycin gene of plasmid pSK
(Tybulewicz et al., 1991). Next, the 6.7-kb SmaI-EcoRV fragment of the
NF-H gene (containing all three introns and coding sequences from amino
acid 34 through most of the KSP repeats in the tail) was ligated just 3' to
the neomycin gene. Finally, a phosphoglycerol kinase-promoted thymidine kinase gene cassette was ligated 3' to this latter NF-H gene segment to yield a final targeting construct. After linearization with KpnI, the targeting DNA was electroporated into embryonic stem (ES) cells (RI cells
kindly provided by Andreas Nagy, University of Toronto, Toronto, Canada), and cells were selected for resistance to 200 µg/ml G418 and 2 µM
gancyclovir. All cell manipulations were performed as described (Joyner,
1994
). To identify homologous recombinants, drug-resistant colonies were
amplified, and DNA was prepared and digested with HindIII or with
EcoRV, separated on 0.8% agarose gels, and transferred to Hybond N+
filters (Amersham Corp., Arlington Heights, IL). NF-H fragments were
identified by hybridization either with the 1.4-kb EcoRV/AatII fragment
of NF-H gene, which encodes some of the KSP multiphosphorylation repeats and part of the NF-H 3'-flanking region, or with the 5' EcoRI/NdeI
fragment (see Fig. 1). DNA labeling was performed by random primer extension using [
-32P]dATP.
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Targeted ES cell clones were injected into C57BL/6J blastocysts to produce chimeric animals. Chimeric animals from two independent ES cell
clones were bred to C57BL/6J mice, and mice heterozygous for the disruption were identified by analysis of mouse tail DNAs (prepared as before [Monteiro et al., 1990]).
Analysis of Neurofilament RNA Levels in Nervous Tissues
Brains and spinal cords of 5-wk-old animals were dissected from NF-H deletion, heterozygous, and wild-type mice and immediately frozen at
80°C. Total cellular RNA was purified as described (Chomcznski and
Sacchi, 1987
). In brief, tissues were homogenized in 4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7, 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol. Protein was removed by addition of an equal volume of phenol
(in 0.2 M sodium citrate, pH 4) and one-fifth volume of chloroform/
isoamyl alcohol (49:1). After mixing well, samples were centrifuged at
10,000 g for 20 min at 4°C. The RNA in the aqueous phase was precipitated (1 h at
20°C) after addition of an equal volume of isopropanol. The precipitate was collected by centrifugation, dissolved in the homogenization solution, and reprecipitated with isopropanol. Final RNA pellets
were washed with 70% ethanol, dried, and dissolved in 10 mM Tris, pH
7.4, 1 mM EDTA, reextracted with phenol/chloroform, and reprecipitated
with ethanol containing 2.5 M ammonium acetate. Finally, the RNA pellets were dissolved in 0.5% SDS, and RNA concentration was determined
by absorbance at 260 nm.
20 µg of total RNA was fractionated on a 1% agarose gel containing
1.85% formaldehyde, transferred to Hybond N+, baked for 2 h at 80°C,
and prehybridized for 1 h in 0.5 M sodium phosphate, pH 7.2, 1 mM
EDTA, 7% SDS, and 1% bovine albumin. RNAs encoding neurofilament
subunits were detected by hybridization with random-primed [-32P]-
dATP-labeled nucleotide probe. Probe to detect NF-H RNA was the
EcoRV-AatII fragment of the NF-H gene (containing sequences for
codons 798-1087). Probe for NF-M was an AccI-EcoRI fragment of the
NF-M gene containing the complete third exon. Probe for NF-L RNA was
a complete NF-L cDNA. For identifying the mRNA encoding type III
-tubulin, sequences corresponding to the final 11 codons and the 3' untranslated region were used as a probe (from mouse EST No. 352879). Filters were then washed in 30 mM Tris, pH 7.4, 300 mM NaCl, for 20 min at
room temperature, followed by two washes (30 min each at 60°C) in 3 mM
Tris, pH 7.4, 30 mM NaCl, 0.5% SDS. RNA bands corresponding to different neurofilament subunits were visualized by autoradiography using
x-ray film (Biomax MS; Eastman Kodak Corp., Rochester, NY) and quantified by phosphorimaging (Molecular Dynamics, Sunnyvale, CA).
SDS-PAGE and Detection of Neurofilament Proteins by Immunoblotting
Sciatic nerves, spinal cords, and brains were homogenized on ice in a
buffer containing 50 mM Tris, pH 7.5, 0.5 mM EDTA, pH 8, and 1 mM
each of PMSF, leupeptin, aprotinin, and chymostatin. An equal volume of
a solution containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 1%
sodium deoxycholate, and 2% SDS was added, and the homogenates were
sonicated for 20 s, boiled for 10 min, and clarified by centrifugation at
16,000 g for 5 min. Protein concentration was determined using the bicinchoninic acid (BCA) assay kit (Pierce Chemical Co., Rockford, IL). Protein extracts, as well as known amounts of neurofilament standards, were
separated by SDS-PAGE using gels containing 6 or 7.5% polyacrylamide.
Proteins separated by SDS-PAGE were transferred to nitrocellulose (Lopata and Cleveland, 1987). The NF-H subunit was identified using an affinity-purified rabbit polyclonal antibody (pAb-NF-HCOOH) prepared to
the COOH-terminal 12 amino acids of mouse NF-H (Xu et al., 1993
), followed by 125I-conjugated protein A. The NF-L subunit was identified using an affinity-purified rabbit polyclonal antibody (pAb-NF-LCOOH) prepared to the COOH-terminal 12 amino acids of mouse NF-L (Xu et al.,
1993
), followed by 125I-conjugated protein A. mAbs to phosphorylated
epitopes in NF-H and NF-M (SMI-31; Sternberger and Sternberger,
1983
), nonphosphorylated NF-H (SMI-32; Sternberger and Sternberger,
1983
), NF-M (RM044; Tu et al., 1995
),
-tubulin (DM1A; Sigma Chemical
Co., St. Louis, MO), or the neuron-specific, class III
-tubulin (TuJ1; Lee
et al., 1990
) were used to identify each subunit, followed by rabbit anti-
mouse IgG (Sigma Chemical Co.) and 125I-conjugated protein A. For plectin, polyclonal antisera (P21; Wiche and Baker, 1982
) and mAb 10F6
(Foisner et al., 1991
) were used as primary antibodies, and binding was detected with goat anti-rabbit and goat anti-mouse secondary antibodies
linked to alkaline phosphatase. Immunoreactive bands were visualized by autoradiography using Kodak Biomax MS film. Quantification was performed by phosphorimaging (Molecular Dynamics) using known amounts
of purified mouse spinal cord neurofilaments as standards.
Tissue Preparation and Morphological Analysis
Mice were perfused transcardially with 4% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M sodium phosphate, pH 7.6, and postfixed overnight in the same buffer. Samples were treated with 2% osmium tetroxide, washed, dehydrated, and embedded in Epon-Araldite resin. Thick sections (0.75 µm) for light microscopy were stained with toluidine blue, and thin sections (70 nm) for electron microscopy were stained with uranyl acetate and lead citrate.
Axons were counted in L5 root cross sections from three to four mice of each genotype and of each age group. Axon diameters from two animals of each genotype and age were measured using the Integrated Morphometry Analysis function from the Image 1/Metamorph Imaging System (Universal Imaging Corp., West Chester, PA). Entire roots were imaged, imaging thresholds were selected individually, and the cross sectional area of each axon was calculated and reported as a diameter of a circle of equivalent area. Axon diameters were grouped into 0.5-µm bins.
Analysis of Filament Spacing: Nearest Neighbor Analysis
To measure nearest neighbor distances between neurofilaments, cross sections of axons larger than 3.0 µm in diameter were photographed at a magnification of 20,000 and enlarged an additional 4.25-fold by printing. Neurofilaments were identified in these end-on views as dots ~10 nm in diameter. Positions of neurofilaments were marked by puncturing the print with a push-pin. By laying the final prints on a light box, neurofilament positions could easily be imaged, and nearest neighbor distances were calculated for each filament using a digital imaging program (Bioquant, Nashville, TN).
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Results |
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Mice Producing no NF-H mRNA or Protein Are Viable, with Elevated Levels of NF-M and Tubulin
To generate mice homozygously deleted for the NF-H gene, a targeting vector was constructed from the mouse NF-H gene (cloned from a mouse 129 SVJ library) by replacing the 1.6-kb segment containing the proximal promoter, methionine initiation codon, and 33 additional codons of the amino-terminal region of NF-H with a neomycin resistance gene (Fig. 1 A; see also Materials and Methods). After electroporation into ES cells and selection for integration by resistance to the neomycin homologue G418, cells in which one NF-H allele was targeted were identified by genomic DNA blotting. With probes corresponding to the gene segments just 5' and 3' to that contained in the gene targeting vector, this revealed that 2 of 70 lines examined had been correctly targeted at both 5' (Fig. 1 C) and 3' (Fig. 1 B) sites of integration. Both clones were injected into C57BL/6J blastocysts, and both lines produced chimeric animals that when mated to C57BL/6J mice transmitted the disrupted allele to their progeny, as revealed by blotting of genomic tail DNAs from such animals (Fig. 1 D).
Mating of heterozygotes to each other produced animals with both NF-H genes disrupted, as well as littermates that retained one or both normal NF-H alleles in the 1:2:1 ratios expected for Mendelian inheritance. Mice with both disrupted genes were viable and fertile, displaying no overt phenotype, at least up to 6 mo of age. To establish that the disruption eliminated NF-H expression, RNA was prepared from brain and spinal cords of animals with NF-H alleles that were normal or heterozygously or homozygously disrupted. Blotting with a probe corresponding to codons 798 to 1087 of mouse NF-H revealed the complete absence of stable mRNAs carrying NF-H coding sequences in the NF-H-deleted mice (Fig. 1 E, lanes 5 and 6). Relative to ribosomal RNA, phosphorimaging demonstrated that mRNAs coding for NF-L and NF-M were not markedly affected by presence or absence of NF-H mRNA (Fig. 1 E, lanes 1-6).
Immunoblotting with an antibody (Xu et al., 1993)
raised to the carboxy terminus of NF-H (Fig. 2 B) or antibodies that recognize either the unphosphorylated NF-H
tail domain (SMI-32; Fig. 2 D) or the same domain when
phosphorylated (SMI-31; Fig. 2 C) confirmed the complete absence of NF-H in extracts from brain, spinal cords, and sciatic nerves of mice with two disrupted alleles (Fig.
2, B-D, lanes 3, 6, and 9). Animals heterozygous for the
disruption had intermediate levels of NF-H, which phosphorimaging revealed to be between ~60-70% of the
level of control samples in brain and sciatic nerves. Parallel immunoblots demonstrated that NF-L levels were unaffected by the presence or absence of NF-H in spinal cord
and sciatic nerve extracts from all three genotypes (Fig. 2
F). In contrast, use of an antibody insensitive to the phosphorylation state of NF-M (RMO44; Tu et al., 1995
) demonstrated that NF-M levels were elevated after diminution
or elimination of NF-H, with phosphorimaging revealing a
twofold increase, relative to normal mice, in spinal cords
(compare Fig. 2 E, lanes 4 and 6) and sciatic nerves (compare Fig. 2 E, lanes 7 and 9) but not brain of NF-H-disrupted animals. These findings provide additional evidence that in lower motor and sensory neurons, NF-M and
NF-H compete in vivo for coassembly with a limiting
amount of NF-L (Wong et al., 1995
; Marszalek et al., 1996
;
Elder et al., 1998
). Using antibody SMI-31 (Sternberger
and Sternberger, 1983
) to detect a phosphorylated determinant on NF-M (Fig. 2 C) further revealed that, in the
absence of NF-H, NF-M phosphorylation levels increase in spinal cord and sciatic nerve. Phosphorimaging demonstrated that the increased phosphorylation quantitatively
matched the increased NF-M subunit abundance; therefore, the proportion of NF-M that is phosphorylated remains unchanged by the loss of NF-H.
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Diminution or loss of NF-H was also accompanied in
sciatic nerve (and to a lesser extent spinal cord) by a significant increase in total tubulin levels, as measured by antibodies that react with all -tubulin (Fig. 2 G) or
-tubulin
(not shown) isotypes. Measuring the content of the class
III, neuron-specific
-tubulin isotype that comprises ~25%
of
-tubulin in normal nervous tissue (Lopata and Cleveland, 1987
) further revealed that the 60% elevation in total
tubulin levels seen in nerve samples (Fig. 2 G) was at least
in part the result of a selective (fourfold) increase in this
neuron-specific isoform (Fig. 2 H). Mechanistically, this increase in tubulin accumulation was due to an approximately twofold elevation in the
III-tubulin mRNA (85%
increase measured by phosphorimaging) in the NF-H-free
animals (Fig. 1 E).
No significant change in the level of the large cytoskeletal cross-linker protein plectin was observed in spinal cord
and nerve samples from NF-H-free mice compared with
normal littermates (Fig. 2 I, compare lanes 4 and 6, 7 and
9). Regardless of the genotype, full-length plectin (~500
kD) was seen in brain and spinal cord samples, while two
plectin antibodies revealed a stable accumulation product
that migrated with an apparent size of less than 200 kD in
all peripheral nerve samples. (Note that lanes 7-9 of Fig. 2
I represent a lower molecular weight portion of the immunoblot than lanes 1-6.) Since it is retained in neurofilament-enriched nerve fractions, this plectin fragment (or
isoform or plectin-related molecule) most probably contains a neurofilament-binding site (such as that found near
the carboxy terminus of typical plectin [Elliott et al.,
1997]).
Absence of NF-H Has Surprisingly Little Effect on Radial Growth of Motor Axons
Since neurofilaments are known to be a principal determinant of axonal caliber (Hoffman et al., 1987; Yamasaki et
al., 1992
; Ohara et al., 1993
; Zhu et al., 1997
) and NF-H
phosphorylation has been tightly linked to neurofilament-dependent control of axon diameter (de Waegh et al.,
1992
; Hsieh et al., 1994
; Nixon et al., 1994
), we examined
how the changes in NF-H content were reflected in
changes in axonal caliber of large myelinated motor axons.
Light microscopic inspection of the L5 ventral roots (Fig. 3
A) revealed the surprising finding that myelinated axons
from wild-type mice, mice heterozygous for NF-H, or mice
with both gene copies disrupted were qualitatively similar
in caliber to those of wild-type mice (compare Fig. 3 A,
left, middle, and right panels).
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To quantify potential caliber changes associated with loss of NF-H expression, cross-sectional areas of every axon within each ventral root (Fig. 4) were measured from 4- and 9-wk-old animals (two animals of each genotype at each time point). Each area was then converted to a diameter corresponding to a circle of the equivalent area. As expected, motor axons of wild-type animals showed a typical bimodal distribution of diameters representing the small and large myelinated axons, with initial diameters centered on 1.5 and 5 µm. Both small and large axons grow considerably in size between 4 and 9 wk of age, achieving diameters of 2.5-3 and 8.5 µm, respectively. Summing the areas of all axons after this 5-wk growth period revealed a threefold increase in axonal volume of the larger axons and a doubling of size for the smaller size class. This bimodal axon size distribution and growth phase was also observed in mice heterozygous or homozygous for the NF-H deletion, although at both time points reducing or deleting NF-H clearly decreased or eliminated the very largest diameter axons (e.g., those larger than 9 µm in Fig. 4, E and F) and shifted the distributions toward smaller sizes (Fig. 4, E and F, arrows). However, by 9 wk of age, loss of NF-H resulted in axonal populations of both large and small motor axons that were only slightly (25 and 20%, respectively) reduced in total axon area relative to wild-type animals. Similar findings were also seen in 15-wk-old animals. Additional radial growth of ~20% continued to equivalent extents in both normal and NF-H-null animals, with a similar loss of the largest population of motor axons found in 15-wk-old NF-H-deleted animals (not shown). Thus, for motor axons, NF-H content (and any interactions provided by it or its long tail domain) are dispensable for the majority of radial growth, despite the fact that it is necessary for achieving the largest diameters.
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Radial Growth of Sensory Axons Requires NF-H
To examine how NF-H contributes to radial growth of sensory axons, calibers were measured in L5 dorsal roots of wild type and animals heterozygous or homozygous for NF-H disruption. While there are many small sensory axons that do not undergo radial growth, light microscopic examination revealed an apparent inhibition of growth of the largest caliber axons (Fig. 3 A). Comparing size distributions of 4- and 9-wk-old wild-type animals demonstrated that radial growth of both larger and smaller axons occurs (Fig. 5 A). For animals with reduced (Fig. 5 B) or no (Fig. 5 C) NF-H content, growth of the smallest axons was eliminated, while growth of the largest class (initially >4 µm) continued, but at a reduced level compared with normal mice, especially for the largest axons (>8 µm in normal mice). Similar findings emerged from analysis of 15-wk-old animals (not shown).
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NF-H Is Required for Survival of the Normal Number of Motor and Sensory Axons
The complete absence of neurofilaments (arising from disruption of the NF-L gene) during initial axon elongation,
targeting, and myelination has been demonstrated to yield
a 13% reduction in the survival of initial motor axons
(Zhu et al., 1997). To determine whether NF-H content affects this developmental survival of motor axons, axons
were counted in the L5 ventral and sensory roots of wild-type mice and mice with one or both copies of the NF-H
gene disrupted. After the initial burst of radial growth, this
quantitation revealed that by 9 wk of age, the total number of surviving motor axons was ~13% lower (n = 6, P = 0.03) in the absence of NF-H than in its presence (Fig. 3
B). This reflects a preferential loss of large (>4 µm in diameter) axons with no significant change in the number of
small axons. A similar trend was seen when the sensory axons were counted (Fig. 3 C). Again, the number of large-
but not small-caliber axons was reduced by ~19% (n = 6, P = 0.03).
Nearest Neighbor Spacing between Neurofilaments Is Unaffected by NF-H Content
To examine whether reduction in NF-H content, and the
corresponding increase in axonal NF-M, affects neurofilament organization in axons, the nearest neighbor spacing
between neurofilaments in cross sections of ventral roots
was compared in 9-wk-old wild-type animals and littermates that were heterozygously and homozygously deleted for the NF-H gene. Qualitative inspection of electron micrographs from wild-type versus NF-H-deleted animals
revealed no consistent differences in neurofilament spacing (compare Fig. 6, A-C), a point reinforced by marking
neurofilament positions in ventral root axons (n = 9, including axons ranging in diameters from 4 to 8 µm) and
calculating nearest neighbor spacings. As seen previously
(Hsieh et al., 1994; Wong et al., 1995
; Marszalek et al.,
1996
; Xu et al., 1996
), measurement of nearest neighbor distances revealed a broad distribution centered on a 45-nm spacing in wild-type mice. Examination of all distances
from 10 axons revealed that neither reduction of NF-H
nor its complete absence markedly affected the distributions of nearest neighbor distances (Fig. 6 D). A different
measure, comparing the mean nearest neighbor distance
calculated axon by axon, again revealed very similar average filament spacing, with only a possible shift (P = 0.05 using a two-tail t test) toward very slightly smaller distances in the NF-H-null axons compared with normal axons (Fig. 6 E). This pattern of filament-filament distances
was confirmed by examination of overall axonal organization in longitudinal sections of motor axons. Although less
organized axoplasm in the NF-H deficient might have
been anticipated, especially in light of the demonstration that dephosphorylated NF-H directly binds to microtubules in vitro (Hisanaga and Hirokawa, 1990
; Hisanaga et
al., 1991
; Miyasaka et al., 1993
), this was not the case. The
absence of NF-H-dependent interactions between adjacent neurofilaments or between neurofilaments and microtubules did not diminish the "straightness" of neurofilaments or their overall orientation along the long axis of
the axon.
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Despite the unchanged spacing and organization of neurofilaments, one difference in axonal organization was apparent by inspection: microtubule density was higher in
the animals with diminished or no NF-H. Counting microtubules in axons of each genotype revealed a 60% increased
density of microtubules in axons from NF-H-deleted animals (Fig. 6 F). A two-tailed paired t test revealed this difference to be highly significant (P < 0.001). The increase
in microtubule number quantitatively corresponds to the
similar increase in tubulin content seen in both the spinal
cord and sciatic nerve homogenates of NF-H-free animals
(Fig. 2, G and H, lanes 6 and 9). In light of the known induction of tubulin expression by nearly an order of magnitude during axonal regeneration (Hoffman et al., 1987) and the reduction in initial survival of motor and sensory
axons in the NF-H-null mice, this increase in tubulin levels
may reflect partial activation of a regenerative program.
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Discussion |
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Several lines of preceding evidence had implicated a primary role for NF-H in crossbridging between adjacent
neurofilaments or between neurofilaments and other axonal structures (Hirokawa et al., 1984; Marszalek et al.,
1996
; Xu et al., 1996
) to promote and maintain axonal caliber. The present analysis of mice with complete absence of
NF-H provides unambiguous evidence that neither NF-H
nor the nearly stoichiometric phosphorylation of its long, repetitive tail domain in the myelinated, internodal axonal
segments are essential features for most of the radial
growth of large motor axons. Moreover, neither neurofilament number nor the organization of those neurofilaments (e.g., nearest neighbor distances or longitudinal orientations within domains of axoplasm) are altered by the
absence of NF-H. As in previous examples (Wong et al.,
1995
; Marszalek et al., 1996
; Xu et al., 1996
), NF-M levels
do undergo a compensatory increase in abundance without NF-H, adding further evidence that NF-M and NF-H
compete for assembly with a limiting amount of NF-L. Perhaps the most surprising feature of these findings is
that they force a reinterpretation of what seemed to be
compelling correlative evidence linking a signaling cascade initiated in the myelinating cell as a trigger for nearly
stoichiometric phosphorylation of the NF-H and NF-M
KSP repeats in myelinated axonal segments to a corresponding increase in neurofilament spacing (de Waegh et
al., 1992
; Yin et al., 1998
). It is now clear that absence of
the nearly 25-50 phosphates added to NF-H by this cascade does not markedly affect neurofilament spacing in internodes nor does it affect the preponderance of radial
growth, at least of motor axons.
While NF-H and its phosphorylation are not as essential
as had been anticipated, it should be emphasized that the
collective evidence still supports a role for NF-H in radial
growth, certainly an important one in sensory axons and
probably a redundant one in motor axons. Indeed, prior
work with transgenic mice expressing elevated levels of
wild-type NF-L (Monteiro et al., 1990), NF-H (Marszalek
et al., 1996
; Xu et al., 1996
), or epitope-tagged NF-M (Wong et al., 1995
) has demonstrated that an excess of any
subunit inhibits radial growth to a greater degree than
does absence of NF-H, while increasing both NF-L
(thereby assembling more filaments) together with either
NF-M or NF-H stimulates radial growth (Xu et al., 1996
).
This lead to the view that radial growth requires contributions from all three subunits: NF-L to support filament assembly and a scaffolding of NF-M and NF-H tails to
crossbridge between neurofilaments and/or other axonal
components (Hisanaga and Hirokawa, 1990
; Xu et al.,
1996
). What is clear now, however, is that changing the
content of NF-H (with a corresponding elevation in NF-M)
does not affect nearest neighbor spacing of neurofilaments, a finding that goes hand in hand with the identical
situation after doubling axonal NF-M and a corresponding
loss in NF-H (Wong et al., 1995
).
The current evidence, along with two earlier efforts
(Wong et al., 1995; Xu et al., 1996
), implicates NF-M (see
schematic in Fig. 7) as the subunit that specifies nearest
neighbor spacing mediated by its 439-amino acid tail that
can extend 30-40 nm from the surface of the filament
(Hisanaga and Hirokawa, 1988
). Indeed, such crossbridges
have been observed after simultaneous expression of NF-L
and NF-M (using baculovirus) in insect cells (Nakagawa et
al., 1995
). Schwann cell-dependent phosphorylation of the
KSP repeats in the NF-M tail may lengthen the effective
cross-link, so as to force wider filament spacing, a feature
potentially necessary, but not sufficient, for radial growth.
It must be noted, however, that since nearest neighbor
neurofilament spacing is also almost unchanged by the
complete absence of NF-M (Elder et al., 1998
), the simplest interpretation is that multiple, redundant elements (including NF-M) establish this spacing.
|
Whatever the interaction between neurofilaments, it
must be an attractive one, not simply a repulsion of the
negatively charged tails, since even in mice with few axonal neurofilaments (as a consequence of disruption of the
NF-M gene [Elder et al., 1998] or trapping most neurofilaments in the cell bodies by expressing high levels of NF-H
[Marszalek et al., 1996
] or NF-M [Wong et al., 1995
]), the
few axonal filaments are found in clusters with spacings
identical to those in normal mice (Xu et al., 1996
). Moreover, since radial growth can be markedly inhibited despite no change in interfilament distance, this gives further
weight to the argument that interactions between nearest
neighbor filaments do not specify radial growth.
Although it appears unlikely that nearest neighbor filament spacing is a key determinant of radial growth, a great
deal of previous evidence still argues for the involvement
of neurofilaments in some other manner. The key properties needed to stimulate this growth must include longer-range interactions between neurofilaments that are not
nearest neighbors or between neurofilaments and other axonal components. Attractive candidates for these additional interactions include the family of proteins such as
BPAG1n/dystonin, proven recently to be a crossbridger
between neurofilaments and actin filaments (Yang et al.,
1996) that is essential for survival of sensory neurons
(Brown et al., 1995
; Guo et al., 1995
). BPAG1n/dystonin, while expressed at highest levels in sensory neurons, is also found in embryonic and postnatal motor axons (Dowling
et al., 1997
), and absence of it leads both to rampant sensory axon degeneration with more modest numbers of motor axon abnormalities, including degeneration (Dowling
et al., 1997
). Another known cross-linker with binding
sites for intermediate filaments, actin filaments, and microtubules is plectin (Wiche, 1989
), a very large (466-kD), essential (Andra et al., 1997
) protein expressed in many
neurons, including motor neurons (Errante et al., 1994
).
While we have not been able to measure BPAG1n/dystonin levels as a function of NF-H content, plectin levels do
not change in the absence of NF-H, a finding consistent
with plectin as one probable linking component in motor
axons that acts in place of NF-H in mediating the bulk
of neurofilament-dependent radial growth. However, we found only a plectin fragment (or much smaller isoform or
plectin-related polypeptide) to accumulate in peripheral
nerves. Definition of precisely what aspects of cross-linking are contributed by this shortened plectin will have
to await determination of whether it oligomerizes and
whether it retains the actin-binding domain normally situated near plectin's amino terminus and/or a microtubule-binding domain (whose position is not yet mapped within
plectin).
Seen from this perspective, a combination of cross-linkers, including NF-H and NF-M, along with members of the
plectin and BPAG1n families, interlink between neurofilaments, microtubules, and cortical actin arrays to determine a three-dimensional, space-filling array of connected
structural elements that expand axonal volume. Contacts
mediated by NF-H are largely, but not completely, dispensable in motor neurons and may, in fact, be compensated by additional interactions mediated by the increased
number of microtubules and additional NF-M. This leads
to a model (Fig. 7) for structuring axoplasm using elements with overlapping function, with each neuronal type
relying upon a different balance of individual components. These findings, along with the discovery of mutations in
plectin as the primary cause of one form of muscular dystrophy accompanied by defects in skin and cardiac cell organization (Gache et al., 1996; Andra et al., 1997
; Smith et
al., 1997
), reinforce the essential nature of correct structuring of cytoplasm through establishment and maintenance
of a flexible, deformable scaffold of interlinked cytoskeletal elements.
![]() |
Footnotes |
---|
Address correspondence to D.W. Cleveland, Ludwig Institute for Cancer Research, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093. Tel.: (619) 534-7811. Fax: (619) 534-7659. E-mail: dcleveland{at}ucsd.edu
Received for publication 4 May 1998 and in revised form 31 July 1998.
We thank Dr.Virginia Lee (University of Pennsylvania, Philadelphia, PA) for providing antibodies to NF-M and Dr. Gerhard Wiche (University of Vienna, Vienna, Austria) for antibodies to plectin. We also thank Ms. Karen Anderson for her expert assistance with some aspects of the microscopy and Mr. Scott Anderson for his assistance with mouse husbandry and genotyping.
This work has been supported by grant NS27036 from the National Institutes of Health to D.W. Cleveland. Salary support for D.W. Cleveland was provided by the Ludwig Institute for Cancer Research. T.L. Williamson was supported, in part, by a postdoctoral fellowship the Muscular Dystrophy Association.
![]() |
Abbreviations used in this paper |
---|
ES, embryonic stem; NF-H, NF-M, and NF-L, heavy, mid-sized, and light neurofilament subunits.
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